SUNDAY-LATE POSTER PRESENTATIONS
LATE POSTER PRESENTATIONS
SUNDAY, DECEMBER 16
Microtubule Cytoskeleton
2539
What makes a good microtubule nucleator? γ-Tubulin is only half the story.
M. Moritz1, J. Kollman2, D. A. Agard3; 1Biochemistry & Biophysics, UCSF, San Francisco, CA,
2
Anatomy & Cell Biology, McGill University, Montreal, QC, Canada, 3Biochemistry & Biophysics,
UCSF, HHMI, San Francisco, CA
γ-Tubulin nucleates microtubules (MTs) in all eukaryotes and acts in complex with an array of
related accessory proteins. It exists in at least two types of complexes inside cells: a ~300kDa
γ-Tubulin Small Complex (γTuSC), which can assemble to form a ~2.2MDa γ-Tubulin Ring
Complex (γTuRC). In vitro MT nucleation assays have shown that the γTuRC is a more potent
nucleator than γTuSC. Interestingly, pure human γ-tubulin can form filaments in vitro that are
also much more active than γTuSC. We have examined the structure-function relationship
between the configuration of γ-tubulins in these complexes and their ability to nucleate MTs
using high-resolution electron microscopic reconstructions of the complexes and in vitro MT
nucleation assays. We find that complexes in which the γ-tubulins make lateral contacts like
those made by αβ-tubulins in the MT wall are the best nucleators. Our finding that the γTuSC
(Tub4 complex) of S. cerevisiae can assemble spontaneously into γTuRC-like rings with 13-fold
symmetry, but without a precise MT-lattice-like arrangement of γ-tubulins, suggested a mode of
regulation for γTuRC activity. The flexibility observed in one of the "arms" of the V-shaped
γTuSC suggests that a conformational change is required to bring the γ-tubulins into an optimal
position for achieving MT nucleation. We are exploring this possibility. We have also found that
the S. cerevisiae γ-tubulin ring complexes show much greater activity with S. cerevisiae αβtubulin than with pig brain tubulin.
2540
Labeling Tubulin with Fluorescent Probes in Live Cells.
K. Mukherjee1, D. L. Sackett2, S. L. Bane1; 1Chemistry, Binghamton University, Binghamton, NY,
2
Program in Physical Biology, NIH, Bethesda, MD
Currently available methods for labeling tubulin in live cells involve recombinant protein
expression, microinjection or the use of taxol-linked probes. Here, we present a technique that
can be employed to visualize tubulin in live cells without genetic manipulation or perturbing the
protein function. Specifically, this technique exploits the mechanism of tyrosination/
detyrosination cycle, a posttranslational modification specific to the C-terminus of α- tubulin, to
covalently attach a chemical probe to it. Cells are first grown in medium supplemented with 3formyltyrosine (3fY). 3fY readily enters the cell and is appended to the C-terminus of α-tubulin
by endogenous tubulin tyrosine ligase. Cells are then treated with a suitably derivatized
fluorophore. Covalent bond formation between the protein bound aldehyde and the reactive
fluorophore takes place instantly in the intracellular milieu. This reaction is accompanied by an
increase in fluorescence that makes this highly dynamic protein suitable for visualization.
Interestingly, 24 hour treatment with 3fY followed by treatment with a fluorophore does not alter
the microtubule network or cell morphology. Thus we present an efficient labeling technique that
SUNDAY-LATE POSTER PRESENTATIONS
can aid in probing this cytoskeletal protein. This approach may also be the basis for site-specific
fluorescent labeling of other proteins in live cells.
2541
A systems analysis of microtubule regulation by +TIP proteins in fission yeast
I. Flor-Parra1, P-J. Ko1, K. Z. Pan1, F. Chang1; 1Microbiology and Immunology, Columbia
University, New York, NY
The dynamic behaviors of microtubules are regulated by a large number of +TIP proteins at
their plus ends. S. pombe is an attractive model organism for studying the MT cytoskeleton in
vivo, as the MT cytoskeleton is relatively simple and amenable for quantitative analysis of MT
dynamics. In contrast to the large number of diverse +TIPs identified in mammalian cells, only
eight +TIPs are known in fission yeast: Mal3 (EB1), Tip1 (CLIP170), Alp14 (XMAP215), Tea2
(kinesin-7), Klp2 (kinesin-14) and Klp5/6 (kinesin-8) and Tea1 and Tea 4 (cell polarity
regulators). Although the +TIPs have been studied individually, still little is known about how all
these proteins work collectively together at the plus end in vivo. Here, we are pursuing a
quantitative systems approach to study all of these eight +TIPs as a group, in order to
understand how they localize and function in MT regulation in vivo. All single null mutants are
viable, allowing us to study true null mutants. Protein localization is assayed using functional
GFP fusions expressed at near endogenous levels. We have analyzed all the single and double
mutant combinations for their effects on MT dynamics. We have also analyzed the effects of
each gene on localization of all the other +TIPs. Our current results suggest that the +TIPs can
be divided into three modules: an EB1 group, an XMAP215 group, and a Kinesin-18 group. MT
growth is strictly dependent on non-overlapping functions of EB1 and XMAP215. Using
quantitative fluorescence intensity measurements, we find that each MT plus end is coated with
50-150 molecules of each +TIP, or about 1000 +TIP molecules total. These studies promise to
provide a framework for studying the in vivo functions of +TIP proteins.
2542
Calcium-dependent reorganization of microtubules in bone marrow mast cells.
Z. Hájková1, V. Sulimenko1, P. Dráber1; 1Laboratory of Biology of Cytoskeleton, Institute of
Molecular Genetics ASCR, Prague, Czech Republic
The key event in an allergic response and inflammation is mast cell activation mediated by the
high-affinity receptor for IgE (FcεRI). Activation of bone marrow-derived mast cells (BMMCs)
leads to changes in the location of microtubules and their accumulation in the cell periphery (J.
Cell Biol. 170: 115, 2005; J. Immunol. 176: 7243, 2006). Recently we found that BMMC
activation stimulates generation of Ca2+-dependent microtubule protrusions (J. Immunol. 186:
913, 2011); however, the molecular mechanisms controlling microtubule rearrangement during
BMMC activation are still unknown. Here we report that Ca2+, through binding to calcium-binding
messenger protein calmodulin (CaM), regulates generation of microtubule protrusions. In
addition, nocodazole wash-out experiments with subsequent quantitative analysis revealed that
microtubule nucleation from centrosome is Ca2+-dependent. Enhanced levels of Ca2+ modulate
interactions of γ-tubulin with γ-tubulin complex proteins GCP2 and GCP4. This might be
important for regulation of microtubule nucleation. Collectively, our data indicate that Ca2+ plays
an important role in the regulation of microtubule (+)-end dynamics and microtubule nucleation
in BMMCs.
SUNDAY-LATE POSTER PRESENTATIONS
2543
Defining the molecular consequences of disease-causing mutations in tubulin using the
model S. cerevisiae.
E. R. Murphy1, A. Luchniak2, C. Baumer1, M. Gupta1; 1Molecular Genetics and Cell Biology,
University of Chicago, Chicago, IL, 2Biochemistry and Molecular Biology, University of Chicago,
Chicago, IL
Microtubules (MTs) are dynamic cytoskeletal filaments that are comprised of α- and β-tubulin
heterodimers. MTs are essential for proper brain and nervous system development and play
critical roles during neuronal cell migration and axon growth. Humans have eight isotypes each
of α- and β-tubulins but the functional significance of these remains unclear. An expanding set
of mutations in the human α-tubulin, TUBA1A, and β-tubulin, TUBB2B, produce disorders
associated with defects in neuronal migration; while mutations in β-tubulin, TUBB3, cause
neurological disabilities characterized by axon guidance and/or maintenance defects,
suggesting process specific functions for these tubulins. However, this paradigm was
challenged by discovery of alternate mutations in TUBB3 that produce neuronal migration
defects reminiscent of those resulting from mutations in TUBA1A and TUBB2B. In order to
understand the molecular etiology of these disorders we characterized the effects of disease
mutations on MT function in vivo using S. cerevisiae. The budding yeast provides an excellent
model system because it contains only two α- and a single β-tubulin isotype in which the
disease-causing regions/residues are highly conserved. Heterozygous mutations were
introduced into diploid cells where, strikingly, all mutations analyzed resulted in increased MT
stability, suggesting they convey dominant effects on MT function, whereas heterozygous
tubulin deletion destabilized MTs. The majority of β-tubulin mutations that disrupt axon guidance
and/or maintenance processes, when modeled in haploid yeast, did not support viability as the
sole source of β-tubulin. In contrast, each mutation associated with abnormal neuronal migration
tested to date supported haploid viability. Additionally, these haploid cells continued to display
stabilized MTs. Together these results demonstrate the mutations do not cause complete loss of
function and likely impart dominant effects on MT stability. Further comparisons of disease
causing mutations are ongoing.
2544
Spatial Regulation of Microtubule Dynamics During Spindle Position Checkpoint
Activation.
Y. Fukuda1, E. Murphy1, M. Gupta1; 1The University of Chicago, Chicago, IL
Microtubules are polar tubulin polymers that undergo dynamic instability, with alternating phases
of growth and shrinkage. The dynamic properties of microtubules are spatially and temporally
regulated to function in various cellular processes. In the budding yeast Saccharomyces
cerevisiae, astral microtubules (aMTs) play a pivotal role in mitotic spindle alignment along the
mother-bud axis, which is essential because cytokinesis will occur at the bud neck. The viability
of cells with misaligned spindles depends on the spindle position checkpoint (SPOC), which
inhibits mitotic exit until spindle alignment is achieved. This SPOC-induced mitotic arrest is
dependent on interactions between aMTs and the bud neck. The average lifetime of yeast aMTs
is <4 minutes. Strikingly we found that the lifetime of aMTs in the bud of cells with misaligned
spindles is on the order of 90 minutes or more, which is consistent with the duration of SPOCinduced mitotic arrest. Interestingly the average lifetime of mother aMTs in the same cells was
<3 minutes. This dramatic increase in lifetime was achieved largely through increased
rescue/catastrophe ratio, from ~0.5 in the mother to 1 in the bud compartment. To maintain
stable bud aMTs of appropriate length the kinesin Kip3 functions as a cortical-associated
depolymerase and may also contribute to aMT stability. Here we present a working model in
SUNDAY-LATE POSTER PRESENTATIONS
which Kip3 and a yet unidentified rescue factor function cooperatively to spatially regulate the
length and lifetime of aMTs to both delay mitotic exit and achieve spindle alignment.
2545
The XMAP215 family drives microtubule polymerization using a structurally diverse TOG
array.
J. N. Campbell1, K. C. Slep2; 1Biochemistry and Biophysics, University of North Carolina, Chapel
Hill, NC, 2Biology, University of North Carolina, Chapel Hill, NC
Microtubules (MTs) are dynamic cytoskeletal polymers. Misregulation of MT dynamics leads to
catastrophic events in the cell including genomic instability and cell death. XMAP215 family
proteins are key regulators of microtubule dynamics, specifically promoting MT polymerization.
Higher eukaryotic members of the XMAP215 family consist of five arrayed TOG domains. TOG
domain structures reveal a series of six HEAT repeats that form an oblong structure. While TOG
domain structures determined to date have yielded insight into the XMAP215 mechanism, how
XMAP215 family members promote MT dynamics remains an outstanding question. In this
study we investigate how TOG domains promote MT polymerization, comparing the functional
and structural properties of XMAP215 arrayed TOG domains from the Drosophila and human
members, Msps and ch-TOG respectively. We report the x-ray crystal structure of Msps TOG4
to 1.65 Å, which reveals a distinct domain architecture as compared to previously determined
TOG domain structures: TOG domains 1-3 from various species. TOG domains 1-3 are
structurally homologous in their arrangement of HEAT repeats. In contrast, the ultimate three
HEAT repeats of Msps TOG4 are angled 45° relative to the corresponding HEAT repeats in
TOG domains 1-3. Extrapolating from the yeast TOG1-αβ tubulin complex structure, we
hypothesize that TOG4 uses its ultimate HEAT repeats to differentially bind α-tubulin. To
confirm the structural differences observed in Msps TOG4, we determined the x-ray crystal
structure of ch-TOG TOG4 to 1.9 Å, representing the first human TOG domain structure. The
ch-TOG TOG4 structure has an identical HEAT repeat conformation as Msps TOG4, confirming
the unique architecture and indicating that this positional differential in the pentameric TOG
array is conserved across species. To analyze how the constituent TOG domains in the array
contribute to MT dynamics we performed in vivo MT polymerization rescue assays on a minimal
Msps construct. Mutating a common determinant in the first HEAT repeat across individual TOG
domains had a similar effect on MT dynamics: failure to rescue MT polymerization. This
suggests that each TOG domain uses conserved determinants in the first HEAT repeat to bind
β-tubulin and that a full, functional array of TOG domains is required to promote MT
polymerization. These data highlight the role of arrayed TOG domains in the XMAP215
mechanism, their common and differential tubulin-binding determinants, and their spatially
distinct, conserved location in the array that likely underlies XMAP215 mechanism.
SUNDAY-LATE POSTER PRESENTATIONS
2546
Mechanism of microtubule array expansion in cytokinetic phragmoplast.
T. Murata1,2, T. Sano3, M. Sasabe4, S. Nonaka2,5, T. Higashiyama4, S. Hasezawa6, Y. Machida4,
M. Hasebe1,2; 1Division of Evolutionary Biology, National Institute for Basic Biology, Okazaki,
Japan, 2Department of Basic Biology, School of Life Science, Graduate School of Advanced
Studies, Okazaki, Japan, 3Faculty of Bioscience and Applied Chemistry, Hosei University,
Koganei, Japan, 4Division of Biological Science, Graduate School of Science, Nagoya
University, Nagoya, Japan, 5Laboratory of Spatiotemporal Regulations, National Institute for
Basic Biology, Okazaki, Japan, 6Division of Biosciences, Graduate School of Frontier Science,
The University of Tokyo, Kashiwa, Japan
In cell division, daughter cells are separated into two. In land plants, the cytokinetic organelle,
the phragmoplast, forms between daughter nuclei and expands centrifugally until reaching the
plasma membrane. The phragmoplast contains microtubules of opposing polarity that abut in
the midzone, where the cell plate is built partitioning the daughter cells. The centrifugal
development of the cell plate is driven by the centrifugal expansion of the phragmoplast
microtubule array. Although factors for microtubule nucleation, stabilization and cross-linking in
the phragmoplast have been characterized, the molecular mechanism underlying the expansion
is unknown.
We analyzed microtubule dynamics in the expanding phragmoplast by spinning disk
microscopy, speckle microscopy and quantitative photobleaching microscopy. We found that the
phragmoplast array comprises stable microtubule bundles and dynamic microtubules. The
dynamic microtubules were nucleated by γ-tubulin on stable bundles. The dynamic microtubules
elongated at the plus ends and formed new bundles preferentially at the leading edge of the
phragmoplast. At the same time, they were moved away from the cell plate, maintaining a
restricted distribution of minus ends. We propose that cycles of attachment of γ-tubulin
complexes onto the microtubule bundles, microtubule nucleation and bundling, accompanied by
minus-end directed motility, drive the centrifugal development of the phragmoplast.
2547
Basal bodies exhibit polarized positioning in zebrafish cone photoreceptors.
B. Perkins1,2, M. Ramsey2; 1Ophthalmic Research, Cleveland Clinic Foundation, Cleveland, OH,
2
Biology, Texas A&M University, College Station, TX
The asymmetric positioning of basal bodies, and therefore cilia, is often critical for proper cilia
function. This planar polarity is critical for motile cilia function but has not been extensively
investigated for non-motile cilia or for sensory cilia such as vertebrate photoreceptors.
Zebrafish photoreceptors form an organized mosaic ideal for investigating cilia positioning. We
report that in the adult retina, the basal bodies of red-, green-, and blue-sensitive cone
photoreceptors localized asymmetrically on the cell edge nearest to the optic nerve. In contrast,
no patterning was seen in the basal bodies of ultraviolet-sensitive cones or in rod
photoreceptors. The asymmetric localization of basal bodies was consistent in all regions of the
adult retina. Basal body patterning was unaffected in the cones of the XOPS-mCFP transgenic
line, which lacks rod photoreceptors. Finally, the adult pattern was not seen in 7 day post
fertilization (dpf) larvae as basal bodies were randomly distributed in all the photoreceptor
subtypes. These results establish the asymmetrical localization of basal bodies in red-, green-,
and blue-sensitive cones in adult zebrafish retinas but not in larvae. This pattern suggests an
active cellular mechanism regulated the positioning of basal bodies after the transition to the
adult mosaic and that rods do not seem to be necessary for the patterning of cone basal bodies.
SUNDAY-LATE POSTER PRESENTATIONS
2548
The ciliary protein Nek8/Nphp9 acts downstream of Inv/Nphp2.
D. Shiba1, H. Fukui1, K. Asakawa2, K. Kawakami2, T. Yokoyama1; 1Department of Anatomy and
Developmental Biology, Kyoto Prefectural University of Medicine, Kyoto, Japan, 2Department of
Genetics, Graduate University for Advanced Studies, Mishima, Japan
Primary cilia have diverse functions in cell signaling pathways, including mechanosensation and
hedgehog signaling. The cilium is structurally divided along its vertical axis into the ciliary tip, the
shaft, the transition zone (TZ) and the basal body (BB). More than 650 proteins are now
identified to localize in the cilia. Nephronophthisis (NPHP) is an autosomal-recessive cystic
kidney disorder that constitutes the most frequent genetic cause of end-stage renal failure in
children. To date, 14 causative genes have been identified. Nphp1, 4, 5, 6 and 8 are localized to
TZ, whereas Nphp2, 3 and 9 are localized to the Inv compartment of renal primary cilia. These
gene products are assumed to act coordinately, but the molecular relationships are poorly
understood. We report here the functional relationship between Inv/Nphp2 and Nek8/Nphp9.
Pull-down assay of Inv/Nphp2 and Nek8/Nphp9 revealed that Nek8 interacts with Inv. Further
study focused on phenotype analysis of nek8 knockdown embryos and the genetic relationship
between nek8 and inv in zebrafish. Knockdown of nek8 exhibited pronephric cysts, curly tail
down and abnormal cardiac looping. The nek8 MO-injected embryos drastically increased
abnormal southpaw expression patterns. Formation of pronephric cysts in Nek8 knockdown
embryos were observed at glomerulus and proximal convoluted tubule regions, where closely
related to inv MO induced-dilation. These results suggested that Nek8 and Inv functioned in the
same pronephric segments, supporting the notion that Nek8 and Inv act in concert in zebrafish
pronephros development. Simultaneous knockdown of nek8 and inv synergistically increased
the incidence of these defects. Interestingly, nek8 mRNA rescued inv morphant phenotypes,
although inv mRNA could not rescue nek8 morphant phenotypes. These results suggest that
Nek8 acts downstream of Inv during pronephros morphogenesis and left-right establishment in
zebrafish.
2549
A conserved sperm flagellar protein Spef1 is required for ordered organelle duplication
in Trypanosoma brucei.
C. Y. He1, L. Gheiratmand1; 1National University of Singapore, Singapore, Singapore
Sperm flagellar protein is identified as a conserved component of motile flagella from mammals
to protozoa. In Trypanosoma brucei, a single-cellular parasite responsible for African sleeping
sickness, a single Spef1 homolog (also known as CMF18) is present and it contains a
conserved domain of unknown function (DUF1042) and a putative calponin homology domain,
which is best known for actin binding. Previous work by others have shown that RNAi depletion
of this protein is lethal in both the procyclic form (proliferates in tse-tse fly vector) and the
bloodstream form (proliferates in the mammalianblood) parasites, causing flagellum motility
defect and cytokinesis arrest. In a recent bi-lobe immunoisolation studies, the TbSpef1 protein
was identified as a bi-lobe associated protein. We have therefore re-examined the cellular
localization of TbSpef1 in T. brucei and its cellular function by inducible RNAi. We show that
TbSpef1 is confined to a small region following a specialized microtubule quartet at the base of
the single flagellum present in T. brucei. Possibly by affecting the nucleation or stability of the
microtubule quartet, TbSpef1 is essential for the sequential duplication of several single copied
organelles, all tethered to a core cytoskeletal network in T. brucei.
SUNDAY-LATE POSTER PRESENTATIONS
2550
The CSC connects three dynein regulating complexes in eukaryotic flagella.
T. Heuser1, E. E. Dymek2, J. Lin1, E. F. Smith2, D. Nicastro1; 1Biology Department, Rosenstiel
Center, Brandeis University, Waltham, MA, 2Department of Biological Sciences, Dartmouth
College, Hanover, NH
Cilia and flagella are eukaryotic organelles that generate motion or sense environmental cues.
They are highly conserved among species – from single cell algae to humans – and defects in
cilia cause various human diseases, called ciliopathies, such as polycystic kidney disease or
primary ciliary dyskinesia [1].
The core structure of cilia and flagella, the axoneme, consists of two single microtubules in the
center that are surrounded by nine doublet microtubules [2]. Each doublet consists of many
copies of a 96 nm long axonemal unit that repeats along the doublet length. Typically, each unit
has three radial spokes, RS1-3, but in some organism, like the algae Chlamydomonas, the most
distal spoke is considerably shorter and called RS3 stand-in (RS3S) [3, 4]. Axonemal movement
is driven by thousands of dynein motor proteins, but the mechanisms that coordinate dyneins’
activity spatially and temporally are not well understood.
Recently, we described a calcium sensing complex, the Calmodulin and Spoke associated
Complex (CSC), that is essential for proper radial spoke assembly and flagellar motility [5].
Using cryo-electron tomography combined with image processing and classification of
heterogeneous data, we analyzed the axonemal structure of CSC mutants in Chlamydomonas.
Interestingly, we found that knockdowns of CSC subunits effect assembly of RS2 and RS3S,
but not RS1. Using a wild type – mutant comparison we present the first 3D localization model
of the CSC and show that it connects three key axonemal complexes that are important for
dynein regulation: RS2, the nexin-dynein regulatory complex (N-DRC) and RS3S.
These results support previous findings of radial spoke heterogeneity, not only between RS3S
and the two full-lengths spokes, but also between RS1 and RS2. The location of the CSC to
only one of the two full-length radial spokes could suggest that calcium sensing might be a
functional difference between them indicating different roles for the radial spokes in axonemal
motility regulation.
[1] Fliegauf et al., 2007 Nat Rev Mol Cell Biol 8:880.
[2] Porter & Sale, 2000 J Cell Biol 151:37.
[3] Barber et al., 2012 Mol Biol Cell 23:11.
[4] Lin et al., 2012 Cytoskeleton 69:88.
[5] Dymek et al., 2011 Mol Biol Cell 22:2520.
2551
Mechanisms of ciliary growth and differentiation in echinoderm embryos.
R. L. Morris1,2,3, L. M. Williams1,2, E. M. Doherty1,2, G. M. Beacham2,4, N. A. Sholi1, A. M.
McCarthy1, K. M. Hewitt1, B. J. Rossetti1,3, R. T. Manguso1, M. L. Keyes1, I. D. Greenstein1,2, T.
Shintaku1,2, A. Hussain1, A. M. Carson1, J. Browne1, A. P. Rawson1, J. J. Nordberg1, C. N.
English1, D. Erkoboni1; 1Biology, Wheaton College, Norton, MA, 2Mount Desert Island Biological
Lab, Salisbury Cove, Maine, ME, 3Marine Biological Laboratory, Woods Hole, MA, 4University of
Maine, Orono, ME
In sea urchin and sand dollar embryos, the onset of ciliogenesis occurs on all cells
simultaneously just prior to embryo hatching and is regulated thereafter in a tissue-specific
SUNDAY-LATE POSTER PRESENTATIONS
manner to generate cilia with different lengths and behaviors. To understand the process of
ciliary formation and differentiation during development, we have begun to characterize the
morphological, behavioral, functional, and molecular differences between cilia subtypes that
arise on echinoid embryos. At hatching, every cell possesses the same type of long motile
cilium to propel the embryo. Such cilia grow in a stepwise manner sometimes to full length
where they linger and sometimes in repeating cycles of incomplete growth and retraction.
Shortly thereafter, while lateral regions continue to produce long motile cilia, other regions
modulate the ciliogenic program in one of three ways. First, skeletogenic primary mesenchyme
cells retract their motile cilia as the cells ingress and then suppress ciliogenesis at least until
spiculogenesis onset. Next, cells of the vegetal plate retract their motile cilia as the cells
invaginate and then grow short cilia of indeterminate motility. Such vegetal cilia come to line the
future gut as invagination proceeds. Finally, cells of the animal plate grow their cilia to
approximately double-length and these apical tuft cilia cease their motility. As with other cilia of
the embryo, apical tuft cilia fully retract before mitosis and are subsequently rebuilt on each
daughter cell.
The ability of heavy metal ions to change cell fates and thereby
“animalizeâ€
or “vegetalizeâ€
echinoderm embryos allowed enrichment and
observation of cilia subtypes that usually form on few cells. Furthermore, morpholino
knockdown of the transcription factor NK2.1 offers the opportunity to examine functions of the
apical tuft cilia separate from other differentiated cilia types. Light microscopic and genomic
approaches suggest that switching of ciliary subtypes may involve retraction of the ciliary
axoneme entirely into the cytoplasm, disassembly of the axoneme, and subsequent regrowth of
a new cilium from a cytoplasm containing an altered suite of ciliary proteins. The echinoderm
embryo therefore helps reveal how the ciliogenic program is modulated in specific embryonic
regions to generate the spectrum of cilia morphologies, motilities, and functions found in
different tissues.
2552
KIF19A controls ciliary length by depolymerizing microtubules at ciliary tips.
S. Niwa1, K. Nakajima1, H. Miki1, Y. Minato1, D. Wang1, N. Hirokawa1; 1University of Tokyo,
Tokyo, Japan
Fluid flow is essential for the development and health of mammals. Each cells have optimal
ciliary length because fluid flow generation is a function of ciliary length. However, the molecular
mechanisms controlling the ciliary length remain largely unknown in mammals. Here, we
suggest that KIF19A, a kinesin superfamily protein, regulates ciliary length by depolymerizing
microtubules at ciliary tips. Kif19a knockout mice showed hydrocephalus and female infertility
phenotypes due to abnormally elongated cilia that cannot generate proper fluid flow. KIF19A
localized to cilia tips in ciliated epithelial cells, and recombinant KIF19A controlled the length of
microtubules newly polymerized from axonemes in vitro. KIF19A had ATP-dependent
microtubule-depolymerizing activity mainly at the plus end of microtubules. Our results suggest
a molecular mechanism of ciliary length regulation in mammals and its importance in the
maintenance of the mammalian body.
2553
Non-mitotic Functions of Centrosomal Scaffolding Protein Cep192.
B. O'Rourke1, D. Sharp1; 1Physiology and Biophysics, Albert Einstein College of Medicine,
Bronx, NY
Cep192 is a centrosomal scaffolding protein studied primarily for its role in mitosis. Our work
has shown the protein persists in interphase and plays a role in several non-mitotic functions.
We have established multiple platforms with which to knockdown Cep192(including a shRNA
SUNDAY-LATE POSTER PRESENTATIONS
cell line), allowing us to assess the impact of Cep192 depletion through a broad range of
assays. Using siRNA encapsulated in nanoparticles, we are able to deliver a siRNA based KD
throughout a tissue. We have shown knockdown of Cep192 has a dramatic impact in altering 3D migration of cells migrating out of tumor explants. Additionally, we have shown that Cep192
depletion strongly impacts cell polarization when cells plated at low density. Using traditional
and nanoparticle based KD, we have shown that knockdown of Cep192 alters centrosomal MT
nucleation (EB3 data) as well as the overall organization of the microtubule cytoskeleton—in
particular, cells lacking Cep192 appear to lose their radial microtubule arrays. MT regrowth
from the centrosome is also changed following nocodazole washout in Cep192 depleted cells.
Studies are currently underway to further characterize this phenotype.Based on these data, we
hypothesize that Cep192 is instrumental for the establishment of a centrosomal microtubule
array in interphase cells which in turn strongly influences the establishment of cell polarity and
cell motility, particularly in 3-D.
2554
Odf2 organizes two distinct appendage structures/functions in centrosomes and
basalbodies.
*K. Tateishi1, *Y. Yamazaki1, K. Kunimoto1, Y. Ogura1, T. Nishida2, H. Ishikawa3, S. Tsukita1;
1
Laboratory of Biological Science, Graduate School of Frontier Biosciences, Osaka University,
Osaka, Japan, 2Research Center for Ultra-high Voltage Electron Microscopy, Osaka University,
Osaka, Japan, 3Department of Biochemistry and Biophysics, University of California, San
Francisco, Japan
* These are equally contributed
Ciliogenesis is regulated by various cell context cues, some of which are transduced through
the appendage structures of anchoring fibers(AFs) and basal feet(BFs) of basal bodies at the
base of cilia. However, the information on molecular architecture and function of appendage
structures are not fully accumulated. We have shown that in the mutant mice, in which exons 6
and 7 of Odf2 are deleted, cilia are formed in the multi-ciliated and non-multi-ciliated cells but
subdistal appendages are lost, and that there are detected expressions of the C-terminal
regions of Odf2 (which are translated by the C-terminal exons between exon 8-22).
We here examined the domains of Odf2 in cultured epithelial cells, which are required for
formation of AFs and BFs of primary cilia, respectively. The results obtained suggested the
different domain usage of Odf2 for distinct functions between AFs and BFs. The domaindependency for the formation of AFs and BFs of basal bodies is closely correlated to that for the
formation of distal and subdistal appendages of centrosomes, respectively, pointing to the
equivalent counterpart role between appendage structures of basal bodies and those of
centrosomes.
2555
HURP is essential for mitosis through its regulation of Kif18A.
F. Ye1, C. Li1, Y. Zhang1, L. Tan1, Q. Yang1, Y. Xia1, Y-C. Liou1; 1DBS, NUS, singapore,
Singapore
Chromosome biorientation and congression during mitosis require precise control of microtubule
dynamics. The dynamics of kinetochore microtubules (K-MTs) are regulated by a variety of
microtubule-associated proteins (MAPs). Recently, a MAP known as HURP (Hepatoma
UpRegulated Protein) was identified. During mitosis, Ran-guanosine 5'-triphosphate (RanGTP)
releases HURP from the importin beta inhibitory complex and allows it to localize to the
kinetochore fiber (k-fiber). HURP stabilizes k-fibers and promotes chromosome congression.
SUNDAY-LATE POSTER PRESENTATIONS
However, the molecular mechanism underlying the role of HURP in regulating chromosome
congression remains elusive. Here, we show that overexpression of the N-terminal microtubule
binding domain (1-278 aa, HURP(278)) of HURP induces a series of mitotic defects that mimic
the effects of Kif18A depletion. In addition, coimmunoprecipitation and bimolecular fluorescence
complementation assays identify Kif18A as a novel interaction partner of HURP. Furthermore,
quantitative results from live-cell imaging analyses illustrate that HURP regulates Kif18A
localization and dynamics at the plus end of K-MTs. Lastly, misaligned chromosomes in
HURP(278)-overexpressing cells can be partially rescued by the overexpression of Kif18A. Our
results demonstrate in part the regulatory mechanism for Kif18A during chromosome
congression and provide new insights into the mechanism of chromosome movement at the
metaphase plate.
Molecular Motors
2556
Fragmentation and Compaction of Membrane-bound Actin Filaments by Myosin Motors.
S. K. Vogel1, Z. Petrasek1, F. Heinemann1, P. Schwille1; 1Cellular and Molecular Biophysics,
Max Planck Institute, Martinsried, Germany
Cortex remodeling during cell division is a result of myofilament-driven contractility of the cortical
membrane-bound actin meshwork. Only little is known about the interaction between individual
myofilaments and membrane-bound actin and the role of the membrane during actin cortex
reorganization. We therefore developed minimal actin cortices, consisting of membrane bound
actin and myosin motors, that allow performing well-defined quantitative experiments, which are
difficult to realize in living cells. By directly visualizing the action of individual myofilaments on
membrane-bound actin using TIRF microscopy, we found that myosins fragment and compact
membrane-bound actin while processively moving along actin filaments. We propose a
mechanism by which tension builds up between the ends of myofilaments, resulting in
compressive stress exerted to single actin filaments, causing their buckling and breakage.
Modeling of this mechanism revealed that indeed sufficient force can be generated by single
myofilaments to buckle and break actin filaments. This mechanism of filament fragmentation
and compaction may contribute to actin turnover and cortex reorganization during cytokinesis in
living cells.
2557
Nuclear Myosin Va: Evidence of its role in transcription and splicing regulation.
M. M. Baqui1, H. Yu2, S. Yokoo3, W. A. Hofmann4, E. Espreafico1, M. D. Gomes3, P. de
Lanerolle2, R. E. Larson1; 1Dept of Cell and Molecular Biology, School of Medicine of Ribeirao
Preto, University of Sao Paulo, Ribeirao Preto, Brazil, 2Dept. of Physiology, University of Illinois
at Chicago, Chicago, IL, 3Dept of Biochemistry and Immunology, School of Medicine of Ribeirao
Preto, University of Sao Paulo, Ribeirao Preto, Brazil, 4Dept. of Physiology and Biophysics,
SUNY at Buffalo, Buffalo, NY
Myosin V is a class of actin-based motor proteins involved in cytoplasmic vesicle transport and
anchorage, spindle-pole alignment, pseudopod extension and mRNA translocation. Recently,
we have shown that, in normal transcribing cells, phosphorylated myosin Va (pMVa) is localized
to the subnuclear compartments referred to as speckles, which are rich in splicing factors such
as SC35 that earmarks these interchromatin particles. However, when transcription is inhibited
by actinomycin D there is a translocation of pMVa to the nucleoli, as well as a separation of a
SUNDAY-LATE POSTER PRESENTATIONS
subpopulation of pMVa from the splicing factor SC35 into near-neighboring particles. Here, we
show by immunofluorescence that pMVa colocalizes with several proteins implicated in RNA
processing within the nucleus, including β-actin, RNA polymerase II and hSNF2H, a major
chromatin remodeling protein. Also, an in vivo transcription assay revealed a partial
colocalization between pMVa and nascent mRNA that incorporated Br-UTP. Antibodies against
MVa blocked the in vitro transcription and splicing of a globin minigene. The in vivo splicing of
an adenovirus E1A mRNA was incomplete in murine S91-6 melanocytes, null mutants for MVa,
compared to B16 cells, but was rescued by stable transfection of full length MVa in S91-6 cells.
Alternative splicing was also affected in MVa-/- human fibroblasts from a Griscelli syndrome
patient. The splicing phenotype was recovered in S91-6 cells when full length MVa was stably
transfected in these cells. Thus, our results indicate a novel role for myosin Va in nuclear
compartmentalization and in RNA processing, suggesting that MVa is a part of the
transcriptional machinery of the cell.
Supported by FAPESP, CNPq, FAEPA
2558
Live analysis of vesicular transport in CAD cells: determining the activity of plus- and
minus-end directed motors.
R. E. Turn1, N. J. Quintyne1; 1Florida Atlantic University, Jupiter, FL
Movement of cargoes along microtubules in cells rely upon the action of two families of motor.
These motors use energy from ATP to generate force to drive various cargoes around the cell in
an anterograde or retrograde direction. Microtubules use two distinct classes of motor: kinesins
and cytoplasmic dynein. The kinesin superfamily is a set of proteins unified by the presence of a
conserved motor domain. Each kinesin family member has other, unique domains that define its
specific function within the cell. Broadly, kinesins are thought of as “plus-end” (anterograde)
motors, that is to say that they move along the microtubule towards the dynamic plus-end which
is usually situated towards the periphery of the cell, or the terminus of the axon. There exist,
however, other classes of kinesin that are minus-end directed, and yet others that are involved
in regulating microtubule dynamics. By contrast, cytoplasmic dynein is a single protein, albeit it
with many subunits that have interchangeable isoforms. Dynein relies upon a second protein
complex, named dynactin, to function properly. The dynein-dynactin complex is the major
minus-end (retrograde) directed motor complex within the cell, traveling from the periphery to
the center. We are interested in investigating the role of KIF9, as well as other kinesins and
dynein in movement of vesicles in CAD cells. CAD are mouse neuroblastoma cells that can be
induced to grow axon-like projections which allow for easy identification of retrograde and
anterograde movements. We have used a baculovirus system to deliver DNA to generate GFPtagged markers for mitochondria and lysosomes and have used the fluorescent dye Nile Red to
mark lipid droplets. Using live analysis of these tagged CAD cells, we have established
baselines for average velocities and processivities in both the plus- and minus-end directions,
as well as ratios of plus-end: minus-end movement events. After this, we have knocked down
motor expression levels in the cells via shRNA. In addition to KIF9 knockdown, we have
examined KIFC1 (a nonessential kinesin, also called HSET), KIF5B (conventional kinesin-1)
and dynamitin (a component of the dynactin complex vital to dynein-based transport). Our initial
data show that knockdown of KIF9 does not affect velocities of movements of vesicles, but we
do see a change in processivity, suggesting that the motor may not be directly involved in
moving cargoes but may play a supporting role in ensuring efficient delivery across greater
distances.
SUNDAY-LATE POSTER PRESENTATIONS
2559
MCAK conformation regulates its function in spindle pole formation.
S. K. Lamb1, H. Zong1, S. G. Hainline1, S. C. Ems-McClung2, C. E. Walczak2; 1Department of
Biology, Indiana University, Bloomington, IN, 2Medical Sciences, Indiana University,
Bloomington, IN
Microtubule (MT) dynamics are critical for proper assembly of the mitotic spindle, and the MT
depolymerizing Kinesin-13 MCAK is a key regulator in this process. Our previous studies
showed that MCAK MT depolymerization activity alone is not sufficient for spindle assembly,
and that there is a complex interplay between the N-terminal domain (NT), neck, and C-terminal
domains (CT) of MCAK. How these non-catalytic domains cooperate is unknown.
We
propose that the NT and/or neck physically interact with the CT to coordinate MCAK
biochemical and physiological activity. To test this idea we performed pull-down assays with
GST-tagged CT (GST-CT), which showed that the CT interacts with MCAK containing the neck
and catalytic domain (MD). Truncation of the last 27 amino acids abolished this interaction,
whereas deletion of the last 14 amino acids (aa 717-730) did not, implicating residues 704-716
in regulating the interaction between the CT and MD. Because the neck domain of MCAK is
positively charged, we hypothesized that this interaction might be electrostatic. MCAK contains
two conserved glutamate residues at positions 715 and 716, and mutation of these residues to
alanine (AA) abolished the interaction of the CT with MD, supporting this idea. To test the
importance of these interactions in the full-length protein, we created FRET biosensors. FRET
analysis of full-length MCAK, NT, and CT deletion constructs indicates that the neck and CT are
in close proximity, supporting the direct interaction seen between the CT and MD in our pulldown assays. Surprisingly, the MCAK(AA) mutant did not exhibit reduced FRET, but rather had
increased FRET. This suggests a more complex interaction in the full-length protein. To test
the physiological significance of E715 and E716, we added MCAK(AA), to cycled spindle
assembly reactions. MCAK(AA) addition decreased spindle assembly and increased the amount
of free chromatin compared to wt MCAK addition, consistent with the idea that MCAK(AA) has
increased MT depolymerization activity. However, ~50% of the MCAK(AA) spindles had
unfocused poles, suggesting that the CT conformation is critical for pole focusing. Together,
these results indicate that the conformation of the CT of MCAK is important for an interaction
with the neck and motor domain in the regulation of spindle pole formation.
2560
Systems Enabled Methods for Single Molecule Investigation.
M. Salapaka1, S. Roychowdhury1, D. Materassi2, T. S. Hays3, T. Aggarwal1, M. Li3; 1Electrical
Engineering, University of Minnesota, Minneapolis, MN, 2Massachusets Institute of Technology,
Cambridge, MA, 3Genetics, Cell Biology, and Development, University of Minnesota,
Minneapolis, MN
Recent technological advances in instruments, such as optical tweezers and atomic force
microscopes (AFMs), have led to significant advances in understanding on how single
molecules behave. These advances have also necessitated newer methods for control,
modeling and interpretation of data. We are studying how tools from systems and controls
engineering can enable higher temporal and spatial resolution methods for single molecule
investigation. These include methods for simulation engines as well as experimental methods.
Given that many biological processes depend crucially on the ability of molecules to change
their state in discrete events such as steps, statistics on the sizes and dwell times related to
these events can provide crucial insights into the dynamics of processes. Key challenges in the
task of estimating the size and related statistics are: dynamics of the probe that convolutes how
molecules behavior appears in the measurement, thermal noise that is often the source of
SUNDAY-LATE POSTER PRESENTATIONS
significant uncertainty in the measurement. Most current methods have a temporal resolution
that is below that of the probe dynamics. Here we report a systems based method based on
Dynamic Programming that can surpass the limitations posed by the time scale of the dynamics
of the probe. The method is capable of detecting events such as steps taken by motor-proteins,
and unraveling of domains in an AFM based single molecule pulling experiments with significant
improvements in temporal as well as spatial resolution. The method also incorporates
mechanisms to learn the statistics of event sizes, steps in particular, which is of crucial
significance in the study of motor-proteins.
Even-though significant strides in temporal and spatial resolution of instruments applicable to
investigation of motor-proteins are being made, often, these capabilities fall short of needs.
Here we report a modeling method which is applicable to studies of how multiple proteins
coordinate the transfer of single cargo. Here, only the relative configuration on how the multiple
motors are arranged with respect to one another forms the state of the system and the absolute
configuration, which is infinite dimensional, is not tracked. We show that it is possible to recover
most of the relevant quantities of biological interest such as run-length, and velocity of cargo,
from the projected state of relative configurations. Furthermore we show that the probability
distribution on the projected space of relative configurations can be computed a priori which we
show enables detection of rare modalities of cargo transport.
2561
Spastic Paraplegia Mutation N256S in the Neuronal Microtubule Motor KIF5A Disrupts
Axonal Transport in a Drosophila HSP Model.
P. Füger1, V. Sreekumar1, R. Schüle1, J. Kern1, D. Stanchev1, C. Schneider1, K. Karle1, K.
Daub1, V. Siegert1, M. Flötenmeyer2, H. Schwarz2, L. Schöls1, T. M. Rasse1; 1Hertie Institute,
University of Tübingen, Tuebingen, Germany, 2Max-Planck Institute for Developmental Biology,
Tuebingen, Germany
Hereditary spastic paraplegias (HSPs) comprise a group of genetically heterogeneous
neurodegenerative disorders characterized by spastic weakness of the lower extremities. We
have generated a Drosophila model for HSP type 10 (SPG10), caused by mutations in KIF5A.
KIF5A encodes the heavy chain of kinesin-1, a neuronal microtubule motor. Our results imply
that SPG10 is not caused by haploinsufficiency but by the loss of endogenous kinesin-1 function
due to a selective dominant-negative action of mutant KIF5A on kinesin-1 complexes. We have
not found any evidence for an additional, more generalized toxicity of mutant Kinesin heavy
chain (Khc) or the affected kinesin-1 complexes. Ectopic expression of Drosophila Khc carrying
a human SPG10-associated mutation (N256S) is sufficient to disturb axonal transport and to
induce motoneuron disease in Drosophila. Neurofilaments, which have been recently implicated
in SPG10 disease manifestation, are absent in arthropods. Impairments in the transport of
kinesin-1 cargos different from neurofilaments are thus sufficient to cause HSP-like pathological
changes such as axonal swellings, altered structure and function of synapses, behavioral
deficits and increased mortality.
SUNDAY-LATE POSTER PRESENTATIONS
2562
A shared binding site for different cargo adaptors in the asymmetric coiled-coil structure
of the dynein co-factor Bicaudal-D.
H. K. Salter1, Y. Liu1, A. N. Holding1, C. Johnson2, E. Stephens1, J. Walshaw3, S. L. Bullock1;
1
Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom, 2Protein &
Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom,
3
University of East Anglia, Norwich, United Kingdom
Microtubule-based motor proteins play critical roles in the sorting and dispersal of cellular
constituents. While there are many members of the kinesin family of plus-end directed motors,
there is only one minus end-directed cytoplasmic dynein. This motor is responsible for the
transport of a wide range of cellular cargoes, including vesicles and mRNA, as well as viral and
bacterial pathogens. How a single motor is able to recognize such a diverse range of cargoes is
poorly understood. Bicaudal-D (BicD) is one of a small number of adaptors known to facilitate
dynein’s linkage to cargoes. The C-terminal domain (CTD) of BicD binds cargo adaptors such
as the GTPase Rab6 and the non-canonical RNA-binding protein Egalitarian (Egl), while the Nterminal domain binds to the dynein motor and its accessory complex dynactin. To further our
understanding of how BicD recognizes such diverse cargoes, we solved the crystal structure of
the Drosophila BicD CTD to 2.2 Å resolution. We found that this domain forms a parallel coiledcoil with an unusual degree of asymmetry. Based on our analysis of the structure, evolutionary
conservation, gel-filtration chromatography and cross-linking studies we have delineated the
minimal binding domain for Rab6GTP in BicD in vitro. We then identified residues needed for
Rab6 binding in this region using recombinant protein binding assays combined with structurebased mutagenesis. Interestingly, we found that the cargo adaptor Egl has the same minimal
binding domain and key residue requirement on BicD as Rab6. These data suggest that
mutually exclusive binding of cargo adaptors to BicD is one method by which cargoes with
distinct patterns of localization are correctly sorted within cells. Using transgenic flies, we
confirmed that residues key for both Egl and Rab6 binding are required for the normal function
of full-length BicD in vivo. We also present results on the relationship between cargo and motor
recruitment to BicD, as well as our findings regarding the molecular basis of the classic bicaudal
mutation BicD1.
2563
Using siRNA to map the contribution of dynactin subunits in anchoring microtubules at
the centrosome.
K. M. Hazellief1, C. S. Turn1, N. J. Quintyne1; 1Honors College, Florida Atlantic University,
Jupiter, FL
Dynactin is a multisubunit protein complex required for proper functioning of the microtubule
motor cytoplasmic dynein. Dynactin serves as a processivity factor for the motor as well as a
cargo adaptor, allowing dynein to function in a wide array of cellular processes. Additionally,
dynactin performs a dynein-independent role, anchoring microtubules at the centrosome during
interphase in fibroblast cells. Our previous work has shown that disruption of the dynactin
complex, by overexpression or knockdown of selected dynactin subunits, leads to a
disorganization of microtubules as they are no longer anchored at the centrosome. The
p150Glued subunit of dynactin is of particular importance to this process, as it possesses
dynactin’s microtubule binding regions, termed the CAP-Gly and Basic domains. These
domains have differential affinity for microtubules, with CAP-Gly binding to microtubules with a
higher affinity than Basic. Using shRNA to the untranslated region of p150Glued’s mRNA, we
have knocked down expression of the endogenous protein and are replacing it with new
versions of the protein which lack either CAP-Gly, Basic or both subunits, showing a differential
SUNDAY-LATE POSTER PRESENTATIONS
effect on anchoring, dependent upon which domain is present. Additionally, we have used
shRNAs to the p24, p27, p62, and Arp1 subunits of dynactin to determine any role those
subunits play in anchoring. Recent work has shown that p62 is required for dynactin complex
integrity, and its loss leads to a breakdown of the complex into its component parts (Yeh et al.,
2012). The loss of integral dynactin from the centrosome is sufficient to disrupt anchoring. On
the other hand, p27, along with its partner p25, are more peripherally localized to the complex
and seem to play a role in membrane selectivity and have little effect on dynactin structure and
therefore anchoring. Our results show that an intact dynactin complex with all of its essential
components in place is necessary to maintain microtubule organization by the anchoring
complex and that while p150Glued is needed to bind the microtubules, other subunits provide
structural support to facilitate the interaction of dynactin with other anchoring components at the
centrosome.
2564
Phosphorylation of a conserved site in the LIC subunit of cytoplasmic dynein is required
for proper mitotic SAC inactivation.
Y. He1, J. Thompson2, T. Xu2, M. Serr1, R. Davison1, M. Li1, J. Yates2, T. Hays1; 1University of
Minnesota, Minneapolis, MN, 2The Scripps Research Institute, La Jolla, CA
In mitosis, the spindle assembly checkpoint (SAC) ensures the proper segregation of
chromosomes by preventing the onset of anaphase until all kinetochores are properly attached
to microtubules. Previous work has shown a requirement for the light intermediate chain (LIC)
subunit of cytoplasmic dynein in releasing the checkpoint, but details of its role are poorly
understood. We have identified seven LIC phosphorylation sites by mass spectrometry of
dynein prepared from Drosophila embryos. One of these sites, LIC Ser190, is evolutionarily
conserved and a putative mitotic cdc2/CDK1 target. Here we report our characterization of this
site in Drosophila. We have raised a phospho-specific antibody against the S190 site to confirm
its in vivo phosphorylation. In cultured S2 cells, we expressed LIC phosphomimetic mutations.
The S190A mutation, but not S190E, causes delays in mitosis. Observed defects include
retention of the checkpoint proteins Rod and Mad2 at aligned metaphase kinetochores; yet,
S190A mutant LIC is still capable of streaming away from mitotic kinetochores. In an alternative
experimental approach, we microinjected antibodies into syncytial embryos. Inhibition of
phosphorylated LIC S190 function by injection of the phospho-specific S190 antibody does not
affect the poleward movement of the dynein complex, but does block the release of Mad2 and
Rod. Blot overlay and co-IP experiments further support a physical interaction between LIC and
Mad2. Our results indicate that phosphoregulation of LIC S190 facilitates the efficient removal of
the checkpoint proteins Rod and Mad2 from attached kinetochores.
Membrane Trafficking
2565
CAPS promotes VAMP2-dependent vesicle docking in neuroendocrine cells.
M. Kielar-Grevstad1, T. F. Martin1; 1Department of Biochemistry, University of Wisconsin Madison, Madison, WI
Exocytic vesicles attached to the plasma membrane are termed docked; however, this term
encompasses multiple attachment states. Here, we describe new techniques for analyzing
vesicle plasma membrane attachment in live cells. Use of TIRFM (total internal reflection
microscopy) in actin depleted PC12 cells revealed vesicles immobile in the x-y dimension that
SUNDAY-LATE POSTER PRESENTATIONS
exhibited distinct attachment states. We show a clear dependence of vesicle attachment on
SNAP25, Syntaxin1a, Rab3/27, and Slp4a and a lack of dependence of vesicle attachment on
the v-SNARE (VAMP2) or the Ca2+-sensors synaptotagmin 1/9. Direct assessment of single
vesicle attachment and fusion was simultaneously monitored, allowing for identification of the
fusion-competent and incompetent attachment states. Interestingly, Slp4a knockdown resulted
in a large reduction in the number of attached vesicles but markedly increased the fusion
probability of remaining vesicles, which is consistent with previous reports that Slp4a inhibits
fusion competence.
Further analysis of the immobile (x-y dimension) vesicles revealed distinct states that were
distinguished by z-dimension movement into tight (docked) and loose (tethered) pools. The
knockdown of Slp4a reduced the number of docked vesicles, thereby reducing the total number
of attached vesicles. By contrast, loss of VAMP2 reduced the number of vesicles in the docked
pool and simultaneously increased the number of vesicles in the tethered pool. Results similar
to those for VAMP2 knockdown were observed for knockdown of the priming factor CAPS, but
not for ubMunc13-2. The effects of VAMP2 and CAPS knockdown were not additive suggesting
the two proteins function on the same pool of docked vesicles. Additionally, unlike knockdown
of Slp4a, the loss of VAMP2 or CAPS reduced vesicle fusion probability.
Taken together our results indicate two distinct docked states in PC12 cells. In the first, docked
vesicles immobilized at the plasma membrane by Slp4a are fusion-incompetent. In the second,
trans SNARE-complexes mediate a fusion-competent docked state whose formation requires
the priming factor CAPS.
2566
Syntaxin 1A-MUNC18a complexes probed by Dipicrylamine FRET.
G. A. Zhu1, J. W. Taraska1; 1Laboratory of Molecular and Cellular Imaging, National Heart,
Lung, Blood Institute, Bethesda, MD
Syntaxin 1A is a ubiquitously-expressed neuronal membrane protein essential for the specific
and timely docking, priming, and exocytosis of syntaptic vesicles. The cytosolic domain of
Syntaxin 1A consists of the alpha helical H3 SNARE (soluble N-ethylmaleimide attachment
protein receptor) domain joined by a flexible linker to a three-helix bundle, termed the Habc
domain. During vesicle priming and exocytosis, the H3 domain of Syntaxin 1A interacts with two
other SNARE proteins, VAMP2 and SNAP-25, to form a compact four-helical bundle that drives
membrane fusion. Regulation of Syntaxin 1A in vivo relies on its propensity to adopt a closed
conformation, in which the Habc domain is thought to fold back on the H3 domain to form a
closed four-helical bundle. A number of Syntaxin 1A-binding proteins have been discovered,
some of which regulate exocytosis by altering the equilibrium between the fusion-competent
“open” conformation and the fusion-incompetent “closed” conformation.
Here, we imaged the structure of Syntaxin 1A in living cell membranes with fluorescence
resonance energy transfer (FRET). Specifically, FRET between fluorescently-tagged Syntaxin
1A and the membrane-resident FRET acceptor dipicrylamine (DPA) was used to map the
relative distances between domains in Syntaxin 1A and the plane of the plasma membrane of
PC12 cells. The N-terminus of Syntaxin 1A was measured to be closer to the plasma membrane
when Syntaxin 1A adopted the closed conformation and farther from the membrane in the open
conformation. Furthermore, the Syntaxin 1A binding protein MUNC18a, which plays an integral
role in vesicle docking and fusion events, promoted Syntaxin 1A to adopt the closed
conformation. Syntaxin 1A carrying a mutation that abolished MUNC18a binding was
SUNDAY-LATE POSTER PRESENTATIONS
indistinguishable from constitutively open Syntaxin 1A. Our results suggest a fundamental role
for MUNC18a in regulating the conformation of Syntaxin 1A by promoting the closed state.
2567
The dynamin PH domain variable loop 1 (VL1) is an assembly-independent sensor of
membrane curvature
J. Nichols1, A. Samanta2, R. Ramachandran2; 1Cedarville University, Cedarville, OH,
2
Physiology and Biophysics, Case Western Reserve University, Cleveland, OH
The role of the membrane-inserting pleckstrin homology (PH) domain variable loop 1 (VL1) in
dynamin-catalyzed membrane fission remains unclear. A membrane bending function has been
proposed based on the bilayer-couple mechanism and the inability of a membrane insertiondefective VL1 point mutant (Dyn1 I533A) to generate curvature from model membrane
templates in vitro. Here using site-directed fluorescence labeling coupled to multiple,
independent spectroscopic techniques and confocal imaging on Giant Unilamellar Vesicles
(GUVs), we have resolved the role of VL1 in dynamin function. Contrary to current
understanding, our characterization of the isolated Dyn1 PH domain in comparison to full-length
dynamin reveals that the PH domain VL1 is primarily a sensor of membrane curvature that
serves in partitioning dynamin to regions of high membrane curvature (i.e. the narrow
membrane neck of an invaginated coated pit) to direct localized dynamin self-assembly. Similar
to full-length dynamin, the isolated PH domain preferentially inserted into highly curved
membrane bilayers in vitro. However, unlike the full-length molecule, the PH domain was
unable to generate curvature from relatively planar membrane templates on its own. Our studies
further reveal that in vivo fission-incompetent Dyn1 I533A fails to distinguish membrane
curvature in vitro and is defective in directing organized self-assembly on curved membrane
templates. Our studies provide critical insights into the role of the PH domain VL1 in dynamin
function and expand the repertoire of PH domain functionality in protein-protein and proteinmembrane interactions.
2568
Lovastatin Disrupts CD14 Trafficking in Murine Macrophages.
S. Silvershein1, J. Turofski2, M. Stekla1, J. Perlmutter1, T. A. Frey1; 1Biology, Dickinson College,
Carlisle, PA, 2Biology, University of Scranton, Scranton, PA
CD14 is the primary binding site on myeloid cells for bacterial lipopolysaccharide (LPS) and can
be found as two protein isoforms: a glycosylphosphatidylinositol (GPI)-anchored membrane
protein (mCD14) and a soluble serum protein (sCD14) that lacks the GPI anchor. Statins are a
class of drugs widely prescribed for the treatment of hypercholesterolemia. In addition to their
ability to lower plasma cholesterol levels, statins also block the cellular production of the
isoprenoid intermediates farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate
(GGPP). FPP and GGPP are lipid moieties that are critical for the proper function of the Rho
and Rab families of small GTP-binding proteins. Therefore, these drugs have the potential to
affect the function of cells in various ways and have been reported to have anti-inflammatory
properties. In particular, treatment of RAW 264.7 macrophages with lovastatin has been shown
to increase mCD14 while decreasing the release of sCD14 following LPS stimulation. In order to
further characterize the decreased release of sCD14 in the presence of lovastatin, we
performed immunostaining for CD14 on RAW 264.7 macrophages following LPS stimulation. In
control cells, CD14 is primarily localized to the plasma membrane and in a concentrated
perinuclear region that partially co-localizes with the Golgi apparatus marker GM-130. However,
while CD14 is still localized to the plasma membrane in lovastatin treated cells, the percentage
of cells exhibiting a concentrated perinuclear staining pattern is decreased. Co-incubation of
SUNDAY-LATE POSTER PRESENTATIONS
lovastatin treated macrophages with either mevalonate or GGPP blocks alterations in the
release of sCD14 indicating that these effects are dependent on inhibition of cholesterol
biosynthesis. Moreover, inhibition of Rho GTPases with either GGTI-298 or Toxin B does not
block the release of sCD14 from these cells indicating that this is likely to be a Rab dependent
effect. Our current working hypothesis is that depletion of GGPP and subsequent inhibition of
Rab proteins by lovastatin disrupts trafficking of CD14 through the Golgi apparatus thereby
dramatically reducing the release of sCD14 from macrophages. Reduced levels of sCD14 may
contribute to the anti-inflammatory effects of statins.
2569
Interaction of Polycystin-1 with ADP Ribosylation Factor (ARF) family G Proteins.
T. Wang1, K. L. Gorelick 1, G. G. Germino1; 1National Institute of Diabetes and Digestive and
Kidney Diseases (NIDDK), NIH, Bethesda, MD
Autosomal dominant polycystic kidney disease (ADPKD) is a multisystem disorder,
characterized by bilateral renal cysts and cysts in other organs. Mutations in PKD1 cause 85%
of ADPKD. The gene encodes polycystin-1 (PC1), a 4072 aa protein with an ~3000aa
extracellular N-terminus, 11 TMs, and a short cytoplasmic C-terminus. The first intracellular loop
of PC1 contains the Polycystin-1, Lipoxygenase, Alpha-Toxin (PLAT) domain, a highly
conserved, signature motif common to the entire PC1-like family of proteins whose function is
unknown. We used the PLAT domain of a related polycystin-1 family member, PKDREJ, as bait
in a yeast two-hybrid screen of a testis library and isolated Arl4, a member of the ADP
ribosylation factor (ARF) family of proteins, as a putative interactor. ARF proteins belong to the
small GTPase superfamily and are suggested to have roles in vesicle formation and trafficking.
We confirmed the interaction using the PLAT domain of PC1 and extended these studies to
demonstrate co-immunoprecipitation between the full length proteins in HEK293 cells. We
further show by co-immunoprecipitation and immunostaining that other members of the ARF
family also associate with PC1. Using an MDCK in vitro tubulogenesis system we have
previously described, we find that shRNA knockdown of at least one of these factors disrupts
PC1-mediated tube formation. In summary, our data suggest that the PLAT domain may
mediate important, functional interactions with members of the ARF family.
2570
Inter-Golgi Transport Mediated by COPI Vesicles Carrying Secretory Cargo and Golgi
Resident Glycosyltransferases.
P. Pellett1, F. Dietrich2, J. Bewersdorf3, J. Rothman3, G. Lavieu 3; 1Department of Chemistry/
Cell Biology, Yale University, New Haven, CT, 2Fakultät für Mathematik, Technische Universität
München, Garching, Germany, 3Department of Cell Biology, Yale University, New Haven, CT
Trafficking of anterograde cargoes and Golgi resident enzymes through the Golgi is central to
the secretory pathway. However, the role of COPI vesicles in anterograde and retrograde
transport has remained controversial. The use of a cell-cell fusion assay, in which we can
evaluate transport between two or more well separated Golgis by fluorescence microscopy, and
super-resolution microscopy reveal that COPI positive transport intermediates contain either
fluorescently tagged anterograde cargo (60 +/- 17%), fluorescently tagged resident Golgi
enzymes (21 +/- 8%) or both cargoes together (18 +/- 6%). The size of these COPI positive
inter-Golgi transport intermediates (124 +/- 16nm) is similar to the size of purified COPI vesicles
(114 +/- 11nm). Our data strongly suggest that COPI vesicles play a role in both anterograde
and retrograde transport.
SUNDAY-LATE POSTER PRESENTATIONS
2571
A housekeeping Golgi-based signalling pathway controls retrograde transport between
Golgi and endoplamic reticulum.
J. Cancino1,2, A. Capalbo1,2, M. Giannotta3, M. Sallese3, A. Luini1,2; 1Istituto di Biochimica delle
Proteine (IBP) Consiglio Nazionale delle Ricerca (CNR), Napoli, Italy, 2Telethon Institute for
Genetic and Medicine (TIGEM), Napoli, Italy, 3Consorzio Mario Negri Sud, Santa Maria Imbaro,
Italy
The eukaryotic intracellular membrane transport is composed of transport stations that are
constantly subjected to large anterograde and retrograde membrane fluxes through specialized
carriers. The extent and composition of these fluxes must be precisely controlled by control
systems to maintain the homeostasis of the endomembrane system. These control systems
have been proposed to depend on either the self-assembly properties of relevant components,
and/or on dedicated signaling circuits or, more likely, on the integration of both. Indeed, several
signal transduction molecules reside on endomembranes, and a variety of kinases have been
shown to potently affect different traffic steps. However, the organizational principles, the full
molecular composition and the flow of information through the endomembrane based signaling
pathways, are poorly understood, despite their physiological importance.
Here we describes the molecular composition and organization of a control circuit operating at
the interface between cis-Golgi and ER that senses the intensity of membrane trafficking
reaching the Golgi from the ER. We use specific cargo synchronization protocol to control timing
and intensity of traffic from endoplasmic reticulum (ER) to the Golgi complex followed by
quantitative phospho-protein and quantitative image analysis to delineate and characterize that
is modulated by the incoming traffic at the Golgi apparatus. We characterize here a KDELRdependent cAMP/PKA pathway located at the Golgi that modulates actin cytoskeleton dynamic
to control the retrograde traffic from the Golgi to the ER. Thus it helps to maintain membrane
homeostasis at the ER-Golgi interface.
2572
Degradation of EDEM1 occurs by selective autophagy.
I. Jang1, S. Park2, J. W. Cho1,2, J. Roth2; 1Department of System Biology, Yonsei University,
Seoul, Korea, 2Department of Integrated OMICS for Biomedical Science, Yonsei University,
Seoul, Korea
The endoplasmic reticulum (ER) has a quality control system monitoring the folding state of
newly synthesized proteins. Misfolded glycoproteins are retained in the ER and subsequently
targeted for degradation by a process called ER-associated protein degradation (ERAD).
EDEM1 is an ERAD component that interacts with misfolded luminal glycoproteins and routes
them for dislocation. This is followed by their ubiqitination and proteasomal degradation.
Although EDEM1 was proposed to be a lectin–like protein and to react with Man8GlcNAc2
oligosaccharides of glycoproteins, its mechanism of action and its fate are still largely unknown.
Previously, we reported that EDEM1 exits the ER by a vesicular non-COPII-mediated
mechanism and becomes rapidly degraded by basal autophagy. Here, we report details of how
EDEM1 becomes degraded. After EDEM1 is dislocated to the cytosol, it is forming complexes
with the selective autophagy cargo receptors p62, NBR1 and Alfy that are removed by selective
autophagy. This was directly demonstrated by double and triple confocal laser scanning
immunofluorescence and double immunogold electron microscopy in HepG2 cells. Following
inhibition of autophagy by wortmannin, the number and size of cytoplasmic clusters composed
of EDEM1 and the selective autophagy cargo receptors dramatically increased and this
aggregate formation was independent of the activity of HDAC6. These changes were fully
reversible upon wortmannin wash-out. When EDEM1 was co-immunoprecipitated with p62, only
SUNDAY-LATE POSTER PRESENTATIONS
its de-glycosylated form was detected and found to be ubiquitinated. We observed that
deglycosylation of EDEM1 occured by the action of the cytosolic peptide N-glycanase since
treatment with inhibitors resulted in a strong increase in the amount of glycosylated EDEM1.
Inhibition of cytosolic peptide N-glycanase also inhibited wortmannin-induced aggregation of
EDEM1 and ist complex formation with p62. This indicates that deglycosylation of EDEM1 is a
prerequisite for subsequent ubiquitination and interaction with selective autophagy cargo
receptors. All together, our data imply that the ERAD component EDEM1 itself undergoes
regulated degradation by ERAD, which involves selective autophagy.
This work was supported by the World Class University program through the National Research
Foundation of Korea funded by the Ministry of Education, Science and Technology (R31-2008000-10086-0) and a grant from the National Research Foundation of Korea by the Ministry of
Education, Science and Technology (2010-0027736). I.J. is a fellowship awardee of the Brain
Korea 21 program.
2573
ERp44, a chaperone involved in ER quality control activates PKA through the KDELR.
J. E. Jung1,2, J. Cancino1,2, A. di Campli1, R. Rizzo1,2, R. Sitia3, A. Luini1,2; 1Istituto di Biochimica
delle Proteine, CNR, Napoli, Italy, 2Telethon Institute of Genetics and Medicine, Napoli, Italy,
3
Università Vita San Raffaele Scientific Institute, Milano, Italy
The secretory pathway is composed of several membrane-bound organelles and compartments
that together are responsible for the delivery of all the proteins inserted into the lumen or the
membrane of the Endoplasmic Reticulum (ER) to their final destinations (about one third of the
total proteins).
Despite the large amount of membrane fluxes that traverse this pathway, the system is able to
maintain its integrity. We propose that this is in part because of the intrinsic properties of its
components (coats, adaptors, etc.) but mainly because there are regulatory circuits that
maintain the systems balance. One of these regulatory circuits has been characterized in our
laboratory and found to be dependent on the KDEL receptor (KDELR). We have demonstrated
that this receptor acts as a G-protein coupled receptor that is able to activate the G proteins Gq
and Gs in response to protein trafficking from the ER to the Golgi. Trafficking from the ER
initiates signaling cascades leading to the activation of two kinases: PKA and Src, at the Golgi
complex. These traffic-activated kinases control traffic itself by regulating anterograde and
retrograde transport; moreover, they might also regulate other cellular functions. Our hypothesis
is that during trafficking some chaperones containing KDEL-like sequences bind and activate
the KDELR, thus generating the signaling events. We tested different KDEL-like sequences and
proteins with regard to their ability to induce PKA and Src signaling at the Golgi and we found
that ERp44, a member of the thioredoxin family of chaperones is able to activate a Golgiassociated PKA. Moreover, cells depleted of ERp44 show defects in membrane trafficking. Our
results show a novel function for ERp44 and highlight the existence of endogenous ligands for
the KDELR-signaling system.
2574
Characterization of early secretory pathway regulators.
F. Verissimo1, C. Tischer1, M. Neukam1, S. Scharaw1, R. Pepperkok1; 1EMBL, Heidelberg,
Germany
A genome-wide siRNA screening revealed several hundred genes with a potential role in the
early secretory pathway (Simpson et al., 2012). Here we selected 35 of these genes with an
effect on COPII organization and/or endoplasmic reticulum (ER) localization and evaluated their
SUNDAY-LATE POSTER PRESENTATIONS
putative role in the regulation of ER export. We used RNA interference and quantitative timelapse imaging of the secretory marker VSVG in living cells to identify genes with a role in ER to
Golgi transport and grouped them according to their phenotypes, establishing the functional
classes: protein folding, carrier formation and cargo selection/concentration. To support the
function of these proteins in the early secretory pathway, the effect of the ectopically expressed
GFP-tagged proteins on VSVG transport was also evaluated. Immunoprecipitation of the GFPtagged proteins from cell extracts was used to reveal a possible interaction of the candidates
with COPII components. Altogether, this work identified 13 proteins with a new role in ER to
Golgi transport, supporting the notion that the COPII coat operates in the context of a network of
proteins involved in regulating the different steps of cargo export from the ER.
Simpson, J. C., Joggerst, B., Laketa, V., Verissimo, F., Cetin, C., Erfle, H., Bexiga, M.
G., Singan, V. R., Heriche, J. K., Neumann, B. et al. (2012). Genome-wide RNAi screening
identifies human proteins with a regulatory function in the early secretory pathway. Nat Cell Biol
14, 764-74.
2575
Role of the Erv41-Erv46 Complex in Endoplasmic Reticulum Quality Control.
N. G. Margulis1, C. Barlowe1; 1Biochemistry, Geisel School of Medicine at Dartmouth, Hanover,
NH
In the endoplasmic reticulum (ER), properly folded and assembled secretory proteins are
selectively exported in COPII vesicles, whereas misfolded proteins are retained and
retrotranslocated to the cytosol for proteasomal degradation via the ER-associated degradation
(ERAD) pathway. Proteomic approaches have identified ER vesicle (Erv) proteins that actively
cycle between the ER and Golgi compartments and function in trafficking through the early
secretory pathway. Interestingly, studies show that Erv proteins also influence ER quality control
of terminally misfolded model ERAD substrates. We are investigating the highly conserved
Erv41-Erv46 vesicle proteins to gain insight on the mechanisms by which ER quality control and
ER export are coordinated. Erv41 and Erv46 form an integral membrane complex that cycles
between the ER and Golgi but has no clear molecular function. The erv41Δ and erv46Δ
deletion mutations in yeast do not produce detectable delays in forward transport of secretory
cargo, however we observe that these mutations accelerate turnover of a diversity of lumenal
ERAD substrates including PrA*, ALP* and Gas1*. Surprisingly, this accelerated turnover
proceeds via an ERAD- and proteasome-independent pathway. Model ERAD substrates are
hyperglycosylated and strongly stabilized in double deletion strains when erv41Δ is paired with
vps10Δ or pep4Δ, indicating that this degradation pathway traverses the Golgi and endosomal
systems and terminates in the vacuole. We also find that combining erv41Δ with deletions in
the N-glycan trimming machinery produces expected glycosylation defects on ERAD substrates
but these substrates are degraded at the accelerated erv41Δ rate, indicating that Erv41-Erv46
functions downstream of initial folding steps in a glycan-independent manner. We propose that
the Erv41-Erv46 complex acts on ER recognition and retention of misfolded secretory proteins
at the junction of ERAD and post-ER quality control pathways.
SUNDAY-LATE POSTER PRESENTATIONS
2576
Spatiotemporal regulation of FMNL2 through PKCα mediates α5β1 integrin internalization
and cancer cell invasion.
Y. Wang1, J. Pouwels2, A. Arjonen2, H. Ta3, T. Hofmann1, J. Ivaska2, R. Grosse1; 1Institute of
Pharmacology, University of Marburg, Marburg, Germany, 2Centre for Biotechnology, University
of Turku, Turku, Finland, 3Department of NanoBiophotonics, Max Planck Institute for Biophysical
Chemistry, Goettingen, Germany
The formin family of actin assembly factors polymerizes linear actin filaments for an array of
cellular functions including cytokinesis, adhesion and cell motility. We have previously shown
that formin-like 2 (FMNL2) is involved in invasive migration of cancer cells. Here we performed
an integrin endocytosis screen of actin cytoskeletal regulators and identified FMNL2 as a
candidate affecting β1-integrin trafficking, which is known to be regulated by PKC. Interestingly,
we find that FMNL2 is regulated through C-terminal phosphorylation, thereby releasing FMNL2
autoinhibition. We identify PKCα as the predominant PKC isoform that associates with and
phosphorylates FMNL2 in vitro and in vivo. Strikingly, stimulation of PKC activity rapidly
translocates FMNL2 from the plasma membrane to α5-integrin positive vesicles. Silencing of
FMNL2 inhibits β1-integrin internalization, suggesting that FMNL2 controls integrin trafficking
necessary for invasive tumor cell migration. In agreement with this, a phosphomimetic mutant of
FMNL2 drives cancer cell invasion, while a non-phosphorylatable mutant inhibits it. Taken
together, we identified a novel mechanism that tightly controls FMNL2 function in a
spatiotemporal manner to regulate integrin trafficking for invasive migration.
2577
Quantitative study of effects of free cationic chains on intracellular trafficking of small
DNA/polymer polyplexes using confocal laser scanning microscopy.
J. Cai1, Y. Yue1, C. Wu1; 1The Chinese University of Hong Kong, N.T., Hong Kong
Our previous studies suggested that nearly all the anionic DNA chains are complexed by
cationic polyethylenimine (PEI) chains to form small polyplexes (~102 nm) when the molar ratio
of nitrogen from PEI to phosphate from DNA (N:P) reaches ~3, but the in vitro gene transfection
efficiency is ~100 fold higher when N:P ≥ 10, indicating that those further added chains free in
the solution mixture play an important role in promoting the gene transfection. Using a
quantitative analysis of three-dimensional confocal images, we found that adding free cationic
chains only slightly increases the cellular uptake rate and amount of the polyplexes. Therefore,
the cellular uptake is not the main reason for the high gene transfection efficiency. In order to
elucidate the mechanism of how the free cationic chains promote the gene transfection, we
have investigated the intracellular trafficking of the polyplexes with or without in the presence of
free cationic chains by labelling DNA and lysosomes with Cy3 and GFP, respectively. Our
results reveal that adding long free PEI chains (M ~ 25,000 g/mol) reduces the entrapment of
the polyplexes inside the lysosomes from 75% to 30%, irrespective of the chain topology (linear
or branched). For short free PEI chains (M ~ 2,000 g/mol), linear free chains also prevent the
polyplexes entrapment, whereas branched free chains have no such an ability. On the basis of
our current results and literature data, we hypothesize that those free cationic chains with a
sufficient length (15-20 nm) are partially embedded inside or adsorbed on the membranes of
different organelles via the electrostatic interaction so that they 1) interfere the inter-vesicular
fusion signals (proteins) and prevent the endosomes-to-endolysosomes development so that
the gene will not be digested inside the lysosomes; and 2) destabilize/weaken the endosome
membrane to promote the escape of the polyplexes entrapped inside so that the gene has a
high chance to be translocated inside the nucleus.
SUNDAY-LATE POSTER PRESENTATIONS
2578
Cellular trafficking and cytotoxicity assessment of cerium oxide nanoparticles in lung
derived cells.
J. Mazzolini1, S. Chen1, J. Lead1, J. Z. Rappoport1; 1The University of Birmingham, Birmingham,
United Kingdom
The increasing potential for exposure to manufactured nanomaterials has created significant
concern regarding the risk of toxicological outcomes. Nanoparticles present in exhaust could
affect cells of the airway, while those applied to the skin or ingested through food and drink
could similarly gain entry in the body. Cerium oxide nanoparticles can be used to increase fuel
efficiency in internal combustion engines. Therefore we have assessed whether these will enter
lung derived cells, and potentially result in cytotoxicity. We have employed confocal reflectance
imaging and UV spectrophotometry to assess the mechanisms of entry of cerium oxide
nanoparticles into A549 cells and have demonstrated an apparently non-saturable linear
increase in uptake with increasing concentrations and times of incubation. Internalisation was
inhibited by Dynasore treatment demonstrating a significant role for a micro-pinocytotic
endocytosis mechanism. Remarkably, even after several days at relatively high concentrations
(e.g. 1mg/ml) no cytotoxicity was observed. Interestingly though cell-to-cell transmission of
cerium oxide nanoparticles was observed via tunnelling nanotubes (TNTs) between adjacent
cells, suggesting that nanoparticles could be transferred across the airway into cells which could
be sensitive to toxicological affects.
Therefore, in summary, we have demonstrated
accumulation of cerium oxide nanoparticles into lung derived cells through a dynamin
dependant mechanism, and while no cytotoxicity was detected we did observe transfer of
nanoparticles between cells via TNTs. Thus, these results suggest the potential for transfer of
nanoparticles across the intact airway and demonstrate the need for further analysis of the
potential for toxicological effects following exposure to cerium oxide through the airway.
2579
Structural and biochemical analysis of the regulation of human and bacterial ArfGEFs by
membranes.
J. Cherfils1, K. Aizel1, V. Biou1, V. Campanacci1, M. Folly-Klan1, M. Zeghouf1; 1Laboratoire
d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette, France
Small GTP-binding proteins of the Arf family (Arf GTPases) interact with multiple cellular
partners and with membranes to regulate intracellular traffic and organelle structure. When and
where they become active in the cell is controlled by guanine nucleotide exchange factors
(GEFs). GEFs stimulate the intrinsically slow GDP/GTP exchange of small GTPases, thereby
converting them into their active, GTP-bound form. It is now accepted that most, if not all, GEFs
are themselves highly regulated. Here, we combined structural studies with in vitro biochemical
assays that reconstitute GEF-stimulated nucleotide exchange on artificial membranes to
analyze the regulation of Brag2, a human ArfGEF associated with EGF signaling in cancer cells
and in receptor endocytosis, and of Legionella RalF, a type IV secreted ArfGEF that diverts host
Arf proteins at the onset of infection. These studies reveal that each GEF monitors a unique
ensemble of physico-chemical properties of membrane, resulting in exquisitely regulated
nucleotide exchange activities.
SUNDAY-LATE POSTER PRESENTATIONS
2580
Proprotein convertase subtilisin/kexin type 9 (PCSK9) can mediate degradation of the
low density lipoprotein receptor-related protein (LRP-1).
M. Canuel1, X. Sun1, M-C. Asselin1, A. Prat1, E. Paramithiotis2, N. G. Seidah1; 1IRCM, Montreal,
QC, Canada, 2Caprion Proteomics Inc., Montreal, QC, Canada
Elevated LDL cholesterol (LDLc) levels are a major risk factor for cardiovascular disease and
atherosclerosis. LDLc is cleared from circulation by the LDL receptor (LDLR). Proprotein
convertase subtilisin/kexin 9 (PCSK9) enhances the degradation of the LDLR in
endosomes/lysosomes, resulting in increased circulating LDLc. PCSK9 can also mediate the
degradation of LDLR lacking its cytosolic tail, suggesting the presence of as yet undefined
lysosomal-targeting factor(s). Herein, we confirm this, and also eliminate a role for the
transmembrane-domain of the LDLR in mediating its PCSK9-induced internalization and
degradation. To identify potential [PCSK9.LDLR] partners, we compared by quantitative mass
spectrometry livers from Pcsk9-/- and wild type mice. While proteomics from Pcsk9-/- mice
revealed a ~2.5-fold increase in LDLR, we detected a minor (~20%) decrease in the levels of
low density lipoprotein receptor-related protein 1 (LRP-1). Western blot analysis of HEK293 and
HepG2 cells showed that PCSK9 can induce the degradation of LRP-1. Furthermore, in B16F1
melanoma cells, PCSK9 induces the degradation of LRP-1 but not LDLR, revealing the
presence of distinct targeting-partners of LRP-1 and LDLR. Furthermore, PCSK9 is still capable
of acting upon the LDLR in CHO 13-5-1 cells lacking LRP-1. Conversely, PCSK9 also acts on
LRP-1 in the absence of the LDLR in CHO-A7 cells, where re-introduction of the LDLR leads to
reduced PCSK9-mediated degradation of LRP-1. Thus, while we have shown that PCSK9 is
capable of inducing degradation of LRP-1, the latter is not an essential factor for LDLR
regulation, but LDLR effectively competes with LRP-1 for PCSK9 activity.
2581
Epsin1 promotes LDLR Internalization via an FXNPXY-Independent Pathway.
Y-L. Kang1, J. Yochem2, L. Bell2, E. B. Sorensen3, L. Chen1, S. D. Conner1; 1Department of
Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN,
2
Department of Molecular Biology, University of Wyoming, Laramie, WY, 3Department of
Biochemistry, University of Wisconsin-Madison, Madison, WI
Low density lipoprotein receptor (LDLR) internalization clears cholesterol-laden LDL particles
from circulation in humans. Defects in clathrin-dependent LDLR endocytosis promote elevated
serum cholesterol levels and can lead to atherosclerosis. However, our understanding of the
mechanisms that control LDLR uptake remains incomplete. To identify factors critical to LDLR
uptake, we pursued a genome-wide RNAi screen using Caenorhabditis elegans LRP-1/megalin
as a model for LDLR transport. In doing so, we discovered an unanticipated requirement for the
clathrin-binding endocytic adaptor epsin1 in LDLR endocytosis. Epsin1 depletion reduced LDLR
internalization rates in mammalian cells, similar to that observed following clathrin depletion.
Genetic and biochemical analyses of epsin in C. elegans and mammalian cells uncovered a
requirement for the ubiquitin-interaction motif (UIM) as critical for receptor transport. As the
epsin UIM promotes the internalization of some ubiquitinated receptors, we predicted LDLR
ubiquitination as necessary for endocytosis. However, engineered ubiquitination-impaired
LDLR mutants showed modest internalization defects that were further enhanced with epsin1
depletion, demonstrating epsin1-mediated LDLR endocytosis is independent of receptor
ubiquitination. Finally, we provide evidence that epsin1-mediated LDLR endocytosis occurs
independently of Dab2, another endocytic adaptor that is critical for LDLR uptake, indicating
distinct mechanisms for LDLR endocytosis.
SUNDAY-LATE POSTER PRESENTATIONS
2582
Polarized sorting of the cyclic nucleotide-gated channel to the light-sensing cilia of
mouse photoreceptor cells requires the endocytic protein Numb.
V. Ramamurthy1,2, C. Jolicoeur1, J. Mühlhans3, D. Koutroumbas4, L. Yun-Zheng5, W. W.
Hauswirth6, A. Giessl3, M. Cayouette1,2,4,7; 1Institut de recherches cliniques de Montreal (IRCM),
Montreal, QC, Canada, 2Div. of Experimental Medicine, McGill University, Montreal, QC,
Canada, 3Department of Biology, Animal Physiology, University of Erlangen-Nuremberg,
Erlangen, Germany, 4Department of Anatomy and Cell Biology, McGill University, Montreal, QC,
Canada, 5Department of Cell Biology, University of Oklahoma Health Sciences Center,
Oklahoma City, OK, 6Department of Ophthalmology and Powell Gene Therapy Center,
University of Florida, Gainesville, FL, 7Department of Medicine, Université de Montréal,
Montreal, QC, Canada
Cell physiology and survival critically depend on mechanisms regulating the development and
maintenance of polarity, which is manifested by the asymmetric distribution of proteins or
organelles to specific sub-cellular compartments. A prime example of this is observed in
mammalian photoreceptor cells, where the proteins involved in the phototransduction cascade
are housed exclusively in a specialized primary cilium called the outer segment (OS). Despite
the importance of polarized protein trafficking in photoreceptor physiology and disease, the
mechanisms regulating this process remain largely unknown. Recent studies have shown that
Numb, an endocytic adapter protein, plays a part in regulating directional protein trafficking in
migrating cells and neurons, raising the possibility that it might be involved in OS protein sorting.
Consistent with this idea, we find that Numb is expressed in a highly polarized manner in the
preciliary region of rod photoreceptors, located between the inner segments (IS), where proteins
are synthesized, and the OS, where proteins involved in phototransduction are targeted. To
study the role of Numb in photoreceptors, we used the Cre/lox system to generate a conditional
knockout (cKO) mouse in which the Numb gene is specifically inactivated in postmitotic rod
photoreceptors. While rod OS disk membrane proteins trafficked normally in Numb cKO
photoreceptors, the cyclic nucleotide-gated channel (CNGC), which normally localizes to the
plasma membrane of rod OS, accumulated in the preciliary region of the IS. Since CGNC was
also observed in this region in wiltype rod photoreceptors, albeit at much reduced levels, these
results suggested a model in which the preciliary domain might constitute an intermediate
sorting station wherein Numb-mediated endocytosis of CNGC would promote trafficking to the
OS. Consistent with this possibility, we found that Numb and CNGC physically interact both in
vitro and in vivo, and that overexpressing Numb in a heterologous cell culture assay increases
the pool of CNGC localized in early endosomes. Together, these results support a new model
implicating polarized endocytosis in OS plasma membrane protein sorting in mouse rod
photoreceptor cells. In addition, since Numb cKO photoreceptors ultimately undergo progressive
degeneration, this study could have important implications in our understanding of the
pathophysiology leading to retinal degeneration in humans.
2583
Quantitative analysis of the dynamics of protein-complex formation during clathrinmediated endocytosis.
D. Boeke1, S. Trautmann-Hoenicke1,2, M. Wachsmuth1, M. Knop3, M. Kaksonen1; 1Cell Biology
and Biophysics, EMBL, Heidelberg, Germany, 2Picoquant GmbH, Berlin, Germany, 3ZMBH,
Heidelberg University, Heidelberg, Germany
Clathrin-mediated endocytosis is a highly conserved trafficking pathway that depends on
dynamic protein-protein interactions between up to 50 different molecules, which are recruited
to the endocytic site from the cytoplasmic pool. However, little is known about the spatio-
SUNDAY-LATE POSTER PRESENTATIONS
temporal regulation of these interactions. We applied fluorescence (cross)-correlation
spectroscopy (FCS/FCCS) to characterize these interactions in vivo. FCS/FCCS allows
quantification of protein concentrations and protein-protein interactions in the cytoplasm of living
cells and yields the diffusion time of proteins as well as the stoichiometry of protein complexes.
Obtaining all of these data simultaneously is unique for an in vivo technique and enables us to
draw conclusions about how protein-protein interactions are spatially regulated.
To perform FCS/FCCS experiments, we tagged the different components of the yeast endocytic
machinery with two spectrally distinct fluorophores. We determined the diffusion coefficient and
the cytoplasmic concentration of 37 endocytic proteins and quantified multiple cytosolic
interactions. Two key endocytic scaffold proteins, Ede1 and Pan1 were further analyzed. While
Ede1 is a crucial factor in organizing the early endocytic pathway, Pan1 is essential for the later
stages of endocytosis. Both proteins contain multiple Eps15 homology (EH) domains through
which they interact with various endocytic proteins. Our FCS/FCCS analysis indicates that Ede1
forms oligomers in the cytoplasm. This property could contribute to its proposed function in
increasing the local concentration of endocytic adaptors, which link cargo to the endocytic coat
at the plasma membrane. Investigation of different truncation mutants of Ede1 revealed a
central coiled coil domain of Ede1 to be essential for its self-interaction. Interestingly, Ede1
mutants lacking the coiled coil domain (ede1ΔCC) failed to properly target Ede1 to the
membrane. Chemically induced artificial dimerization of ede1ΔCC could rescue its localization.
Pan1 does not appear to oligomerize in the cytoplasm. However, using FCCS, we could show
that Pan1 interacts tightly with another EH-domain-containing protein End3 in the cytoplasm.
Oligomerization of Ede1 and stable binding of Pan1 to End3 might indicate that interaction
between EH-domain-containing proteins is a common mechanism in helping to organize the
endocytic coat.
2584
G-Clathrin regulation of cargo and receptor trafficking and signaling.
Y. Luo1, J. Keen1; 1Dept. of Biochemistry and Molecular Biology, Thomas Jefferson University,
Philadelphia, PA
We have previously identified gyrating- “G-†clathrin, a subgroup of clathrin-coated
membranes exhibiting rapid and localized movement at the periphery of cells. Recently, we
further demonstrated that G-clathrin is predominately regulated by Arf6, a member of the family
of small Ras-related GTPases. Here, we further study the function of G-clathrin in intracellular
trafficking. Using fluorescently-tagged epidermal growth factor receptor “EGFR†,
transferrin receptor “TfR†, low density lipoprotein receptor “LDLR†and mannose6-phosphate receptor “M6PR†constructs at low expression levels, we find that the
fluorescent receptors are generally present in the cell periphery as vesicular and short tubular
structures, as others have observed. However, rapid “gyrating†movement of a fraction
of these receptors can also be visualized by live cell imaging at ≥27 Hz exposure rate.
Moreover, co-expression of mCherry-clathrin reveals that all of these “G-receptors†show
some degree of co-localization with G-clathrin. Especially coincident with G-clathrin are EGFR:
almost all G-EGFRs have corresponding G-clathrin in the absence of EGF ligand. We also find
that, as with G-clathrin, G-EGFR is regulated by Arf proteins. Depletion of both Arf6 and Arf1,
which resulted in almost complete disappearance of G-clathrin, also almost abolishes G-EGFR
as well. However, relatively stationary or less dynamic EGFR structures, almost of which also
contain clathrin, persist in the absence of Arf6 and Arf1. Also, overexpression of wild-type Arf6
increases G-clathrin/EGFR while expression of Arf6(Q67L), a GTPase-deficient mutant which
dramatically increases G-clathrin levels, does not increase G-EGFR. This suggests that the full
SUNDAY-LATE POSTER PRESENTATIONS
Arf6 cycle is critical for EGFR sorting into G-clathrin structures and/or that Arf6 plays multiple
functions in EGFR trafficking. Previously we also demonstrated that expression of GGA1
increases the levels of G-clathrin, and the two proteins are virtually completely coincident.
Interestingly, overexpression of GGA1 results in reduction of G-EGFR, although the levels of Gclathrin increase as we have previously reported. This suggests that the presence of GGA1 in
G-clathrin structures may block the recruitment of EGFR. Consistent with this interpretation, the
levels of G-M6PR and its colocalization with G-clathrin were increased in GGA1-expressing
cells. In this context, the partial colocalization of G-TfR with G-clathrin may also explain the
limited reduction of Tf recycling observed (about 30%) by inhibiting G-clathrin upon depletion of
both Arf1 and Arf6. As EGFR shows a much higher co-localization with G-clathrin, the effect of
G-clathrin in EGFR recycling will be important to evaluate, as will the identification of the
adaptor used in recruitment of EGFR to G-clathrin structures.
2585
Ubiquitin-specific protease TRE17/USP6 targets clathrin-independent cargo to recycling.
Y. Funakoshi1,2, Y. Kanaho2, J. G. Donaldson1; 1Laboratory of Cell Biology, National Heart,
Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 2Department of
Physiological Chemistry, Graduate School of Comprehensive Human Sciences and Institute of
Basic Medical Sciences, University of Tsukuba, Tsukuba, Japan
The density and distribution of plasma membrane proteins at the cell surface are regulated
through endocytosis. Following endocytosis, internalized proteins are sorted either to late
endosomes/lysosomes for degradation or recycled back to the plasma membrane. Therefore,
the control of cargo protein sorting plays a pivotal role in the maintenance of quality and quantity
of plasma membrane proteins. It has been shown that the addition of ubiquitin targets cargo
proteins to lysosomal degradation. Previously, we demonstrated in HeLa cells that expression of
the membrane-associated RING-CH (MARCH) ubiquitin ligases alters trafficking of a set of
proteins that are internalized through clathrin-independent endocytosis (CIE) from recycling to
degradation. In the present study, we find that the ubiquitin-specific protease TRE17/USP6 can
couteract the effect of MARCH. TRE17 was originally identified as a potential oncogene from
Ewing’s carcinoma. It harbors a deubiquitinating (DUB) domain which catalyzes the hydrolysis
of ubiquitin. The DUB domain of TRE17 has been implicated in tumorigenesis of osteoblasts
and NIH3T3 cell line. Here we report a novel function of TRE17 mediated through the DUB
domain.
Expression of MARCH ubiquitinates some of CIE cargos and targets them to late
endosome/lysosome, resulting in a decrease of their surface level. When we co-expressed
TRE17 with MARCH, the targeting of the cargos to late endosome/lysosome was suppressed
and the cell surface level was partially recovered. Ubiquitination of the cargo by MARCH was
also reversed by the expression of TRE17 accompanied by stabilization of the cargo protein.
The above effects of TRE17 were DUB-dependent since the TRE17 mutant that lacks DUB
activity could not suppress the MARCH-dependent trafficking alteration and ubiquitination of
cargos. These results indicate that TRE17 is a novel deubiquitinating enzyme that targets
membrane proteins to a recycling pathway through the control of ubiquitination. TRE17 and
MARCH may balance the ubiquitination status of cargo proteins to determine whether they are
degraded or recycled.
SUNDAY-LATE POSTER PRESENTATIONS
2586
The PKD domain distinguishes the trafficking and amyloidogenic properties of the
pigment cell protein PMEL and its homologue GPNMB.
A. C. Theos1, B. Watt2, S. C. Theos2, K. E. Herman2, D. C. Harper2, M. S. Marks2; 1Human
Science, Georgetown University, Washington, DC, 2Pathology & Laboratory Medicine and
Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA
Proteolytic fragments of the pigment cell-specific glycoprotein, PMEL, form the amyloid fibrillar
matrix underlying melanins in melanosomes. The fibrils form within multivesicular endosomes to
which PMEL is selectively sorted and that serve as melanosome precursors. GPNMB is a
tissue-restricted glycoprotein with substantial sequence homology to PMEL but no known
function, and was proposed to localize to non-fibrillar domains of distinct melanosome
subcompartments in melanocytes. Here we confirm that GPNMB localizes to compartments
distinct from the PMEL-containing multivesicular premelanosomes or late endosomes in
melanocytes and HeLa cells, respectively, and is largely absent from fibrils. Using domain
swapping, the unique PMEL localization is ascribed to its PKD domain, whereas the
homologous PKD domain of GPNMB lacks apparent sorting function. The difference likely
reflects extensive modification of the GPNMB PKD domain by N-glycosylation, nullifying its
sorting function. These results reveal the molecular basis for the distinct trafficking and
morphogenetic properties of PMEL and GPNMB, and support a deterministic function of the
PMEL PKD domain in both protein sorting and amyloidogenesis.
2587
Tracking membrane dynamics and fusion during plasma membrane repair.
R. T. Pedersen1, N. Davenport1,2, A. Audhya3, W. Bement1; 1Laboratory of Cell and Molecular
Biology, University of Wisconsin, Madison, WI, 2Program in Cell and Molecular Biology,
University of Wisconsin-Madison, Madison, WI, 3Department of Biomolecular Chemistry,
University of Wisconsin-Madison, Madison, WI
Physical insults to individual cells are a regular occurrence for any organism. Upon wounding,
the cell must respond quickly to repair the plasma membrane in order to stop unregulated
exchange between the cell interior and exterior. This repair process depends on extracellular
calcium and has been thought to rely on exocytosis as well as vesicle-vesicle fusion to seal off
the site of PM damage. However, the identity of the compartments involved in resealing PM
wounds remains controversial. I set out to determine which vesicles patch PM wounds. For this
work, I employed Xenopus laevis oocytes, an ideal model system for investigation of single cell
wound healing: their large size allows for easy microinjection with mRNA and subsequent
imaging. By expressing fluorescently tagged Rab5, Rab7, Rab11, and Lamp2B in oocytes, I
have labeled early endosomes, late endosomes, recycling endosomes, and lysosomes,
respectively. I have also used fluorescent dextrans to label compartments derived from
exocytosis. Using 4D imaging and laser ablation, I have imaged vesicle dynamics during cell
wound healing in vivo. Thus far, all vesicle populations tracked have become concentrated at
the wound to some degree. I have found that several vesicle populations localize to wounds in
distinct patterns and that different compartments fuse to contribute to the PM patch. I also
describe novel behavior of vesicles at the wound site.
SUNDAY-LATE POSTER PRESENTATIONS
2588
The autophagosome forms at ER−mitochondria contact sites.
M. Hamasaki1, N. Furuta2, A. Matsuda3,4, A. Nezu1, A. Yamamoto5, H. Oomori6, T. Haraguchi3,
Y. Hiraoka4, T. Yoshimori1, A. Amano2,7; 1Graduate School of Medicine, Osaka University,
Osaka, Japan, 2Dep. of Oral Frontier Biology, Center for Oral Frontier Science, Osaka, Japan,
3
National Institute of Information and Communications Technology, kobe, Japan, 4Biomolecular
Networks Laboratories Group, Graduate School of Frontier Biosciences, Osaka University,
Osaka, Japan, 5Department of Cell Biology, Nagahama Institute of Bio-Science and
Technology, Nagahama, Japan, 6Research Institute for Microbial Diseases, Osaka, Japan,
7
Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, Osaka,
Japan
Autophagy is a tightly regulated intracellular bulk degradation system that plays fundamental
roles in cellular homeostasis. Autophagy is initiated by nucleation of small flattened vesicles,
termed the isolation membrane. Isolation membranes engulf portions of the cytoplasm,
organelles, invading pathogens etc and elongate to become autophagosomes, which then fuse
with lysosomes to degrade the contents. The origin of autophagosomal membranes has been
controversial among ER, Mt or plasma membrane.
Upon initiation of autophagy by starvation, Atg14L (a component of autophagosome formation
machinery) relocalizes adjacent to the ER-mitochondria junction and found in the mitochondriaassociated ER membrane (MAM) fraction. Atg5 (a component marks only autophagosomes)
emerge next to ER-mitochondria junction as autophagosome formation start and disappear
when formation complete. Disruption of the ER-mitochondria junction prevents formation of
Atg14L punta that appears upon starvation induction. The ER-resident SNARE protein Syntaxin
17 (Stx17) is bound to Atg14L and also located in ER-mitochondria junction. Knockdown of
Stx17 fails to bring Atg14 to ER-mitochondria junction and to complete the autophagosome
formation indicate the Stx17 is a new component involved in autophagosome formation.
These results suggest ER-Mitochondria junction plays an important site for the autophagosome
formation.
2589
Visualizing internalized cargo acidification in live cells with novel pH-sensing fluorescent
dyes.
C. Langsdorf1, K. Chambers1, D. Beacham1, S. Jaron1, M. Janes1; 1Molecular Probes, part of
Life Technologies, Eugene, OR
The basic cellular internalization processes of endocytosis and phagocytosis are important to
many areas of cell biology including receptor internalization, pathogen response, and apoptotic
cell clearance. However, the ability to study these processes has historically been limited by the
lack of quality tools to measure the acidification of internalized cargo. Here we present an
assortment of tools to enable research into cellular internalization pathways. Two fluorogenic,
pH sensitive probes, one green and one red, are presented. These compounds are nonfluorescent at neutral pH outside the cell, but become brightly fluorescent in acidic cellular
compartments, permitting direct monitoring of the internalization process. We present
experimental data in which these dyes were used to label microorganisms including bacteria
and yeast. Flow cytometry and live cell imaging were then used to study phagocytosis and
acidification of these labeled particles. Additionally, we investigated the ability of these dyes to
monitor phagocytic clearance of apoptotic cells. As with bacteria and yeast, live cell imaging
was used to observe the phagocytosis of primary or cultured apoptotic cells. We anticipate that
these tools will enable additional research into the fields of receptor trafficking, drug delivery,
host-pathogen response, and clearance of apoptotic cells.
SUNDAY-LATE POSTER PRESENTATIONS
Organelles and Membrane Biology
2590
Ouabain modulates ciliogenesis in epithelial cells.
M. I. Larre1, A. Castillo1, R. G. Contreras1, C. E. Flores1, M. Cereijido1; 1Physiology, Cinvestav,
Mexico, Mexico
The exchange of substances between higher organisms and the environment occurs across
transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular
space, and polarity, which enables cells to transport substances vectorially. In a previous study,
we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity
as well. We gauge polarity through the development of a cilium at the apical domain of MadinDarby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an
ERK1/2-dependent manner. Comparing wild-type and ouabain-resistant MDCK cells, we show
that ouabain acts through Na(+),K(+)-ATPase. Taken together, our previous and present results
support the possibility that ouabain constitutes a hormone that modulates the transporting
epithelial phenotype, thereby playing a crucial role in metazoan life.
2591
Understanding the Stacked Morphology of the Golgi by GRASP55 Knock-Sideways
M. Dunlop1, J. Rothman1; 1Cell Biology, Yale University, New Haven, CT
The stack of flattened membranes is a highly conserved morphological feature of the Golgi from
yeast to humans. Despite the huge amount of secreted proteins that traffic from the
endoplasmic reticulum through this obligate intermediate into the plasma membrane and
beyond, the structural integrity of the Golgi remains unperturbed and surprisingly stable. While it
has become clear that a composite of proteins that reside between the Golgi membranes holds
the stack together, it is unclear how these factors cooperate to maintain the stacking as well as
to accommodate cargo transport. In order to better understand the stacked morphology in
relation to the function of the Golgi, we have established a knock-sideways system in HeLa cells
for GRASP55, a component of the Golgi matrix. The addition of a rapamycin analog induces the
rapid redistribution of GRASP55 from the Golgi to the mitochondria. Interestingly, we observed
that not only GRASP55, but also the Golgi membranes as well migrate to the mitochondria.
Furthermore, when the Golgi ribbon is fragmented into mini-stacks by nocodazole, GRASP55
knock-sideways induced the unstacking of Golgi membranes into single cisternae that
sandwiched between mitochondria. These artificial Golgi-mitochondria stacks were capable of
performing conventional secretory functions of the Golgi, such as secretion of sec-HRP or
plasma membrane arrival of VSVG-GFP, suggesting that Golgi function is surprisingly
independent of its stacked organization.
2592
ER-Septin Tethering Polarizes the Endoplasmic Reticulum.
J. T-C. Chao1, S. Tavassoli1, A. K. Wong1, B. P. Young1, A. Chrusciki2, N. N. Fang2, L. J. Howe2,
T. Mayor2, L. Foster2, C. J. Loewen1; 1Cellular and Physiological Sciences, University of British
Columbia, Vancouver, BC, Canada, 2Biochemistry and Molecular Biology, University of British
Columbia, Vancouver, BC, Canada
Polarization of the plasma membrane (PM) into domains is an important mechanism to establish
cell polarity. Polarization requires formation of diffusion barriers that prevent mixing of proteins
between domains. Recent studies have uncovered that the endoplasmic reticulum (ER) is
polarized by diffusion barriers, which control cellular aging in budding yeast and glutamate
SUNDAY-LATE POSTER PRESENTATIONS
signaling in neurons. The identity of these diffusion barriers is currently unknown. We have
found that direct interactions between ER and septins create the ER diffusion barrier in yeast.
The ER diffusion barrier prevents integral ER proteins from diffusing from mother to bud through
the bud neck. This mechanism controls the timing of nuclear migration by preventing the
diffusion of the microtubule cortical capture protein Num1 from mother to bud until its translation
in the bud in M phase. Thus, the ER diffusion barrier in coordination with bud-specific translation
of ER proteins controls the timing of appearance of ER proteins in the bud.
2593
SPO71 mediates prospore membrane size and maturation during sporulation in
Saccharomyces cerevisiae.
E. M. Parodi1, C. S. Baker1, C. Tetzlaff1, S. Villahermosa1, L. S. Huang1; 1Biology, University of
Massachusetts Boston, Boston, MA
We are interested in understanding how the shapes and sizes of membranes are regulated, and
use the Saccharomyces cerevisiae prospore membrane as a model to study this question.
Sporulation is a developmental program that occurs upon starvation in yeast, resulting in the
creation of four haploid spores (or gametes) from a diploid mother cell. The process of
sporulation involves meiosis followed by spore morphogenesis. During spore morphogenesis, a
new membrane structure, a prospore membrane, grows de novo from the spindle pole body to
surround each of the four meiotic nuclei. The prospore membrane ultimately determines the
size and shape of each newly formed spore, as the prospore membrane acts as the template for
spore wall deposition and the membrane’s inner leaflet becomes the plasma membrane of the
new haploid cell. We find that SPO71, which encodes a Pleckstrin-homology domain containing
protein, is required for proper development of the prospore membrane. Cells lacking SPO71
produce prospore membranes that are smaller than those found in wild type cells. These
mutant prospore membranes do not properly localize the sporulation-specific septin, Spr28. We
find that SPO71 genetically interacts with SPO1, a gene with homology to phospholipase B and
important for the proper shape of the prospore membrane. These data suggest a critical role for
SPO71 in prospore membrane morphogenesis.
2594
Neuronal activity-dependent Golgi fragmentation.
D. A. Thayer1, S-B. Yang1, L. Y. Jan1; 1Physiology Department, University of California, San
Francisco, San Francisco, CA
The Golgi complex is essential for many aspects of cellular function, including trafficking and
sorting of membrane and secretory proteins and post-translational modification by glycosylation.
We observed fragmentation of the Golgi complex in cultured hippocampal neurons during
increased neuronal activity by prolonged treatment with bicuculline (Bic) or withdrawal of 2amino-5-phosphonovaleric acid (APV). Bic blocks GABAA-mediated inhibition, thereby
increasing neuronal activity, while APV is a selective NMDA receptor antagonist, and removal of
APV after extended exposure results in increased neuronal activity. Golgi fragmentation has
been reported in hyperexcitable motor neurons in an amyotrophic lateral sclerosis mouse
model, and we also observe Golgi fragmentation in hyperexcitable hippocampal neurons. Thus,
Golgi fragmentation in neurons may be a general cellular effect that deserves further study in
neuronal excitability and neurodegenerative diseases.
SUNDAY-LATE POSTER PRESENTATIONS
2595
Seipin Promotes Lipid Droplet Assembly.
D. D. Binns1, B. R. Cartwright1, C. L. Hilton1, S. Han1, J. M. Goodman1; 1UT Southwestern
Medical Center, Dallas, TX
Mutations in the integral membrane protein seipin cause severe generalized lipodystrophy - the
absence of adipose tissue. The molecular function of seipin, however, has not been
determined. In model systems the absence of seipin leads to several changes in cellular lipid
composition and results in aberrant cytoplasmic lipid droplets. To determine whether seipin
promotes the assembly of droplets per se, we developed a system in the yeast Saccharomyces
cerevisiae in which neutral lipid synthesis is regulated by the galactose promoter. Droplets are
initially absent and then are rapidly produced upon incubation of cells in galactose medium.
However, in the absence of seipin, formation of droplets is severely attenuated and neutral lipids
appear to accumulate in the endoplasmic reticulum. This occurs regardless of whether
galactose drives the induction of the diacylglycerol acyltransferase DGA1 or the steryl
acyltransferase ARE1. When droplets are formed in the absence of seipin, they are in clusters
of tiny droplets with membrane fragments, as though they are blebbing uncontrolled from the
ER. To understand further the function of seipin, deletion mutants were constructed. In the
absence of the amino-terminal domain, supersized droplets are formed that cannot be
suppressed by the addition of phospholipid precursors, unlike the seipin-knockout cells. This
mutant seipin assembles into oligomers and is localized to the ER as is the wild type protein.
Our data indicate that seipin plays an important role in lipid droplet formation and suggest that
the amino terminus promotes the spooling of phospholipids into droplets.
2596
Peripheral serotonin activates brown adipose tissues of mice on the high-fat diet.
R. Saito1, H. Watanabe1, T. Nakano1, K. Sumiyoshi1, Y. Nagasawa1, X. Chen1, S. Terada1, N.
Okada1, K. Watanabe1, H. Aso1; 1Tohoku University, Sendai, Japan
Brown adipose tissue (BAT) acts as a thermogenesis organ, which oxidizes fatty acid and
wastes energy produced as heat, because BAT has numbers of mitochondria including
uncoupling protein-1 (UCP-1) and multilocular lipid droplets. BAT is stimulated sympathetic
nerve activity, which is mediated the activation of serotonin (5-hydroxytryptamine; 5HT)
receptors in the spinal cord. 5HT is a neurotransmitter in central nervous system and an
intestinal hormone in peripheral tissue. Since 5HT is incapable of crossing the brain blood
barrier, 5HT is thought to have independent function in central and peripheral. As noted
previously, the function of central 5HT is well known, but the effect of peripheral 5HT on BAT
remains poorly understood. In this study, we investigated the role of peripheral 5HT in the
activity of BAT.
Mice (C57BL/6, male) are fed chow diet or high-fat diet (HFD) from 5 weeks to 14 weeks of
age. Mice were intraperitoneally administrated with 1 mg 5HT or PBS twice a week. BAT was
obtained from fasted mice at 14 weeks of age. HE staining sections show that 5HT prevents
BAT of HFD mice from decreasing the cell density. The transmission electron microscopy
analyses show that 5HT increases the total length of mitochondrial cristae in BAT of HFD mice.
Next, we investigated the number of mitochondria in BAT using the mitochondrial DNA / nuclear
DNA quantification. The DNA copy numbers of mitochondrial component proteins were
significantly increased in HFD mice by 5HT. These results suggest that 5HT may increase the
mitochondrial quantity in HFD mice. The histological immunostaining of UCP1 was detected at
the largest amount in HFD mice with 5HT. The western blot analyses revealed that the protein
expression of UCP1 was also markedly elevated in BAT of HFD mice with 5HT. These data
indicated that 5HT activated the mitochondrial biosynthesis in BAT of HFD mice. Finally, BAT
SUNDAY-LATE POSTER PRESENTATIONS
mitochondria were isolated and then examined the mitochondrial capacity to oxidize fatty acid
using a Clark type oxygen electrode. 5HT dramatically increased the mitochondrial oxidative
capacity depended on proton leak of UCP1 in HFD mice. These findings show that peripheral
5HT contributes to activate the mitochondrial biosynthesis and respiratory capacity in BAT of
HFD mice.
2597
New tools for engineering the mitochondrial genome content of mammalian cells.
Y-W. Yang1, M. D. Koob1,2; 1Department of Laboratory Medicine and Pathology, University of
Minnesota, Minneapolis, MN, 2Institutes for Translational Neuroscience (ITN) & of Human
Genetics (IHG), Minneapolis, MN
Objective: The goal of this study was to develop technology to modify the mitochondrial
genome content of mammalian cells.
Method: A mitocytoplast cell fusion method was developed to deliver isolated mitochondria into
mammalian tissue culture cells. Isolated mitochondria were directly injected into rat oocytes and
mitocytoplasts carrying the injected mitochondria were generated by withdrawing part of the
oocytoplasm within a sealed membrane. These mitocytoplasts were then fused to mouse tissue
culture cells via viral-mediated membrane fusion.
Result: Mitochondria introduced into mouse cells using the mitocytoplast cell fusion method
quickly fused to the endogenous mitochondria, as indicated by the diffusion of fluorescent
markers. The mitochondrial genomes transferred by these fusion events were functional and
rescued mitochondrial respiration in the mitochondria-deficient recipient cells. We used the
mitocytoplast cell fusion method to generate mouse cells that carry only rat mitochondrial
genomes as a model system to develop new selection markers for engineering mammalian
mitochondrial genomes. Transient expression of mitochondrial-targeted XhoI restriction
endonuclease in these mouse cells was sufficient to shift the mitochondrial genome
heteroplasmy toward 100% (homoplasmic) mitochondrial genomes that do not contain any
XhoI-recognition sites.
Conclusion: 1) Introducing isolated mitochondria and the associated genomes into mammalian
tissue culture cells is now achievable using a mitocytoplast cell fusion method. 2) Mouse cells
carrying only rat mitochondrial genomes are useful tools for investigating new selection markers
for engineering mammalian mitochondrial genomes.
2598
Microtubule Dependent Anomalous Diffusion of Chloroplasts in Moss.
G. Arpağ1, Z. Shen2, K. P. Lemoi1, L. Vidali2, E. Tüzel1; 1Department of Physics, Worcester
Polytechnic Institute, Worcester, MA, 2Department of Biology and Biotechnology, Worcester
Polytechnic Institute, Worcester, MA
Organelle motility via molecular motors plays an important role in eukaryotic cell functioning. In
the model plant, Physcomitrella patens, reorganization of the chloroplasts to adapt to changes
in light intensity and quality is driven by the actin or microtubule (MT) cytoskeleton. In this work,
we investigate the motility of chloroplasts, in the absence of the actin cytoskeleton. To
characterize this motility, we analyze the mean squared displacement (MSD) of chloroplasts in
moss cells at steady state and constant illumination, and show that they are actively transported
via the MT cytoskeleton. Our results show that while the apparent diffusion coefficient is
dependent on the intensity of blue light, the super-diffusive nature of the movement determined
by the MSD exponents is not. In order to develop a mechanistic understanding of this process,
we developed a coarse-grained model of chloroplast motility. The model incorporates various
MT network topologies, consistent with experimental data, and uses point like cargo to mimic
SUNDAY-LATE POSTER PRESENTATIONS
chloroplasts. Our simulations show a strong dependence of MSD exponents to the MT network
topology, while the apparent diffusion coefficient is mainly influenced by the MT density and the
presence of passive cross-linkers.
2599
Microscopy-based genetic screen for peroxisome biogenesis mutants.
L. N. Dimitrov1,2, Y. Klug3, L. Wang1, M. Schuldiner3, R. Schekman1,2; 1Molecular and Cell
Biology, University of California, Berkeley, CA, 2Howard Hughes Medical Institute, University of
California, Berkeley, CA, 3Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
Peroxisomes are essential cellular organelles involved in lipid metabolism. Recent evidence
implicates the endoplasmic reticulum (ER) as the source of pre-peroxisomal vesicles (PPVs)
that carry peroxisomal membrane proteins (PMPs) to the mature organelle. Except for Pex19p,
the cytosolic factors required for PPV budding from the ER are unknown. Their discovery has
been difficult because only two mutants in S. cerevisiae, pex3 and pex19 have no detectable
peroxisomal membranes. To look for additional mutants with phenotypes similar to pex3 and
pex19, we conducted a microscopy-based genetic screen. We created a strain with a GFPtagged PMP (GFP-Ant1p) and mCherry fused to a peroxisomal targeting sequence (mCherrySKL). Both fluorescent markers co-localize to peroxisomes. Using SGA technology, we
introduced these markers into the deletion collection of non-essential S. cerevisiae genes and
the DAmP collection of hypomorphic mutants in essential yeast genes. We obtained highresolution images of the ~6,000 resultant strains on an automated microscopy platform. Manual
analysis of the images and secondary screening identified at least 4 mutant strains - pex25,
spf1, rvs161, and sec3, in which the majority of cells had mislocalized GFP-Ant1p and mCherrySKL. We are currently following up on these top hits of the screen using a de novo peroxisome
biogenesis assay and an in vitro budding assay for PPV formation from microsomal
membranes.
2600
Bioinformatic probing of the Arabidopsis thaliana chloroplast outer envelope
.interactome.
G. C. Harris1, E. Shortt1; 1Biological Sciences, Wellesley College, Wellesley, MA
Chloroplasts perform a variety of critical functions in plant cells, including lipid metabolism,
protein synthesis, and photosynthesis. For the cell to operate smoothly, these important
functions must be effectively integrated and regulated with other processes in the cell. This
necessitates the existence of extensive intracellular communication networks. Recent work in a
variety of organism has revealed that networks of protein-protein interactions form the core of
most intracellular communication grids. In an effort to determine how the chloroplast plugs into
the cell’s protein-protein interaction network we have used bioinformatic tools to generate a
virtual chloroplast outer envelope interactome.
Using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database and
other sources for plant interactome data (Biogrid, PAIR, BAR, or APID), we have compiled
interactions for each of 30 proteins currently known to compose the Arabidopsis thaliana
chloroplast outer envelope proteome. Using Cytoscape to visualize and analyze these
predicted interactions, we have constructed an interactome for the chloroplast outer envelope
that consists of 325 nodes and 2835 edges. Topological observations indicated that degree
distribution (number of interacting partners) ranged from two to fifty, with an average of 17.4
interacting partners and a characteristic path length of 3.5. Interestingly, these interactions
have included proteins that are expressed in the nucleus, mitochondria, nucleolus and
peroxisome.
SUNDAY-LATE POSTER PRESENTATIONS
In addition to predictions from STRING and the other plant interaction databases, textmining
and interolog predictions were used to improve the predicted interactome. Actin interactions
were of particular interest as several organelles in the plant cell have been shown to interact
with actin. For example, chloroplast positioning is known to be actin dependent and to involve
an actin-binding protein called CHUP1 (Schmidt von Braun and Schleiff 2008). However, neither
STRING nor other plant interactome databases predicted interactions between CHUP1 and
actin. This highlights the necessity of annotations based on literature mining for accurate
annotation and enrichment of the predicted interactome.
Schmidt von Braun, S. and Schleiff, E. (2008) The chloroplast outer membrane protein
CHUP1 interacts with actin and profiling. Planta 227: 1151-1159.
2601
Mitochondrial localization of Hsp22 dictated by its N-terminal domain confers
cardioprotection.
E. R. Rashed1, H. Dai1, X. Sui1, D. Vatner1, C. Depre1, H. Qiu1; 1UMDNJ, Newark, NJ
Introduction. H11 kinase/Hsp22 (Hsp22) is a small heat shock protein that confers
cardioprotection equivalent to ischemic preconditioning. One of the cytoprotective effects of
Hsp22 involves its transfer to the mitochondria, where it promotes the activity of the oxidative
phosphorylation chain and prevents the caspase 9-mediated pathway of apoptosis. The
mechanism of mitochondrial transfer of Hsp22 in mammals is unknown, although it has been
shown in Drosophila to be mediated by its N terminal domain. Hypothesis. Here, we tested
the hypothesis that the mitochondrial transfer of Hsp22 mediated by its N-terminal domain is
necessary for its cardioprotective effects in mammalian myocytes. Methods and Results. Two
adenoviruses were generated that harbored either the full coding sequence of Hsp22 (Ad-WTHsp22) or a dominant negative (DN) mutant lacking the N-terminal 20 amino acids (Ad-N20-Hsp
22), and these were infected at 20 moi in neonatal rat cardiomyocytes. Expression of both
proteins was increased to the same extent (about 2.5-fold) as compared to the β-Gal control
(P<0.05) in total protein extracts. Using sub-cellular fractionation and western blotting,
expression of Hsp22 was increased by about 2-fold in mitochondria of myocytes infected with
Ad-WT-Hsp22 (P<0.05 versus β-Gal), but was left unchanged in myocytes infected with AdN20-Hsp 22. Using a Clark-type oxygen electrode, the oxygen consumption rate, which reflects
the capability of ATP generation during stress, increased by about 3-fold in myocytes infected
with Ad-WT-Hsp22 compared to β-Gal, as measured by the ratio of uncoupled to coupled
respiration rates (4.5± 0.1 versus 1.8±0.1; P<0.01), but it was not significantly affected in
myocytes infected with Ad-N20- Hsp22 (2.1±0.1, NS versus β-Gal). Over-expression of WTHsp22 provided a 2.2-fold reduction in chelerythrine-induced apoptosis as compared to β-Gal
(P<0.01), while no significant protection was observed upon over-expression of Ad-N20-Hsp 22.
Conclusion. Therefore, localization of Hsp22 to the mitochondria in mammalian cells depends
on its N terminus, where it is critical to stimulate mitochondrial respiration and to protect against
the mitochondrial pathway of apoptosis.
SUNDAY-LATE POSTER PRESENTATIONS
2602
Endoplasmic Reticulum-Plasma Membrane Contact Sites Regulate Phosphatidylcholine
Synthesis.
S. Tavassoli1, J. T. Chao1, B. P. Young1, W. A. Prinz2, A. I. de Kroon3, C. J. Loewen1; 1Cellular
and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada, 2National
Institutes of Health (NIDDK), Bethesda, MD, 3Utrecht University, Utrecht, Netherlands
Synthesis of phospholipids, sterols and sphingolipids is thought to occur at contact sites
between the ER and other organelles because many lipid synthesizing enzymes are enriched in
these contacts. In only a few cases have the enzymes been localized to contacts in vivo and in
no instances have the contacts been demonstrated to be required for enzyme function. Here,
we show that plasma membrane (PM) - endoplasmic reticulum (ER) contact sites in yeast are
required for phosphatidylcholine synthesis and regulate the activity of the
phosphatidylethanolamine N-methyltransferase enzyme, Opi3. Opi3 activity requires Osh3,
which localizes to PM-ER contacts where it may facilitate in trans catalysis by Opi3. Thus,
membrane contact sites provide a structural mechanism to regulate lipid synthesis.
2603
Understanding the Glycosphingolipid pattern establishment.
D. Russo1, S. Capasso1, G. D`Angelo1; 1Institute of Protein Biochemistry-, Naples, Italy
Glycosphingolipids (GSLs) are ubiquitous lipid constituents of the eukaryotic plasma
membranes involved in various aspects of cell and developmental biology. The balance
between different GSLs is crucial to determine cells responsiveness to external environment
and define their transcriptional state. The mechanisms responsible for GSL pattern
establishment in specific cells and tissues is not understood. Taking advantage of the property
of Shiga and Cholera toxins B subunits (ShTxB, and ChTxB) to bind specifically the globoside
Gb3 and ganglioside GM1 respectively, we have devised a fluorescence-based assay for the
evaluation of GSLs expression profile in HeLa cells. Single-cell analysis showed a nearly
complete mutual exclusion of Gb3 and GM1 expression in Hela cells. We have noticed that both
ShTxB and ChTxB positive cells tent to appear in clusters whose size grows with time as a
function of cell division, suggesting that cells inherit their GSLs profile through cell generations.
qPCR approaches revealed that GSL synthases mRNA expression is sensitive to GSLs either
endogenously produced or exogenously added to cells. The resulting picture suggests the
existence of a complex circuitry in GSL metabolism (based on cross-inhibitory feedback loops)
whose topology accounts for mutual exclusion between specific GSL expression patterns and
self-sustained persistence of a given GSL pattern across cell generations. Altogether these
observations might ascribe an unreported property to GSLs to specify for inheritable nongenetic information and thus to participate to the epigenetic control of fundamental biological
processes such as differentiation and tissue patterning.
2604
Neutral Sphingomyelinase-3 is essential for TNF-stimulated redox signaling in skeletal
muscle.
J. S. Moylan1, S. A. Stasko1, J. D. Smith1, E. M. Wolf1, J. B. McLean1, M. B. Reid1; 1Physiology,
University of Kentucky, Lexington, KY
Sphingolipid and oxidant signaling affect similar skeletal muscle processes including glucose
uptake, TNF-induced muscle atrophy, force production, and fatigue. These similarities suggest a
connection between systems. Neutral sphingomyelinase (nSMase) is an important effector
enzyme that initiates sphingolipid signaling. In this report, we demonstrate that in skeletal
SUNDAY-LATE POSTER PRESENTATIONS
muscle and myotubes, the predominant nSMase mRNA is nSMase3 (Smpd4 gene). The Smpd4
gene is expressed as at least two splice variants, termed here nSMase3a and nSMase3b. A cterminal transmembrane domain is intact in both variants; however, they appear to localize to
different membrane types. nSMase3a localized with proteins that are resistant to extraction by
mild detergent (1% Triton) such as nuclear lamin and giantin. In contrast, nSMase3b cofractionated with detergent soluble membrane proteins, including TNF receptor 1 and
mitochondrial ATP synthase. Correspondingly, immunofluorescence microscopy demonstrates
that nSMase3 localizes to different cellular structures. nSMase3 is found surrounding
mitochondria and nuclei, and near the plasma membrane. Finally, we show that knockdown of
nSMase3 reduces medium chain ceramides and abolishes TNF-induced oxidant production.
These findings identify nSMase3 as the first known intermediate between TNF receptor
activation and skeletal muscle oxidant production. In conclusion, our data show that nSMase3 is
an important skeletal muscle nSMase that is essential for TNF-stimulated oxidant activity.
2605
Raft-mediated Endocytosis Shares Mechanistic Features, Including PI(4,5)P2
Involvement, with Endocytosis of Clustered Non-Raft Proteins.
D. A. Brown1, A. Ostermeyer-Fay1, C. Damiani1; 1Biochemistry & Cell Biology, Stony Brook
University, Stony Brook, NY
Raft-mediated endocytosis results from clustering of cell-surface proteins or lipids that have a
high affinity for liquid-ordered (lo) phase lipid rafts. This clustering can form or stabilize rafts.
The specific role of rafts in this endocytic process is not known. We used a well-characterized
raft model system: clustering of raft-loving glycosyl phosphatidyl inositol-anchored proteins
(GPI-AP) on the surface of live cells with antibodies. Clustered GPI-AP recruited actin
filaments, colocalized with the caveolar marker caveolin-1, and underwent actin- and
cholesterol-dependent endocytosis. To determine the role of rafts in these events, we
compared the behavior of a non-raft protein, the interleukin 2 receptor alpha subunit (IL2RA),
after antibody clustering. Surprisingly, clustered IL2RA also recruited actin, colocalized with
caveolin-1, and was endocytosed by an actin- and cholesterol-dependent mechanism.
Clustering of either protein recruited both PI(4)P-5 kinase I (PIPK1), which makes PI(4,5)P2 at
the plasma membrane, and PI(4,5)P2 itself, and PIPK1 affected endocytosis. Clustering of
cholera toxin B subunit enhanced its endocytosis by this pathway, and reduced internalization
by clathrin-mediated endocytosis. We conclude that raft-mediated endocytosis shares
mechanistic features - including a role for PI(4,5)P2 - with endocytosis triggered by clustering of
non-raft proteins.
2606
Ceramides are required in cell division.
E. Muro1, G. E. Atilla-Gokcumen2, J. Relat-Goberna1, S. Garcia-Manyes1, U. S. Eggert1,2;
1
King's College London, London, United Kingdom, 2Dana-Farber Cancer Institute, Harvard
Medical School, Boston, MA
The plasma membrane undergoes dramatic structural rearrangements during cell division.
However, little is known about how or if changes in its composition occur during this process.
Only few lipids have been shown to localize to the cleavage furrow or are known to perturb cell
division. The specific composition of the membrane at this phase of the cell cycle and the
function of most of the lipids involved is still unknown.
To address these questions we performed an RNAi screen of lipid biosynthetic enzymes in
parallel with a lipidomic analysis. We used the formation of binucleated cells to systematically
identify enzymes, and therefore lipids, necessary for the successful completion of cell division.
SUNDAY-LATE POSTER PRESENTATIONS
We scored 29 enzymes, out of which 12 participate in the sphingolipid biosynthetic pathway.
More precisely, out of the enzymes involved in sphingolipid modifications, the hits giving strong
phenotypes were mostly enzymes with ceramides or dihydroceramides as substrates or
products.
To understand the importance of sphingolipids in cytokinesis, we have coupled an analysis of
the biophysical properties of the specific membrane composition during cell division to the
localization at high-resolution level of ceramides during different phases of cell division. We
conclude that ceramides are critical to successfully complete cell division and we identified the
specific enzymes that are involved in this process.
2607
Glutamate modulates alpha2 adrenergic receptors through NMDA receptors in the
medulla oblongata of newborn rats.
S. M. da Silva1, D. R. Fior-Chadi1; 1Departament of physiology, Institute of Biosciences,
University of São Paulo, São Paulo, Brazil
Introduction: The neural control of the cardiovascular system is performed mainly through nuclei
located in the medulla oblongata. This control is performed by several neurotransmitters, among
them glutamate (glu) and the catecholamines norepinephrine and epinephrine. The control of
the cardiovascular system through the catecholaminergic system is realized mainly through the
alpha2 adrenoreceptors (alpha2a). It is well addressed that glutamate is capable of modulating
the catecholaminergic system, but little is known about the role of each glutamate receptors.
Thus, the objective of this project was to evaluate the modulation of alpha2a by glu and its
ionotropic receptor agonists in cell cultures of the medulla oblongata of newborn rats.
Methods: All the procedures and protocols were performed in accordance with the Institutional
Guidelines for Animal Experimentation (CEA/IB-USP: 084/2008). Cell culture was prepared from
the medulla oblongata of newborn rats (n=30). Cells were enzymatically and mechanically
dissociated, and plated in culture dishes pre-treated with Poly-D-Lisine and maintained for
seven days in appropriated culture medium. Cells were characterized by immunohistochemical
techniques, employing fluorescent antibodies against neurons and astrocytes.Cultures were
treated with different concentrations of Glu, NMDA, MK801, kainate and DNQX and protein
levels of the alpha2a were analyzed 24 hours after treatment by Western Blotting. All data were
evaluated by one-way analysis of variance (ANOVA) followed by Bonferroni post test.
Results: Results were expressed as percentage of control ± standard deviation. Glu treatments
increased alpha2a protein levels, but no significant augment was observed with the combined
treatment of glu with NMDA antagonist MK801 (control: 100 ±0; 10µM glu: 177.8 ± 26.7*; 10µM
glu + 100µM MK801: 142.2 ± 69.2), or combined with kainate receptor antagonist DNQX
(control: 100; 10µM glu: 123.4 ± 11**; 10µM glu + 10µM DNQX: 111.9 ± 15.2*). The level of
alpha2 protein was upregulated by NMDA treatment (control: 100; 10µM NMDA: 202.3*), but not
by kainate. (control: 100 ± 0; 1µM: 126.5 ± 44.2; 10µM: 94,95 ± 16,0; 100µM: 95,1 ± 33,2)
(*p<0.05; **p<0.1).
Discussion: We showed that both NMDA and kainate receptors are required for glutamate to
modulate alpha2 adrenoceptor at the protein level in cells from the medulla oblongata of
newborn rats, while only kainate fails to induce modulation. We suggest that this might be
important to the neural mechanisms of blood pressure control. This study was supported by
grants from FAPESP and CNPq.
SUNDAY-LATE POSTER PRESENTATIONS
2608
Effect of peripheral 5-HT on cholesterol metabolism.
H. Watanabe1, Y. Nagasawa1, R. Saito1, T. Nakano1, K. Sumiyoshi1, S. Someya1, N. Okada1, X.
Chen1, K. Watanabe1, H. Aso1; 1Tohoku University, Sendai, Japan
Serotonin (5-HT) is synthesized by two distinct tryptophan hydroxylase (TPH) rate-limiting
enzyme in brain (TPH2) and in peripheral (TPH1). As 5-HT is inability to cross the blood-brain
barrier, there are two 5-HT systems in brain and periphery with independent functions. 5-HT is a
monoaminergic neurotransmitter with activities that modulate central and peripheral functions.
5-HT affects food intake, sleep, anxiety, sexual behavior and mood in the central nervous
system. On the other hand, the functions of 5-HT in peripheral tissue have not yet been fully
elucidated. Recently, peripheral 5-HT is known to be associated with glucose metabolism
mainly because of its regulation of the secretion of insulin in pancreatic B cells. Additionally, the
concentration of 5-HT in the blood of mice fed a high fat diet was much higher than that of lean
control mice. These studies suggest that 5-HT may play important roles with regard to glucose
and lipid metabolism. In this study, in order to investigate the role of peripheral 5-HT in
cholesterol metabolism, we examined that the effect of the injection of 5-HT in periphery on
plasma and hepatic cholesterol contents.
Fasted mice were intraperitoneally injected with 1 mg 5-HT. The concentrations of plasma total
cholesterol, HDL-cholesterol and LDL-cholesterol were significantly decreased at 60 min after
the injection of 5-HT. Mice significantly increased hepatic cholesterol content at 60 min after the
administration of 5-HT. At 15 min after 5-HT injection, 5-HT induced the translocation of lowdensity lipoprotein receptor (LDLR) and scavenger receptor class B type 1 (SR-B1) to the
cytoplasmic membrane. These lipoprotein receptors also were co-localized with caveolin-1,
such as the main component of the caveolae plasma membranes. Additionally, the expressions
of 5-HT receptor type (HTR) 1D, 2A and 7 were observed in the liver. The pre-treatment of
methysergide (HTR1, 2 and 7 antagonists) inhibited the decrease of plasma cholesterol
concentration induced by 5-HT injection. However, the reduction of plasma cholesterol level
induced by 5-HT was not prevented by pre-treatment of ketanserin (HTR2A antagonist) and SB269970 (HTR7 antagonist). These results suggest that 5-HT increases the cholesterol uptake to
the liver and then may decrease its plasma concentration through the HTR1D.
2609
Microscopy based determination of the activation of signalling pathways through the
Angiotensin II type 1 receptor in live cells.
E. A. Christensen1, C. B. Lagerholm2, J. L. Hansen3; 1Department of Molecular Biology and
Genetics, Aarhus University, Aarhus, Denmark, 2Department of Physics, Chemistry and
Pharmacy, University of Southern Denmark, Odense, Denmark, 3Novo Nordisk, Måløv,
Denmark
The Angiotensin II type 1 receptor (AT1R) is involved in cardiovascular physiology at the
molecular level. Its role is as key regulator of directing the cardiac cells in one of two pathways
leading to either muscle contraction or proliferation of cells and hypertrophy of the heart. AT1R
signalling in cardiac myocytes allows the heart to adapt and maintain normal cardiovascular
function under conditions of increased workload. The actions of the cells that are controlled by
AT1R include contraction, survival, growth, and differentiation. Prolonged angiotensin
stimulation is however detrimental, implicating the receptor in the pathogenesis of
cardiovascular diseases, such as hypertension, cardiac hypertrophy, and congestive heart
failure. The AT1R belongs to the super family of 7TM receptors, and act as a molecular switch
that upon agonist binding activates diverse downstream signalling pathways.
SUNDAY-LATE POSTER PRESENTATIONS
I have to developed a microscopy based live cell assay for the determination of the activation of
signalling pathways through the Angiotensin II type 1 receptor. This receptor signals through
two pathways, a G protein-dependent pathway and a β-arrestin2-dependent pathway. Spinning
disk and TIRF microscopy as well as the advanced image analysis tool k space image
correlation spectroscopy (kICS) has been used to perform a comparative quantification of the
activation of the two pathways through the receptor in the mammalian cell membrane in live
cells. This has enlightened us with novel and deeper understanding of the signalling pathways
through the angiotensin receptor. I also plan to use spatial intensity distribution analysis (SpIDA)
to perform a comparative quantification of numbers of monomers / dimers of the receptor as a
function of the signalling pathway.
2610
Aquaporin-5 plasma membrane regulation.
J. S. Koffman1,2, E. A. Christensen1,2, S. Marlar1,2, L. N. Nejsum1,2; 1Department of Molecular
Biology and Genetics, Aarhus University, Aarhus, Denmark, 2Interdisciplinary Nanoscience
Center, Aarhus University, Aarhus, Denmark
Aquaporin-5 (AQP5) belongs to a family of closely related transmembrane water channels that
facilitate transepithelial water flow along osmotic gradients in many tissues. AQP5 is found in
secretory epithelial cells of salivary glands, sweat glands, and the lungs. Water transport
through AQP5 is believed to play a functional role in secretion. AQP5 is primarily localized in the
apical plasma membrane, but there is also evidence that AQP5 undergoes translocation from
intracellular sites in response to secretory stimuli. We speculate that the dynamics of AQP5
within the plasma membrane is tightly regulated in order to facilitate the necessary water flow in
response to specific secretory stimuli.
The dynamics of AQP5 within the plasma membrane has not previously been studied. We use
two different approaches for live cell imaging and analysis of AQP5 membrane mobility in an
MDCK epithelial cell model. By inserting an extracellular c-myc-tag in AQP5-EGFP (AQP5-mycEGFP), we are able to label AQP5 only at the cell surface using specific antibody and Fab
fragments conjugated with quantum dots. This approach allows us to track the movement of
single proteins within the membrane, but requires a tedious labeling step. Using the EGFP
signal in AQP5-myc-EGFP we can perform fast and simple live cell imaging experiments.
However, the entire pool of AQP5 within the cell is visualized at once, thus we look at global
dynamics averaged over the entire cell. We use k-space image correlation spectroscopy (kICS)
to analyze the diffusion coefficient in image sequences of AQP5-myc-EGFP labeled by quantum
dots or by imaging the EGFP signal. We are able to measure steady state diffusion as well as
changes upon drug stimulation with both approaches.
Forskolin is known to increase the total AQP5 protein level as well as induce protein
translocation to the plasma membrane. We speculated that forskolin may also affect the mobility
of AQP5 within the plasma membrane. We found that forskolin reduced the diffusion of AQP5
by approximately 25 % both when we used quantum dots and the EGFP signal, thus validating
either approach for measuring AQP5 membrane dynamics. Secretory release is mediated by
intracellular signaling pathways that lead to release of calcium from intracellular stores. An
increase in intracellular calcium is known to raise AQP5 translocation to the plasma membrane,
and we speculated that it could also alter AQP5 membrane dynamics. Using quantum dot
labeling, we find that treatment with A23187 calcium ionophore might only cause a slight
increase in the diffusion of AQP5. We will continue to study AQP5 membrane regulation by
kICS analyses and a combination of other microscopy and biochemical techniques. Diffusion of
AQP5 in the presence of forskolin potentially involves a distinct pattern of protein movements in
SUNDAY-LATE POSTER PRESENTATIONS
the membrane, as AQP5 may interact differently with specific complexes that control its
retention, turnover, and recycling.
2611
TGF-beta-induced Shc-Erk-MAP kinase pathway signaling in epithelial to mesenchymal
transition.
B-P. Muthusamy1, R. Derynck1; 1The Eli and Edythe Broad Center of Regeneration Medicine
and Stem Cell Research, Department of Cell and Tissue Biology, University of California San
Francisco, San Francisco, CA
Transforming growth factor-β(TGF-β) plays a critical role in both cancer initiation and
progression. In cancer progression, TGF-β induces an epithelial to mesenchymal transition
(EMT) that results in de-adhesion, increased motility and invasion. EMT of carcinoma cells is
thought to be at the basis of cancer invasion, and of the properties of carcinoma stem cells, and
is therefore an important step in cancer progression. TGF-β has been shown to induce EMT,
and increased production of TGF-β by cancer cells, resulting in increased autocrine TGF-β
signaling, is thought to initiate and drive cancer invasion and metastasis. TGF-β acts through a
heteromeric cell surface complex of “type II” and “type I” receptor kinases, and induces Smad
and non-Smad signaling that results in activation of Smad and MAP kinase pathways,
specifically Erk MAP kinase, p38 MAP kinase and JNK signaling, and the PI3 kinase-Akt-mTOR
pathway. However, the relative contributions of these non-Smad signaling pathways in TGF-βinduced EMT have been poorly characterized. Previous research has identified ShcA as an
adaptor protein that associates with receptor tyrosine kinases and initiates Erk MAP kinase
signaling. We found that, in response to TGF-β, the type I TGF-beta receptor (TβRI) interacts
with ShcA in epithelial HaCaT and NMuMG cells. Further, silencing ShcA expression promoted
a mesenchymal phenotype, characterized by actin reorganization, increased expression of
vimentin and N-cadherin, and reduced expression of epithelial E-cadherin. Cell invasion,
assessed using a modified Boyden chamber assay, was also increased in cells with silenced
ShcA expression. This compound phenotype reversed to epithelial when the kinase of the type I
receptor was pharmacologically blocked by SB431542. ShcA may modulate the Smad signaling
pathway either by competing with elements of the Smad pathway, or by controlling the
availability of receptors that activate Smad signaling in the plasma membrane and endosomes.
2612
Coupling between ryanodine receptors and Ca2+ release-activated Ca2+ channel
machinery regulates store-operated Ca2+ entry in human T lymphocytes.
P. Thakur1, S. Dadsetan1, A. F. Fomina1;
1
Department of Physiology and Membrane Biology, University of California, Davis, Davis, CA
T lymphocytes play a central role in cell-mediated immunity. Elevation in cytosolic Ca2+
concentration is crucial for T cell activation. We investigated the expression and functional
significance of ryanodine receptors (RyR), the intracellular Ca2+ release channels, in resting and
activated primary human T cells. Transcripts of all three RyR isoforms (RyR1, RyR2, and RyR3)
were detected in human T cells with RyR1 appearing to be the most abundant. RyR1/2
transcript levels increased, whereas those of RyR3 decreased after T cell activation. RyR1/2
protein immunoreactivity was detected in activated, but not in resting T cells. The RyR agonist
caffeine evoked Ca2+ release from the intracellular store in activated T cells but not in resting T
cells, indicating that RyR are functionally upregulated in activated T cells compared with resting
T cells. In the presence of store-operated Ca2+ entry (SOCE) via plasmalemmal Ca2+ releaseactivated Ca2+ (CRAC) channels, RyR blockers reduced the Ca2+ leak from the endoplasmic
reticulum (ER) and the magnitude of SOCE, indicating that a positive feedback relationship
SUNDAY-LATE POSTER PRESENTATIONS
exists between RyR and CRAC channels. Overexpression of fluorescently tagged RyR2 and
stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor gating CRAC channels, in HEK293
cells revealed that RyR are co-localized with STIM1 in the puncta formed after store depletion.
We conclude that in primary human T cells, the RyR are coupled to CRAC channel machinery
such that SOCE activates RyR via a Ca2+-induced Ca2+ release mechanism, which in turn
reduces the Ca2+ concentration within the ER lumen in the vicinity of STIM1, thus facilitating
SOCE by reducing store-dependent CRAC channel inactivation. Treatment with RyR blockers
suppressed activated T cell expansion, demonstrating the functional importance of RyR in T
cells.
2613
Title: Cloning and characterization of the Rat and Human Sodium hydrogen exchanger
domain containing protein-1(NHEDC1).
P. Kumar1, P. James1; 1Zoology, Miami University, Oxford, OH
Na+/H+exchangers (NHEs) are a family of integral membrane proteins that mediate
electroneutral exchange of one sodium ion for one proton across the plasma membrane. As a
result they are known to play important roles in a variety of physiological processes such as pH
regulation, cellular differentiation, cell volume control and electrolyte transport. Regulation of
intracellular pH as a result of activity of sodium hydrogen exchangers has been shown to be
important for normal sperm function. NHE1, NHE5 and NHE10 are the three isoforms of the
Na+/H+ exchanger known to be expressed in the mouse sperm. Targeted deletion of NHE1
results in mice that have no sperm motility defects indicating that the loss of NHE1 does not
affect sperm motility. On the other hand, knockout of NHE10 results in infertile males due to
severe sperm motility defects. The role of NHE5 in sperm motility is yet to be determined. More
recently, a testis-specific putative sodium hydrogen exchanger was identified in the mouse and
named Sodium Hydrogen Exchanger Domain Containing Protein1 (NHEDC1). It has been
suggested that NHEDC1 plays a role in maintaining normal sperm function as incubation of
mouse sperm with an antibody against NHEDC1 results in depressed sperm motility. The
human ortholog of NHEDC1 has been identified and RT-PCR data suggests testis-specific
expression. The rat ortholog has not been identified or characterized. Importantly, no Na+/H+
exchange activity has been associated with any NHEDC1 protein. In this study, we have cloned
the rat NHEDC1 and human NHEDC1 cDNAs. Multi-tissue western blot data suggests that the
human NHEDC1 is a testis-specific protein. Multi-tissue northern blot analysis suggests that,
like in humans, the rat NHEDC1 is also specifically expressed in the testis. In rat sperm,
NHEDC1 is localized to the principle piece and the acrosome. In order to characterize NHEDC1
as a functional NHE we have used an NHE-deficient skin fibroblast cell line. Using these cells,
we have generated cell lines stably expressing hNHEDC1. Functional characterization of this
protein from humans may allow for the development of compounds (either inhibitors or
activators) that may be useful as male contraceptives or for the treatment of male infertility.
2614
Beta1-subunit of Na,K-ATPase is involved in the stability and membrane localization of
the Sodium/Calcium exchanger NCX1.
S. P. Barwe1, S. Balasubramaniam1, A. Rajasekaran1; 1Alfred I. duPont Hospital for Children,
Wilmington, DE
The Na,K-ATPase actively transports three Na+ out and two K+ into the cell, and provides the
driving force for the Na+/Ca2+ exchange. Na,K-ATPase consists of two essential subunits,
alpha-subunit and beta-subunit. The alpha-subunit is the catalytic subunit with 10
transmembrane domains, and the beta-subunit is the regulatory subunit, which also acts as a
SUNDAY-LATE POSTER PRESENTATIONS
chaperone for the efficient transport and stabilization of the alpha-subunit on the plasma
membrane. Na+/Ca2+ exchanger (NCX1) also has 10 transmembrane domains and it
transports one Ca2+ out and three Na+ in. Thus the Na,K-ATPase and NCX1 together maintain
the charge balance. We showed earlier that knockout mice with heart-specific ablation of beta1subunit have drastically diminished levels of NCX1 (Barwe et al., J Mol Cell Cardiol, 47:552-560,
2009). Although a small amount of NCX1 was present in beta1-subunit knockout hearts, the
mice were completely insensitive to ouabain, similar to NCX1 knockout mice. We now provide
evidence that beta1-subunit regulates NCX1 at the post-transcriptional level in Madin-Darby
canine kidney (MDCK) cells. MDCK cells with RNA interference mediated knockdown of beta1subunit showed equivalent amounts of NCX1 transcripts but reduced NCX1 protein levels.
Replenishing beta1-subunit levels led to a concomitant increase in NCX1 levels, implying the
specificity of beta1-subunit in regulating NCX1 levels. Lysosomal inhibitors prevented
degradation of NCX1 in beta1-subunit knockdown cells. Furthermore, knockdown of beta1subunit resulted in almost complete loss of NCX1 on the surface of MDCK cells, and this
phenotype was rescued by restoration of beta1-subunit levels in knockdown cells. This data
suggests that beta1-subunit is required for the membrane targeting of NCX1. Further, coimmunoprecipitation assay showed that beta1-subunit and NCX1 are associated with each
other in a complex. Thus, these observations reveal a previously unknown molecular link
between beta1-subunit and NCX1.
2615
Quantitative immunoproteomics of MHC class I repertoire upon cellular stress induced
by Azetidine-2-carboxylic Acid (Aze).
V. S. Rao1, A. N. Scharf1, J. E. Elias1; 1Chemical and Systems Biology, Stanford University,
Stanford, CA
Multiple sclerosis is an autoimmune disease affecting brain and spinal cord communication. It
has been shown that dietary factors play a significant part in developing this disease.
Interestingly, prevalent geographic areas with people diagnosed with multiple sclerosis rely
heavily on livestock, which are fed the byproducts of sugar beets, as their source of dairy. Sugar
beets naturally produce azetidine-2-carboxylic acid (aze), an amino acid analog of proline,
which can be misincorporated into the proteins of humans and is known to induce protein
misfolding. Misfolded proteins undergo degradation and select peptides are displayed on the
cell surface, via major histocompatability complex (MHC) I molecules, for the immune system. In
this project we are investigating the effect of aze treatment on the MHC I peptide repertoire as
well as aze misincorporation in the peptides displayed on the MHC I molecules, using
quantitative mass spectrometry. Here we show that aze affects cell growth rates, having about a
1% aze incorporation rate in B-lymphocyte cells. Additionally we identified over 350 peptides
displayed on the MHC class I molecules (pMHCI)s without aze treatment. To identify pMHCI
specifically displayed upon treatment we used a chemical labeling approach prior to mass
spectrometry. This quantification method allowed identification of several pMHCI specific for aze
treatment, such as T.L.I.D.L.P.Q.I.T.K.V (derived from DNM2) and L.T.D.I.T.K.G.V.Q.Y (EEF2)
with a high binding score for the MHC class I isoforms HLA-A*03 and HLA-A*26 respectively.
Having identified a pMHCI repertoire specific for aze exposure is a major step towards
identifying possible antigens triggering the immune system response and determining aze
involvement in autoimmune diseases like multiple sclerosis.
SUNDAY-LATE POSTER PRESENTATIONS
Cell Division I: G1, G1-S, and S Phase Regulation, Cytokinesis, Meiosis, and
Spindle Checkpoints
2616
Redefining the Mammalian Restriction Point with Single-Cell Analysis.
A. Johnson1, C. Schwarz1, J. Skotheim1; 1Stanford University, Stanford, CA
Cell cycle progress occurs through stages demarcated by irreversible transitions that are
essential for reliable cell duplication. In the G1 phase of the cell cycle, animal cells commit to
division when certain environmental requirements are fulfilled. The commitment process is
viewed as a single restriction point in late G1 that governs passage into S phase; once this point
is passed, a cell progresses through the cell cycle independent of mitogenic signaling. The
prevailing model of the restriction point is centered on inactivation of the retinoblastoma protein
(Rb) by phosphorylation, which allows weak activation of the E2F transcription factor. E2F
activates its own expression and thereby titrates out its key negative regulator Rb. Once
threshold levels of active E2F are reached, the cell becomes insensitive to stimulatory or
inhibitory signaling. In contradiction to the prevailing model which was largely established
through population-based analyses, recent single cell studies of G1 concluded that the
restriction point might occur several hours before Rb hyperphosphorylation. This suggests that
upstream events define commitment. Given these conflicting results, the prevailing view of the
restriction point would benefit from a thorough reexamination; here, I propose an unbiased,
high-resolution approach toward understanding the molecular basis of commitment. The goal of
this study is to answer three questions: 1. When exactly does commitment occur? 2. What is the
identity and timing of gene expression that establishes the restriction point? 3. What other G1
signaling pathways are potentially involved in determining the restriction point? By revisiting the
restriction point and answering these fundamental questions, I will precisely resolve the
mechanism underlying G1 commitment.
2617
The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at
mitotic exit.
S. L. Spencer1, F-C. Tsai1, T. Meyer1; 1Chemical and Systems Biology, Stanford University,
Stanford, CA
Tissue homeostasis in metazoans is controlled by regulated transitions between quiescence
and proliferation. Cells emerging from quiescence commit to the cell cycle at a "restriction
point" in late G1 that involves up-regulation of Cyclin-dependent kinase 2 (CDK2) activity. Here
we develop a live-cell sensor for CDK2 activity and show that this restriction point model only
applies to cells emerging from quiescence. In contrast, in proliferating cells, we discovered a
bifurcation point at mitotic exit where cells either immediately build up CDK2 activity and commit
to the cell cycle or suppress CDK2 activity and enter a transient state of quiescence. This
bifurcation is under the control of mitogenic stimuli prior to mitosis and is regulated by the CDK
inhibitor, p21, and the CDK2 activator, Cyclin A. Thus, in cycling cells, the critical event in cell
cycle commitment is the activation or suppression of CDK2 at mitotic exit.
SUNDAY-LATE POSTER PRESENTATIONS
2618
Characterizing the cell cycle progression in differentially polyploid cells.
T. A. Potapova1, R. Li1; 1Stowers Institute, Kansas City, MO
Chromosomal instability and the resultant aneuploidy can drive cancer evolution. Aneuploid
cells may arise from polyploid cells through unequal chromosome segregation during mitosis,
because mitosis in polyploid cells is error-prone. Many factors that lead to polyploidy, such as
viral infections and chronic tissue injury, are associated with carcinogenesis. A biologically
sensible response of a multicellular organism to polyploidization is to prevent polyploid cells
from entering another cell cycle. Indeed, in many instances non-transformed cells stop dividing
upon polyploidization. The nature of the mechanism or mechanisms that suppress proliferation
in non-transformed polyploid cells is unknown, but this machinery apparently is lost or not
working in cancer.
This study investigated the cell cycle progression of non-transformed and cancer-derived
human cells that became polyploid by two distinct routes. First route was cytokinesis failure that
leads to the formation of binucleate cells. Second route was mitotic slippage that leaves cells
with one distorted nucleus and micronuclei. The cell cycle progression was assayed by live
imaging, EdU incorporation and flow cytometry.
Our preliminary data suggest that most non-malignant hTERT immortalized cells arrest in G1
with tetraploid or octaploid DNA content, while cancer-derived cells tend to progress to higher
ploidy. The mechanism(s) arresting the cell cycle in polyploid cells likely depends on the route of
polyploidization and involves p53.
Elucidating the nature of mechanisms that stop polyploid cells from proliferating thus averting
the forthcoming evolution of aneuploid progenies may provide better understanding of
carcinogenesis and potentially translate into new targets for anti-cancer drug development.
2619
Critical targets of Cyclin D/Cdk4 in the control of cell cycle entry.
I. The1, S. Ruijtenberg1, J. Munoz2, A. Heck2, S. van den Heuvel1; 1Developmental Biology,
Utrecht University, Utrecht, Netherlands, 2Biomolecular Mass Spectrometry and Proteomics
Group, Utrecht University, Utrecht, Netherlands
Based on extracellular signals and cell intrinsic information, cells decide whether to enter the
next division cycle or to withdraw from cell division. Ultimately, these decisions depend on the
activity of Cyclin-dependent kinases (Cdk’s) in the G1 phase of the cell cycle. Active Cdk4 (or
Cdk6) in association with a D-type cyclin promotes cell-cycle entry and triggers commitment to
undergoing a full cell-division cycle. In this process, Cyclin D/Cdk4 phosphorylates the
Retinoblastoma (Rb) tumor suppressor protein, which inhibits transcriptional repression by pRB
and allows expression of Cyclin E and other cell cycle genes. It is currently unknown if
additional Cyclin D/Cdk4 substrates are critical in the control of G1 progression.
We study cell-cycle entry and exit in the nematode C. elegans, an animal with cell-cycle control
mechanisms that are highly conserved but less redundant compared to mammals. We have
previously shown that C. elegans uses single cyd-1 Cyclin D and cdk-4 Cdk4/6 genes that are
essential for G1 progression. Inactivation of the sole Rb family member partly overcomes the
need for Cyclin D/CDK-4 in C. elegans. However, cell proliferation remains limited in double
mutants of Rb and cyd-1 Cyclin D, or Rb and cdk-4, which indicates that Cyclin D/CDK-4 has
critical target(s) in addition to the pRB protein family.
To identify such targets, we performed a genetic screen for mutations that restore cell
proliferation in the absence of cyd-1 Cyclin D and Rb function. The screen identified a single
SUNDAY-LATE POSTER PRESENTATIONS
mutant with normal development and cell proliferation patterns. We found that the mutation
results in incomplete loss-of-function of fzr-1 Cdh1, a substrate specificity factor of the APC/C
complex. The APC/CCdh1 E3 ubiquitin ligase targets proteins for degradation and is critical for
exit from mitosis and inhibition of cell cycle entry in G1.
Using in vitro kinase assays, we found that Cyclin D/CDK-4 phosphorylates C. elegans Rb on a
specific site that corresponds to Rb inactivating phosphorylations in mammalian cells. In
addition, Cyclin D/CDK-4 phosphorylated 7 different serine residues in the N-terminal region of
FZR-1 Cdh1. In other systems, phosphorylation of the Cdh1 N-terminus by mitotic Cdk’s has
been shown to prevent association of Cdh1 with the APC/C. Similarly, phosphorylation by CDK4/Cyclin D may overcome APC/CCdh1 activity in G1 and thereby promote cell cycle entry. Our
data support that the pRB and FZR-1 Cdh1 proteins are the two critical phosphorylation targets
of Cyclin D/CDK-4 in the control of cell-cycle entry.
2620
Integration of mechanical forces by E-cadherin underlies epithelial cytokinesis.
C. Guillot1, T. Lecuit1; 1Ibdml Cnrs UMR 7288, Marseille, France
Cytokinesis entails cell invagination by a contractile actomyosin ring. In epithelia, E-cadherin
mediated adhesion connects the cortices of contacting cells, thus it is unclear how invagination
occurs in this context and how tissue integrity is preserved.
Investigations in Drosophila embryos first show that apico-basal cleavage is polarized:
invagination is faster from the basal than from the apical side. Tension in the contractile ring is
controlled by septin filaments but it is not polarized. Polarized cleavage is due instead to
mechanical anchorage of the ring to apical E-cadherin complexes. Formation of the new
junction requires local adhesion disengagement in the cleavage furrow, followed by de novo Ecadherin complex formation. E-cadherin levels control the timing of disengagement, and also
sensitize dividing cells to tension from neighbours. Although cell adhesion and cell division are
considered uncoupled processes, we show that their interplay is essential in epithelia. We
uncover a pivotal role for E-cadherin in integrating intrinsic and extrinsic tensions during
cytokinesis and present a framework for understanding how tissue cohesion is preserved during
epithelial division.
2621
Roles of actin-depolymerizing factor Adf1 in fission yeast cytokinesis.
E-I. Uyeda1, J. Kashiwazaki1, I. Mabuchi1; 1Department of Life Science, Faculty of Science,
Gakushuin University, Tokyo, Japan
Animal cells and fungal cells form the contractile ring at a division site during cytokinesis. The
ring contracts to divide the cell into two by virtue of interaction between myosin and actin
filaments. It has not well been known how the contractile ring is formed. Cells of the fission
yeast Schizosaccharomyces pombe are genetically tractable and appropriate for microscopic
observations. It has been known from analyses of cytokinesis mutant cells of fission yeast that
a number of proteins in the contractile ring in fission yeast cells and animal cells are common.
Therefore, fission yeast cells have been used as good models to study mechanisms of
cytokinesis. In this study we aimed to elucidate roles of the ADF (actin-depolymerizing
factor)/cofilin Adf1 in formation and contraction of the contractile ring. ADF is involved in
severing actin filaments and also in depolymerization of actin from the minus end of the filament
in vitro. A ts-mutant strain adf1-1 expresses mutated Adf1 and has been known to be unable to
form the contractile ring at a restrictive temperature 36°C (Nakano and Mabuchi, MBoC 2006).
We analyzed behavior of actin in live adf1-1 cells by expressing mRuby-tagged Lifeact (Riedl et
SUNDAY-LATE POSTER PRESENTATIONS
al., Nat. Methods 2008). In mitotic adf1-1 cells actin patches and cables were clustered and the
clusters moved from the ends to the mid region of the cell at 36°C. However, the clusters were
never reorganized into a ring structure at the mid region. When Adf1 was inactivated during
formation of the ring, the ring disintegrated and the cell did not undergo cytokinesis. On the
other hand, once the ring formation was completed, it contracted and cytokinesis was
completed. The contraction speed of the ring in adf1-1 cells was a little but significantly slower
than that of wild-type cells. In summary, Adf1 is required in formation and maturation of the
contractile ring, and also in its effective contraction.
2622
Increase of Polyploidization by Cytokinesis failure via Reactive Oxygen Species Induced
Chromosome Bridges and Mis-localization of Aurora B in HeLa Cells.
M-G. Cho1,2, I-J. Lee1,2, Y-Y. Park1,2, J-H. Lee1,2; 1Department of Biochemistry and Molecular
Biology, Ajou Unviersirty, Suwon, Korea, 2Department of Molecular Science and Technology,
Ajou University School, Ajou Unviersirty, Suwon, Korea
Aneuploidy can arise from tetraploid cells resulting from a failure of the cytokinesis. Reactive
oxygen species (ROS) have been implicated cancer formation. However, the role of ROS in
cytokinesis progression has not been studied well. In our data, H2O2 treatment during mitosis
induced cytokinesis failure increased and the number of binucleated cells (BNCs) in HeLa cells.
Our observation revealed that major molecules working in cytokinesis progression did not
participate in H2O2 induced cytokinesis failure. Instead, significantly increase in chromosome
bridges was observed. Moreover the BNCs resulted from the cytokinesis failure by ROS also
contained nucleoplasmic bridges (NPBs) between two nuclei. Next, we examined whether
activity of aurora B, a which in known abscission checkpoint protein, is decreased by ROS. We
found that activity of aurora B was not decreased, but localization of aurora B was changed by
ROS. Taken together, we propose that increase of polyploidization by cytokinesis failure via
reactive oxygen species induced chromosome bridges and mis-localizaiton of aurora B in HeLa
cells.
2623
Multiple Pools of F-Actin Contribute to Cytokinetic Ring Formation, Ingression, and
Tension Generation.
C. Conte1, M. Arocena1, E. R. Griffis1; 1Wellcome Trust Centre for Gene Regulation and
Expression, University of Dundee, Dundee, United Kingdom
The cytokinetic ring that constricts to physically divide a cell in two at the end of cell division is
formed of actin and myosin. Previous work from other labs has shown that for the cytokinetic
furrow to fully ingress, formin proteins must nucleate unbranched actin filaments. Using TIRF to
visualize cortical myosin filaments in formin depleted cells, we noticed that the initial
accumulation of myosin into a uniform equatorial band was normal. However, this equatorial
band rapidly broke and was compacted into smaller non-contiguous patches on the cortex,
suggesting that unbranched actin filaments may be required to resist the tension generated at
the cortex by myosin. Interestingly, in monopolar spindles we observed that formin depletion did
not prevent the symmetry breaking of myosin distribution and polar bundling of microtubules
that normally occurs shortly after anaphase onset. When we observed the anaphase distribution
of Aurora B in these cells, we also found that the depletion of formin reduced the stability of
Aurora B on the cortex, but in monopolar cells, Aurora B, still localized on microtubules tips and
somewhat further out towards the edge of the cell. When all actin filaments were depleted by
treating cells with cytochalasin, we observed that myosin formation at the equatorial cortex still
initiated with similar kinetics, but that the protein was rapidly dispersed from the equator and
SUNDAY-LATE POSTER PRESENTATIONS
formed small punctae, similar to what has previously been shown by Wang and colleagues. The
total lack of actin filaments prevented the symmetry breaking we observe in monopolar
anaphases, suggesting that this process is not driven solely by an asymmetric distribution of
signaling molecules (all microtubules retained equivalent amounts of Aurora B in the presence
of cytochalasin), but that it requires feedback from the formation of the contractile network.
Curiously cytochalasin treatment also changed the shape of the Aurora B accumulated on
microtubule tips from slender comets to more circular structures.
2624
Investigating INCENPICP-1 function during C. elegans cell division using temperature
sensitive mutants.
T. Davies1, S. N. Jordan1, J. C. Canman1; 1Department of Pathology and Cell Biology, Columbia
University, New York, NY
Cell division is a dynamic process controlled by a number of proteins, some of which are
multifunctional and are required at multiple steps throughout mitosis and cytokinesis. A difficulty
in studying these master-regulators is that loss-of-function alleles generated by classic genetic
approaches lead to pleotropic phenotypes due to defects in multiple cellular processes. One of
these key regulators is the multi-component Chromosomal Passenger Complex (CPC), which
plays essential roles during meiosis, mitosis, and cytokinesis. Disrupting CPC function by
traditional forward and reverse genetics causes catastrophic chromosomal segregation defects
and cytokinesis failure. In order to elucidate the role of the CPC during mitosis and cytokinesis,
a forward genetics approach has been used in C. elegans to isolate conditional alleles which
phenocopy the defects. This strategy generated three mutant C. elegans strains that are
completely disrupted in chromosome segregation and cell division. Traditional mapping,
complementation analysis and DNA sequencing showed that the three strains are mutated in
icp-1, the C. elegans homologue of the CPC component INCENP.
Each allele contains a
single missense mutation, resulting in a single amino-acid change. All three of these alleles are
temperature sensitive, showing rapid inactivation of protein function and a fully penetrant lossof-function phenotype within seconds after shifting to restrictive temperature. To further
characterize these mutants we have examined cytokinesis at a range of temperatures between
fully restrictive and fully permissive, using fluorescent membrane and histone markers.
Additionally, by combining these icp-1 alleles with rapid temperature shifts during cell division
we will be able to investigate the role of the CPC in cytokinesis, independent of meiosis and
mitosis.
2625
In Mouse Oocyte Cyclin A2 Is Required for Sister Chromatid Segregation In Meiosis II.
S. Touati1, D. Cladière1, L. Lister2, I. Leontiou1, J. Chambon1, A. Rattani3, F. Böttger4, O.
Stemmann4, K. Nasmyth3, M. Herbert2, K. Wassmann1; 1UPMC-CNRS, Paris, France,
2
Newcastle fertility Centre-Newcastle University, Newcastle, UK, 3Department of BiochemistryUniversity of Oxford, Oxford, UK, 4Department of Genetics-University of Bayreuth, Bayreuth,
Germany
Progression through female meiosis has to be well controlled to produce healthy, fertilizable
oocytes. Errors in chromosome segregation cause the generation of oocytes harboring the
wrong number of chromosomes that can give rise to aneuploid embryos. In meiosis, two
specialized cell divisions allow the separation of paired chromosomes first, and then of sister
chromatids. Separase removes the Cohesin complex holding sister chromatids together in a
step-wise manner from chromosome arms in meiosis I, and then from the centromere region in
meiosis II. Using mouse oocytes our study reveals an unexpected role for Cyclin A2, namely its
SUNDAY-LATE POSTER PRESENTATIONS
requirement for Separase-dependent sister chromatid separation in meiosis II. Untimely Cyclin
A2 associated kinase activity in meiosis I leads to precocious sister separation, whereas
inhibition of Cyclin A in meiosis II prevents it. Accordingly, endogenous Cyclin A is localized to
kinetochores throughout meiosis II, but not in anaphase I. Furthermore we found that Cyclin B1,
but not Cyclin A2 inhibits Separase in meiosis I. These findings indicate that Separasedependent Cohesin removal is differentially regulated by Cyclin B1 and A2 in mammalian
meiosis.
2626
Regulation of meiotic double strand breaks in C. elegans.
C. V. Kotwaliwale1, S. A. Langley2, A. C. Dose1, A. F. Dernburg1; 1University of California,
Berkeley, Berkeley, CA, 2Lawrence Berkeley National Labs, Berkeley, CA
Meiotic recombination generates crossovers, which physically link homologous chromosomes,
and direct their segregation to opposite poles. Despite the benefits, crossovers are restricted
both in number as well as spatially. Crossovers in the nematode C. elegans occur most
frequently within the distal regions (or arms) of the chromosomes, which comprise ~50% of the
genome, but ~90% of the genetic map length. Meiotic recombination initiates with the formation
of programmed double strand breaks (DSBs) catalyzed by the SPO-11 enzyme. It has been
unclear whether the crossover bias in C. elegans is a consequence of DSB distribution, or
instead reflects a mechanism that biases the downstream repair outcome of DSBs. In order to
address this, we generated a genome-wide map of meiotic DSBs by mapping the distribution of
the sole C. elegans RecA-like recombinase, RAD-51, by ChIP-seq. We have found that the
RAD-51 binding pattern is strikingly similar to the known recombination pattern in C. elegans.
The majority of RAD-51 binding sites occur on chromosome arms. In addition, we find that the
histone modification H3K36me1, which is associated with introns of active genes, is most
predictive of DSB sites in the worm genome. This suggests that local gene expression activity
may contribute to DSB formation. Although the DSB pattern is highly correlated with crossover
frequency, there are interesting exceptions. In particular, the sub-telomeric regions are
completely devoid of crossovers but are active DSB sites. We have also identified a DNA
sequence motif that appears to be enriched within DSB sites suggesting that sequence specific
factors may have a role in DSB specification in C. elegans. Surprisingly, this sequence motif is
highly over-represented in C. elegans, but not in other Caenorhabditis species raising the
possibility that recombination hotspots are rapidly evolving in nematodes.
In a parallel study, we have developed an Illumina Golden Gate SNP array to measure genomewide recombination rates in mutants defective in synapsis and/or recombination of specific
chromosomes and in translocation and inversion heterozygotes. This analysis has revealed
striking examples of inter-chromosomal effect in C. elegans. We will discuss these findings
further at the conference. Taken together, our data provide insights into the role of
recombination on the evolution of the C. elegans genome.
2627
Spindle attachment and kinetochore arrangement in apyrene spermatocytes.
D. E. Wilson1, L. V. Paliulis1; 1Biology Department, Bucknell University, Lewisburg, PA
Lepidopteran spermatogenesis differentiates spermatozoa into two types, eupyrene and
apyrene, near the time of pupation. Eupyrene cells undergo typical meiosis, separating a single
chromatid of each type into each sperm. Apyrene cells undergo an anomalous meiosis in which
chromosomes fail to congress to the metaphase plate and produce aneuploid sperm. Though
abundant, the smaller apyrene cells do not appear to play any role in activating the egg.
Although their function is unknown, apyrene cells make ideal model systems for investigating
SUNDAY-LATE POSTER PRESENTATIONS
chromosome congression errors leading to aneuploidy because of the frequent asymmetric
distribution of chromosomes in these cells.
Congression failure is a common proposed
contributor to aneuploidy in humans—a leading cause of miscarriage and birth defects. We
hypothesize that failure to congress may be caused by anomalous construction of bivalents in
apyrene spermatocytes. The element of chromosome construction we focus on in this study is
kinetochore positioning. If kinetochores are incorrectly positioned, chromosome attachment to
the spindle should be different from eupyrene spermatocytes, and the positioning of kinetochore
proteins should be different in the two cell types. Our experiments show that apyrene
kinetochores are able to attach to spindle microtubules, but using anti-kinetochore antibodies,
we find that apyrene kinetochores are positioned differently on bivalents than eupyrene
kinetochores.
2628
Molecular mechanisms of Spindle Assembly Checkpoint inactivation during kinetochore
capture.
N. I. Krefman1, D. Drubin1, G. Barnes1; 1Dept. of Molecular and Cell Biology, University of
California, Berkeley, Berkeley, CA
Mistakes in kinetochore-microtubule attachments are detected by a conserved biochemical
surveillance system known as the Spindle Assembly Checkpoint (SAC) that delays
chromosome segregation until proper attachments are established. The SAC is a crucial cellular
mechanism for preserving genomic integrity across mitosis. Much is now known about how the
SAC delays anaphase, yet little is known about how the SAC is inactivated, despite the fact that
SAC silencing is an essential cellular process. Key SAC proteins, including Mad1, localize
specifically to kinetochores with incorrect attachments. Thus, their kinetochore localization is a
marker of SAC activity at kinetochores. Indeed, inappropriate targeting of Mad1 to kinetochores
is sufficient to induce a SAC-dependent cell-cycle arrest, so depletion of Mad1 from
kinetochores must be a key step towards SAC inactivation. We are exploiting an assay that
allows the observation of individual detached centromeres in living budding yeast cells in order
to analyze the events preceding Mad1 removal from kinetochores. We find that Mad1 rapidly
associates with detached sister centromeres and continuously colocalizes with them until after
their capture by microtubules. Detached centromeres dwell at the nuclear periphery and
continue to track the nuclear periphery during retrieval while Mad1 is still present, suggesting
the existence of some high-affinity interaction between centromeres and the nuclear periphery,
potentially mediated by Mad1. Pairs of sister kinetochores also associate with the nuclear
periphery in cells treated with nocodazole and in asynchronous cells transiting S phase,
revealing that this may be a general behavior of detached yeast centromeres. Mad1 is usually
depleted from detached centromeres before they are completely retrieved to the spindle, often
being unloaded from centromeres to the nuclear periphery during transport. Deletion of the gene
encoding the Mad1-binding nuclear pore protein Nup60, but not Nup53, delays Mad1 removal
from captured centromeres until after retrieval is complete. These and other data suggest that
the Mad1:Nup60 interaction at the nuclear periphery may contribute to SAC inactivation at
individual centromeres by helping to deplete Mad1 from kinetochores. Our ongoing work seeks
to further refine the molecular and structural requirements for Mad1 depletion from kinetochores
through analysis of additional mitotic mutants.
SUNDAY-LATE POSTER PRESENTATIONS
2629
Sirt1 Controls Microtubule Dynamics through negative regulation of Plk1-Kif2a pathway
in Mitosis
J. Kim1, C-Y. Jang2, H. Cha1; 1Sogang University, Seoul, Korea, 2Sookmyung Women's
University, Seoul, Korea
The role of Sirt1 in growth or maintaining the genomic stability has been previously
demonstrated, implying undetermined roles of Sirt1 during mitosis. However, the molecular
mechanisms of Sirt1 to govern mitotic events remain unclear. Herein, we demonstrated that
Sirt1 regulates the enzymatic activity of Plk1, which is critical for both the mitotic progression
and the spindle dynamics during mitosis. Sirt1 depletion as well as chemical inhibition of
enzymatic activity results in an increase of unaligned chromosome, a substantial reduction of
spindle formation, which concurrently occurs with activation of Plk1 mediated Kif2a microtubule
depolymerase. Transient interaction between Plk1 and Sirt1 during mitosis appeared to
determine acetylation of Plk1 and its enzymatic activity under SAC. Alteration of Plk1 and
following Kif2a activity under spindle damage may account for susceptibility to the mitotic arrest
according to Sirt1 expression.
Thereby, Sirt1 functions as a novel mitotic regulator to govern the spindle dynamics and
chromosome movement through Plk1 and Kif2a pathway under spindle damage.
Development and Morphogenesis I: Tissue Development and Morphogenesis
2630
GATA-1 suppresses osteogenesis by binding and inhibiting Runx2.
K-L. Lin1, B-Y. Hsiao1, H-C. Liao1, F-F. Wang1; 1National Yang-Ming University, Taipei, Taiwan
GATA-1 and Runx2 are key transcription factors of hematopoiesis and osteogenesis,
respectively. It has previously been shown that mice expressing low levels of GATA-1 (GATA1low) are associated with osteosclerosis, the mechanisms remain uncharacterized. Here, we
show that the BMP2-stimulated ALP activity was lower in bone marrow cells isolated from
GATA-1low, relative to those from wild-type mice. Overexpression of GATA-1 decreased
osteocalcin mRNA expression, while knockdown of GATA-1 increased the promoter activities of
osteoblastic genes, including osteocalcin, osteopontin and collagen, in cultured osteoblastic
cells. The GATA binding-sequence (-156/-122 bp) juxtaposed to the Runx2 response-element in
the osteocalcin promoter was required for GATA-1-mediated transcriptional inhibition. GATA-1
bound to Runx2, mutagenesis studies showed that the lysine residues located at the tail of the
C-terminal Zn-finger domain were essential for Runx2 interaction and osteocalcin suppression.
In addition, GATA-1 inhibited the complex formation between Runx2 and its co-activator CBFβ
and promoted the degradation of Runx2 via ubiquitination-mediated mechanisms. Together we
have uncovered a novel mechanism indicating that GATA-1 directly inhibits osteogenesis by
suppressing Runx2-mediated transcriptional activity.
2631
Role of Syntaxin-1 in regulating epithelial morphogenesis.
S. Mg1, M. Narasimha1; 1Dept of Biological Sciences, Tata Institute of Fundamental Research,
Mumbai, India
Dorsal closure (DC) is a late step in morphogenesis in Drosophila that involves the bridging of
discontinuity in the dorsal side of epidermis, initially filled by a second tissue, the amnioserosa
SUNDAY-LATE POSTER PRESENTATIONS
(AS), not present in the larva. DC has served as a good model system to study epithelial
morphogenesis and also neural tube closure and wound healing. During DC, the amnioserosa
undergoes a reduction in area, accompanied by the extension of the flanking epidermis towards
the dorsal midline, and finally, fusion of the epidermis. While the involvement of cytoskeleton
and adhesion components in this process has been studied quite extensively, the role of
intracellular trafficking in tissue reorganization and cell shape changes, has not been explored
as much. SNARES, critical molecules in membrane fusion have been implicated in
developmental processes such as cellularization and polarized growth. Here we examine the
role of the plasma membrane SNARE, Syntaxin 1A in the dorsal epidermis during Drosophila
DC. Depletion of Syx 1A using an epidermal Gal4 expressed in alternate stripes resulted in a
failure of leading edge cells of the dorsal epidermis to exhibit polarized elongation (measured as
a ratio of the DV/AP interfaces) compared to control embryos. Additionally, changes in stripe
morphology and thickness were observed. Finally, microtubule organization (normally polarized
along the axis of elongation) also appeared to be disrupted in some LE cells of stripes depleted
of Syx1A.These results suggest a role for Syx1 mediated membrane delivery in mediating
epidermal cell elongation and epithelial organization during Dorsal closure.
2632
Hypoxia Stimulates Cementum Protein 1 Expression and Potentiates Cementum
Formation in Developing Tooth Roots.
H. Choi1, H. Jin1, H. Choung1, J-Y. Park 1, P-H. Choung1; 1Department of Oral and Maxillofacial
Surgery and Dental Research Institute, School of Dentistry, Seoul National University, Seoul,
Korea
Oxygen availability is a critical signal for proper development of many tissues, however there is
limited knowledge of its role in the root formation of tooth. Cementum protein 1 (CEMP1) is
recently known to be highly expressed in cementoblast-like cells. We found that mouse tooth
germ development occurs in a hypoxic state at some point, especially at the root forming area,
and demonstrated that hypoxia stimulates CEMP1 expression in human periodontal ligament
stem cells (PDLSCs), and periapical follicular stem cells (PAFSCs) suggesting that hypoxia
potentiates cementum formation in developing tooth. Hypoxia also increases mineral deposition
and alkaline phosphatase activity in human PDLSCs and PAFSCs while inhibiting their
proliferation. Since the oxemic state is transduced by the transcription factor, hypoxia-inducible
factors (HIFs), experiments were performed to determine if this protein was responsible for the
observed changes in CEMP1 expression. Ectopic expression of HIF1 α increased CEMP1
expression and chemical inhibition of HIF1 α accumulation by YC-1 decreased the expression in
vitro. These findings indicate that hypoxia has potential for further tissue engineering strategy
for cementum regeneration.
2633
Comparison of Osteogenic Potential of Cells Derived from Human Iliac Crest and
Mandible.
E. P. Ferraz1, R. R. Fernandes1, C. E. Sverzut1, A. E. Trivellato1, M. M. Beloti2, A. L. Rosa1;
1
Deartment of Oral and Maxillofacial Surgery, School of Dentistry of Ribeirao Preto, University of
Sao Paulo, Ribeirao Preto, Brazil, 2Department of Morphology, Physiology and Basic Pathology,
School of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil
Reconstruction of jawbone defects is achieved by using either iliac crest or mandible autografts
with satisfactory clinical results. As the osteogenic potential of cells derived from these sites
plays a key role in the clinical outcomes, here we compared the proliferation and osteoblast
marker expression of cells derived from iliac crest and mandible in a donor-matched way. After
SUNDAY-LATE POSTER PRESENTATIONS
harvesting, cells were cultured in osteogenic medium for up to 21 days for evaluating
proliferation (MTT), alkaline phosphatase (ALP) activity (thymolphthatein release), extracellular
matrix mineralization (alizarin red staining) and gene expression (quantitative RT-PCR) of runtrelated transcription factor 2 (Runx2), ALP and osteopontin (Opn). Data were compared by
ANOVA, Kruskal-Wallis and Mann-Whitney tests when appropriate and p value was set at 0.05.
Cell proliferation increased along the culture time-course, being greater for mandible-derived
cells at 7, 10 and 14 days. Despite displaying distinct patterns, ALP activity was detected in both
cultures. Mandible-derived cells exhibited higher ALP activity peaking at 10 and 14 days, while it
peaked at 14 days in iliac crest-derived cells. Extracellular matrix mineralization detected at day
21 was higher in mandible cell cultures. Gene expression was also affected by cell source but in
a more complex way. Runx2 expression was higher in cultures from mandible at 10 and 14 days
with no change along the time while for cultures from iliac crest it increased from 10 to 14 days.
ALP expression decreased in mandible-derived cells and increased in iliac crest-derived cells
from 10 to 14 days. ALP expression was higher at day 10 and lower at day 14 in mandiblederived cells compared with iliac crest. Opn expression was constant in iliac crest-derived and
decreased in mandible-derived cells from 10 to 14 days being higher in the former only at day
10. Our results showed that mandible-derived cells remarkably display higher proliferating
capacity and osteogenic potential compared with iliac crest-derived cells. These differences
may result from the distinct embryonic origin and/or ossification process of these bone sites.
Thus, if it is possible to take into account the osteogenic potential, mandible should be the first
choice as donor graft site.
Financial support: FAPESP and CNPq
2634
Effects of gravitational exercise on bone minerals in rats.
T. Sato1, A. Ohashi1, A. Sakaguchi2, S. Igawa2, S. Yamada1; 1Jissen Women's University,
Tokyo, Japan, 2Nippon Sport Science University, Yokohama, Japan
The constant influence of weightlessness during space flight leads to modifications of several
physiological processes. Degradation of physical performance of astronauts after long-duration
space missions in one of the topical but still unresolved problems of space medicine. The
phenomenon is commonly interpreted as a consequence of deconditioning of not only antigravity muscle but also bone structure because of long absence or reduction of mechanic and
gravitational loads. It is well known that osteoporosis are affected by aging and insufficiency
gravity stumulus as the human walking. The present study investigate the influence of such
gravitational exercise as drop jump compare to swimming exercise in improving of bone
minerals.
The study was carried out with 21 Wister rats divided into three groups each 7 rats, i.e.
gravitational exercise(drop jump), low gravitational exercise(swimming) and control.
The experiments were performed after basically 5weeks breeding. During experiments periods
for 8 weeks all animals were given standard laboratory diet and water avaiable freely per day. It
was increased gravitational effects on drop jump from 25cm(1 week), 30cm(2 week), 35cm(3
week) to 40cm(4-8 weeks) as gravitational training. In swimming group as low gravitational
exercise were performed swimming 1 hour per day in 5 times per week by automatically
pumping wave. The present study was carried out in accordance with the Japanese law, which
allows experiments on laboratory animals in Nippon Sport Science University accordance to the
Princeples of laboratory animal care.
After each one week exercise, hole femoris bone was isolated, bone density diagnosis both
bone mineral content(BMC,g) and bone mineral density(BMD, g/cm^2).One-way ANOVA was
used for statistical analysis of data between difference group, and P<0.05 was accepted for
significance.
SUNDAY-LATE POSTER PRESENTATIONS
No significant changes in both BMD and BMC in response from inital week to until 5 weeks
were observed. Significant change in increaseing was observed in 6 and 7 weeeks training both
BMD and BMC(p<0.05).Overall there were not show the trend in irder of decending both BMC
and BMD for jump, swimming and control, respectively from inital week to 5 weeks.We found
good exercise condition as gravitational effect for intensity and periods. The above-mentioned
results indicate that the jump training can increase both BMC and BMD over 6 weeks, but not
under 5 weeks.The gravity load but not swimming, is more susceptible to bone minerals transit
time than the exercise intensity.
2635
Regulation of the actin cytoskeleton during early morphogenesis in Drosophila.
M. Soares e Silva1, K. Kasza2, J. Zallen1; 1Howard Hughes Medical Institute and Developmental
Biology Program, Sloan-Kettering Institute, New York, NY, 2Developmental Biology Program,
Sloan-Kettering Institute, New York, NY
The actin cytoskeleton is responsible for dynamic changes in cell shape and behavior
throughout development. During morphogenesis of the fruit fly Drosophila melanogaster, cells
rearrange and change shape to give rise to an elongated body axis. This elongation is
immediately preceded by the emergence of planar polarized patterns of protein localization, and
actin polarization is the first evidence of planar polarity during Drosophila axis elongation. The
contraction of cell interfaces perpendicular to the elongation axis results in a narrowing of the
body in that direction, while new interface formation effectively elongates the embryo along the
anterior-posterior axis. While adherens junction proteins stabilize cellular interfaces parallel to
the axis of elongation, filamentous actin (F-actin) and myosin II assemble into contractile cablelike structures perpendicular to this axis. These contractile cables are under myosin-induced
tension and shrink to form multicellular rosettes, groups of cells in contact at a common vertex.
During the resolution of rosette structures, new interfaces form between cells parallel to the axis
of elongation. These interfaces transiently accumulate F-actin and ultimately recruit Baz/Par-3,
which stabilizes adherens junctions at new cell contacts. The net result of these actindependent cellular rearrangements is the doubling of the length of the body axis. Mechanical
tension contributes to this narrowing by promoting an additional, force-dependent stabilization of
myosin in contractile actomyosin cables. However, the mechanisms by which actin regulators
and mechanical tension coordinate to organize the actin cytoskeleton during axis elongation are
not well understood. In addition, little is known about the mechanisms that recruit F-actin to new
cell contacts during rosette resolution. To address these questions, we are studying the
localization and function of actin regulators during axis elongation in Drosophila embryos. To
investigate whether the actin cytoskeleton or its regulators are responsive to mechanical tension
and define their order of recruitment, we are developing biophysical assays to apply stress to
the apical surface of this tissue with a micropipette. This combination of cell biological and
biophysical approaches will provide an opportunity to identify molecular and mechanical inputs
that influence polarized actin organization during development.
2636
Analysis of Gpr126 in peripheral nerve development and myelination.
A. Mogha1, A. E. Benesh1, K. R. Monk1; 1Developmental Biology, Washington University School
of Medicine, Saint Louis, MO
Myelin, the multilayered membrane synthesized by glial cells, provides insulation and trophic
support to axons. In the peripheral nervous system (PNS), Schwann cells produce myelin by
iteratively wrapping their membranes around axons. Reciprocal signaling between axons and
Schwann cells is required for proper myelination to occur, but the exact signaling mechanisms
SUNDAY-LATE POSTER PRESENTATIONS
regulating myelination are poorly understood. An orphan adhesion G protein-coupled receptor,
Gpr126, is required for the initiation of myelination in zebrafish Schwann cells. In mouse, loss of
Gpr126 leads to complete amyelination in the PNS as well as multiple defects in peripheral
nerves. In zebrafish, we previously showed that forskolin treatment to elevate cAMP levels
suppresses the mutant phenotype and restores myelination. We hypothesized that elevation of
cAMP levels would similarly suppress mutant phenotypes in mouse mutants. Indeed, cAMP
elevation or PKA activation in DRG explant cultures from Gpr126 mutant mice rescued the
myelin defects, providing further support that cAMP and PKA are involved in the Gpr126mediated pathway initiating myelination. Although Gpr126 is predominately expressed in
Schwann cells, systemic Gpr126 knockouts show pronounced axon loss. Therefore, we have
begun to analyze conditional Gpr126 mouse mutants, and our analysis supports the hypothesis
that Gpr126 is required autonomously in Schwann cells for myelination in mammals.
2637
Dynamic three-dimensional imaging of developing zebrafish heart reveals changes in
cellular shapes and gene expressions.
V. Trivedi1, T. V. Truong2, L. A. Trinh2, D. B. Holland3, M. Liebling4, S. E. Fraser2;
1
Bioengineering, California Institute of Technology, Pasadena, CA, 2Biology, California Institute
of Technology, Pasadena, CA, 3California Institute of Technology, Pasadena, CA, 4University of
California Santa Barbara, Santa Barbara, CA
We present our results in dynamic three-dimensional (3D) imaging and quantification of the
cellular shape changes and gene expressions of the developing zebrafish heart, in the effort to
understand the mechanisms of the embryonic construction of this critical organ. The vertebrate
heart is built up through a series of steps taking two flat layers of cells to a hollow heart tube to a
multi-layered, multi-chambered, chirally twisted structure of the mature organ. Additionally, the
heart is the first organ in the developing embryo to function, through its beating and pumping of
the blood, shortly after the formation of the heart tube. Despite the intrinsic dynamic 3D nature
of the developing heart, previous works documenting its development consist of largely 2D
and/or static imaging (utilizing pharmacological means to stop the beating of the heart), due to
the challenges in achieving fast, high 3D-resolution with conventional imaging modalities. To
overcome these challenges, we employ 2-photon light sheet microscopy and a wavelet-based
synchronization and registration method to achieve the required spatial and temporal resolution
to capture the 3D motion of the heart. The high speed 3D imaging and analysis is carried out on
several transgenic zebrafish lines that have been recently generated in our lab where proteins
important for heart development are fluorescently tagged at their endogenous loci. We thus
document not only cellular morphology but also critical genes' expression, with sub-cellular
resolution, of the developing heart, over its beating cycle and at different development times.
These results provide the necessary groundwork to start deciphering the process where the
dynamic changes in cellular shapes, gene expressions, and cellular physical properties
participate, in concert with the genetic program, in the development of the vertebrate heart.
2638
Role of cAMP-dependent protein kinase A in regulation of mammary epithelial
morphogenesis in vitro.
P. I. Nedvetsky1, S-H. Kwon1, J. Debnath2, K. E. Mostov1; 1Anatomy, UCSF, San Francisco, CA,
2
Pathology, UCSF, San Francisco, CA
Epithelial cells form tubular and acinar structures notable for a hollow lumen. In threedimensional (3D) culture models utilizing MCF10A mammary epithelial cells, hollow acini form
due to integrin-dependent polarization and survival of cells contacting extracellular matrix
SUNDAY-LATE POSTER PRESENTATIONS
(ECM), and the apoptosis of inner cells of acini lacking ECM contact. Here, we report that
cAMP-dependent protein kinase A (PKA) promotes hollow acini formation via two mechanisms.
First, cAMP accelerates the polarization of outer cells due to ERK/MAPK inhibition. Second,
cAMP, promotes the death of inner cells occupying the lumen. In the absence of cAMP,
apoptosis is delayed resulting in profound luminal filling. cAMP-dependent apoptosis is
accompanied by a posttrascriptional increase in the proapoptotic protein BIM, which is at least
partially ERK-independent. These data demonstrate that cAMP regulates lumen formation in
mammary epithelial cell cultures both through acceleration of polarization of outer cells and
apoptosis of inner cells of the acinus.
2639
Identification of surface marker and functional heterogeneity in cultured primary human
tracheal basal cells.
M. A. Clifford1, S. Hasenoeder2,3, N. Vu4, T. K. Waddell2, N. Moghal2; 1Medical Biophysics,
University of Toronto, Toronto, ON, Canada, 2University Health Network, Toronto, ON, Canada,
3
Helmholtz Zentrum Munchen, Institute of Stem Cell Research, Neuherberg, Germany,
4
Graduate Program in Genetic Counciling, University of Utah School of Medicine, Salt Lake City,
UT
The upper airways of the lungs include the trachea and the large bronchi, which are lined with a
pseudostratified mucociliary epithelium. Understanding how the epithelium is renewed is
important for understanding the origins of certain lung cancers, and to potentially develop
regenerative strategies to treat diseases like cystic fibrosis. Although it is known that the
epithelium is renewed by basal cells, which collectively comprise ~30% of upper airway cells,
how stem/progenitor cell activity is organized within the basal cell compartment is poorly
understood. While clonal marking of human basal cells transplanted into tracheal xenografts
suggests there may be functional heterogeneity within the basal cell compartment, definitive
evidence is lacking. To investigate functional and phenotypic heterogeneity within the human
basal cell compartment, we used a combination of limiting dilution assays and surface marker
profiling of primary cultures of human basal cells. Although these cells were expanded on
plastic after tracheal digestion, they were not passaged, and we verified the presence of
progenitor activity in differentiation assays on Transwell® filters (air-liquid-interface culture) and
in rat tracheal xenografts. In limiting dilution assays on Transwell® filters, we determined that
there was functional heterogeneity in the ability of basal cells to repopulate a filter and form a
barrier that effectively maintained an air-liquid interface. The frequency of this activity varied
between strains of basal cells isolated from different donor tracheas and was on the order of
0.08%-1% of the total basal cell population. We also performed a large-scale comprehensive
surface marker profiling of basal cells isolated from two different donor tracheas, using 339
antibodies. Hits common to both strains were then validated by single tube FACS in a third
strain of basal cell, yielding 76 markers showing heterogeneous staining within the bulk
population of basal cells. The subpopulations vary in frequency from <1% to ~50%. Preliminary
analyses suggest there may be functional differences between some of the subpopulations.
This work supports the notion that the basal cell compartment may be functionally
heterogeneous, and provides a new arsenal of molecular tools for the directed investigation of
heterogeneity among human basal cells.
SUNDAY-LATE POSTER PRESENTATIONS
2640
O-GlcNAc modification of the transcription factor myocyte enhancer factor 2c regulates
terminal differentiation of skeletal myogenesis.
J. Cho1, H. Kim1, J. Roth1; 1Integrated OMICS for Biomedical Science, Yonsei University, Seoul,
Korea
β-O-linked N-acetylglucosamine(O-GlcNAc) modification is one of the post-translational
modification to occur in nucleus and cytosol. This modification is dynamically regulated by OGlcNAc transferase(OGT) and O-GlcNAcase. There is the interplay between O-GlcNAc
modification and phosphorylation .The source of O-GlcNAc modification is the UDP-GlcNAc,
final product of the hexosamine biosynthesis pathway to takes about 5% of glucose in total
glucose flux. Many proteins, such as transcription factors, enzymes, cytoskeletal proteins,
ribosomal proteins and chaperones have been identified to be modified with O-GlcNAc. OGlcNAc modification modifies at their Ser/Thr residues and affects their functions. O-GlcNAc
modification regulates cellular function in responses to the nutrient change, stress and cell
signals. O-GlcNAc modification is reported to associate with the development of skeletal muscle
atrophy and with muscle metabolism. Particularly, terminal differentiation of skeletal muscle is
repressed by increased O-GlcNAc modification. We observed that the expression of myogenin,
a key regulator for skeletal muscle terminal differentiation, decreased after NButGT treatment
which inhibits O-GlcNAcase to increase O-GlcNAc modification. This repression of myogenin
expression is due to decrease in mRNA level of myogenin. In addition, we confirmed that the
promoter activity of myogenin decreased after NButGT treatment using luciferase reporter gene
assay. To find the promoter region affected by O-GlcNAc , we used shorter length promoter
gene and found that at least 169 base pair upstream region of myogenin is affected by OGlcNAc. We also found that Mef2c protein can be an important transcription factor regulated by
O-GlcNAc via avidin-biotin complex DNA binding assay on 169 base pair upstream region. We
performed immunoprecipitation and mass spectrometry to confirm that Mef2c is O-GlcNAcylated
and to find O-GlcNAcylated sites on Mef2c. As we expected, mutagenesis of these serine or
threonine to alanine resulted in a decrease in O-GlcNAc modification of Mef2c. Additionally, we
found that O-GlcNAc modification could regulate DNA binding affinity but not the complex
formation with HDAC4.
2641
Establishing the epidermis: Formation of a Primary Outer Epithelial Barrier in Zebrafish
Embryos.
P. R. Nano1, J. A. Klimko1, J-A. Fleming2, A. C. Levken2, D. S. Wagner1; 1Dept. of Biochemistry
and Cell Biology, Rice University, Houston, TX, 2Dept. of Biology, Texas A&M University,
College Station, TX
The epidermis is a complex organ that enables organisms to withstand external injury and adapt
to various environments. Recent work has shown molecular homology between epidermal
differentiation in mammals and the differentiation of the zebrafish enveloping layer (EVL), the
superficial layer of the zebrafish embryonic blastoderm. We aim to determine the as-yet
unknown mechanism by which the EVL is initially specified. Our preliminary results have
suggested that this developmental program is mediated by the Wnt pathway. Inducing overactive Wnt signaling in zebrafish embryos led to arrested epiboly and a decreased expression of
keratin-4, a marker of differentiated EVL. However, these embryos could still maintain an outer
barrier with a similar cell morphology and cellular adhesion as mature EVL. We thus
hypothesize that Wnt signaling mediates the formation of a nascent, outer epithelial barrier
under maternal control and that Wnt signaling must be countered for mature EVL to
differentiate. We are currently testing the relationship between Wnt signaling and the Ikk1
SUNDAY-LATE POSTER PRESENTATIONS
signaling pathway, a known mediator of EVL maturation. Results from these experiments have
the potential to advance the understanding of the complete mechanism underlying EVL
development. Our findings in zebrafish embryonic epithelia, known to be similar to mammalian
embryonic epithelia, can ultimately be applied to the study of normal skin morphogenesis and
abnormal skin development in squamous cell carcinoma.
2642
Blm-s, a proapoptotic molecule expressed in postmitotic immature neurons, is a direct
target gene of p53 and AP1 after DNA damage.
P-H. Huang1; 1Graduate Institute of Pathology, College of Medicine, National Taiwan University,
Taipei, Taiwan
Programmed cell death (PCD) is an essential feature for the developing nervous system to
mature into a functional adult brain. Bcl-2 family members are major components for PCD, as
elucidated by previous genetic ablation studies. Blm-s, a CNS-enriched Bcl-2 family member
that we identified via mRNA differential display, functions as a BH3-only apoptosis
sensitizer/derepressor to induce apoptosis via the mitochondrial pathway. Blm-s is specifically
expressed in postmitotic migratory neurons of the developing cortex but rarely detected in adult
brain. Yet little are known about how the expression of Blm-s is developmentally regulated and
whether the apoptosis occurs in Blm-s expressing neurons. Here we demonstrate that Blm-s is
transcriptionally induced by DNA double strand breaks (DSBs) in postmitotic neurons at the
intermediate zone and cortical plate of mouse embryonic brain. Assays including chromatin
immunoprecipitation, promoter reporter assay, DNase I hypersensitivity and gel motility shift
together indicate that Blm-s is a direct target gene of p53 and AP1, both of which are activated
in the ATM signaling pathway triggered by DSBs.
2643
A Role for Small Molecule Wnt modulators in Cardiovascular Therapeutics.
A. N. Russo1, A. Frist2, C. Hong1,2; 1Cell and Developmental Biology, Vanderbilt University,
Nashville, TN, 2Cardiovascular Medicine, Vanderbilt University, Nashville, TN
Wnt/β-catenin signal transduction plays critical roles in development, adult tissue homeostasis,
and regeneration. In vertebrate embryos, Wnt signaling is critical for the regulation of embryonic
patterning. Recent studies have shown that Wnt signaling is activated following myocardial
infarction (MI), but the functional significance is unclear. We hypothesize that post-MI Wnt
signaling plays an active role in myocardial injury response but that this process is usually
insufficient to prevent heart failure, characterized by myocardial cell loss and dysfunction as well
as scarring of the heart tissue. Thus, small molecules that selectively augment Wnt/β-catenin
signaling are would be valuable as potential therapeutics to promote myocardial repair. Despite
intense efforts, there are no chemical Wnt potentiators that have been rigorously validated in in
vivo animal models. Through chemical genetic screens for small molecules that perturb
embryonic patterning in zebrafish, we have identified several classes of novel small molecules
that either augment or inhibit Wnt signaling in zebrafish and human cells. This project will
explore the effects of these small molecules on embryonic pattern formations well as whether or
not modulation of canonical Wnt signaling promotes myocardial repair.
SUNDAY-LATE POSTER PRESENTATIONS
2644
Tramtrack69 regulates epithelial tube expansion in the Drosophila ovary through Paxillin,
Dynamin, and the homeobox protein Mirror.
N. C. Peters1, C. A. Berg1; 1Department of Genome Sciences / Molecular and Cellular Biology
Program, University of Washington, Seattle, WA
Epithelial tubes are the foundation of many organs, and tube formation requires precise
orchestration of cell signaling, shape change, polarity, migration, and adhesion. Follicle cells
that encase developing egg chambers in the Drosophila ovary form a pair of epithelial tubes, the
apical lumens of which act as molds for the eggshell respiratory filaments, or dorsal
appendages (DAs). This system is a robust and accessible model for studying the patterning,
formation, and expansion of epithelial tubes. The Tramtrack69 (TTK69) transcription factor
controls DA lumen volume by regulating apical surface area and tube expansion; the twin peaks
(twk) mutation reduces TTK69 levels specifically late in oogenesis, inhibiting tube expansion
and stunting the DAs. Microarray analysis of wild type and twk ovaries, followed by in situ
hybridization and RNAi of candidate genes, identified the focal adhesion scaffold protein
Paxillin, the endocytotic regulator Shibire (Dynamin), and the homeodomain transcription factor
Mirror, as important TTK69 effectors of DA-tube expansion. These genes display enriched
expression in DA-tube-cells, reduced expression in twk mutants, and RNAi phenotypes that are
enhanced in a twk/+ background, indicating twk genetic interactions. Although previous studies
show that Mirror patterns the follicular epithelium prior to DA tubulogenesis, we show that Mirror
regulates DA-tube expansion independently of patterning, revealing a novel role for this
transcription factor in tubulogenesis. Additionally, we show that Mirror, as well as TTK69,
positively influences the expression of paxillin, providing evidence that these TTK69 effectors
are in the same pathway. Finally, our studies implicate numerous other genes, including
shibire, as tube expansion effectors downstream of TTK69, and we are beginning to examine
their regulatory interactions with TTK69, Mirror, and each other. Thus, our effort to identify and
characterize twk-differentially expressed genes has identified novel tubulogenic regulators and
has begun to elucidate the network of TTK69 effectors required for epithelial tube expansion.
2645
A Novel Mechanism for Epithelial Cell Height Regulation.
H. Wu1, T. Mikawa1; 1Cardiovascular Research Institute, University of California San Francisco,
San Francisco, CA
During development epithelial cells dynamically change their height to fulfill their function. While
the mechanisms for cell size and cell polarity determination have been extensively studied, little
is known about how cell height is regulated. Here we show that within an established epithelial
monolayer, cell volume varies dramatically while cell height remains constant. These
observations suggest that cell height is determined through a novel mechanism that is distinct
from cell size regulation. We examined the distribution of microtubules, a cytoskeletal element
involved in cell shape regulation, in cells of different height including squamous and cuboidal
epithelium. Both cell types expressed alpha tubulin at the similar level but the distribution was
significantly different. Confocal microscopy analysis revealed that the microtubules were evenly
distributed along the apicobasal axis in squamous and subconfluent shorter cuboidal cells. As
cuboidal cells increase their height, microtubules became enriched in the apical region, i.e.,
taller cells exhibited a polarized alpha tubulin distribution. We also examined a potential role for
lipid incorporation into the lateral membrane during cell height regulation. Pharmacological
inhibitors of PI3Kinase, which is the enzyme that converts PIP2 into PIP3 at the lateral domain,
significantly reduced epithelial cell height while apical microtubule enrichment remained. These
results suggest that microtubules undergo dynamic reorganization during cell height
SUNDAY-LATE POSTER PRESENTATIONS
establishment, but do not appear to provide the driving force for this process. Instead, regulation
of membrane lipid incorporation is implicated for cell height regulation. Supported in part by the
grant from NIH to TM and T32 HL007731 training grant to HW.
2646
Hedgehog interacting protein regulation of the Sonic Hedgehog pathway.
L. Kwong1, M. F. Bijlsma1, H. Roelink1; 1Molecular and Cell Biology, University of California,
Berkeley, Berkeley, CA
A target of the Sonic Hedgehog (Shh) pathway activation is the upregulation of genes that
negatively regulate Shh signaling, which includes the Shh receptor Patched1 (Ptch1) and
Hedgehog interacting protein (Hhip). How these negative regulators are involved in the
generation of differential Shh responses in a responding tissue is not completely understood.
Unlike Ptch1, little is known about Hhip. The main function of Hhip seems to be in restricting
Shh signaling by sequestering Shh ligands at the cell surface, which then leads to a cell
nonautonomous inhibition of Shh signaling. In order to understand the mechanism by which
Hhip regulates the Shh pathway, we examined more closely the cell autonomous and cell
nonautonomous contribution to the regulation of the Shh response. We ectopically coexpressed Hhip and activated Smoothened (Smo) in the neural tube, and found that Hhip
cannot inhibit the Shh response in cells expressing activated Smo, indicating that Hhip acts
upstream of Smo activation. Moreover, the coexpression allowed us to clearly demonstrate that
Hhip inhibits the Shh response in nearby cells, where Hhip was not expressed. Because Hhip is
most highly expressed near the source of Shh, it raises the question of why the normal sites of
Hhip expression have an apparently unimpaired response to Shh. We found that activated Smo
causes a significant loss of cell associated Hhip, thus negating the cell-autonomous inhibitory
activity of Hhip. These results provide a mechanism by which Hhip is able to act despite being
near the Shh source, where Shh is able to activate the pathway, while Hhip can still induce cell
nonautonomous effects, adding a new dimension to Hhip and Shh regulation.
2647
Evaluation of testosterone’s effects in prostatic complex of Artibeus planirostris
(Chiroptera: Phyllostomidae).
C. I. Puga1, M. R. Beguelini1, F. F. Martins1, E. Morielle-Versute1, P. L. Vilamaior1, S. R.
Taboga1; 1UNESP, São José do Rio Preto, Brazil
Previous studies evaluating - seasonal modifications in the prostatic complex (PC) of bats
showed variations along the year linked to serum testosterone levels. The aim of this study was
to evaluate the effect of ablation and replacement of testosterone in PC of Artibeus planirostris.
Eight groups were formed: two groups for 3 (CA3) and 15 (CA15) days castrated, with their
respective controls (CO3 and CO15); and castrated groups which received testosterone
cypionate (1µg/Kg/day) for 3 (T3) or 7 (T7) days, whereas control group received only mineral
oil (O3 and O7). All groups (n=4 and p≤0.05) were submitted to the following analyses:
histological, estereological (hematoxylin-eosin, periodic acid-Schiff and picrosirius red stained
sections) and immunohistochemistry for proliferating cell nuclear antigen (PCNA). In both
regions of the PC, ventral (VR) and dorsal (DR), the CO3, CA3 and CO15 groups presented a
normal morphology compared to CA15 which observed an accentuated glandular atrophy with
an increase in cells aggregates, cell debris regions, alterations in glandular secretion, increased
stromal compartment and decrease in luminal region. In DR was observed a significant increase
in epithelial height at CA15 group. The O3, T3 and O7 groups presented a similar morphology
to CA15. Only in T7 was observed a decline in stromal compartment and increased luminal
SUNDAY-LATE POSTER PRESENTATIONS
region. These data are consistent with findings in a previous work from our laboratory, which
showed that along seasons, testosterone levels and epithelial height of DR are inversely
proportional. However in morphometric analysis of DR we observed that there was an increase
in epithelial height in all groups under testosterone replacement (T3 and T7), showing that after
castration the hormonal replacement helps the recuperating of glandular activity.
Immunohistochemistry indicated that the VR of castrated and control groups exhibited no
difference regarding cell proliferation, as well as the treated groups with oil. The remodeling of
the gland mediated by testosterone appears to increase cellular proliferation, this increase was
observed in T3 and T7 groups. We observed a constant in the number of proliferating cells,
which indicates that DR seems not to change their rate of proliferation. These data indicated
that both regions of PC are responsive to testosterone, but both respond differently to - ablation
and replacement of testosterone, being that the VR is more sensitive than the DR. Approved by
Ethics Committee of UNESP - Protocol: 036/2010 – CEUA. Financial Support: FAPESP, CNPq
and CAPES.
2648
Targeted Disruption in Mice of a Neural Stem Cell-Maintaining, KRAB-Zn FingerEncoding Gene That Has Rapidly Evolved in the Human Lineage.
H-C. Chien1,2, H-Y. Wang3, Y-N. Su4, K-Y. Lai1, L-C. Lu1, P-C. Chen5, S-F. Tsai6, C-I. Wu7, W-S.
Hsieh8, C-K. J. Shen1,2; 1Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan,
2
Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University,
Taipei, Taiwan, 3Institute of Clinical Medicine, Taipei, Taiwan, 4National Taiwan University
Hospital, Taipei, Taiwan, 5Institute of Occupational Medicine and Industrial Hygiene, Taipei,
Taiwan, 6National Health Research Institute, Taipei, Taiwan, 7Department of Ecology and
Evolution, University of Chicago, Chicago, IL, 8Department of Pediatrics, National Taiwan
University Hospital, Taipei, Taiwan
Understanding the genetic basis of the physical and behavioral traits that separate humans from
other primates is a challenging but intriguing topic. The adaptive functions of the expansion
and/or reduction in human brain size have long been explored. From a brain transcriptome
project we have identified a KRAB-Zn finger protein-encoding gene (M003-A06) that has rapidly
evolved since the human-chimpanzee separation. Quantitative RT-PCR analysis of different
human tissues indicates that M003-A06 expression is enriched in the human fetal brain in
addition to the fetal heart. Furthermore, analysis with use of immunofluorescence staining,
neurosphere culturing and Western blotting indicates that the mouse ortholog of M003-A06,
Zfp568, is expressed mainly in the embryonic stem (ES) cells and fetal as well as adult neural
stem cells (NSCs). Conditional gene knockout experiments in mice demonstrates that Zfp568 is
both an NSC maintaining- and a brain size-regulating gene. Significantly, molecular genetic
analyses show that human M003-A06 consists of 2 equilibrated allelic types, H and C, one of
which (H) is human-specific. Combined contemporary genotyping and database mining have
revealed interesting genetic associations between the different genotypes of M003-A06 and the
human head sizes. We propose that M003-A06 is likely one of the genes contributing to the
uniqueness of the human brain in comparison to other higher primates.
2649
Analyzing cellular dynamics in a proliferating epithelial explant to assess the influence of
morphogen signaling on tissue mechanics and growth orientation.
N. A. Dye1, S. Eaton1; 1MPI-CBG, Dresden, Germany
We are using the Drosophila imaginal wing disc to study how morphogen gradients regulate the
growth and division of cells within a proliferating epithelium to ultimately control tissue shape
SUNDAY-LATE POSTER PRESENTATIONS
and size. Spatially homogeneous proliferation in the wing disc is under the control of three
regulatory signaling pathways (Dpp, Wingless, and the atypical cadherins Fat and Daschous)
with graded distributions of activity throughout the tissue. Perturbing any one of these gradients
results in spatially inhomogeneous proliferation, altering the growth axis and tissue shape. We
believe that the regulation of tissue size and shape may be influenced not only by biochemical
signaling but also by the distribution of physical forces within the epithelium. Furthermore, it has
been shown in other systems that anisotropic stresses in an epithelium can orient the axis of
cell divisions and rearrangements. Thus, we hypothesize that the three signaling centers in the
wing disc, while regulating cell proliferation, can also affect local mechanical stresses in the
epithelium to orient growth. To test this idea, we are quantitatively and systematically analyzing
tissue growth at a cellular resolution in live proliferating explanted imaginal discs. We have
developed culture conditions that support growth and division of isolated imaginal discs for ~6
hours while imaging at high spatial and temporal resolution. We are using custom developed
software to measure and follow cell shape changes, division rate and orientation, cell
rearrangements and extrusions throughout the entire tissue. Combining these measurements
with laser ablation data and physical modeling should enable us to assess the relationship
between morphogen signaling, epithelial stress patterns and growth orientation. Progress on the
analysis of growth under wild type and mutant conditions will be presented.
2650
Analysis of cell shape changes and cell migration required for morphogenesis in
evolution of volvocine algae.
P. V. Elvira1, I. Nishii1; 1Temasek Life Sciences Laboratory, Singapore, Singapore
The transition from multicellular to unicellular organisms evolved a variety of forms through
morphogenesis in that they became more complex as cell number increased. As the simplest
model for studying such evolutionary steps, volvocine green algae including Volvox offer an
array of colonial species with cells ranging from four to thousands. During the development of
volvocine embryos, a single cell cleaves to produce a certain number of daughter cells that are
interconnected via cytoplasmic bridges, formed by incomplete cytokinesis, that coherently hold
a whole embryo. After cleavage stage, most of these algae end up with cells having flagella
oriented inside the bowl-like cell sheet, impeding colony locomotion. The embryo corrects this
situation through ‘inversion’ wherein the cell sheet attains an opposite curvature so that the
flagella direct externally. This inversion is achieved when cells change their shape from conical
(flagella facing the colony interior), to spindle, and finally to inverted-conical or elongated flaskshape, while cells simultaneously migrate relative to the cytoplasmic bridges to be linked at the
thin end. Inversion has been extensively studied in this algal group. It has been shown that in
Volvox carteri cell shape changes depend on microtubules (MTs), while a kinesin, InvA,
localized around cytoplasmic bridges has an essential role in cell migration. Since previous
studies on the related species showing inversion indicated similar events in their
morphogenesis, here we intended to analyze them in detail based on MTs and InvA localization
to elucidate the evolutionary steps of morphogenesis in this group. MT staining revealed
increasing degrees of cell shape changes during inversion as cell number increased in Gonium
(16 cells), Pandorina (16), Eudorina (32) and Pleodorina (128). InvA was localized right above
the cell equatorial region before inversion, then gradually moved below the equatorial region
and finally reached the elongated thin end at completion of inversion in Pandorina, Eudorina
and Pleodorina. These findings confirmed that translocation of InvA also occurs in these species
as in V. carteri, suggesting a similar role of InvA in cell migration. These cellular and molecular
bases of inversion in V. carteri and the related species add more evidence that similar
frameworks for cell shape changes and cell migration facilitate morphogenesis in this algal
SUNDAY-LATE POSTER PRESENTATIONS
group. Here, we will also discuss how increasing degrees of cell shape changes may indicate
an evolutionary path for inversion in a group of species with rising number of embryonic cells.
2651
Gerbil female prostate under effect of short exposure to exogenous prolactin.
M. Zanatelli1, D. A. da Silva1, F. C. dos Santos2, S. R. Taboga3; 1Cell Biology, Unicamp,
Campinas, Brazil, 2Morphology, UFG, Goiânia, Brazil, 3Biology, UNESP, São José do Rio Preto,
Brazil
The prostate is an accessory gland of genital system present not only in males, but also in
females of various species, such as humans and rodents. In female rodents, the prostate is
composed by a set of acini and ducts inserted into musculature surrounding the urethra. The
acini are lined by epithelial secretory and basal cells and are embedded in a fibromuscular
stroma. Prolactin (PRL) is a polypeptide hormone with more than 300 biological activities in the
body, among them, functions involved with reproduction. The exact role of prolactin in female
prostate is not well defined, so this study aimed to evaluate its effects on the prostate of female
gerbil (Meriones unguiculatus), normal and castrated. For this, three experimental groups were
formed (n=6): Co (females who have not suffered any type of intervention), PRL (females
subjected to subcutaneous administration of exogenous prolactin, 0.3mg/Kg, diluted in 0.1ml of
saline, once a day, from 111 to 113 days of age) and CaPRL (females subjected to bilateral
ovariectomy at 90 days of age and administration of exogenous prolactin, in the same way as
above). Females from all groups were killed by decapitation after inhalation of CO2, on 114 days
of age. The prostatic complex were fixed and processed to morphological and
immunohistochemistry (IHC) analysis. The sections obtained were subjected to stereology, premalignant lesion counts and immunoreation for cell proliferation marker (PCNA). The prostates
from PRL group demonstrated, according to stereology, a significant increase in epithelial
volume. Despite the IHC of proliferating cells has not been altered in relation to Co group, the
prostates showed lots of prostatic intraepithelial neoplasia (PIN) with cribriform arrangement,
accompanied by intense stromal inflammation. It is already known exist a relationship between
the rise in prolactin levels and the increase of prostatic diseases in men, as well, that PRL is
responsible for development of inflammations in male gland. This study shows evidence that
these same effects are also found in female tissue. In contrast, females from CaPRL group
showed some characteristics of atrophy, as acini with short epithelium, presence of cell debris
and low amounts of proliferative cells. However, the PRL administration after castration seems
to have avoided that this process was aggravated. The stereology demonstrated no changes in
volume of different prostatic compartments when compared to Co group. Some studies have
shown that PRL acts as a growth factor for prostate tissue, with role in the survival of prostate
cells after castration. It is believed that this function is also active in female gland, as well as its
importance in regulation and maintenance of the organ. Financial support: FAPESP, CNPq,
CAPES.
2652
Gestational and postnatal exposure to high-fat diet increases the incidence of malignant
lesions in the prostate of Mongolian gerbil.
M. M. Jesus1, A. C. Negrin1, R. M. Góes2; 1Institute of Biology, Unicamp, Campinas, Brazil,
2
Department of Biology, Ibilce/Unesp, São José do Rio Preto, Brazil
The inadequate maternal nutrition can result in obesity or metabolic syndrome during fetal and
neonatal development. Some clinical data have correlated the obesity with a higher incidence of
pathological lesions and adenocarcinome in prostate gland, however the mechanisms
responsible for these pathological alterations are unclear. This study examined the influence of
SUNDAY-LATE POSTER PRESENTATIONS
high-fat diet exposure, from gestation to sexual maturity, on the histology and physiology of
male Mongolian gerbils (Meriones unguiculatus) ventral prostate (VP). Male gerbils (4w old),
born from mothers fed with high-fat (20% satured fat, 4.8kcal/g; high-fat group - HF) or balanced
diet (4% satured fat, 3.16kcal/g, control group - C), were fed with the same diet as their
mothers, during 10 weeks. After the experimental period, one gerbil (14w old) per family was
randomly chosen (n=12 per group), killed and the VP was removed, weighed and processed for
paraffin embedding. Microscopic alterations in the VP were evaluated in sections stained with
H&E and complemented by stereological analysis of main gland tissue components. The whole
VP of all animals was scanned for the incidence of premalignant and malignant lesions (PIN,
microinvasive carcinoma, atrophy and prostatitis). Proliferating activity was estimated using
immunohistochemistry for proliferating cell nuclear antigen (PCNA) and smooth muscle cells
were differentiated using immunofluorescence for alpha-actin. Tissue lesion areas were not
included in stereological and cell proliferation analysis. The exposure of mothers to HF did not
alter the body weight of litters at weaning, but increased 9.5% at adulthood (p=0.02) in
comparison with control gerbils. The adiposity index was unaltered after HF exposure. The
relative and absolute weights of VP increased 38.5% (p=0.03) and 50.7% (p=0.02), respectively
in HF in relation to C. One third of HF animals showed epithelial atrophy, exhibiting acinus with
wide lumen and very low secretory cells, data which were confirmed by stereological analysis.
30% of control gerbil exhibited one to three foci of low grade PIN whereas 55.6% of HF gerbils
showed multiple foci of high grade PIN in VP. The incidence of chronic prostatitis increased 1.7
fold in HF. Furthermore, 50% of HF gerbils exhibited microinvasive carcinoma against 10% of
control group. The immunofluorescence for alpha-actin revealed the interruption and
disorganization of fibromuscular stroma both in adenocarcinome and PIN foci. The density of
proliferating cells was unchanged both in stroma and epithelium of HF gerbils. Previous reports
indicated that Mongolian gerbil has a greater propensity to spontaneous development of
malignant lesions during aging. The present data show that continuous exposure of this rodent
to HF in gestation and postnatal development is associated with inflammatory processes of
prostate and increase of PIN and adenocarcinome, which suggests a premature aging of the
gland. Financial support: Fapesp, Capes and CNPq.
2653
The BMP regulator BMPER is necessary for normal coronary artery formation.
L. A. Dyer1, C. Patterson1; 1McAllister Heart Institute, University of North Carolina at Chapel Hill,
Chapel Hill, NC
Connection of the coronary vasculature to the aorta is one of the last essential steps of cardiac
development. However, little is known about the signaling events that promote normal coronary
artery formation. The bone morphogenetic protein (BMP) signaling pathway regulates multiple
aspects of endothelial cell biology but has not been specifically implicated in coronary vascular
development. BMP signaling is tightly regulated by numerous factors, including BMP-binding
endothelial cell precursor-derived regulator (BMPER), which can both promote and repress
BMP signaling activity. In the embryonic heart, BMPER expression is limited to the endothelial
cells and the endothelial-derived cushions, suggesting that coronary vascular defects may be
present. Histological analysis of BMPER-/- embryos at embryonic day 16.5 revealed that the
coronary arteries were either atretic or connected distal to the semilunar valves. However,
analysis of earlier embryonic stages showed that the coronary plexus began differentiating
normally and that apoptosis and cell proliferation were unaffected in BMPER-/- embryos. In vitro
tubulogenesis assays showed that isolated BMPER-/- endothelial cells had impaired tube
formation compared to wild-type endothelial cells. Together, these results indicate that BMPERregulated BMP signaling is critical for the migration of coronary endothelial cells and normal
coronary artery development.
SUNDAY-LATE POSTER PRESENTATIONS
2654
Development of a chemical screening platform to identify molecular mechanisms of rod
photoreceptor outer segment renewal.
A. M. Jensen1,2, L. J. Campbell1, J. J. Willoughby1; 1Biology, University of Massachusetts,
Amherst, MA, 22Molecular and Cellular Biology Program, University of Massachusetts, Amherst,
MA
The long-term goal of our research is to identify and define the molecular pathways and cellular
mechanisms that regulate retinal photoreceptor maintenance. Retinal photoreceptors are unique
cells in that they are continually renewing a part of themselves– their outer segments, the light
receptive organelle. Although this renewal process is central to the function of photoreceptor we
still know very little about how these cells renew their outer segments through the combined
processes of growth at the base of the outer segment and shedding from the tip. It is observed
that the outer segments of photoreceptors progressively shorten before photoreceptors die
during photoreceptor degeneration diseases and the question remains as to whether these cells
might be dying because they lose their outer segments. We have developed a screening
platform for screening small molecules to identify molecular pathways and mechanisms
regulating rod photoreceptor outer segment renewal. However, this platform offers the added
opportunity to screen at the same time for small molecules affecting phagocytosis and digestion
of shed rod outer segment material by the neighboring retinal pigmented epithelium, rod outer
segment disc integrity, Rhodopsin trafficking and localization, and rod differentiation and
morphogenesis. We will present data showing that this platform using our recently developed
Tg(hsp70:mCherryTM) line allows us to quantitatively measure rates of rod outer segment
growth and shedding, as well as the ability to assay phagocytosis and digestion of shed rod
outer segment material by the neighboring retinal pigmented epithelium, rod outer segment disc
integrity, Rhodopsin trafficking and localization. If, as expected, chemical compounds are
discovered that stimulate outer segment growth, these can then be tested in mouse models of
human retinal degeneration disease for their ability to stimulate outer segment growth, promote
photoreceptor survival and prolong vision.
2655
Similar behavior of male and female prostate gland after progesterone and testosterone
treatment.
R. A. Fochi1, R. M. Góes2, S. R. Taboga2; 1Structural and Functional Biology, Campinas State
University - Unicamp, São José do Rio Preto, Brazil, 2Department of Biology, São Paulo State
University - Ibilce, São José do Rio Preto, Brazil
In this work, we investigated how the male and female prostate of Mongolian gerbils (Meriones
unguiculatus), responds to surgical castration at the beginning of puberty, as well as the effects
of progesterone (P4) and testosterone (T) replacement. To perform this investigation, male and
female gerbils were castrated at prepubertal period (45 days). After adulthood (90 days), some
of the castrated animals were killed, forming the control group (CaC), while others were treated
with subcutaneous doses (1 mg/kg b.w.) of P4 (CaP) and T plus P4 (CaPT), during 14 days. For
the intact control group (NC) non-castrated adult gerbils were used. In the prostate of castrated
animals of both sexes, an intense glandular regression, disorganization in the stromal
compartment, and abundant hyperplasia were noted. Besides such alterations, it was observed
a decrease of androgen receptor (AR) and also an increased number of PCNA-positive cells. In
the CaP group, the P4 stimulated a recovery of epithelial compartment accompanied by an
intense hyperplasia and a moderate hypertrophy. There was also an increase of the AR,
progesterone (PR) and estrogen receptors (ERα) in the stromal cells of female and male
prostate. The secretory cells became larger compared to CaC group and also presented a
SUNDAY-LATE POSTER PRESENTATIONS
higher number of PCNA-labeled cells. Although this group has exhibited epithelial dysplasia, it
was still possible to observe the presence of basal cells strongly reactive to the P63. In CaP
group the glycoprotein secretion became more intense in the lumen of male and female
prostate. The replacement of P4 associated to T in CaPT group did not promote important
changes in the secretory pattern. However, the epithelial compartment of this group presented
drastic changes, either in males as in females. The secretory cells were larger than those
observed in CaP animals, but without epithelial hyperplasia and dysplasia. In this group, the
p63-positive cells at basal layer are still present, but in smaller number. In addition, epithelial
cells continued to show the same PCNA expression pattern, while the apoptotic index has risen.
CaPT group of both sexes showed an increase of AR-positive cells in the epithelium, but in
males the stromal ERα-positive cells reduced, whereas in female gland these cells revealed the
same immunoreaction observed in CaP. In summary, these results show that the physiological
interaction between the hormones P4 and T is essential for the maintenance of the prostate
morphophysiology in males and females. Moreover, the female prostate has a responsive
pattern very similar to that found in male prostate gland regarding the steroid hormones action
in the reproductive system. Financial support: FAPESP; Capes; CNPq.
2656
Identification and characterization of a new gene required for axis elongation in
Drosophila.
A. Vichas1, J. Zallen 2; 1Developmental Biology Program, Sloan-Kettering Institute, New York,
NY, 2HHMI and Developmental Biology Program, Sloan-Kettering Institute, New York, NY
During axis elongation in the Drosophila embryo, polarized cell behaviors cause the germband
epithelium to double in length along the anterior–posterior axis and narrow in width along the
dorsal–ventral axis, establishing the structural basis for the body plan. These cell
rearrangements are dependent on the asymmetric distribution of actomyosin contractility and
adherens junction proteins. An oriented actomyosin network provides the spatial information
that guides cell rearrangement, while differential adhesion influences local interactions between
cells. In a forward genetic screen, we identified a maternally provided gene on chromosome 3R
that is required for polarized cell rearrangements during axis elongation. In the absence of this
gene, the adherens junction regulator Bazooka/Par-3 forms planar-polarized aggregates that
occupy a fraction of its wild-type domain. In addition, centrosomes fail to downregulate their
association with pericentriolar material and do not relocalize basal to the adherens junctions,
instead remaining in close association with cortical Baz aggregates. These observations
suggest a role for this gene in regulating interactions between centrosomes and adherens
junctions that are important for cell rearrangement. Current experiments are focused on the
molecular identification of this gene and investigating the relationship between centrosomes,
microtubules, and adherens junctions during cell rearrangement.
2657
MicroRNA regulation of eye development in Xenopus laevis: effect of miR-26.
R. A. Ritter1, A. K. Sater1, B. Brzezinska2, H. M. El-Hodiri2; 1University of Houston, Houston, TX,
2
Nationwide Children's Hospital Research Institute, Columbus, OH
MicroRNAs are key regulators of early eye development. However, much still remains to be
known regarding how specific microRNAs may regulate gene expression within the embryonic
eye field. We generated transgenic Xenopus laevis expressing myc-tagged Argonaute2 (Ago)
under the control of the Retinal Homeobox (RX1A) promoter. This allows tissue specific
expression of Argonaute in the eye field, optic cup, and retina from early neurula stages onward.
In parallel experiments, myc-Ago was injected into the dorsal blastomeres of 16cell stage
SUNDAY-LATE POSTER PRESENTATIONS
Xenopus embryos, which are fated to make a large contribution to the eye field. Using a
ribonucleoprotein immunoprecipitation (RNP-IP) strategy to isolate myc-Ago and associated
mRNAs, we can isolate and identify eye-field transcripts that co-immunoprecipitate with Ago and
are thus candidates for microRNA-dependent regulation. Q-RT-PCR assays of Ago RNP-IP
RNA show enrichment in both injected and transgenic IP samples over uninjected IP samples
for LHX2. However, Otx2 and Tbx3 were not enriched in the Ago-RNP IP pool. In parallel
studies, we are investigating the role of miR-26 as a regulator of Smad1 in eye development. In
Xenopus, miR-26 is additionally predicted to target prominent eye field transcription factors
RX1A, Six3, and Lhx2, as well as the pan neural transcription factor Sox3 (PITA, ddG <= -5). Insitu using RAX2A as an eye field marker showed that targeted injection of miR-26-Smad1
Target Protector Morpholino (TPMO) broadened the lateral anterior edge of the early eye field,
compared to embryos injected with the Target Protector MisMatch (TP MM). Ongoing studies
will characterize the effect of miR-26 on the early eye field, and assess how this regulatory
network may be modulated at specific nodes by microRNAs, specifically miR-26.
2658
A requirement for Islet1 in thymus development.
Y. Hu1, B. G. Condie1; 1University of Georgia, Athens, GA
Thymic epithelial cells (TECs) play essential roles in promoting many aspects of T cell
maturation within the thymus, including T cell differentiation, proliferation, and apoptosis.
Although many studies have been focused on studying the functions of TECs, our knowledge of
the transcriptional network that controls TEC development is still rather limited. To address this
gap in our knowledge of thymus biology, we performed a focused in situ hybridization screen of
a list of transcription factors that might be required for TEC development. This screen identified
the LIM-Homeodomain transcription factor Isl1 (Islet1) as a potential regulator of TEC
development. Isl1 expression initiates in the 3rd pharyngeal pouch at E9.5, well before the
expression of Foxn1, and continues to be expressed in TECs to postnatal stages. Our analysis
of mice in which Isl1 was inactivated only in TECs revealed a requirement for Isl1 for
development of the normal architecture of the TEC network. Also, we found that the expression
levels of several functional markers of TEC differentiation were changed in Isl1 conditional
knockout mice. Our analysis indicates that Isl1 is required for normal TEC differentiation and
thymus development.
2659
P114RhoGEF regulates lumen formation via ROCK-myosin II pathway during 3D
epithelial tubulogenesis.
M. Kim1, A. Shewan2, K. Mostov1; 1Anatomy, UCSF, san francisco, CA, 2The University of
Queensland, Brisbane, Australia
Tubulogenesis is fundamental to the development of the kidney and many other epithelial
organs. Although cell polarization is essential for lumen morphogenesis, little is known about the
molecular mechanism of epithelial tubulogenesis underlying lumenogenesis. Here we examine
the real-time behavior of cells during HGF-induced tubulogenesis. Time-lapse imaging reveals
that basal protrusions from MDCK cysts by HGF treatment transform into tubes enclosing a
central lumen, which is connected with the cyst lumen. Interestingly, loss of p114 RhoGEF does
not block basal protrusion but induces abnormal tubules with discontinuous and multiple
lumens. The inhibition of Rho or Rho kinase by pharmaceutical inhibitors after chain formation
also impairs tubular lumen formation. Furthermore, knockdown of myosin II phenocopies the
effect of ROCK inhibition. However, this is not resulted from the loss of apicobasal polarity or
defective mitotic spindle orientation. We propose that HGF induces MDCK tubulogenesis via the
SUNDAY-LATE POSTER PRESENTATIONS
combined mechanism of cell movement and rearrangement and that tubular lumen formation is
regulated by a p114 RhoGEF-ROCK-myosin II pathway.
2660
Comparative effects of high-fat diet exposure during different developmental periods on
adult rat prostate structure and cell proliferation.
E. Z. Pytlowanciv1, M. G. Gobbo1, R. M. Góes2; 1Biology Institute, State University of Campinas
(Unicamp), Campinas, Brazil, 2Department of Biology, Univ Estadual Paulista (Unesp), São
José do Rio Preto, Brazil
Obesity causes neuroendocrine and hormonal changes that may be associated with
predisposition to several diseases including reproductive problems. Experimental data show
that obesity promotes hypogonadism and rat ventral prostate growth. However, there are no
studies comparing the consequences of the obesogenic environment during different
developmental stages on prostate histophysiology. The present study compared the ventral
prostate structure and cell proliferation levels of adult Wistar rats treated with balanced chow
(Control group - C; 3% satured fat, 3.5Kcal/g) with those exposed to obesogenic environment
induced by high-fat diet (20% satured fat, 4.9kcal/g) from gestation to weaning (GW), from
gestation to adulthood (GA) and from post weaning to adulthood (A). Rats (19 week old) were
killed, blood samples were collected and the ventral prostate was removed, weighed and
processed for paraffin embedding. Immunohistochemistry for proliferating cells (PCNA) and
stereological analysis were performed in prostate sections. All periods of obesogenic
environment exposure increased the prostate weight, however this growth was more
pronounced in GW (2.7x) and GA (2.5x) in comparison with A (2x).
Microscopic and
stereological analysis indicated that obesity promotes marked stromal tissue enlargement, due
to increment in collagen fibers and blood vessels. Obesogenic environment also led to epithelial
alterations such as increase in epithelium height and nuclei density, resulting in foci of
stratification and epithelial dysplasia. In addition, epithelial cell proliferation levels raised 2.4x in
GW, 2x in A and 2.3x in GA. The increase in epithelial cell proliferation observed here for all
obesogenic groups is contradictory with testosterone decline (GW: 1.4x; A: 1.4x; GA: 2.9x), but
may be explained by increase in estrogen levels (GW: 1.8x; A: 1.6x; GA: 1.7x), which may have
acted as a cell proliferation stimulator. Interestingly, histological and proliferative alterations
were more pronounced in GW, even in comparison with longer diet exposure group (GA). In this
same group, the levels of estrogen were significantly higher, showing that estrogen may have
played a major role in these findings. In conclusion, the obesogenic environment induced by
high-fat diet interferes on ventral prostate histology and epithelial cell proliferation kinetics at
adulthood but the effects are more pronounced in gestational and lactation exposition.
2661
Development of a mESC Reporter Line to Identify Cardiac Conduction System Cells.
F. See1, J. Lu1, K. Maass1, S. D'Souza2, G. I. Fishman1; 1NYU School of Medicine, New York,
NY, 2Mount Sinai School of Medicine, New York, NY
Background: We have previously demonstrated that the cell adhesion protein contactin2
(Cntn2) is enriched in Purkinje cells of the cardiac conduction system (CCS). Objective:
Generation of a mouse embryonic stem cell (mESC) reporter line that allows identification of
Purkinje-like cardiomyocytes in vitro. Methods and Results: mESC were generated from
transgenic mice carrying a BAC Cntn2-eGFP reporter gene and were subsequently transduced
with lentivirus coding for a selectable MHCα-mCherry cardiomyocyte reporter gene.
Immunostaining analysis confirmed that mESC expressed markers of pluripotency (Oct3/4; Klf4)
and spontaneously differentiated into cells of all three germ layers in the absence of LIF (α-
SUNDAY-LATE POSTER PRESENTATIONS
smooth muscle actin; β-tubulin; α-fetoprotein). Spontaneous or serum-free directed cardiac
differentiation resulted in generation of double positive, spontaneously beating cardiomyocytes
after three weeks. Yield of double positive cells could be increased by adding endocardialderived factors (Nrg1; ET-1). FACS isolated double positive cells were enriched in transcripts of
cardiomyocytes (Tbx5; Nkx2.5) and the CCS (Cntn2; Cx40). Action potential recordings of
eGFP positive cardiomyocytes demonstrated distinct plateau phase and elongated action
potential duration (APD50=79.9±10.4ms, APD90=170.2±17.5ms; n=11) compared with eGFP
negative cardiomyocytes (APD50=53.4±9.4ms, APD90=120.6±17.3ms; n=15). Conclusion: We
have established a mESC reporter line for the identification of CCS-like cells. This model should
be useful for downstream studies of CCS development and pathology. Cntn2 may also be a
useful marker of CCS-like cells derived from human ES and/or iPS cells.
New Technologies and Frontiers
2662
High Resolution Optical DNA Mapping.
M. Baday1, A. Cravens2, A. Hastie3, H. Kim2, D. Kudeki4, M. Xiao3, P. Selvin1,2; 1Biophysics,
University of Illinois at Urbana-Champaign, Urbana, IL, 2Physics, University of Illinois at UrbanaChampaign, Urbana, IL, 3BioNano Genomics Inc, San Diego, CA, 4Computer Science,
University of Illinois at Urbana-Champaign, Urbana, IL
Many types of diseases including cancer and autism are associated with copy-number
variations in the genome. Most of these variations could not be identified with existing
sequencing and optical DNA mapping methods. We have developed Multi-color Superresolution technique, with potential for high throughput and low cost, which can allow us to
recognize more of these variations. Our technique has made 10–fold improvement in the
resolution of optical DNA mapping. Using a 180 kb BAC clone as a model system, we resolved
dense patterns from 108 fluorescent labels of two different colors representing two different
sequence-motifs. Overall, a detailed DNA map with 100 bp resolution was achieved, which has
the potential to reveal detailed information about genetic variance and to facilitate medical
diagnosis of genetic disease
2663
3D EM reveals the secrets of structural hierarchy of tissues and organelles.
I. Belevich1, H. Vihinen1, E. Jokitalo1; 1Institute of Biotechnology, University of Helsinki, Helsinki,
Finland
Detailed morphological analysis of cellular organelles, cells, and tissues is almost impossible
without understanding of their 3D organization. In order to facilitate the 3D imaging a number of
methods in electron microscopy (EM) have been developed during the recent years. The best
methods that provide 3D information at the cellular level are electron tomography (ET) and
serial block face (SBF) or focused ion beam (FIB) scanning electron microscopy (SEM). ET is
best suited for obtaining 3D volumes with extremely high resolution, whereas the two later
methods extend the 3D imaging to much larger volumes while preserving the relatively high
resolution factor. The SEM techniques are based on automated sequential imaging of block
faces of resin-embedded specimens using a back scatter detector yielding images resembling
transmission EM images of thin sections. A fresh block face is sequentially created by a
microtome inserted in the imaging chamber (SBFSEM) or by focused ion beam (FIBSEM).
SUNDAY-LATE POSTER PRESENTATIONS
We apply a combination of ET and SBFSEM for imaging of biological specimens ranging in size
from tens of nanometers to hundreds of micrometers. In this poster, we present some of our
results and share our experience in using SBFSEM alone and in correlation with ET for imaging
and analysis of cellular organelles such as smooth and rough endoplasmic reticulum (ER), lipid
droplet/ER contacts, and tissues (islets of Langerhans and inner ear cochlea of transgenic mice,
and differentiation of phloem sieve elements in the roots of Arabidopsis thaliana).
The results indicate that ET is effective in resolving small cellular features and structures like
membrane connections, intermediate filaments and ribosomes, while SBFSEM gives a
possibility to understand and model organization of larger organelles such as mitochondria, lipid
droplets or ER within whole cells. The large volumes obtained with SBFSEM can be used as a
screening tool to find a phenotype in larger number of specimens and to increase statistical
strength of the results. To complement SBFSEM, the discovered target phonotype can be
further characterized by ET, which provides higher resolution and allows the use high pressure
freezing and freeze substitution methods for sample preparation.
In summary, the combination of ET and SBFSEM seems to be an extremely reliable method
that allows from one side imaging of large volumes and statistical analysis, and from another
high resolution imaging of specific features prepared with the best preservation methods.
2664
Magnetically Enhanced Gene Delivery Using Polyethyleneimine-Conjugated Magnetic
Nanoparticles.
L. Zhang1, J. C. Yu1; 1Chemistry, CUHK, Hong Kong, Hong Kong
Gene transfection is a non-viral therapy on gene-based diseases by delivering nucleic acids into
the nucleus of target cells1. Magnetofection can often enhance gene transfection efficiency
when the system involves appropriate magnetic nanomaterials (MNPs) under a magnetic field2.
To combine nucleic acids with nanoparticles and to protect them from degradation after
endocytosis, MNPs are usually coated with cationic compounds, such as 25 kDa branched
polyethylenimine (PEI) 3,4. Luciferase assay and flow cytometry experiments were carried out. It
was found that the PEI-conjugated MNP was a more stable and efficient gene transfection
vector than the PEI-coated MNP. The optimal N/P ratio was 20.
The triplex (plasmid DNA, PEI-conjugated MNP, and free PEI) has been considered a reliable
system for tracking the intracellular fate of each component. We labeled the magnetic particles
with different fluorescent dyes and found that the magnetic nanoparticles accumulated near the
cellular membrane, while the free PEI/DNA existed around the nuclear region before entering
the nucleus. We also used a flow cytometer to determine the uptake of each component. When
a magnetic field was applied, more magnetic nanoparticles could enter the HepG2 cells. The
best conditions were appropriate amount of free PEI (N/P =20) bind to nanoparticles before
entering cells, and delivering plasmids into the nucleus after the endocytosis process.
SUNDAY-LATE POSTER PRESENTATIONS
2665
Improved dynamic range of genetically encoded FRET sensors with new, bright green
and red fluorescent proteins.
A. Lam1, F. St-Pierre1, Y. Gong2, J. D. Marshall2, M. R. McKeown3, J. Wiedenmann4, M.
Schnitzer2, R. Y. Tsien3, M. Z. Lin1; 1Bioengineering and Pediatrics, Stanford University,
Stanford, CA, 2James H. Clark Center, Stanford University, Stanford, CA, 3Pharmacology and
Chemistry, University of California, San Diego, La Jolla, CA, 4National Oceanography Centre,
University of Southampton, Southampton, United Kingdom
FRET between fluorescent proteins allows for real-time study of the spatiotemporal dynamics of
biochemical processes in genetically defined cell populations. This technique is appropriate as
the length scale of FRET is well matched to protein conformational changes and protein-protein
interactions. Although most FRET-based reporters rely on CFPs and YFPs, aspects of CFPs
and YFPs are problematic for FRET. CFPs and YFPs can undergo rapid multirate and
reversible photobleaching, YFPs can photoconvert into cyan fluorescent species, CFPs can
photoactivate at YFP excitation wavelengths and the violet CFP excitation light can be
phototoxic. Furthermore, many CFP- and YFP-based FRET reporters produce small changes in
FRET, creating detection challenges when the imaged structures are small or when biochemical
responses are subtle or transient. In this work, we have developed new fluorescent proteins with
properties better suited to a wide range of FRET applications.
Starting from Aequorea victoria GFP and the RFP mRuby, we engineered a green fluorescent
protein Clover and a red fluorescent protein mRuby2, with spectral properties better matched for
FRET. Clover has an extinction coefficient (ε) of 111,000 M-1 cm-1 and quantum yield (φ) of
0.76, making it the brightest fluorescent protein yet characterized. mRuby2 has better
brightness compared to mRuby (ε = 113,000 vs. 112,000 M-1 cm-1, φ = 0.38 vs. 0.35) and
shows negligible reversible photobleaching.
Clover and mRuby2, as a FRET pair, conferred greater dynamic range and photostability to four
existing FRET reporter designs. Among the improved reporters is a voltage sensor that allows
more reliable detection of single action potentials than do previous sensors and an improved
RhoA reporter able to detect local and rapid RhoA activation in neuronal growth cones during
ephrinA- stimulated retraction.
2666
oCellaris, a new software for image analysis of fluorescently labeled yeast cells to
evaluate localized fluorescence (granularity) and intracellular pH
Z. Kosour1, P. Jagos1, J. Hasek2, I. Malcova2, H. Sychrova3, T. Grousl2, J. Zahradka3, O.
Zimmermannova3, I. Malec1, L. Malek1; 1Del, a.s., Zdar nad Sazavou, Czech Republic, 2Inst.
Microbiollogy ASCR,v.v.i., Prague, Czech Republic, 3Inst. Physiology ASCR, v.v.i., Prague,
Czech Republic
The new software oCellaris was developed for analyses of fluorescence images of living yeast
cells. It is based on Hough transformation to identify yeast cells in the bright field and to
compute cell’s contours with an active contour algorithm. A set of algorithms enable fitting
fluorescence images to cell contours and computing various parameters of fluorescence for
each cell. These parameters describing fluorescence images are suitable not only for evaluation
of total fluorescence intensity within the cell area but also for comparison of various
experimental conditions applied to one particular strain or for comparison of various strains in
one parameter either at the single cell level and/or within the population.
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We used this software to distinguish between two types of granular fluorescence – P-bodies and
stress granules (SGs) represented by Dcp2 (marker of P-bodies) and Rpg1/Tif32/eIF3a (marker
of SGs). From plots either in form of histograms or distribution graphs showing behavior of
individual cells, the difference in structure of localized fluorescence/patches/granules is clearly
visible. P-bodies labeled by Dcp2-GFP represent a very distinct form of patches that display
very high intensity peaks with an important slope in contrast to stress granules marked by Rpg1GFP fluorescence and described by a gradual slope. oCellaris also enables to estimate the
internal pH and its changes directly in living yeast cells with use of pHluorin (pH-sensitive
variant of green fluorescent protein) The newly developed technique was validated by
estimation of intracellular pH in pairs of strains differing in their intracellular pH in comparison
with the classical approach using spectrofluorometer. Use of microfluidic flow chambers
(CellASIC) appeared to be advantageous for a precise assessment of pH in individual living
yeast cells and a subsequent analysis of the whole cell population.
The development of this software was supported by the TA 01011467 grant from the
Technology Agency CR and more information will be available at www.ocellaris.cz.
2667
Near-infrared fluorescent proteins for imaging of mammalian cells and whole animals.
D. M. Shcherbakova1, V. V. Verkhusha1; 1Department of Anatomy and Structural Biology, and
Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY
Spectrally resolvable fluorescent proteins (FPs) with excitation/emission in a near-infrared range
of spectrum are required for multicolor whole-body imaging. In a “near-infrared window” (from
650 nm to 900 nm) mammalian tissue has reduced autofluorescence, low absorbance and low
light scattering. Recently, several FPs based on bacterial phytochromes have been developed
for this spectral region. So far only iRFP (excitation/emission at 690 nm/713 nm) has been used
for imaging in mammals without a supply of exogenous biliverdin, which serves as a
chromophore. We developed four bacterial phytochrome based FPs such as iRFP670,
iRFP682, iRFP702 and iRFP720. Together with iRFP, they span the range of 645-702 nm in
excitation and 670-720 nm in emission wavelengths. Similarly to iRFP they utilize endogenous
biliverdin, which is present in mammalian tissues. Their effective brightness in transiently
transfected HeLa cells and stably expressing adenocarcinoma MTLn3 cells is similar to iRFP.
Comparing to conventional far-red GFP-like FPs, iRFP670 (excitation/emission at 645 nm/670
nm) produces several fold larger fluorescent signal in the HeLa cells upon excitation with
common 635-640 nm red lasers. Cytotoxicity of FPs was tested as a change of brightness of the
stably expressing MTLn3 cells during prolonged culturing. All developed FPs exhibit a low
cytotoxicity comparable to iRFP and conventional EGFP. Two the most spectrally distant FPs,
iRFP670 and iRFP720 (excitation/emission at 702 nm/720 nm), were successfully used for twocolor deep-tissue imaging. Up to four FPs could be resolved using linear spectral unmixing
algorithms of commercial imaging systems. The developed FPs were tested for imaging in living
mammals. MTLn3 cells stably expressing FPs formed tumors in mammary glands of
immunocompromised mice. Tumors growth was monitored with fluorescence imaging, starting
from early stages. The designed near-infrared FPs will extend multicolor labeling in microscopy
and enable two-color deep-tissue imaging in mammals.
SUNDAY-LATE POSTER PRESENTATIONS
2668
Photoactivatable near-infrared fluorescent protein designed from bacterial phytochrome
for in vivo imaging.
K. D. Piatkevich1, F. V. Subach2, V. V. Verkhusha1; 1Anatomy and Strucutural Biology, Albert
Einstein College of Medicine, Bronx, NY, 2National Research Centre "Kurchatov Institute",
Moscow, Russia
Fluorescence imaging is a powerful and widely used technique for biological research.
However, lack of genetically-encoded probes for in vivo imaging is the major limitation in this
field. Mammalian tissues are relatively transparent in a near-infrared window of 650-900 nm due
to significant decrease in hemoglobin and melanin absorbance and still low water absorbance.
Therefore, probes with fluorescence spectra within the near-infrared range are preferable for
imaging in mammalian tissues. On the basis of a bacterial phytochrome we have developed a
photoactivatable near-infrared fluorescent protein, named PAiRFP. PAiRFP utilizes a heme
derived biliverdin, which is ubiquitous in mammalian tissues, as a chromophore. In an initial
state PAiRFP is weakly fluorescent, but undergoes 16-fold increase in fluorescence brightness
after a brief irradiation with red light. After photoactivation PAiRFP slowly reverts back into the
initial state with a half-time of 55 min. In the photoactivated state PAiRFP exhibits fluorescence
excitation/emission maxima at 690 nm/717 nm, extinction coefficient of 67,000 M-1cm-1, pKa
value of 5.2, and fluorescence quantum yield of 4.8%. PAiRFP can be readily expressed in
mammalian cells without supply of exogenous biliverdin. Effective brightness of PAiRFP in HeLa
cells is 31% of that of iRFP, which currently is the brightest bacterial phytochrome based nearinfrared protein. To test the performance of PAiRFP in vivo we have utilized a mouse xenograft
model of breast cancer. A tumor expressing PAiRFP was photoactivated noninvasively via skin
of a living mouse using a low 26 mW/cm2 intensity of red light. The 5.5-fold photoactivation of
the whole tumor has been achieved within a second. Focused red light of the similar intensity
enabled a spatially selective photoactivation of the PAiRFP expressing tissue with a millimeter
resolution. In vivo applications of PAiRFP could include short-term tracking of cells and
monitoring of rapid inflammations inside tissues or organisms.
2669
Improved array tomography imaging for conjugate proteometric and ultrastructural
analysis of central nervous system synapses.
J. Buchanan1, F. C. Collman1, K. Phend2, K. D. Micheva1, R. J. Weinberg2, S. J. Smith1;
1
Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, 2Dept
of Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC
Array tomography (AT) is a volumetric imaging method based on acquiring images of planar
arrays of serial ultrathin sections followed by computational volume reconstruction and analysis.
Arrays can be imaged either by high-resolution, high-dimensional immunofluorescence (IF) or
by field emission scanning electron microscopy (FESEM). AT by IF provides information on the
molecular composition of neuronal structures, including proteometric analysis of millions of
synapses. AT by FESEM provides detailed ultrastructure of membranes and tracing of neural
wiring and synaptic contacts.
Until now, conjugate imaging of individual specimens by both IF and FESEM has been limited
by conflicting requirements for preservation of tissue antigenicity and tissue ultrastructure. Here
we report that aldehyde-fixed freeze-substituted mouse brain samples embedded in Lowicryl
allow high quality AT using both IF and FESEM on the same serial sections. IF imaging using
up to 18 different antibodies to identify the molecular profile of synaptic structure was followed
by FESEM imaging to examine the ultrastructure of the same neuronal structures, visualizing
synaptic vesicles, pre- and post-synaptic densities, and the synaptic cleft.
Neuronal
SUNDAY-LATE POSTER PRESENTATIONS
membranes and cytoskeletal elements including microtubules appear well defined, thus allowing
tracing of the neuronal connections.
FESEM backscatter imaging operating at 5-7 kV accelerating voltage provided good contrast of
adequately post-stained 70 nm sections. However, beam damage to the resin sections affected
the quality of captured images and adversely affected sections for further imaging. Secondary
electron imaging using the in lens mode of the Zeiss Supra FESEM was performed at 2kV with
a 30 micron aperture. This mode proved superior to backscatter imaging, due to reduced beam
damage of the thin sections and improved resolution. Correlated images of IF and FESEM
images were easily registered and ultrastructural details were easily resolvable.
Information obtained by this conjugate proteometric and ultrastructural analysis is critically
important for providing molecular context to neural circuit mapping, allowing one to identify the
neurotransmitters and receptors present at each synapse. It is also of general biological interest
as a technique to measure the molecular composition of structures that are best visualized in
the electron microscope.
2670
Substrates for Improved Live-Cell Fluorescence Labeling of SNAP-tag.
I. R. Correa Jr. 1, B. Baker1, A. Zhang1, L. Sun1, C. Provost1, G. Lukinavicius2, L. Reymond2, K.
Johnsson2, M-Q. Xu1; 1New England Biolabs, Ipswich, MA, 2Ecole Polytechnique Federale de
Lausanne (EPFL), Lausanne, Switzerland
The development and application of tagging tools that give scientists access to the dynamic
functioning and localization of proteins in living cells is an active, interdisciplinary area of
research. Among the most prominent site-specific self-labeling tags is the SNAP-tag, an
engineered variant of the human repair protein O6-alkylguanine-DNA alkyltransferase (hAGT)
that covalently reacts with benzylguanine (BG) or chloropyrimidine (CP) derivatives bearing a
chemical or optical probe. The SNAP-tag labeling approach integrates the convenience and
specificity of genetically encoded systems with the versatility of small molecule probes. Recent
applications include its use for the analysis of protein-protein interactions, single molecule and
super-resolution microscopy, drug target identification, and the determination of protein half-life
in animals. In the course of our studies to improve the properties of SNAP-tag fluorophore
conjugates for cell imaging applications, we discovered that SNAP-tag has a strong preference
for the 6-positional regioisomer of carboxyrhodamine dyes (carboxyrhodamines are typically
available as mixtures of 5- and 6-regioisomers in respect to the position of their carboxyl groups
in the phenyl ring). We will present and discuss our initial results for the labeling of SNAP-tag
fusion proteins both in vitro and in cell culture with regioisomerically pure carboxyrhodamine
dyes. While it is not completely clear why these structural differences affect so dramatically the
reaction rate, it is evident from our labeling experiments in distinct organelles and cell lines that
SNAP-tag is predominantly labeled with the 6-isomer, whereas the 5-isomer mainly contributes
to background fluorescence.
2671
Imaging proteins, cells, and tissues dynamics during embryogenesis with two-photon
light sheet microscopy.
T. V. Truong1,2, V. Trivedi1,2, D. B. Holland1, S. E. Fraser1,2; 1California Institute of Technology,
Pasadena, CA, 2University of Southern California, Los Angeles, CA
Two-photon light sheet microscopy combines nonlinear excitation with the novel sheetillumination, orthogonal to the detection direction, to achieve high penetration depth, high
acquisition speed, and low photodamage, compared with conventional imaging techniques.
These advantages allow unprecedented observation of the processes that govern
SUNDAY-LATE POSTER PRESENTATIONS
embryogenesis, where the ability to image fast the dynamic three dimensional structure of the
developing embryo, over extended periods of time, is critical. We present a comparison of twophoton light sheet microscopy with other conventional techniques in live imaging of embryos,
demonstrating its outstanding performance. We also present a selection of applications where
two-photon light sheet microscopy is utilized to observe the dynamics of proteins, cells, and
tissues during embryogenesis, toward an understanding of the construction program of the
developing embryo.
2672
Automated wound delineation in Drosophila embryos for high-throughput screening.
T. Zulueta-Coarasa1, R. Fernandez-Gonzalez1,2; 1Institute of Biomaterials and Biomedical
Engineering and Department of Cell and Systems Biology, University of Toronto, Toronto, ON,
Canada, 2Developmental and Stem Cell Biology Program, The Hospital for Sick Children,
Toronto, ON, Canada
Epidermal wound repair in embryos is a conserved process mediated by the assembly and
contraction of an actin-myosin purse string in the cells adjacent to the wound. To determine the
mechanisms of purse string assembly, we are screening the localization and dynamics of GFPfused proteins around epidermal wounds in Drosophila embryos. High-throughput screening
requires automated identification of proteins that localize at the purse string from time-lapse
imaging data. The first step in this process is wound delineation.
We propose to use snakes, an active contour model, to automatically delineate the wound
margin in time-lapse images. A snake is a curve that evolves in an image to achieve energy
minimization. The energy functional is a combination of internal forces that minimize the
perimeter and roughness of the contour, and external forces that attract the contour towards
bright structures indicated by high image gradients. We initialize the snake in an intermediate
time point of the sequence using the LiveWire algorithm, a semi-automated segmentation
method based on Dijkstra’s minimal path search algorithm. This initial curve is allowed to evolve
under the forces described above. The resulting contour is automatically propagated to the
adjacent time points by calculating the average optic flow or local signal displacement, and the
propagated contours are used to initialize the snakes in those time points.
In preliminary experiments, we compared the time necessary to delineate wounds in a wound
healing movie in which time points were acquired every 15 seconds for a total of 150 time
points. Manual annotation of the wound in all time points required 150 minutes. Using the
LiveWire algorithm alone, took 114 minutes. Initializing snakes every 5 time points using the
Live Wire allowed us to segment the entire movie in just 61 minutes. Taking the manual
segmentation as a reference, we measured a percentage of variation of wound area of 1.6%
using the Live Wire and 4.3% using snakes, and a variation of wound perimeter of 14% and
0.7%, respectively. Finally, we measured the variation of the fluorescence under the wound
perimeter, a key parameter to detect molecules enriched at the wound margin, and we found it
to be 0.47% for LiveWire-only segmentation and 0.27% for snakes. These data demonstrate
that our method is able to rapidly delineate wounds with minimal user interaction, without
compromising the quality of the results. We are currently extending these tools delineate
wounds in three dimensions, and applying them to the automated screening of molecular factors
implicated in purse string assembly around embryonic wounds.
SUNDAY-LATE POSTER PRESENTATIONS
2673
FRET/FLIM Method for NADH binding proteins using the Phasor Analysis.
J-H. Chiang1, S. Jahid1, M. A. Digman1, E. Gratton1; 1Biomedical Engineering, University of
California, Irvine, Irvine, CA
The ratio of the free/bound NADH is used to measure the metabolic state of live cells; however,
it does not provide specific information about which protein is involved with NADH binding. In
vitro, FRET between proteins with tryptophan residues (excitation wavelength: 280-300nm) and
the NADH co-factor (emission wavelength: 400-450nm) has been exploited to determine the
affinity of NADH to specific proteins. In vivo, this approach is difficult to implement because UV
excitation damages live cell nucleus. If a specific protein that interacts with NADH has a
fluorescent moiety that can act as an acceptor, this will result in the quenching of the NADH
fluorescence. Due to large amounts of NADH in live cells, this result will be very difficult to
associate with the specific protein. Therefore, with a fluorescent tag on the target protein, the
tagged protein and NADH will show a sensitized emission of the acceptor molecule, which is
easy to detect via fluorescence lifetime imaging microscopy (FLIM). Here, we develop an in vivo
FLIM/FRET assay in which we use FRET between NADH (using 2-photon excitation at 740nm)
and the green fluorescent tag protein attached to an NADH binding protein. In our assay, we
use Histone 2B-EGFP construct localized in the nucleus and it has been shown that this protein
tag does not modify histone activity. This FRET/FLIM assay can be used to determine
transcription factor binding partners of NADH if the transcription factor is labeled with a
fluorescent protein. The phasor lifetime map of histone bound NADH is generated to show
areas of increased chromatin activity.
2674
Visualization of inter-subunit ribosome FRET and β-actin mRNA in living cells.
Y. J. Yoon1, H. Park1, E. M. Schuman2, R. H. Singer1; 1Anatomy and Structural Biology, Albert
Einstein College of Medicine, Bronx, NY, 2Max Planck Institute for Brain Research, Frankfurt,
Germany
We have developed a FRET-based assay that assesses the association of the small and large
subunits of the ribosome to visualize the intracellular landscape of protein synthesis together
with mRNA in living cells. We provide evidence that our ribosomal protein FRET pair is able to
incorporate into functional ribosomes and mirror the behavior of endogenous counterparts. In
addition, we observed inter-subunit FRET between our labeled ribosome subunits and we show
that it is possible to visually correlate FRET with the translation state of β-actin mRNA in living
cells. Our results demonstrate that our FRET assay can be used to dynamically visualize
assembled ribosomes in living cells, and when imaged in conjunction with endogenous β-actin
mRNA can reveal the translation state of an mRNA with spatial and temporal resolution.
2675
A Method for the Delivery of Small Molecules and Proteins Into Algal Cells using
Molecular Transporters.
J. M. Hyman1, E. Geihe Stanzl2, B. Trantow2, P. Wender2, B. Parvin1; 1Life Sciences Division,
Lawrence Berkeley National Laboratory, Berkeley, CA, 2Chemistry, Stanford University,
Stanford, CA
Algae present the potential for producing industrially useful and biochemically interesting
secondary metabolites, biofuel precursors, synthetic building blocks, recombinant proteins,
vaccines and food additives using CO2 or simple carbon sources, and photosynthesis for
energy. Because of the broad range of habits algae are able to occupy, the relative efficiency of
SUNDAY-LATE POSTER PRESENTATIONS
photosynthesis in algal species, and their tolerance in some cases for high levels of CO 2,
metals, and other potential waste products, algae can be used in niches that do not directly
compete with agricultural land or for fresh water. However, in order to fully realize the
possibilities for using algae species in constructive ways, simple, scalable and robust methods
for delivery of chemical, radiological and biological agents (from small molecules to large pieces
of genetic material) would be greatly beneficial. We have examined the potential for using
guanidinium-rich molecular transporters (also known as cell-penetrating peptides) for the
delivery of cargoes into Chlamydomonas Reinhardtii and several other algal species. Here we
present a general method for bringing a variety of molecular cargoes into algal cells.
2676
Validation of Bird Flu and Human Flu Viral Haemagglutinin (HA) Multimers as Probes for
the Detection of 2,3- and 2,6-Sialic Acid Bond Structures.
K. Partyka1, M. McDonald1, D. D. Nowack2, B. B. Haab1; 1Cancer Immunodiagnostics, Van
Andel Institute, Grand Rapids, MI, 2Chemistry and Biochemistry, Andrews University, Berrien
Springs, MI
The carbohydrate (CHO) moieties attached to glycoproteins are proving to be important loci for
detection and regulation of cellular activity1. Changes to the CHO moieties are linked to the
presence of pre-cancerous and cancerous changes in the cells and tissues2. In particular,
changes in sialic acid bonds between 2,3 and 2,6 linkages may be important indicators of
disease3, but unfortunately an effective way of precisely measuring these linkages in biological
samples does not yet exist. The various HA proteins (bird flu, swine flu, human flu) bind to 2,3and 2,6-sialic acid bonds as a preliminary step for infection within the host4. We hypothesized
that HA proteins could be used to detect specific presentations of sialic acid moieties on
glycoproteins captured from biological samples. In this study we used a simple method of
multimerizing the HA protein to increase its binding avidity5, and applied the HA proteins to the
detection of sialic acids on biological glycoproteins. Using that specificity to detect changes in
glycoprotein CHO structures, we discovered that the HA probes can differentiate between 2,3and 2,6- sialic acid bond types in an efficient and reproducible manner. Studies also are
underway to determine the relationship between sialic acid linkages and the presence of
pancreatic cancer. This approach may provide robust and sensitive measurements of specific
sialic acid presentations in biological samples.
1
“Phosphofructokinase 1 Glycosylation Regulates Cell Growth and Metabolism,” Yi, W., et.al.,
Science, Vol. 337, p. 975, Aug., 2012.
2,3
“α-2,3-Sialyltransferase ST3Gal III Modulates Pancreatic Cancer Cell Motility and Adhesion
In Vitro and Enhances Its Metastatic Potential In Vivo,” Perez-Garay, M., et.al., PLoS ONE, Vol.
5, p. e12524.
4
“Influenza Hemagglutinin and Neuraminidase Membrane Glycoproteins,” Gamblin, S.J.,
Skehel, J.J., J. of Biological Chemistry, Vol. 285, No. 37, p. 28403, Sept., 2010.
5
“Glycan Micro Analysis of the Hemagglutinins from Modern and Pandemic Influenza Viruses
Reveals Different Receptor Specificities," Stevens, J., et.al. J. Mol. Biol. Vol 355, p. 1143, 2006.
SUNDAY-LATE POSTER PRESENTATIONS
2677
Elucidate the molecular bases of neurogenesis by isolation and characterization of
protein assemblies at neurogenic genomic loci.
A. Das1, R. Bajpai1; 1CCMB, University of Southern Caifornia, los angeles, CA
Whereas neural fate specification is considered a default mechanism in vertebrates, the
molecular bases of this fundamental process remains poorly understood. Identifying
macromolecules mediating neural specification will reveal long sought basic concepts in
developmental biology and help gain a better control over directed differentiation of pluripotent
cells into neural fates for their use in regenerative therapies. Previous studies have shown that
lineage outcomes of pluripotent cells are regulated via genomic enhancer regions that integrate
signaling with transcriptional circuitry and chromatin remodeling and cause well-orchestrated
gene expression profiles. Using a group of genomic enhancers that we find undergo rapid
transitions from ‘poised-to-active’ chromatin state during neural differentiation, we plan to
undertake a specific enrichment of DNA and protein complexes from these cells by utilizing a
novel technique, validate the newly identified candidates, and characterize function to elucidate
the molecular bases of neural specification.
2678
Novel assay system for isolation and quantification of rare cells using fluorescent
magnetic nanoparticles.
Q. Han1, B. Lin1, A. Fu1; 1Nvigen Inc, Sunnyvale, CA
Cell isolation and identification are widely needed in biomedical research and clinical
diagnostics. Due to the low frequency of some rare cell population, cell separation and
quantification could be very challenging under certain circumstances, for example, the isolation
of circulating tumor cells (CTCs) from whole blood samples. While most of cell-sorting
experiments utilize flow cytometry, it is limited by the sensitivity and higher cost of facilities.
Magnetic separation provides a quick and cost-effective way to isolate cell subpopulation. In
addition, fluorescence could be incorporated within the magnetic nanoparticles allowing
multiplexed fluorescent identification. In this work, we innovated the fluorescent magnetic
nanoparticle technology and demonstrated the application of these nanoparticles for rare cell
isolation, identification, and quantification.
These fluorescent magnetic nanoparticles are biocompatible, with high magnetic moment,
small particle size (~200 nm), and bright stable fluorescence. The surface of these
nanoparticles could be functionalized with streptavidin, which can further bind to biotinconjugated antibodies to magnetically select target cells from mixed cell populations. These
nanoparticles showed very high binding specificity of antibodies and cells. Furthermore, we
developed a flow system to spread magnetically captured cells onto a 2D surface that allows
fast isolation, identification, and quantification of target cells. Our fluorescent magnetic
nanoparticles expand the potential of magnetic cell separation, and provide a better way to
perform cell-based assays in a high-throughput and multiplexed format.
SUNDAY-LATE POSTER PRESENTATIONS
2679
Fully Automated Cell Culture Systems Increase the Efficiency and Consistency of
Downstream Viral Plaque Assays.
J. Michelotti1, N. Deiuliis1, K. Lamberton1, S. Mazur1, S. Agar1, D. Freeburger1, J. Kuhn1;
1
Contractor, IRF-Frederick, NIAID, Frederick, MD
The Cell Culture Section at the NIAID Integrated Research Facility at Fort Detrick (IRFFrederick) provides cell biology expertise, mammalian cell maintenance, and assay-ready cells
to investigators involved in translational virology research. To consistently produce large
quantities of plates containing mammalian cells for such use, we incorporated a cell culture
robot (CompacT SelecT™) into our laboratory workflow. This robot presently maintains up to 90
flasks of four different African Green Monkey kidney (AGM) cell lines (MA-104 clone 1, MARC145, CL2621, BS-C-1) in a controlled environment. The robot automatically passages the
adherent cells and dispenses a specific number of cells into multi-well plates as required for
downstream experiments (e.g., TCID50 and plaque assays). In a study evaluating the
comparative growth of two arteriviruses, these AGM cell lines were provided weekly. The four
AGM lines were inoculated with arteriviruses, and cell supernatants were harvested 6-144 hours
later and quantified by plaque assay in either MA-104 clone 1 or MARC-145 cells using 6-well
plates. Robotic plating technique was compared to standard manual plating technique using the
MA-104 clone 1 cell line. Analysis of the time course of arterivirus 1 replication was similar in
cells plated by the robot and those plated by the standard manual cell culture technique. In
addition, the cell monolayers and plaques appeared identical using either of the two plating
techniques. Thus, the robotic plating technique is capable of automatically generating high
quality plates. The consistency of robot performance over 3 months was evaluated using the
MARC-145 cell line. The low variation of virus titer of arterivirus 2 using the robotic plating
technique indicates that cell density and plaque morphology were consistent over the course of
the study.
2680
A novel rapid apoptosis assay based on thiol redox status.
M. E. Skindersø1, L. D. Johansen1, S. Kjærulff1; 1ChemoMetec A/S, Allerod, Denmark
We have developed two novel probes for determining the level and distribution of free thiols,
such as reduced glutathione (GSH), in a cell population. GSH provides a large proportion of the
reducing power available in the cell, thus its oxidation status largely determines the thioldisulfide status of the cell by interchange reactions. The concentration of GSH, has been found
to decrease upon induction of apoptosis due to extrusion of GSH, also when using nonoxidative apoptogenic agents. We have investigated the use of two novel thiol reactive agents
as well as a well established GSH probe; monochlorobimane to explore the changes in the level
of free thiols in response to apoptosis induction. Jurkat cells were induced to undergo apoptosis
using camptothecin, and apoptotic traits such as phosphatidylserine externalisation, caspase
activity and mitochondrial potential were investigated at different time points after induction.
Along with detection of the classical apoptotic features we also used the three thiol probes to
determine changes in the thiol level. Upon addition to the cells the probes permeate the cell
membrane and react with intracellular thiols, causing the cell to fluoresce. Quantification of the
cell fluorescence after staining (without washing) can then be used to determine the
population's cellular thiol level at the single cell level. We found that though all three thiol probes
could be used to detect changes in the level of free thiols correlating well with apoptotic
markers, other properties such as detection of early versus late apoptosis and staining kinetics
differed among the three probes. However, we suggest adding examination of the level of free
SUNDAY-LATE POSTER PRESENTATIONS
thiols to the list of phenotypes which may be measured in order to detect apoptosis, as this is a
reliable and easy way of assaying apoptosis.
2681
Engineering of a protein tag for small-molecule assisted shut-off of specific protein
production.
H. K. Chung1, Y. Huo2, C. Jacobs1, J. Yang3, R. K. Plemper4, R. Y. Tsien3,5, M. Z. Lin2,6;
1
Department of Biology, Stanford University, Stanford, CA, 2Department of Bioengineering,
Stanford University, Stanford, CA, 3Department of Pharmacology, University of California, San
Diego, La Jolla, CA, 4Department of Pediatrics, Emory University, Atlanta, GA, 5Department of
Chemistry and Biochemistry and Howard Hughes Medical Institute, University of California, San
Diego, La Jolla, CA, 6Department of Pediatrics, Stanford University, Stanford, CA
A method to quickly shut off the production of a specific protein at a post-transcriptional step
would be desirable for understanding protein function, measuring protein turnover, or controlling
therapeutic protein production. Here, we develop a novel technology to shut off specific protein
expression with a small-molecule drug. This system, termed Small-Molecule Assisted protein
Shut-off (SMASh), uses an engineered site-specific HCV NS3 protease and its small-molecule
inhibitors to control retention of a degradation signal on a protein of interest. We fused target
control protein to a protease cleavage site followed by protease1 and a strong degron. In the
absence of drug, the protease cleaves its substrate and separates the degron from the protein
of interest immediately after translation and protein folding. Importantly, this protein therefore is
essentially unmodified except for a few amino acids and maintains its function. In the presence
of protease inhibitor, however, the degron cannot be removed from the target proteins thereby
inducing rapid degradation. To demonstrate the capabilities of this system, we engineered the
oncolytic measles vaccine virus2 that expresses the viral replication protein P under the control
of the SMASh tag (MV-PSMASh). We observed tight drug-induced inhibition of MV-PSMASh
replication by virus infectivity test in the standard host Vero cell line and in various cancer cell
lines. As a general strategy, SMASh should allow rapid drug control of specific protein
production with minimal perturbation of protein structure and function.
1. Lin, M. Z., Glenn, J. S. & Tsien, R. Y. A drug-controllable tag for visualizing newly
synthesized proteins in cells and whole animals. PNAS (2008)
2. Russell, S. J., Peng, K. W. Measles virus for cancer therapy. Current topics in microbiology
and immunology (2009)
2682
Application of high throughput screening in the development of a CHO production
medium.
M. Wight1, E. Garner1, A. Elwood1, J. Manwaring1; 1Thermo Fisher Scientific, Logan, UT
The Chinese hamster ovary (CHO) cell line is used extensively in the manufacture of
recombinant human therapeutics. The nutritional requirements of this line vary specific to the
individual recombinant clones isolated for this purpose. This property often necessitates the
development of a clone-specific production media in order to achieve optimal productivity in a
bioprocess. Traditionally, such media development efforts have primarily incorporated shake
flask cultures, which limit the number of conditions, the number of replicates and the ability to
incorporate advanced design of experiments (DoE) statistical methods. In order to overcome
these limitations, we investigated the use of various high throughput screening (HTS) equipment
in the development and testing of a medium designed for multiple CHO clones. HTS allowed for
the inclusion of more individual cell lines, increased component factor-levels and mixture design
treatments, and all with a higher number of replicates yet less labor-intensive methods. Nearly
SUNDAY-LATE POSTER PRESENTATIONS
all developmental work was performed at the 96-deepwell scale, with total culture volume at just
over 1mL. Subsequent scale-up and comparison of promising treatments, at shake flask scale,
revealed exceptionally similar trends in both cell density and product quantity, validating the
plate level design approach. Experimental data comparing HTS to shake flask results show that
25 of 26 conditions demonstrated agreement between the two methods. Optimized media
developed with this approach demonstrate consistently higher protein yield across multiple
clones. Comparisons of results at bothcales are presented, along with a (DoE) approach to
mixture design.
2683
Comparison of Mesenchymal Stem Cells Derived from Bone Marrow, Adipo tissue and
Umbilical Cord Blood as cell therapy source.
M. Kim1, H. Jin1, Y. Bae 1, S. Kwon 1, S. Choi 1, W. Oh 1, J. Chang 1; 1Medipost Co.,Ltd, Seoul,
Korea
Human mesenchymal stem cells (hMSCs) have multi-lineage differentiation potential and selfrenewal, thus highlighting the feasibility of using MSCs as a valuable source of stem-cells for
tissue engineering. Although bone marrow (BM) is a main source of MSCs, the limitations of
using BM-MSCs for clinical application, including invasive procedure, low rate of cell
proliferation, and the stringent requirements for donor age, have forced researchers to look for
alternative sources of MSCs, such as umbilical cord blood (UCB) and adipo tissue (AT). Here,
we isolated the hMSCs derived from BM, AT and UCB, and compared their effects on the stem
cell characterization or injured model in vitro. Using hMSC obtained from BM and AT as
reference controls, we observed that UCB-MSCs exhibited small cell size, high proliferation
rate, and the expansion of large scale required for cell therapies. Functionally, UCB-MSCs could
be more readily induced to differentiate into chondrocytes than BM-MSCs and AT-MSCs. UCBMSCs showed immune-suppressive activity equal to that of BM-MSC and AT-MSC.
Interestingly, in pathological condition, there were significant differences in functional
regeneration among the MSCs groups. For example, UCB-MSCs showed the highest increase
cell survival and anti-inflammation activity. Our data suggest that UCB should be reconsidered
as a practical source for cell protection and less inflammation compared to other MSCs.
Therefore, it may be crucial to predetermine the most appropriate MSCs source for future stem
cell therapy.
2684
The Effects of recombinant ice-binding protein from the Arctic yeast Leucosporidium sp.
on the cryopreservation of Umbilical cord blood-derived Mesenchymal stem cell.
J. Seo1, J. Oh2, J. Chang1, W. Oh1, S. Kwon 1; 1Medipost Co.,Ltd, Seoul, Korea, 2Sunkyunkwan
University, Suwon, Korea
Human mesenchymal stem cell (MSC) hold much promise for cellular therapy. But, one of the
challenges in realizing this goal is the preservation method of living cells. The cryopreservation
solution usually contains FBS and DMSO, which limit the administration of stem cells for
therapeutic purpose. Recently, many studies are being conducted to remove or reduce these
components for the safety issues. We will describe the effects of recombinant ice-binding
protein (pLeIBP) isolated from Arctic yeast Leucosporidium sp. for the cryo-preservation of
umbilical cord blood (UCB)-derived MSC. To investigate its effects on the cryopreservation,
various concentrations and recombinations of pLeIBP, DMSO and FBS were tested based on
the typical compositions of cryopreservation solution. The addition of pLeIBP enabled the
reduction of DMSO concentrations in the cryopreservants via increasing the proliferative
capacity of thawed MSCs compared to that in the usual compositions. The cryopreserved MSCs
SUNDAY-LATE POSTER PRESENTATIONS
maintained the differentiation capacity and surface marker profiles. Also, this protein showed the
possibility of the administration into vitrification methods. The viability of UCB-MSCs was highly
enhanced when pLeIBP was added into some composition of vitrification solutions. Our data
suggests the cryoprotectant effects of this ice-binding protein and the possibility of adaptation
into the development of new cryopreservation compositions.
2685
12-Channel High-Throughput Microscope for Cancer Cell Mechanics.
L. D. Osborne1, J. Cribb1, L. Vicci2, T. O'Brien1, J. Hsiao2, R. Taylor2, R. Superfine1; 1Dept of
Physics & Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, NC, 2Dept of
Computer Science, University of North Carolina at Chapel Hill, Chapel Hill, NC
Understanding how cell behavior and function relates to physical structure is a major goal of
current cell mechanics studies. Elucidating this relationship is especially important in the study
of cancer biology related to dramatic phenotypic changes as seen in epithelial to mesenchymal
transitions. Although much interest is given to studying the signaling pathways that govern how
normal cell function and structure become abnormal during cancer progression, these changes
convolute already difficult and highly variable mechanical measurements of single cells. To
address the need of minimizing single measurement variability as well as the desire to explore
the large signaling-protein parameter spaces of cancer biology, we have developed an
automated high throughput microscope system that utilizes passive microbead diffusion to
characterize cell mechanics. Here, we report on the instrumentation advances of our system,
including 12 independently controllable optical paths – each of which is capable of video rate
image acquisition in brightfield and two-channel fluorescence, and is equipped with
electronically tunable autofocus. In all, video data collection across a 96-well plate takes as little
as 10 minutes. A data analysis pipeline then identifies and tracks microbeads, filters and applies
statistical analysis to mechanical measurements, and occurs completely unsupervised. We
show that the thermal diffusion of micron-sized beads connected to integrin surface receptors
via fibronectin can distinguish ovarian, pancreatic and skin cancers with varying metastatic
potentials. With sampling sizes in the thousands, we report elastic moduli differences between
cancer cell types as well as the effect of pharmaceutical treatments aimed to alter actin
cytoskeletal structures. Our results support previously published work describing the inverse
relationship between mechanical stiffness and invasion behavior, and ultimately, demonstrate
the value of our high throughput instrument and passive rheology assay as a screening tool for
studying relevant signaling pathways involved in cancer cell mechanics.
SUNDAY-LATE POSTER PRESENTATIONS
2686
Enhancement of Small Fragment Homologous Replacement (SFHR)-Mediated Gene
Targeting with Nucleases (TALENs).
S. Suzuki1,2, T. J. Cradick3, M. Muench4, R. G. Sargent1, A. Lee1, M. Porteus5, G. Bao3, D. C.
Gruenert1,6,7; 1Otolaryngology-Head and Neck Surgery, University of California, San Francisco,
San Francisco, CA, 2Molecular Medicine, Graduate School of Pharmaceutical Sciences,
Kumamoto University, Kumamoto, Japan, 3Biomedical Engineering, Georgia Institute of
Technology and Emory University, Atlanta, GA, 4Blood Centers of the Pacific Research Institute,
San Francisco, CA, 5Pediatrics, Stanford University, Stanford, CA, 6Laboratory Medicine, Eli and
Edythe Broad Institute for Regenerative Medicine and Stem Cell Research, Institute for Human
Genetics, Cardiovascular Research Institute, and Helen Diller Family Comprehensive Cancer
Center, University of California, San Francisco, San Francisco, CA, 7Pediatrics, University of
Vermont School of Medicine, Burlington, VT
Sequence-specific modification of mammalian genomic DNA has been achieved through
homologous recombination (HR) with plasmids that contain sequences homologous to the
genomic target. While that event occurs at low frequency (<10-6), recent studies with
oligo/polynucleotides (small DNA fragments - SDFs) have shown that this frequency of
homologous exchange can be enhanced. In addition, studies using chimeric zinc finger
nucleases (ZFNs) have been shown to further enhance the efficiency through the introduction of
sequence-specific double strand breaks (DSBs) in conjunction with plasmid carrying donor
targeting sequences. More recently, transcription activator-like (TAL) effector nucleases
(TALENs) have also been developed as a new class of artificial nucleases capable of cleaving
specific DNA sequences and editing genomic DNA.
This study demonstrates that small fragment homologous replacement (SFHR)-mediated
correction with SDFs can be enhanced by co-transfection with TALEN pairs that cleave a
specific sequence in a mutant green fluorescent protein (GFP). Relative HR frequencies with
the SDFs, in presence or absence of TALENs, were measured as the correction of the mutant
GFPgene integrated into HEK293 cells. Transient expression of the TALENs significantly
enhanced HR in HEK293 cells. This approach was then applied to correct the mutation in cystic
fibrosis (CF) induced pluripotent stem (CF-iPS) cells. The studies showed a higher correction
efficiency of the most common CF transmembrane conductance regulator (CFTR) mutation (del
F508) with combination of SDFs and TALENs than with SDFs alone on day 3 after transfection
in CF-iPS cells that have a phenylalanine deletion at codon 508 in the CFTR gene (delF508)
and established from patient derived cells. Correction was detected both on day 3 and day 7
after transfection, indicating that the TALENs can enhance SFHR-mediated exchange and
suggest the combination of SDFs and TALENs have the potential as a genetic therapy for
inherited diseases.
2687
Multiparametic resolution of population heterogeneity through single cell FACS isolation
and identification correlated with gene expression profiling.
D. Shehu1, K. Livak1, K. Datta1, D. Soni2, G. Reinin2, J. Trotter2, C. Mason2; 1Fluidigm
Corporation, South San Francisco, CA, 2BD Biosciences, San Jose, CA
This paper describes a complete workflow for single-cell analysis from a heterogeneous mixture
of cells. Our method achieves accurate isolation and characterization of rare cell populations,
from a heterogeneous sample that contained both KG2A and Jurkat cells. Subsequent single
cell gene expression analysis demonstrated an exact correlation with cell types isolated by flow
cytometry.
SUNDAY-LATE POSTER PRESENTATIONS
Cells were rapidly identified and isolated by single-cell deposition into 96-well plates using a
flow cytometer equipped with index sorting software, followed by single-cell gene expression
analysis of each sorted cell. Index sorting software enables the collection of robust phenotype
information of the sorted cells and their exact location on the 96 well plate. and also allows the
subsequent correlation with the single cell gene expression profiles.
This data demonstrates
∙The ability to identify, isolate and profile cells from a complex sample that enables studies at
the granular level of a single-cell.
∙These results provide performance metrics about the workflow regarding its accuracy in
isolating and identifying and cells of interest.
∙The combination of flow cytometry and gene expression data on single cells provides a more
complete description of the cell biology.
2688
A novel FRET-based glycocalyx compression sensor reveals forces in the extracellular
interface.
A. C. Wijekoon1, M. J. Paszek1, V. M. Weaver1; 1Department of Surgery, Center for
Bioengineering and Tissue Regeneration, University of California, San Francisco, San
Francisco, CA
Coordinated cellular movements underscore important physiological processes, including tissue
morphogenesis, wound healing, and durotactic communication. While new genetically encoded
tension sensors permit the measurement of tensile “tugging” forces on intracellular proteins,
such as focal adhesion components, visualizing the compressive “pushing” forces imposed on
the cell surface remains challenging. To address this limitation, we developed a glycocalyx
compression sensor, consisting of a FRET-based force-sensing module embedded between the
extracellular stalk of a type 1 transmembrane protein and a rigid force-bearing rod domain. We
applied the sensor to measure compressive forces in the interface between the cell membrane
and extracellular substrate. Interestingly, we found high levels of compression in thick
glycocalyxes frequently associated with tumor cells, whereas there was minimal compression in
thin glycocalyxes associated with normal cells. Regions of highest compression in the
glycocalyx were immediately adjacent to cellular adhesion complexes, indicating direct
mechanical coupling between the glycocalyx and the integrin adhesion system in tumor cells.
Based on our initial findings, we believe our sensor is an important new tool for understanding
the mechano-biology of diverse physiological processes in normal and disease-specific
contexts.
(Work funded by the US National Institutes of Health (NIH)/ National Cancer Institute (NCI)
grant U54CA163155-01 and NCI grant U54CA143836-01and UCSF Program for Biomedical
Breakthrough)
2689
Studying protein conformational change using genome-wide misincorporation proton
alkyl exchange.
J. G. Caldwell1, P. B. Harbury1; 1Biochemistry, Stanford University School of Medicine, Stanford,
CA
The conformational changes experienced by proteins are integral to their functions, but existing
structural methods have limited ability to observe these dynamic motions in the context of the
relevant cellular partners. Misincorporation proton alkyl exchange (MPAX) is a method that
provides information at millisecond resolution about the motions of proteins in complex
SUNDAY-LATE POSTER PRESENTATIONS
mixtures—rather than in highly purified forms—by monitoring the solvent exposure of every
residue of the protein of interest. This process is made possible by mutated tRNAs that
misincorporate cysteine in place of all other amino acids at single hit levels during E. coli
translation; the reactivity of the resulting thiol serves as a probe for the residue’s solvent
exposure. For full information about the conformational changes of a particular protein,
misincorporation should be possible at all codons, but previous work (Kierstin Schmidt,
unpublished work) has demonstrated that only half of E. coli codons can misincorporate
cysteine at useful levels.
Standard survival-based selections are incompatible with
misincorporation, as particularly strong misincorporator tRNAs wreak havoc on the proteome.
We have developed a pulse-chase method to select for better misincorporation that will allow us
to test our hypotheses about how the translational machinery prevents misincorporation at
particular codons. This will also expand the range of codons that can misincorporate cysteine,
allowing us to examine more completely the conformational changes underlying the function of
individual proteins involved in processes ranging from vesicle trafficking to protein folding to cell
division.
Full codon coverage will also allow us to take advantage of translational
misincorporation as a method of “mutagenesis” when a problem is intractable by traditional
cloning or gene synthesis. This opens up questions of genome-wide scope, such as identifying
functional phosphorylation sites and identifying the set of proteins whose conformations change
when a cell is physically stretched.
Normal and Diseased Organs and Therapeutics
2690
Fatty Acid Metabolism Profiling in Cardiomyocytes of Non-Obese, Type 2 Diabetes Rats.
S. Devanathan1, S. Nemanich1, N. Fettig1, K. I. Shoghi1; 1Radiology - Radiation Sciences,
Washington University School of Medicine, Saint Louis, MO
Lipotoxicity of the cardiomyocyte has been implicated as a leading cause of morbidity in Type 2
Diabetes Mellitus (T2DM). Numerous reports have demonstrated increased fatty acid (FA)
utilization in cardiac cells of obese T2DM animal models. The phenotype of FA metabolism in
the cardiomyocyte of non-obese animals, however, has yet to be demonstrated. Therefore, the
present study investigates the interplay between cardiomyocyte FA metabolism and cardiac
function in a non-obese animal model of T2DM. Goto-Kakazaki (GK) rats at the age of 24 weeks
were studied with respect to cardiac function and the myocardial expression of 84 genes
involved in fatty acid metabolism. Echocardiograms (ECHOs) were performed to assess cardiac
function. In addition, plasma levels of triglycerides (TG) and free fatty acids (FFA) were
measured. ECHO data showed that GK rats have significantly (P<0.05) higher left ventricle
mass index (LVMI) compared to Wistar rats suggesting increased contractile impairment and
diastolic dysfunction. Furthermore, there was a significant (P<0.05) reduction in peak mitral
annular velocity (E’), indicating further reduced diastolic function. While there were no significant
differences in peripheral FFA concentrations between GK and Wistar rats, 16 of the 84 pathway
specific genes involved in FA metabolism were up-regulated between 1.5 and 14 fold (P<0.05)
in GK rat cardiomyocytes. A majority of these genes were from the Acyl-CoA Dehydrogenase
(Acad), Acyl-CoA Thioesterase (Acot), Acyl-CoA Synthetase (Acsl) and fatty acid binding
protein (Fabp) gene families, which indicates that the cardiac cells of non-obese diabetic rats
have enhanced fatty acid utilization. Similarly, while we observed increased expression of genes
encoding Lipase (Lipe) and Lipoprotein Lipase (Lpl), which are involved in TG catabolism, there
were no measurable differences in plasma TG levels. Overall, our data suggests that the
phenotype of increased FA metabolism is present in a non-obese animal model of T2DM,
SUNDAY-LATE POSTER PRESENTATIONS
suggesting cardiac-specific abnormalities in FA handling related to the diabetic state are at play
independent of obesity.
2691
Skeletal muscle atrophy is controlled by improving a redox balance by taking aspartic
acid Mg and aspartic acid Zn.
S. Yamada1, E. Kizaki1, A. Ohashi1, A. Ozeki1, T. Sato1, R. Matsuda2; 1Graduate School of
Human Life Science, Jissen Women's University, Hino, Japan, 2Graduate school of Arts and
Sciences, The University of Tokyo, Meguro-ku, Japan
Recent advances in cellular and molecular biology have led to an improved understanding of
those factors that contribute to muscle atrophy during both disuse and wasting pathologies. In
this regard, growing evidence implicates oxidative stress as an important regulator of pathways
leading to muscle atrophy during periods of disuse. For example, redox disturbances (i.e.
oxidant stress) in skeletal muscle myotubes increase the expression of key components of the
proteasome proteolysis system. This important proteolytic system is a prominent contributor to
protein breakdown in skeletal muscle during periods of inactivity. Moreover, additional signaling
pathways exist between muscle redox imbalance and loss of muscle protein and nuclei. In
addition, the imbalance of the mineral might induce the oxidant stress. Actually, Mg decreased
in muscle atrophy in tail suspension. Though this cause is uncertain, Mg deficiency increases
intracellular Ca2+concentration. As a result, calpain might be activated, and a skeletal muscle
protein is resolved. Then, we examined effects of the mineral (Mg and Zn) intake on the
inhibition of skeletal muscles atrophy.
Male's aged mice were used for the laboratory animal. The tail suspension model was used as
a model of the skeletal muscle atrophy. As a result of these experiment, the aspartic acid zinc or
the aspartic acid magnesium intake inhibited the atrophy of skeletal muscles by tail suspension.
In particular, the intake of the aspartic acid zinc was effective in reducing slow muscle atrophy.
And aspartic acid Mg intake reduced the muscle cell atrophy significantly. In addition, the intake
of the aspartic acid magnesium and the aspartic acid zinc also controlled a decrease of the
muscle protein. Such these effects were not observed only by the aspartic acid. Then, MyoD
and Myogenin that controlled the gene expression of muscle protein first were paid to attention
to clarify the mechanism of these phenomenathe appearance of the master gene was different
with the fast muscle and the slow muscle. The amount of appearance of the master gene in the
slow muscle was almost the same as a normal group obviously.
2692
Conditional knockout of Vps34 causes murine muscular dystrophy with impaired
autophagy.
A. Reifler1, X. Li2, J. McDade3, A. Archambeau3, D. E. Michele3,4, J. J. Dowling1,2,5;
1
Neuroscience Program, University of Michigan, Ann Arbor, MI, 2Pediatrics, University of
Michigan, Ann Arbor, MI, 3Molecular and Integrative Physiology, University of Michigan, Ann
Arbor, MI, 4Internal Medicine, University of Michigan, Ann Arbor, MI, 5Neurology, University of
Michigan, Ann Arbor, MI
Childhood neuromuscular disorders span a range of heterogeneous and prevalent diseases that
have early onset and lead to morbidity and premature death. Not all of the conditions have a
known etiology, and there are currently no cures and few treatments for the symptoms. Our
work investigating myotubular myopathy, which is caused by mutations in the MTM family of
genes—phosphatases targeting the 3’-phosphate of phosphatidylinositols (PtdIns), lead us to
investigate the consequences of PtdIns imbalances in muscle. PtdIns phosphorylation is crucial
in regulating many cellular processes, and the class III phosphatidylinositol 3-kinase,
SUNDAY-LATE POSTER PRESENTATIONS
PIK3C3/Vps34, has been shown to regulate endosomal and vacuolar trafficking, including
playing a key role in (macro)autophagy.
We generated a muscle specific Vps34 knockout mouse in order to examine the effects of
dysregulation of PtdIns(3)P complementary to effects seen in MTM mutations. What we
observed was a novel muscular dystrophy with aberrant autophagy.
Methods: Mice with a floxed allele of the PIK3C3 (Vps34) gene were crossed with a line of mice
expressing Cre recombinase under the muscle-specific promoter for muscle creatine kinase
(MCK). We verified genotype, transcript and protein knockout, characterized the survival and
gross phenotypes of the mutant mice, performed standard histological and immunochemical
analyses of sectioned muscles, examined ultrastructural characteristics, and assayed potential
mechanisms of action for Vps34, including membrane repair protocols using isolated myofibers.
Results: We observed a significant impact from loss of Vps34 in mutant mice, leading to severe
loss in muscle integrity and premature death. There were consistent abnormalities in muscle
ultrastructure and histology, and the mice shared many symptoms found in human muscular
dystrophies.
Conclusions: Dysregulation of Vps34 has serious consequences for muscle health. The
mutants provide a unique murine model of muscular dystrophy, and will provide further
clarification on the role of Vps34 and PtdIns regulation in skeletal muscle function and
homeostasis.
2693
Dual specificity phosphatase 4 activates AKT-mTOR in cardiomyopathy caused by LMNA
gene mutation.
J. C. Choi1, W. Wu1, A. Muchir1, S. Iwata1, S. Homma1, J. Morrow1, H. Worman1; 1Columbia
University, New York, NY
A-type lamins (lamin A/C) are a fundamental component of the nuclear lamina, a proteinaceous
meshwork of intermediate filaments lining the nuclear envelope inner membrane. Mutations in
the lamin A/C gene (LMNA) cause a diverse spectrum of tissue-selective diseases termed
laminopathies. The most prevalent form affects striated muscles as dilated cardiomyopathy
(hereinafter referred to as LMNA cardiomyopathy) with variable skeletal muscle involvement,
which includes autosomal Emery-Dreifuss muscular dystrophy. Emerging evidence suggests
the nuclear envelope functions as a signaling node and that abnormalities in the nuclear lamina
lead to dysregulated signaling pathways that underlie disease pathogenesis. We previously
showed that hyperactivated ERK1/2 in hearts of a mouse model of LMNA cardiomyopathy
(LmnaH222P/H222P mice) contributes to disease but the complete molecular pathogenesis remains
poorly understood. Here we uncover a pathogenic role of dual specificity phosphatase 4
(Dusp4), which is transcriptionally induced by ERK1/2. Dusp4 is overexpressed in hearts of
LmnaH222P/H222P mice and transgenic mice with cardiac-selective overexpression of Dusp4
display heart dysfunction similar to LMNA cardiomyopathy. In both primary tissue and cell
culture models, overexpression of Dusp4 positively regulates AKT-mTOR signaling, resulting in
abnormal metabolism and impaired autophagy. Notably, reactivation of autophagy with a
rapamycin analog temsirolimus prevents cardiac function deterioration, implicating defective
autophagy as a pathogenic mechanism of cardiomyopathy arising from LMNA mutation. These
findings identify a pathogenic role of Dusp4 in LMNA cardiomyopathy and provide a rationale for
a novel treatment using rapamycin and its analogs.
SUNDAY-LATE POSTER PRESENTATIONS
2694
Molecular basis of yeast prion nucleation and propagation.
Z. A. Ben Musa1; 1Cell and molecular biology, colorado state university, fort collins, CO
Introduction: Many diseases arise from the conversion of soluble proteins into amyloid
aggregates. Amyloid fibers are associated with a myriad of fatal human and animals diseases
including Alzheimer’s disease and Creutzfeldt–Jacob disease. Prions are a subset of amyloids,
distinguished by their infectivity. In the yeast, amino acid composition, not primary sequence,
drives prion formation. Nine different amyloid-based yeast prions have been identified; for eight
of these, a glutamine/asparagine (Q/N) rich prion domain drives prion formation. Aggregation of
similar prion-like domains has recently been linked to several neurological diseases, including
ALS. Therefore, our goal is to develop improved methods to predict the aggregation propensity
of Q/N-rich domains. We previous developed a quantitative mutagenesis method that allows for
estimation of the prion propensity of each amino acid in vivo; we then used this to develop an
algorithm capable of estimating the prion propensity of Q/N-rich domains.
This project will extend this work, seeking to provide a better understanding of the events
that drive prion formation and propagation. For a protein to act as a prion in yeast, the protein
must not only be able to form prion aggregates, but also propagate these aggregates over
multiple generations. We have demonstrated that prion formation and propagation have distinct
compositional requirements. However, our original prion propensity scoring method does not
separately examine formation and propagation. For the yeast prion protein Sup35, separate
regions of the prion domain are responsible for prion formation and propagation. The first 40
amino acids, termed the nucleation domain, are highly enriched in Q/N residues, and are
primarily responsible for driving prion formation. The remainder of the prion domain, termed the
oligopeptide repeat domain (ORD), contains a series of imperfect nine amino acid repeats, and
is primarily involved in prion propagation. Therefore, to rigorously define the compositional
requirements for these two activities, we have separately repeated our mutagenesis
experiments on nucleation domain and ORD of Sup35.
Methods: PCR with degenerate oligonucleotides was used to generate a library of sequences in
which a 24 nucleotide segment from either the nucleation domain or ORD of Sup35 was
replaced with a random sequence. These mutants were expressed in yeast as the sole copy of
Sup35. This library was then screen for the subset of mutants capable of forming and
propagating prions. By comparing the frequency of occurrence of each amino acid in the
starting library to the frequency of occurrence among the prion-forming subset, we were able to
estimate the extent to which each amino acid promotes/inhibits prion activity within the context
of the nucleation or propagation domain.
Results: The general patterns within the nucleation domain largely resembled those from our
previous mutagenesis screen, with hydrophobic and aromatic residues promoting prion activity,
and charged residues and proline inhibiting prion activity. Interestingly, within the ORD, while
aromatic residues were still strongly prion-promoting and charged residues were strongly prioninhibiting, non-aromatic hydrophobic residues were neutral. These results highlight the contextspecific effects of amino acids on prion activity.
Conclusions: These experiments represent a critical first step towards defining the sequence
requirements for prion formation and propagation.
Our results will facilitate accurate
identification of additional prions in both yeast and other organisms, and provide preliminary
insights into the mechanism of prion formation and propagation.
SUNDAY-LATE POSTER PRESENTATIONS
2695
Targeting Galectin-1 and Galectin-3 Attenuates VEGF-A-induced Angiogenesis.
W-S. Chen1, H. Leffler2, U. J. Nilsson3, N. Panjwani1,4; 1Sackler School of Graduate Biomedical
Sciences, Tufts University, Boston, MA, 2Departments of Laboratory Medicine, University of
Lund, Lund, Sweden, 3Organic Chemistry, University of Lund, Lund, Sweden, 4New England
Eye Center and Department of Ophthalmology, Tufts University, Boston, MA
Vascular endothelial growth factors (VEGFs) signaling though VEGF receptor-2 (VEGFR-2) is
the primary angiogenic pathway. Published studies from our and other laboratories have shown
that both galectin-1 (Gal-1) and galectin-3 (Gal-3) are important modulators for VEGF/VEGFR-2
signaling pathway. TDX is a novel high-affinity carbohydrate compound against Gal-1 (Kd = 12
nM) and Gal-3 (Kd = 14 nM). To date, besides TDX, no compound has been developed to
specifically target both Gal-1 and Gal-3. In addition, how targeting both Gal-1 and Gal-3 affects
VEGF-A/VEGFR-2 signaling has not been fully elucidated. Our goal is to study TDX’s efficacy
against pathological angiogenesis and we hypothesize that TD139 treatment interferes with
VEGF-A/VEGFR-2 signaling and abrogates VEGF-A-induced angiogenesis.
Here, we demonstrate that (i) subconjunctival injection and eye-drop application of TDX reduces
cautery-induced corneal neovascularization in vivo, (ii) Gal-1 and Gal-3 interact with VEGFR-2
in a carbohydrate-dependent manner, (iii) TDX attenuates VEGF-A-induced sprouting of human
umbilical vein endothelial cells (HUVECs) as assessed by a three-dimensional sprouting assay,
(iv) TDX efficiently abrogates VEGF-A-induced HUVEC chemotaxis in a dose-dependent
manner, (v) TDX does not affect HUVEC viability as assessed by two different reagents, Calcien
AM and WST-1, and (vi) TDX treatment has little effect on total protein expression of VEGFR-2.
Our data provide proof of concept that targeting Gal-1 and Gal-3 by the novel, small molecule
inhibitor, TDX, ameliorates pathological angiogenesis. The findings provide a new therapeutic
strategy for a variety of disease conditions including inflammation, diabetes, cancer metastasis,
age-related macular degeneration and corneal neovascularization.
2696
Preserving mitochondrial function using L-carnitine prevents the pulmonary endothelial
dysfunction associated with increased pulmonary blood flow (PBF).
S. M. Black1, S. Sharma1, A. Field-Ridley2, S. Datar2, R. Keller2, J. Fineman2; 1Vascular Biology
Center, Georgia Health Sciences University, Augusta, GA, 2Pediatrics, UC San Francisco, San
Francisco, CA
Background: Congenital heart disease (CHD) with increased pulmonary blood flow results in
progressive pulmonary vascular endothelial dysfunction and increased perioperative morbidity.
Using our model of CHD with increased PBF (Shunt), we have demonstrated progressive
pulmonary endothelial dysfunction and altered vascular reactivity. These changes are
associated with disruptions in carnitine homeostasis. Potential alterations in carnitine
homeostasis in patients with CHD have not been investigated nor has the therapeutic potential
of L-carnitine supplementation on endothelial function been evaluated.
Objective: To test the hypothesis that children with pre-operative increased PBF have altered
carnitine homeostasis compared to children with CHD not associated with increased PBF.
Further, we wished to evaluate the affect of L-carnitine therapy on pulmonary endothelial
function in our Shunt model.
Design/Methods: We determined and compared plasma levels of total carnitine (TC), free
carnitine (FC), and acylcarnitine (AC) in patients with CHD associated with increased PBF
(ventricular septal defects, VSD), and patients without increased PBF (Tetralogy of Fallot, TOF)
prior to and immediately after surgical repair. Plasma samples from 20 subjects with VSD and
18 with TOF were analyzed by HPLC. In addition, 13 late gestation fetal lambs underwent in
SUNDAY-LATE POSTER PRESENTATIONS
utero placement of an aortopulmonary graft (Shunt). Immediately following spontaneous
delivery, lambs received oral L-carnitine (n=7, 100 mg/kg/day) or its vehicle (n=6) daily. At 4weeks of age, the lambs were instrumented to measure vascular pressures and reactivity, and
lung biopsies and blood was obtained for biochemical analyses.
Results: Preoperative AC levels (25.7±13 vs. 12.7 ± 8.3, p=0.0007), and the AC:FC ratio
(0.8±0.1 vs. 0.3±0.05, p=0.0003) were higher in VSD than TOF, consistent with aberrant
mitochondrial function. After surgery, TC and FC levels increased in both groups (P<0.05), while
AC levels were unchanged, normalizing the AC level and the AC:FC ratio between groups. In
Shunt lambs, L-carnitine restored carnitine homeostasis as manifested by increased plasma
and lung tissue TC and FC, and decreased AC (P<0.05). Mitochondrial function was also
improved as manifested by a decreased lactate/pyruvate ratio (P<0.05). L- carnitine also
increased eNOS/Hsp90 interactions (P<0.05), decreased eNOS-derived lung tissue superoxide
levels (P<0.05), increased NOS activity and NOx levels (P<0.05), and preserved endotheliumdependent pulmonary vasodilation (P<0.05).
Conclusions: Increased PBF from VSD results in impaired carnitine homeostasis and these
alterations are corrected following surgical repair implicating the key role of increased PBF in
this process. L-carnitine supplementation may attenuate the endothelial dysfunction associated
with increased PBF, and warrants further investigation.
Supported in part by HL067841 (to SMB), HL61284 (to JRF) and a Transatlantic Network
Development Grant from the LeDucq Foundation (to SMB & JRF).
2697
Overexpression of ezrin in Sjogren's syndrome minor salivary glands can be attributed to
the observed downregulation of microRNA Hsa-mir183.
P. J. Perez Riveros1, L. Teos1, M. Tandon1, A. Gallo1, I. Alevizos1; 1MPTB, NIDCR, National
Institute of Health, Bethesda, MD
Sjogren’s syndrome (SS), an exocrinopathy that affects more than 2-4 million of Americans, is
mainly characterized by salivary gland (SG) hypofunction. SGs from SS patients present with
dilated lumen and loss of microvilli in the acinar cells. Previously, we reported that these
changes correlate with an aberrant location in the basal pole of the acinar cells and an
overexpression of ezrin (Rheumatology 49:915,2010). Ezrin is a cytoplasmic peripheral
membrane protein (80 kDa) that regulates the microvilli organization and secretion of exocrine
cells. The control of ezrin gene expression is poorly understood. In other tissues hsa-mir-183
and hsa-mir-22 target ezrin but nothing is known about their role in ezrin expression control in
SGs. Our aim was to determine if the overexpression of ezrin can be explained by the
downregulation of hsa-mir-183 and/or hsa-miR-22, two microRNAs that using microRNA
microarray have been found to be downregulated in SGs of SS. In SG biopsies of SS patients
(n=17) and healthy volunteers (n= 3), we analyzed the levels of miRNA by qRT-PCR. We
determined the effects of the overexpression of hsa-mir-183 and their specific inhibitor on the
protein levels of ezrin on human salivary glands (HSG) cells cultured in 3D. To further confirm
the function of hsa-mir-183 in SGs, we performed in vivo transfection of LNA antimir-183 on
mice parotid glands. After transfection we measured the levels of ezrin by western blot and
immunofluorescence. Hsa-miR-183 and -22 levels were 4.9 and 6.5 times (??C?: 2.3 and 2.7)
lower respectively in SS compared with healthy volunteers. The levels of hsa-miR-22 and -183
did not correlate with the focus score (a measure of inflammation) in SS. When HSG cells
cultured in 3D were transfected with mimic microRNA for hsa-miR-183, ezrin protein levels
decreased, and an opposite behavior was observed when inhibitors were transfected. The in
vivo transfection of the LNA-antimir in parotid glands of BALB/c mice produced an increase of
protein levels of ezrin and significantly affected the granule secretion of acinar cells, as imaged
with confocal imaging. These experiments suggest that in SG of SS patients the overexpression
SUNDAY-LATE POSTER PRESENTATIONS
of ezrin is caused by the downregulation hsa-miR-183 and/or 22 and propose a role of ezrin in
the mechanism of SG hyposecretion.
2698
Alteration and adaptation of endothelial and cardiovascular disease biomarkers in
placenta and foetal liver of Goto- Kakizaki (GK rats).
J-C. Irminger1, S. Dupuis1, J. Coulaud2, P. A. Halban1, F. Homo-Delarche2; 1Department of
Genetic Medecine and Development, University of Geneva, Geneva, Switzerland, 2Inserm
UMRS 872, Université Pierre et Marie Curie, Paris, France
Background/Aims. Maternal hyperglycaemia and hypercholesterolaemia are known to induce
placental inflammation and vascular and lipid metabolism alterations. In the GK rat model of
spontaneous type 2 diabetes, we recently showed that embryonic day 21 (E21) foetuses
exhibited in addition to hyperglycaemia, hypercholesterolaemia, high circulating total
cholesterol/HDL-cholesterol ratio and hepatic disturbances of HDL-linked variables. We
therefore looked in this study for signs of endothelial alteration and CVD (cardiovascular
disease) biomarkers in both placenta and liver of GK foetus.
Materials and methods. At E21, foetuses were taken from pregnant rats fed ad libitum with a
standard diet and processed after maternal anaesthesia with pentobarbital sodium (35 mg/kg
body weight). Gene expression was analysed in placenta and liver of GK and control Wistar rat
foetuses using Oligo GEAarray hybridization targeted at endothelium, CVD biomarkers and
angiogenesis (128 genes, each). Selected genes were confirmed by quantitative RT-PCR.
Results. Microarray analysis from both E21 GK rat placenta and foetal liver showed mostly
upregulation of nearly 90 genes belonging to the following categories: renin-angiotensin system
(RAS), inflammation (cytokines, chemokines, cellular adhesion molecules, extracellular matrix,
oxidative stress, peroxisome proliferator-activated receptor (PPAR), apo-lipoproteins (Apo) and
lipid metabolism, haemostasis (platelet aggregation, coagulation and fibrinolysis), and
apoptosis. In the liver, 2 genes involved in RAS, angiotensinogen and angiotensin 1, and 2
genes (Nppa and Nppb) encoding natriuretic factors were upregulated. In the placenta, the gene
for 11-β-hydroxysteroid deshydrogenase type 1 (Hsd11b1) was overexpressed, suggesting
increased glucocorticoid activity. Nine genes belonging to the Apo family appeared to be
upregulated (5 were common to both organs). In foetal GK liver we confirmed the increased
expression of most Apo genes with quantitative RT-PCR. In addition, lecithin: cholesterol
acyltransferase (Lcat) expression was upregulated in placenta and liver. Among various
inflammatory genes, Casp1 and Il1b1 were upregulated in the liver, suggesting early
involvement of the inflammasome in the disease. Signs of oxidative stress and/or antioxidant
defence were observed in both GK organs: increased Pon2 (paroxonase 2) and Sod
(superoxide dismutase) and genes involved in modulation of liver apoptosis. Finally, 50% of the
12 factors involved in haemostasis and present in both organs are known to induce primary and
secondary haemostasis, while the remaining factors are known to inhibit coagulation or
activated fibrinolysis.
Conclusion. These data highlight the inflammatory reaction that occurs during GK gestation in
response to maternal hyperglycemia and hypercholesterolaemia. The concomitant upregulation
of RAS and the natriuretic system may suggest pro- and antihypertensive effects. Attempted
adaptation was also observed for haemostasis, inflammatory process, oxidative stress and
apoptosis. Apolipoprotein gene upregulation could be aimed at moderating inflammation and
hyper-coagulation. Finally, upregulated RAS and hyper-coagulation might suggest a trends
towards maternal pre-eclampsia.
SUNDAY-LATE POSTER PRESENTATIONS
2699
Temporal Changes in Cell Marker Expression and Cellular Infiltration in a Controlled
Cortical Impact Model in Adult Male C57BL/6 Mice.
J. Xuemei1, I. Hiroshi1, I. Takahide1, Y. Toshihide1; 1Molecular Neuroscience, Graduate of
Medicine, Osaka University, Suita, Japan
Background: Traumatic injury to the central nervous system (CNS) triggers a robust
inflammatory response that leads to axonal damage and secondary degeneration of spared
tissue. In contrast, some immune responses have neuroprotective effects. However, detailed
information regarding the dynamics of immune responses after traumatic CNS injury is still
unavailable.
Methods: In the present study, changes in the immune cells present in the injured brain,
spleen, and cervical lymph nodes (CLNs), which are draining lymphatic organs from the CNS,
were analyzed after controlled cortical impact (CCI) by flow cytometry and
immunohistochemistry.
Results: The number of neutrophils and macrophages that infiltrated the injured brain
immediately increased 1 d post-injury and declined rapidly thereafter. In the injured brain,
resident microglia showed a bimodal increase during the first week and in the chronic phase (≥3
weeks) after injury. Increase in the Iba-1+ microglia/macrophages was observed around the
injured site. Morphologic analysis showed that Iba-1+ cells were round at 1 week, whereas those
at 3 weeks were more ramified. Furthermore, CD86+/CD11b+ M1-like microglia increased at 4
weeks after CCI, whereas CD206+/CD11b+ M2-like microglia increased at 1 week. These results
suggest that different subsets of microglia increased in the acute and chronic phases after CCI.
Dendritic cells and T cells increased transiently within 1 week in the injured brain. In the CLNs
and the spleen, T cells showed dynamic changes after CCI. In particular, the alteration in the
number of T cells in the CLNs showed a similar pattern, with a 1-week delay, to that of microglia
in the injured brain.
Conclusion: The data from this study provide useful information on the dynamics of immune
cells in CNS injuries.
2700
Role of aldolase A in transcytosis of intestinal M cells.
Y. Nagasawa1, Y. Takahashi1, T. Hondo1, H. Watanabe1, S. Terada1, S. Someya1, K.
Watanabe1, S. Ohwada1, H. Kitazawa1, M. Imamura2, T. Yokoyama2, S. Sakaguchi3, N.
Nishida4, S. Mohri2, H. Aso1; 1Tohoku University, Sendai, Japan, 2National Institute of Animal
Health, Tsukuba, Japan, 3Tokushima University, Tokushima, Japan, 4Nagasaki University,
Nagasaki, Japan
Peyer's patches represent the primary site for uptake and presentation of ingested antigens in
the intestine. Microfold cells (M cells) are a kind of intestinal epithelial cell in the follicleassociated epithelium of Peyer's patches. This cell can transport antigens and microorganisms
into underlying lymphoid tissues. Bovine spongiform encephalopathy(BSE) is a fatal
neurodegenerative disorder in humans and animals. Although abnormal prion proteins known
as BSE agents first have to cross the M cells, the exact mechanisms for intestinal invasion still
are unclear. Our laboratory identified glycolytic enzymes aldolase A as one of PrP-binding
proteins in M cells, which was expressed in epithelia of nasopharynx and intestinal M cells, well
known as PrP uptake sites. Therefore, we investigated the function and role of aldolase A in
intestinal M cells using a bovine intestinal epithelial cell line (BIE cells).
SUNDAY-LATE POSTER PRESENTATIONS
Immunofluorescent staining shows that M cell-differentiated BIE (M-BIE) cells express aldolase
A in their apical part. We added non- (FITC-), prion protein- (PrP-) or BSA-conjugated (BSA-)
particles onto M cell-differentiated BIE (M-BIE) cells. After 9 hours, the uptake and
internalization of each particles was observed and co-stained with aldolase A in M-BIE cells. MBIE cells transported particles to the basal chamber of the insert. The total number of
transported PrP- or BSA-particles by M-BIE cells was significantly greater than that of FITCparticles. Surprisingly, the treatment of anti-aldolase A antibody clearly inhibited the
transportation of PrP and BSA-particles by M-BIE cells. These results suggest that aldolase A
may play an important role in the incorporation of antigens by intestinal M cells.
2701
Glia maturation factor suppression by RNA interference leads to reduced inflammation
and demyelination in experimental autoimmune encephalomyelitis.
S. Zaheer1, Y. Wu1, M. M. Khan1, A. Zaheer1; 1Neurology, VA Health Care System and
University of Iowa, Iowa City, IA
Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system
(CNS). The complex process of chronic inflammation resulting in demyelination in MS is poorly
understood. Glia Maturation Factor (GMF), discovered and characterized in our laboratory, is a
highly conserved brain-specific protein. Our recent findings indicated that GMF plays a critical
role in regulating the myelin oligodendrocyte glycoprotein peptide 35- 55 (MOG 35-55)-induced
experimental autoimmune encephalomyelitis (EAE), an animal model of MS. We have also
reported earlier that GMF-deficient mouse failed to develop EAE. In the presentstudy we show
the therapeutic efficacy of the administration of a lentiviral vector carrying the GMF-shRNA in
EAE. We induced active EAE in C57BL/6 mice by MOG 35–55 immunization and relapsingremitting-EAE in SJL/J mice immunized with myelin proteolipid protein (PLP) 139-151. Mice
were treated intravenously with four injections of GMF-shRNA, given every second day
beginning 5 days after immunization, time at which the brain-blood barrier is breached. In MOGinduced EAE mice, GMF-shRNA treatment significantly reduced the disease severity as
compared to controls. The mean paralysis score in the treated group was 1.3 ± 0.5 versus 3.5 ±
0.5 in the controls, a significant decrease. Significantly reduced inflammatory infiltrating cells
and reduced inflammation, and demyelination were detected in the CNS of GMF-shRNA-treated
mice when compared to controls. Additionally, GMF-shRNA significantly suppressed production
of inflammatory cytokines/chemokines in the CNS of both actively-induced and relapsingremitting EAE mice.
2702
Comparative analysis of ER stress response into HIV protease inhibitors: Lopinavir but
not Darunavir induces potent ER stress response.
M. Taura1, R. Kariya1, E. Kudo1, H. Goto1, S. Okada1; 1Division of Hematopoiesis, Center for
AIDS Research, Kumamoto University, Kumamoto, Japan
HIV-1 protease inhibitors (PIs)-induced ER stress has been associated with adverse effects.
Although it is a serious clinical problem for HIV/AIDS patients, exhaustive comparison of ER
stress induction by clinically used PIs has not been done. Especially, there is no report on the
differential ER stress response between Lopinavir (LPV) and Darunavir (DRV), the most
clinically used PIs. Our objectives are to determine the ER stress induced by FDA-approved
PIs, and to compare the effects of LPV and DRV on ER stress induction as a safety parameter.
We examined PIs-induced ER stress in several human epithelial cells and mouse embryonic
fibroblast. Next, we selected Ritonavir (RTV), LPV and DRV to compare ER stress and
apoptosis induction, and clarified its mechanism. Finally, we analyzed ER stress induction in
SUNDAY-LATE POSTER PRESENTATIONS
tissues of mice interperitoneally injected with RTV, LPV and DRV. LPV showed the most potent
ER stress induction and cytotoxity among the PIs. Comparison between RTV, LPV and DRV
revealed that LPV potently and RTV moderately induced ER stress via ROS-dependent JNK
activation rather than proteasome inhibition. In contrast, DRV did not induce ER stress and had
no cytotoxic effects. In summery, we firstly identify LPV as the most potent ER
stress&#8211;inducing PI among the nine FDA-approved PIs, and although clinical verification
is necessary, we show here the safety of DRV compared with LPV by focusing on ER stress in
vitro and in vivo.
2703
Mesenchymal Stem Cells as Vehicles for Intercellular Delivery of RNAi to Treat
Huntington's Disease.
J. D. Anderson1, S. Olson1, G. Mitchell1, K. Pollock1, K. Pepper1, J. Nolta1; 1Internal Medicine,
Stem Cell Program, UC Davis, Sacramento, CA
Huntington's disease (HD) is a terminal neurodegenerative autosomal dominant genetic disease
without any treatment options. RNAi technology is an ideal therapy for HD as has been
demonstrated in transgenic mouse models (DiFiglia, et al, 2007); however, effective and
prolonged delivery of RNAi is a major obstacle. Mesenchymal stem cells (MSCs) have
extensive therapeutic properties and are being explored as a treatment for neurodegenerative
diseases due to their ability to modulate the immune system and secret a variety of growth and
anti-apoptotic factors. MSCs maintain a strong safety profile when injected systemically and are
now approved as a drug for IV administration in several.. Additionally, MSCs have been shown
to transfer molecules and organelles to neighboring cells (Spees, Olson, et al 2005). For these
reasons, we are seeking to show that MSCs expressing RNAi against mutant huntingtin protein
can be a novel treatment for HD, decreasing the expression of mutant huntingtin protein while
providing a number of innate healing effects to nurture the damaged microenvironment. In an in
vitro co-culture system, we have found decreased expression of an exogenous mutant
huntingtin fragment in target neural cells following co-culture with MSCs expressing a short
hairpin RNA targeting huntingtin mRNA. This finding supports the idea that the efficacy of MSCs
might be enhanced to directly treat Huntington's disease and other dominant genetic diseases.
2704
Dendritic Potassium Channel Regulation by Fragile X Mental Retardation Protein (Role
of Potassium Channels in Fragile X Syndrome).
H. Lee1, Y. Jan1, L. Y. Jan1; 1UCSF, San Francisco, CA
Fragile X syndrome (FXS) is a common form of mental disability and one of the known causes
of autism. The mutation responsible for FXS is a large expansion of the trinucleotide CGG
repeats that leads to DNA methylation of the fragile X mental retardation gene 1 (FMR1) and
transcriptional silencing, resulting in the absence of fragile X mental retardation protein (FMRP),
an mRNA binding protein. Although it is widely known that FMRP is critical for metabotropic
glutamate receptor (mGluR)-dependent long-term depression (LTD), which has provided a
general theme for developing pharmacological drugs for FXS, specific downstream targets of
FMRP may also be of therapeutic value. Since alterations in potassium channel expression
level or activity could underlie neuronal network defects in FXS, we will describe our recent
findings on how these channels might be altered in mouse models of FXS and the possible
therapeutic avenues for treating FXS.
How transmitter receptors modulate neuronal signaling by regulating voltage-gated ion channel
expression remains an open question. Recently we reported dendritic localization of mRNA of
SUNDAY-LATE POSTER PRESENTATIONS
Kv4.2 voltage-gated potassium channel, which regulates synaptic plasticity, and its local
translational regulation by FMRP linked to FXS, the most common heritable mental retardation.
FMRP suppression of Kv4.2 is revealed by elevation of Kv4.2 in neurons from fmr1 knockout
(KO) mice, and in neurons expressing Kv4.2-3’UTR that binds FMRP. Moreover, treating
hippocampal slices from fmr1 KO mice with Kv4 channel blocker restores long-term potentiation
(LTP) induced by moderate stimuli. Surprisingly, recovery of Kv4.2 after N-methyl-D-aspartate
receptor (NMDAR)-induced degradation also requires FMRP, likely due to NMDAR-induced
FMRP dephosphorylation, which turns off FMRP suppression of Kv4.2. Our study of FMRP
regulation of Kv4.2 reveals a novel aspect of NMDAR signaling and a new FMRP target of
potential relevance to FXS.
2705
Identify novel genes that specific expressed in testis and generate knock-out mice by ES
cells.
K. Kim1, S. Shim1, S-W. Jee1, S-H. Lee1, S. Park1, J. Lee1, C-J. Lim1; 1Korea Food and Drug
Administration, Osong, Korea
Normally, infertility is defined as a failure to get pregnant after a year of regular intercourse
without contraception. The number of infertility patients was increased by 25 percent between
2005 and 2009 and is still steadily increasing. Male infertility accounts for 40 percent of total
infertility cases. To find out a novel gene related with male infertility, we found 14 candidate
genes using in silico analysis. As a result of PCR, all of candidate genes were expressed in
testis and epididymis specially. For making knock-out mice using embryonic stem cells, we
chose two genes (E24, F07) in 14 candidate genes. After we made antibodies which detect E24
and F07 proteins, we confirmed that two proteins were expressed in mice testis. Both the
number of in-vitro fertilized embryos and the size of testis were reduced in knock-out mice. On
the other hand, phosphorylation of JNK and p38 MAP kinase was increased in knock-out mice.
These findings suggest that the novel genes can be associated with male infertility.
2706
3-Aryl-2-Quinolone: characterization of an integrins antagonist small molecule that
targets the cytoplasmic tails.
S. Fiorucci1, X. Lin2, K. Sadoul1, D. Bouvard1, C. Albiges-Rizo1, O. Vinogradova2, B. Joseph3, M.
R. Block1; 1Institut A. bonniot inserm U823, Grenoble cedex 09, France, 2Pharmaceutical
Sciences, University of connecticut, School of Pharmacy, Storrs, CT, 3Equipe Smith, Institut de
chimie et de biochimie moléculaire et supramoléculaire, Villeurbanne, France
Flavonoïds have been studied for years for their potential chemopreventive and
chemotherapeutic action. Several mechanisms might account for their anticancer activity,
among which inhibition of cell adhesion and spreading, or inhibition of tumor cell invasion. 3Aryl-2-Quinolone derivatives are chemical structures close to flavonoïds and were first designed
as anti-migratory agents (Joseph et Al., J.Med.Chem, 2002). As cell migration is highly
dependent on the cell adhesion machinery, we decided to investigate the action of these
molecules on focal and fibrillar adhesions. These large protein complexes include heterodimeric
transmembrane proteins, the integrins, and their cytoplasmic interactors able to link to the
cytoskeleton. Integrins allow microenvironment sensing and cellular response to it. Adhesive
structures containing integrins are also able to control cell microenvironment (matrix
degradation, fibrillogenesis…). Our studies show that 3-Aryl-2-Quinolone derivatives are able
not only to prevent cell spreading but also to disrupt already well-established focal adhesions in
a reversible and ECM composition independent manner. The activity of the molecule is closely
linked with its structure, as very slight modification of the lateral chain of the compound can
SUNDAY-LATE POSTER PRESENTATIONS
totally impair its activity. Our work is focused on establishing a Structure-Activity Relationship of
3-Aryl-2-Quinolone derivatives and on investigating the molecular mechanisms underlying this
activity. Pre-osteoblasts treatment by 3-Aryl-2-Quinolone derivatives triggers a rapid
disassembly of focal and fibrillar adhesions. NMR experiments show a direct interaction
between the lead compound of the family and β3 integrin cytoplasmic tail and pull-down assay
show that it is able to reduce the interaction between β3 integrin and kindlin, one of its
coactivator. As platelet activation is an archetype of β3 integrin activation, we tested the activity
of 3-Aryl-2-Quinolone on this physiological process. Under treatment, platelets are neither able
to become activated nor to trigger thrombus formation, providing an interest to the 3-Aryl-2Quinolone derivatives as potential anti-thrombotic agents.
2707
CCL5 derived from bone marrow stem cells ameliorates Alzheimers disease in mice.
J-S. Bae1, J. Lee1, H. Jin2; 1Kyungpook National University School of Medicine, Daegu, Korea,
2
College of Veterinary Medicine, Kyungpook National University, Daegu, Korea
Microglia have the ability to eliminate amyloid β (Aβ) by a cell-specific phagocytic mechanism,
and bone marrow (BM) stem cells have shown a beneficial effect through endogenous microglia
activation in the brains of AD mice. However, the mechanisms underlying BM-induced activation
of microglia have not been resolved. We show that BM-derived mesenchymal stem cells
(MSCs) induced the migration of microglia when exposed to Aβ in vitro. Cytokine array analysis
of the BM-MSC media obtained after stimulation by Aβ further revealed elevated release of the
chemoattractive factor, CCL5. The CCL5 was increased when BM-MSCs were transplanted into
the brains of Aβ-deposited AD mice, but not normal mice. Interestingly, alternative activation of
microglia was associated with elevated CCL5 expression. Furthermore, by generating a
chimeric mouse, we ascertained that the activated microglia resulted from endogenous BM cells
that were recruited into the brain by CCL5. Additionally, we observed that neprilysin (NEP) and
IL-4 derived from the alternative microglia were associated with a reduction in Aβ deposition and
memory impairment in AD mice. These results suggest that the recruitment of the alternative
microglia into the brain is driven by CCL5 secretion from the transplanted BM-MSCs, which
itself is induced by Aβ deposition in the AD brain.
2708
BM-MSCs promote proliferation and neuronal differentiation of Niemann-Pick type C
mouse neural stem cells by upregulation and secretion of CCL2.
H. Jin1, H. Lee1, J-S. Bae2; 1College of Veterinary Medicine, Kyungpook National University,
Daegu, Korea, 2Kyungpook National University School of Medicine, Daegu, Korea
Niemann-Pick type C (NP-C) disease is a neurodegenerative disorder characterized
neuropathologically by ballooned neurons distended with lipid storage and widespread neuronal
loss. Neural stem cells (NSC) derived from NP-C disease models have decreased ability for
self-renewal and neuronal differentiation. With the investigation of neurogenesis in the adult
brain, hopes that NP-C disease can be overcome, or at least ameliorated, by generation of new
neurons have increased. Due to their ability to promote neurogenesis, bone marrow-derived
mesenchymal stem cells (BM-MSCs) are regarded as potential candidates for use in treatment
of neurodegenerative disorders. However, the underlying mechanisms of BM-MSCs-induced
promotion of neurogenesis have not been resolved. The aim of the present study was therefore
to examine the mechanism of neurogenesis by BM-MSCs in NP-C disease. Here we found that
embryonic NSCs from NP-C mice exhibit impaired ability for self-renewal and decreased rates
of neuronal differentiation. Co-culture of these cells with BM-MSCs resulted in an enhanced
capacity for self-renewal and an increased ability for differentiation into neurons or
SUNDAY-LATE POSTER PRESENTATIONS
oligodendrocytes. In addition, results of in vivo studies have demonstrated that transplantation
of intracerebral BM-MSCs resulted in stimulated proliferation and neuronal differentiation of
NSCs within the subventricular zone. Of particular interest, enhanced proliferation and neuronal
differentiation of endogenous NP-C mouse NSCs showed an association with elevated release
of the chemokine (C-C motif) ligand 2 (CCL2) from BM-MSCs. These effects suggest that
soluble CCL2 derived from BM-MSCs can modulate endogenous NP-C NSCs, resulting in their
improved proliferation and neuronal differentiation in mice.
2709
The biological pacemaker derived from mouse embryonic stem cells for treatment of
bradycardia.
Y. Shirayoshi1, K. Morikawa1, I. Hisatome1; 1Institute of Medical Science, Tottori University,
Yonago, Japan
Cardiac pacemaker cells can be extracted from differentiating mouse embryonic stem cells
(mESCs) through basis on their specific expression of ion-channel HCN4 at sinus node. We
have established green fluorescent protein (GFP) knock-in mESCs at HCN4 locus (H7 clone)
and characterized pace-making cells derived from H7 ES cells by isolation of GFP-positive cells
using embryoid body (EB) differentiating system.
The expression of GFP was specifically restricted at their contracting region in differentiating H7
EBs. Cell sorting revealed that a few cells (0.1~0.5%) of H7 EBs were GFP positive, of which
approximately 80% of HCN4+ cells showed the spontaneous beating activity. Sorted GFP+ cells
had the essentially same properties as cardiac pacemaker cells at sinus node: 1) the common
gene and protein expression profiles with cardiac pacemaker cells, for examples, pacemaker
specific makers such as HCN4, cav3.1 and connexin43 as well as cardiomyocyte specific
maker, tropmyosinC, 2) cesium sensitive spontaneous action potentials, which responded to
adrenergic stimulation and cholinergic repression, 3) the electrophysiological properties such as
If currents, calcium currents, relatively high sodium currents and ERG currents. Finally, we
investigate whether H7 derived HCN4+ cells can restore myocardial electromechanical
properties. Using imaging techniques, we demonstrated that HCN4+ cells established electrical
coupling with HL-1 cells, a cadiac muscle cell line derived from the mouse atrial cardiomyocytes
tumor, to induce rhythmic electrical and contractile activities in vitro. Similarly, transplanted
HCN4+ cells paced the hearts of rat with complete atrioventricular block, indicated that HCN4+
cells could substitute for pacemaker cells and elicit an ectopic rhythm.
These results demonstrate the potential of HCN4+ pace-making cells to act as a rateresponsive biological pacemaker and for future myocardial regenerative medicine to
bradycardia.
2710
Long-term Proteasome Inhibition by Epoxomicin Confers Cardioprotection against
Ischemia/Reperfusion Injury.
D. Li1, G. Babu1; 1umdnj, newark, NJ
Background: Cell death (both apoptosis and necrosis) is the most important cause of the
infarct formation resulted from ischemia/reperfusion injury, which eventually leads to heart
failure. However, there is still no effective therapy that reduces cell death. In this study, we
demonstrate that epoxomicin, a proteosome inhibitor formerly used as an anti-cancer drug,
confers effective cardiac protection against Ischemia/Reperfusion injury. Methods and Results:
The C57 mice (3.5 months, n=7) were intraperitoneally injected daily with epoxomicin (0.5
mg/kg) or vehicle for 2 weeks. Mice were then subjected to 30 min ischemia followed by 24 hour
reperfusion. Myocardial infarct size was measured by TTC staining. Our data show that
SUNDAY-LATE POSTER PRESENTATIONS
epoxomicin reduced the infarct size by 50% (p<0.05), despite similar area-at-risk. To investigate
the mechanism behind the cardiac protection, in vitro study was performed on neonatal rat
cardiac myocytes (NRCM). NRCM were treated with epoxomicin (0.01μM) or vehicle for 3, 10,
or 24 hours, followed by hypoxia for 36 hrs. Proteasome activity was completely blocked by
epoxomicin within 3 hr of treatment. Epoxomicin treatment for 24 hrs reduced the hypoxia
induced cell death by 50% (n=6; p<0.01) as measured by TUNEL and PI staining, whereas
epoxomicin treatment for 3 and 10 hrs did not prevent any cell death, indicating the protection is
protein expression dependent. Western blotting showed 3.5 fold (p<0.01) increase in iNOS,
which was eliminated by NF-κB inhibitor SN50. The increase in iNOS was in accordance with 3
fold (p<0.01) elevated expression of H11K in epoxomicin treated NRCMs. Silencing of H11K in
NRCMs abolished the epoxomicin induced iNOS expression as well as the protection against
cell death. Conclusion: Long-term epoxomicin treatment increases iNOS expression, through
the activation of NF-κB by upregulated H11k expression. Thus provides NO-dependent
cardioprotection, in vivo equivalent to preconditioning, against ischemia/reperfusion injury. Our
data point to a novel role of epoxomicin as a promising therapeutic agent in protecting the
myocardium against ischemic heart disease.
2711
Estradiol and Estradiol plus Testosterone lead to the differentiated expression of AR,
Erα, ERβ and PCNA in gerbil female prostate (Meriones unguiculatus).
D. L. Silva1, M. Zanatelli1, F. Z. Shinohara1, F. A. Santos2, R. M. Goes3, S. Taboga3; 1Institute of
Biology, State University of Campinas (Unicamp), Campinas, Brazil, 2Department of Histology
and Embriology, Federal University of Goiás (UFV), Goiânia, Brazil, 3Biology Department, Univ
Estadual Paulista (UNESP), São José do Rio Preto, Brazil
The female prostate (FP) is a functionally gland found in several mammal species. Its
morphophysiology in the estrous cycle phases is influenced by estradiol and progesterone
oscillations. This study aimed to evaluate the effect of estradiol and estradiol plus testosterone
administration on the FP of ovariectomized gerbils. Five experimental groups were employed:
control (C), castrated (CA), castrated plus oil (CO), castrated plus estradiol (CE) and castrated
plus estradiol plus testosterone (CET). Except C group, all adult females had ovaries surgically
removed. After 10 days recovering, CE and CET females received subcutaneous doses of the
hormones (0.1 mg/kg) and CO received oil vehicle, each 48h. All females were killed 130 days
old. Gland sections were submitted to ERα, ERβ, AR and PCNA immunohistochemistry.
Although there is evidence that ERβ expression decreases with castration in adult rats or
advanced degrees of intraepithelial neoplasia, these features were not observed here, since the
expression pattern remained similar in all animals. Despite being commonly associated to the
stroma in men and rodents prostates, ERα was also found in FP epithelial cells with significant
labeling particularly in animals from CET. ERα also showed a decrease in CA stroma labeling.
When it comes to AR, we did not observe epithelial labeling differences among groups, but
stromal immunoreaction seems to be increased in CE and CET, which permits to associate this
steroid with glandular remodeling process. The PCNA showed a decreased reactivity in CA and
CO epithelia compared to C, which was recovered with both hormonal treatments. In the
stroma, we noted that there are more marked nuclei in CE and CET compared to other groups.
These findings prove the involvement of the steroids used in this study with the proliferative
activity of FP epithelial cells. Therefore, the coexistence of immunostaining for ERα and PCNA,
especially in the treated groups, may indicate a predisposition to the development of malignant
or premalignant lesions. In conclusion, it can be inferred that the FP is influenced by steroid
hormones in a similar way as male glands and its morphophysiology depends on the hormonal
balance, which reinforces the homology between these structures.
SUNDAY-LATE POSTER PRESENTATIONS
2712
Effect of minocycline treatment: a dorsal and ventral hippocampus
immunohistochemical analysis after traumatic brain injury of rats.
A. Blokhin1, N. E. Grunberg2, D. V. Agoston3, C. E. Kasper1; 1Department of Veterans Affairs,
Bethesda, MD, 2Medical & Clinical Psychology, USUHS, Bethesda, MD, 3Anatomy, Physiology,
& Genetics, USUHS, Bethesda, MD
Traumatic brain injury (TBI) consist of two phases: an immediate phase in which damage is
caused as a direct results of the mechanical impact; and a late phase of altered biochemical
events that results in delayed tissue damage and is therefore amenable to therapeutic
treatment. Our purpose for this research is following: what the effects of minocycline treatment
as intervention to aide de novo neuroregeneration at the molecular and cellular levels in
different part of hippocampus. Minocycline is a member of the tetracycline class of molecules
with broad-spectrum antibiotic activity. Aside from its antimicrobial properties, minocycline has
been found to have beneficial effect on inflammation, microglial activation and apoptotic cell
death. A rodent model of blast TBI is used. Rats were randomly divided into sham and injury
group with or without minocycline treatment. Neuronal apoptosis of dorsal and ventral
hippocampus of rats was observed by immunocytochemistry staining in each group. Results
Caspase 3 staining showed that the number of apoptotic neurons in minocycline injury group
was significantly less than in control injury group: 6.33±1.91 (dorsal) & 8.1±1.83 (ventral) vs
15.1±2.75 (dorsal) & 16.7±2.8 (ventral) for injury with minocycline and without minocycline
treatment correspondently. Also we found significant reduced apoptosis for different conditions
of minocycline treatment: 3.9±1.2 (dorsal) & 4.95±1.73 (ventral) vs 6.33±1.91 (dorsal) &
8.1±1.83 (ventral) for early and late minocycline treatment correspondently. These data suggest
that, after bTBI, minocycline treatment improved functional recovery in the injury rats. This
approach may provide a therapeutic intervention enabling us to reduce cell death and improve
functional recovery after bTBI. Supported by VA Merit B5044R.
2713
Cell Viability and Proliferation in Scaffolds Developed for Intervertebral Disc Tissue
Regeneration.
K. Toomer1, J. P. Sheehan1, A. M. Branda1, C. Wiltsey2, N. Albertson1, A. Nitzl1, T. Wooden1, A.
Vernengo2, C. Iftode1; 1Biological Sciences, Rowan University, Glassboro, NJ, 2Chemical
Engineering, Rowan University, Glassboro, NJ
The goal of our research is to develop a minimally invasive stem cell-based therapy treatment
for intervertebral disc degeneration (IVDD) using tissue engineering. Current procedures only
manage symptoms of IVDD including surgical disc removal or spinal fusion along with various
steroidal injections. Tissue engineering approaches make use of scaffolds to promote
regeneration and to provide sustenance to cells as they proliferate and differentiate into discspecific chondrocytes in vivo. The objective of the study is to compare the biocompatibility and
bioadhesive properties of three formulations of polymer scaffold enveloping adipose-derived
stem cells (ASCs). A scaffold polymer consisting of 5% Poly-N-isopropylacrylamide grafted
chondroitin sulfate (PNP-CS) was examined because the thermosensitive properties of the
polymer allow the formation of a hydrogel at human body temperature. An adhesive composed
of 3% oxidized chondroitin sulfate (CS-Ox) was also incorporated into the polymer scaffold and
tests investigating maximum stress and work of adhesion were preformed in order to optimize
the polymer’s mechanical properties. At a concentration of 4 mg/mL, Gelatin-loaded liposomes
(CS-Ox-liposomes) were incorporated to combat the cytotoxic effects of the CS-Ox adhesive. In
order to test the effectiveness of gelatin, PicoGreen measurements of double stranded DNA
were done using extracted DNA from adherent cultures of HEK-293 cells grown in CS-Ox
SUNDAY-LATE POSTER PRESENTATIONS
containing 0.3, 0.6, and 1.22 mg/ml of free gelatin. Data showed an increase in DNA amounts
from cells seeded with all gelatin concentrations, suggesting that the gelatin combats the
cytotoxic effects of CS-Ox. To evaluate biocompatibility of the three polymers, cells were grown
and in vitro XTT-based toxicology and Live/Dead chemical assays were performed. Data from
XTT showed similar viability in both PNP-CS and CS-Ox-liposomes, but decreased viability in
CS-Ox, as expected. Live/Dead results showed a higher ratio of live cells to dead cells in PNPCS. Results also showed an even distribution of cells in this polymer, a requirement for cell
proliferation and differentiation. Additional Live/Dead tests are in progress to confirm
biocompatibility of the polymers and that all polymers support uniform cell distribution. Future
studies hope to show viability, differentiation, and proliferation of ASCs in the same polymers.
2714
Shh inhibition improves renal function in Pkd2 mutant mice.
Y. Li1,2, W. wang1, A. Li1, H. Liu1, E. A. Green2, D. liang3, G. Wu3; 1State Key Laboratory of
Molecular Oncology, Cancer Hospital and Institute, Chinese Academy of Medical Sciences,
beijing, China, 2medicine, Vanderbilt University, nashville, TN, 3medicine, vanderbilt university,
nashville, TN
ADPKD is one of the most common monogenic diseases in the kidneys and is characterized by
numerous fluid-filled renal cysts. ADPKD is caused by mutations in either PKD1 or PKD2, each
of which respectively encodes the proteins, polycystin-1 (PC1) and polycystin-2 (PC2). Recent
studies indicate that PC1 and PC2 form a protein complex through interaction between their
COOH-terminals, and loss of either polycystins dysregulates multiple cell signaling pathways.
Such disruption may lead to aberrant ionic transportation, polarity, and proliferation/apoptosis in
renal epithelial cells and eventually cause renal cyst formation.
Since Shh protein has been reported to closely associate with the cilia of diverse epithelial cells
including those of the kidneys, we hypothesized that polycystins and Shh may have epistatic
effects and contribute to cyst formation in ADPKD. To this end, we employed Pkd2 and Shh
mutant mice to study their functional relationship in vivo. In this study, we have crossed
heterozygous Shh mice (Shh+/-) and Vil-Cre-derived Pkd2 conditional knockout mice (VilCre:Pkd2f3/f3) to generate compound Vil-Cre:Pkd2f3/f3:Shh+/- mutant mice. We performed a
cohort study for Vil-Cre:Pkd2f3/f3 mice with and without Shh+/- mutant alleles (n>20 for each
group). By examining their survival rate, kidney or liver/body ratio, renal cystic index, and renal
function of these mice, we found that there are no significant differences in lethal phenotypes or
renal cystic severity between mice with Vil-Cre:Pkd2f3/f3:Shh+/- and Vil-Cre:Pkd2f3/f3 alleles.
However, significantly improved renal function (BUN and Cr) can be seen in VilCre:Pkd2f3/f3:Shh+/- mice compared to Vil-Cre:Pkd2f3/f3 mice. We have also investigated
PC2-deficient effects in Shh+/- mice. Interestingly, we found that the lack of PC2 induces
solitary kidney in Shh+/- mice (20.6%). These results indicate that downregulation of Shh may
have a protective role in ADPKD models/patients and PC2 may engage in Shh-associated
kidney development
MONDAY-LATE POSTER PRESENTATIONS
MONDAY, DECEMBER 17
Science Education
2715
Science research-based collaboration between a high school teacher and university
scientists results in multiple benefits to students.
J. R. Sider1, W. M. Bement2, G. von Dassow3; 1Science Department, Amundsen High School,
Chicago Public Schools, Chicago, IL, 2Department of Zoology, University of Wisconsin-Madison,
Madison, WI, 3Oregon Institute of Marine Biology, University of Oregon, Charleston, OR
Here we describe an innovative long-standing collaboration between one high school teacher
and two cell biologists. This collaboration has exposed students to basic research questions,
applications, and process via university lab workshops, guest scientist lectures, class lessons
incorporating current cell image data, and an interactive website. One challenge of teaching
high school science, especially in under-performing urban schools, is exposing students to the
particulars and importance of basic research. Teaching science as process has always been
part of best practice pedagogy, and is now embedded in new national and state science
standards.
The four aspects of this collaboration are described and analyzed: 1) A hands-on cell imaging
lab workshop serving a total of 160 students over 4 years, including preparatory and follow up
lessons. 2) Guest cell biologist lectures, prepared in collaboration with the high school teacher,
at three different Chicago public high schools, serving all biology students at each school. 3)
High school biology lessons that incorporate current cell imaging data. 4) An interactive website
called “1 Cell 2 Cell” that teaches about cell division and big picture research questions and
process.
This collaboration has additional benefits: The high school teacher remains in constant touch
with current scientific developments and educational issues, and shares with other teachers.
The university lab students and scientists experience preparing for and working with high school
students. Details of the collaboration are shared with other teachers and scientists seeking
models for outreach partnerships.
2716
Think Globally Learn Locally: Introducing scientists into science classrooms.
K. A. Moore1, S. Gaukler1, H. Godsey2, D. Feener1; 1Biology, University of Utah, Salt Lake City,
UT, 2Geology and Geophysics, University of Utah, Salt Lake City, UT
There is currently a gap between how science is taught in the classroom and how science is
accomplished in the laboratory. This gap often leads to student misconceptions about what a
scientist’s job entails on a daily basis and how research is successfully performed. The NSF
GK-12 program Think Globally, Learn Locally (TGLL) aims to bridge part of this gap by
introducing graduate students in the biological, atmospheric and geophysical sciences into K-12
classrooms in an effort to improve graduate student communication skills, link teachers to
resources and research opportunities, and allow students a chance to interact with scientists on
a personal level. TGLL fellows are paired with a K-12 teacher throughout the year, and engage
in lesson planning and teaching within the classroom. In addition to working one-on-one with a
teacher in a classroom setting, TGLL fellows are also responsible for planning a cross-
MONDAY-LATE POSTER PRESENTATIONS
disciplinary module of lesson plans based on a rotating yearly theme. These objective-driven
modules often include a field experience, which provides an opportunity for higher-level
cognitive learning, and the chance for students to explore science in the world around them.
Here we present resources generated for a module based on infectious diseases and student
outcomes from module based activities. We will also share several general successes and
challenges of the program.
2717
From Motors to Proteins: A High School Physics Lesson.
J. Y. Sheung1,2, J. M. Scott3, S. Denos2; 1Physics, University of Illinois, Urbana, IL, 2Center for
Physics of Living Cells, University of Illinois, Urbana, IL, 3Normal Community High School,
Normal, IL
Biophysics has been an exciting, active field of research for the past three decades, yet to this
day few physics classes below the graduate level devote any time to this topic. We present a
lesson developed in collaboration between researchers at the University of Illinois and local high
school science teachers, which introduces students at the high school physics and AP physics
levels to molecular motors. During this lesson, which fits nicely into the typical unit on work and
energy, students compare the efficiency of themselves with that of a motor protein. They
perform a guided, hands-on experiment where they use their knowledge of physics to make
measurements directly analogous to those first made by biophysicists to determine the stall
force and step size distributions of motor proteins. Most students at the local high school
physics classes expressed surprise at the end of the lesson; it had never occurred to most of
them that physics and biology could be intertwined.
2718
Lessons learned from undergraduate students in designing a science course in
bioethics.
J. D. Loike1,2, B. Rush3, R. L. Fischbach2; 1Physiology and Cellular Biophysics, Columbia
University, New York, NY, 2Center for Bioethics, Columbia University, New York, NY, 3Postbac
Premed, Columbia School of General Studies, New York, NY
Columbia University offers two innovative bioethics undergraduate courses in the biosciences
and pre-health studies. The goals of these courses are to introduce future scientists and
healthcare providers to ethical questions they will no doubt confront in their professional lives,
and to enable them to begin to develop their own strategies to address bioethical dilemmas.
These courses incorporate innovative pedagogical methods, case studies, and class
discussions to stimulate students to think creativity about bioethical issues emerging from a
variety of new biotechnologies, such as stem cell science, cloning, and genetic engineering. To
assess the effectiveness of these courses, students are required to submit original one-page
strategies at the end of the semester describing how they would resolve bioethical dilemmas.
Two collective outcomes from the students’ responses are that these courses spark interest in
both science and bioethics and improve competence in analyzing bioethical challenges arising
from emerging biotechnologies. Based on a qualitative analysis of these student “strategies”, we
also present specific recommendations for creating or improving an undergraduate science
course in bioethics. Recommendations include: a) integrating the science of emerging
biotechnologies, their ethical ramifications, and contemporary bioethical theories into dynamic
and interactive class sessions, b) structuring discussion-based classes to stimulate students to
consider the impact of their moral intuitions in contemplating bioethical issues, and c) using
current and futuristic case studies to highlight bioethical issues and to develop creative student
problem-solving skills.
MONDAY-LATE POSTER PRESENTATIONS
2719
An assessment of student learning in traditional vs. on-line hybrid offerings of an
introductory molecular and cell biology course at a regional state university.
M. A. Daggett1; 1Biology, Missouri Western State University, St. Joseph, MO
Many faculty are under increasing pressure to provide students with alternatives to the
traditional course format by offering more of their courses on-line. One concern for biology
faculty is whether on-line introductory courses prepare students for further upper level courses.
During the past spring and summer semesters, students enrolled in a traditional introductory
molecular and cell biology course for majors at a regional state institution have been assessed
and compared with students enrolled in the same class offered as a on-line hybrid. This on-line
hybrid course did not include the posting of faculty presented lectures, but did require students
to read the text and complete on-line assessments and tutorials. Results from pre- and postcourse testing using a validated diagnostic assessment administered in a proctored testing
setting, indicate that there was no significant difference in the mean learning gains of the
important concepts as reported in previous studies. This research does not address all the
concerns faculty have in moving away from traditional course formats, but does suggest that
students are able to acquire important conceptual understanding in an on-line format that does
not include faculty lectures.
2720
Concrete approaches to link Research and Teaching in a large 2nd year undergraduate
classroom.
A. Ashok1; 1University of Toronto Scarborough, Toronto, ON, Canada
Science educators repeatedly recognize the importance of introducing the ever-changing and
somewhat murky waters of cutting-edge science in classroom teaching and yet, there always
remains the challenge of executing this process, especially in large undergraduate classes
(defined here as over 200 students). With the exception of incorporating data from recent
papers into lecture slides, other methods of engaging students with research findings have been
less prevalent. This could be due to the logistics involved in handling student groups or the
evaluation of the outcomes of such practices within large classes. I would like to share two
strategies that link student learning in fundamental courses to recent research (primary
literature) that have been incorporated into a large 2nd year Cell Biology course (BIOB10Y) at
the University of Toronto, Scarborough. The first strategy, which emphasized supervised, yet
self-directed learning, was a student assignment in which student groups picked a research
paper and generated specific questions, which they would ask during a recorded interview with
the corresponding author of the publication. The students would then produce an audiocast,
which included a summary of the paper and clips from the interview. The second strategy, which
emphasized peer-led learning, involved students in “Thinking Tutorials” that was led by a peer
who presented on-going research in areas only touched upon in lectures. Students were
encouraged to ask questions of the presenter and develop a good understanding of the
numerous unanswered questions in Cell Biology and the importance of fundamental research to
the future of medicine. The qualitative feedback from students was very positive on both
strategies. The audiocast assignment allowed students to easily conceptualize the link between
topics in the lecture to current research. Students were very excited to speak to the authors and
their interactions with these leading scientists gave them an appreciation of the human side of
science. In several interviews, the conversations seemed to naturally progress to the career
paths of the scientists, providing students with information on scicareer trajectories. The
Thinking Tutorials provided a relaxed environment in which students were able to reflect deeply
about current evidence for a specific model or therapy and relate it back to the classroom
MONDAY-LATE POSTER PRESENTATIONS
concepts. Students also became more open to the idea of reading scientific research per se, as
they observed their peers (who led the tutorials) able to delve through the scientific literature
around a topic of interest. The audiocast assignment required significant student effort, but the
finished product (a 5 minute audioclip) was a manageable grading expectation from the
instructor’s perspective. The Thinking Tutorials were exceptionally amenable to large classes,
as supervision of the peer student leader was the major requirement. Both strategies posed
some challenges and in addressing concerns, such as assessing individual contributions in the
audiocast group assignment, I hope to further refine what appears to be significant student
interest in the cross-talk between teaching and research in Cell Biology.
2721
Nanoparticle-based cell-targeting therapeutic systems: a golden opportunity for
innovation in cell biology.
D. J. Matthes1; 1Genetics, Cell Biology and Dev, University of Minnesota, Minneapolis, MN
Student learning is enhanced when students see how the concepts they encounter in a class
apply to issues beyond the classroom, issues that are authentic, of broad significance, and of
interest to the students themselves. The nanoparticle-based cell-targeting therapy design
project that will be described here is collaborative from beginning to end, spans an entire
semester of a senior-level cell biology course, is scaffolded to provide multiple opportunities for
peer review and instructor feedback, requires students to use much of what they learn in the
course, and involves verbal, written, and graphical communication of evidence, ideas, and
anticipated results.
Cell biology students are introduced to an emerging technology – therapeutic nanoparticles –
and asked to design a multi-layer nanoparticle therapeutic that, for a given human disease, (a)
targets a tissue with high specificity using pairs of target-specific surface proteins, (b) avoids
getting entangled in the extracellular matrix or stuck in body compartments lacking the target
cells, (c) enters the target cells without being destroyed by the lysosomal degradation system,
(d) exploits the cell’s available motor proteins to rapidly deliver inner layers of the nanoparticle
to the nucleus, (e) gains access to the nucleus with appropriately utilized nuclear localization
signals, (f) delivers DNA that is safely integrated into pre-determined chromosomal targets, and
(g) encodes one or more therapeutic agents as well as secreted reporter peptides capable of
informing the investigator/clinician how effectively the nanoparticle payload has arrived and is
being expressed.
While students in this course don’t work with nanoparticles in a wet lab setting and understand
that the nanoparticles as envisioned are beyond state-of-the-art, the design project requires
them to do the vital intellectual work of innovation-oriented scientists, gets them to see how a
knowledge of cell biology could contribute to biotechnological solutions for unmet medical
needs, and asks them to go beyond simple memorization, recall, and answering of isolated
problem set questions and instead to innovate, collaborate and communicate at a level that
transforms their experience of cell biology.
MONDAY-LATE POSTER PRESENTATIONS
2722
Using the SWoRD peer review system in an introductory biology course to improve
scientific literacy among undergraduates.
B. Steinwand1, K. Hogan1; 1Biology, University of North Carolina, Chapel Hill, NC
Engaging students in higher order thinking allows them to gain a deeper understanding of
scientific concepts and practices in critical thinking. We have integrated essay writing into a
large introductory biology course, Principles of Biology, to improve scientific literacy among
mixed majors. Our goal was improve our student’s ability to think critically about science as it
relates to society and make informed decisions on biological issues. The objectives of this
approach required students to use higher order thinking skills. We challenged them to analyze
and interpret scientific findings, evaluate news media articles for a bias, and support an opinion
with facts from credible sources. To facilitate the grading of essays in a large lecture course of
over 400 students, we used the web-based peer review system SWoRD. Students used
SWoRD to upload their essays, evaluate their peer’s written work, and revise and improve their
essays for a final review and grade. By allowing students to become their own teachers and
reviewers, SWoRD actively engaged students in the learning process. Here we present our
approach and the results that we obtained from pre/post SWoRD surveys on student
perceptions and beliefs of biological issues.
2723
A multi-session laboratory on temperature-sensitive transport to the lysosome for
intermediate level college molecular cell biology students.
A. C. Theos1; 1Human Science, Georgetown University, Washington, DC
We have developed a multi-session laboratory series that seeks to prepare intermediate-level
undergraduate molecular cell biology students for biomedical research internships. The series
involves learning a specific set of research skills including a basic set of techniques utilized
broadly throughout biomedical research. Students also gain first hand experience of the
sequential process of conducting a scientific investigation though experiments designed to
address the molecular basis of temperature-sensitive albinism caused by a missense mutation
in tyrosinase. The students are engaged throughout the semester as each session leads on
from one to the next and parallels central concepts including gene expression, conditional
mutations and protein structure and folding, integral membrane protein biosynthesis and
membrane transport to the melanosome/lysosome. The results of pre- and post-series analysis
of student learning conducted over three class iterations support the effectiveness of our
approach to facilitate student understanding of both the scientific concepts as well as the
techniques employed in their investigation. Open-ended course evaluations indicate that similar
format multi-session laboratory investigations involving “real world” research are likely to provide
a highly engaging learning environment as well as prepare students for subsequent immersion
in biomedical laboratory research environments.
2724
Development of Core Research Facility Encourages Shift in Institutional Focus Toward
Enhanced Student Research.
D. Cooke1, K. Pulliam1, V. Haftel1, M. Demetrikopoulos2; 1Morehouse College, Atlanta, GA,
2
Institute for Biomedical Philosophy, Dunedin, FL
Introduction: Morehouse College is an independent, fully accredited, historically black, liberal
arts college for men and has been a leader in the education of African American men in science
and mathematics. The College has developed a Core Scientific Instrumentation Facility (CSIF)
MONDAY-LATE POSTER PRESENTATIONS
within the Division of Science and Mathematics that has enhanced interdisciplinary teaching and
student research efforts across the Division. Methods: CSIF was conceptualized to include
both varied equipment as well as staffing necessary to maintain and coordinate the scheduling
of this resource including a full time Core Facilities Manager. Results: This core facility serves
both student research and teaching needs in interdisciplinary sciences such as bioinformatics
and neuroscience and has been integrated into existing STEM courses. This facility has served
as a mechanism to leverage the recent growth in opportunities for students to be engaged in
research on campus. This has allowed the Division to take the next step towards providing a
truly integrative research experience for students and to provide a successful mechanism for the
development of collaborative research experiences. Success of CSIF was partly responsible for
the ability of the College (Interdivisional) to obtain a NIH-funded Building Research
Infrastructure and Capacity (BRIC) grant. This has allowed the College to hire a full time
laboratory technician to support faculty and student collaborative research efforts occurring
within the HHMI supported CSIF. In addition, the efforts led to the development of an additional
shared facility, the Survey and Virtual Research Center (SVRC). Discussion: CSIF has
facilitated collaborations to further enhance student development by offering research
experiences on campus. Students benefit by receiving mentoring with varied theoretical focus.
Co-mentoring at an undergraduate liberal arts institution such as Morehouse College allows
faculty members to be involved in more substantive research. Thus their shared expertise,
time, and effort synergizes to provide a mechanism to conduct full research programs on
campus.
2725
The Vermont Genetics Network Outreach Program: Building a Culture of Research in
Vermont.
J. M. Murray1; 1Biology, University of Vermont, Burlington, VT
The Vermont Genetics Network (VGN) Outreach Program works with undergraduate college
faculty throughout the state to implement and integrate cutting edge technology into
undergraduate college science curricula. Thereby, increasing the skills of undergraduates and
encouraging them as future researchers in biomedical science.
The Vermont Genetics Network has developed three core facilities at the University of Vermont
(UVM); microarray, bioinformatics and proteomics. The VGN outreach program has developed
educational modules based on these technologies. Through these modules, the VGN outreach
team has worked with over 20 faculty from 8 baccalaureate colleges and over 560
undergraduate students. Seven of the eight colleges have integrated one or more modules into
their curricula and all eight BPIs have reported other curricula change influenced by VGN
outreach interactions.
Outreach surveys show that the educational and career goals of undergraduates are influenced
by our outreach programs. For instance, in 2012, of the 39 students who responded to our
online surveys, 66% said outreach increased their interest to study science or medicine. When
asked about their interest in pursuing a career in science and medicine, 69% said participation
increased their interest.
The outreach team also directly supports faculty and student research including working with
faculty to bring their research into the classroom by designing novel experiments for integration
into the modules, establishing relationships with the core facilities at UVM, working with
undergraduate students on independent senior projects as technical advisors and/or thesis
committee members, and offering technical support for project design, training and
troubleshooting, and extensive bioinformatics support for faculty. Through all the VGN
programs, a stronger culture of research is being developed at our partner institutes and around
the state.
MONDAY-LATE POSTER PRESENTATIONS
This work was sponsored by Grant Number P20 RR16462, from the IDeA Networks of
Biomedical Research Excellence (INBRE) Program of the National Institute of General Medical
Sciences (NIGMS) a component of the National Institutes of Health (NIH).
2726
The cell biology of Trypanosoma cruzi: how to teach using animations.
D. E. Teixeira1,2, M. Benchimol1,2, P. Crepaldi1,2, W. de Souza2,3; 1Cecierj Foundation, Rio de
Janeiro, Brazil, 2National Institute of Metrology, Quality and Technology, Rio de Janeiro, Brazil,
3
Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
Parasitic protozoa are important agents of human and veterinary diseases not only in Brazil but
also all over the world. The life cycle of Trypanosoma cruzi, the causative agent of Chagas
disease, is taught in different levels of education, from fundamental to graduate schools. The
aim of this work is to develop didactic multimedia material using three-dimensional (3D)
animations to visualize the life cycle of the parasite T. cruzi, which is a good model to analyze
the basic cell biology of parasites. This work also include (1) cell division, (2) endocytosis, (3)
flagellar beating, (4) the interaction of the protozoan with a vertebrate host cell, and (5) the
behavior of the protozoan in the digestive tract of the invertebrate host. In addition, this material
improves scientific accuracy and provides insights concerning the overall cell biology of this
parasite. All animations were produced by designers working at the CECIERJ
Foundation/CEDERJ Consortium, using software such as 3Ds Max, Maya, Hexagon, Blender,
Poser and Flash. Animations are a powerful tool to communicate quickly and easily scientific
ideas that are difficult to understand when described only with words or using static images.
This material is derived from scientific publications and previous experience provided by the
authors, and includes light and electron microscopy. The present multimedia material is useful
for a broad audience, which includes students, teachers, and any member of the general public
that may be interested in parasites.
2727
Team based learning for structured analysis and discussion of research papers in a
graduate course.
M. S. Risley1, T. U. Meier1, D. Cox1; 1Department of Anatomy and Structural Biology, Albert
Einstein College of Medicine, bronx, NY
Critical analysis of journal articles is a skill essential to science.
Both graduate and
undergraduate science curricula employ a "journal club" format for students to acquire these
skills. In our experience, whether papers are presented by faculty or students, this traditional
format often has weaknesses including uneven student participation and uncertainty regarding
the degree of mastery achieved by each student. To address these issues, we applied teambased learning (TBL) for small group research paper analysis in a graduate Molecular Cell
Biology course. For two separate iterations of the course, students were sorted into teams of
5-7 that were maintained throughout course.
In each repeat of the course, teams were
assigned the task of reading four research articles chosen to reinforce major course concepts
and research strategies. Individual Readiness Tests (IRATS) and Group Readiness Tests
(GRATS) consisting of five multiple choice questions were administered to each individual and
team for each of the four assignments. Then each team was asked to solve a set of significant
problems relevant to the research article. The IRAT and GRAT test data were compared to
each other and to data for four major problem oriented exams based on lecture content. Overall
IRAT averages (80.3) were 16% higher than exam averages but individual’s IRATS did not
correlate with their exam scores. Students with poor exam scores often performed well on
IRATS. Overall GRAT averages (96.7) were higher than IRATS and team GRAT scores were
MONDAY-LATE POSTER PRESENTATIONS
not dependent on the exam scores or IRAT scores of individual team members. This indicates
that cooperative learning between team members led to the increase in correct answers on
GRATS. All teams cooperatively and effectively solved the final set of significant problems, as
revealed by discussion with faculty facilitators. The data suggest that team commitment
motivates each student to prepare adequately for paper discussions. Comments from student
evaluations indicated a high level of satisfaction with the TBL approach. Faculty facilitators
were all highly satisfied with the overall student performance, team cooperative efforts at
problem solving and the quantification of these parameters. Overall, TBL based research
paper analysis and discussion were viewed as more effective than traditional journal club style
approaches.
Actin Cytoskeleton
2728
Cooperativity and redundancy in the mechanics of compositely crosslinked cytoskeletal
networks
M. Das1, D. Quint2, J. M. Schwarz3; 1Physics and Astronomy, Rochester Institute of Technology,
Rochester, NY, 2Physics, University of California, Merced, Merced, CA, 3Physics, Syracuse
University, Syracuse, NY
The actin cytoskeleton contains many types of crosslinkers---for example, crosslinkers that
allow for free rotations between filaments and others that constrain the angle between crossing
filaments. The mechanical interplay between different crosslinkers is an open issue in
cytoskeletal mechanics. We develop a theoretical framework to study such a compositely
crosslinked actin network to address this issue. We use an effective medium theory and
computational modeling to carry out our investigations, with very good agreement between the
two.
We find that the addition of angle-constraining crosslinkers, acting cooperatively with freelyrotating crosslinkers, allows the network to attain mechanical rigidity (a nonzero shear modulus)
as soon as there exists a connected percolating cluster. This finding suggests the optimal
conditions for the actin cytoskeleton to become rigid and be able to bear and transmit forces
with the minimal amount of material possible. Without angle-constraining crosslinkers, the
rigidity percolation threshold requires a higher density of filaments such that the transmission of
forces is not as efficient. We further demonstrate that the introduction of angle-constraining
crosslinks in flexible filament networks results in mechanical response similar to just freelyrotating crosslinked semiflexible filaments indicating redundancy. Our results also impact upon
tissue engineering by providing design principles for developing filamentous scaffolds with
tunable mechanical properties.
Reference: Redundancy and cooperativity in the mechanics of compositely crosslinked
filamentous networks, M. Das , D. Quint and J.M. Schwarz, PloS one 7, 35939 (2012).
MONDAY-LATE POSTER PRESENTATIONS
2729
p90RSKdelays cell cycle at G2/M in response to actin disruption in the primary fibroblast
IMR90 cells.
D. Shrestha1, D. Choi1, K. Song1; 1Biochemistry, Life Science and Biotechnology, Yonsei
University, Seoul, Korea
Actin, one of the three major components of the cytoskeleton, plays a role at the entry of
mitosis, in addition to its function in cytokinesis. In previous studies, we had shown that in
presence of actin disruption, the sustained activation of ERK1/2 delays mitotic entry by
maintaining the inhibitory phosphorylation of Cdc2 (Y15) in primary mammalian cells. Further,
we studied the mechanism of this mitotic entry delay by actin dysfunction in IMR90 cells to
search for the downstream targets of ERK1/2 kinase that promote and maintain the inhibitory
phosphorylation of Cdc2 (Y15). We show here that depolymerization of actin with cytochalasin
D constitutively activates p90RSKand induces inhibitory phosphorylation of Cdc25C (S216) at
G2/M phase in IMR90 cells. The effect of cyotochalasin D leading to the sustained activation of
p90RSK was observed only in primary IMR90 cells but not in cancerous Hela cells. To confirm,
p90RSK as a downstream target of ERK1/2 kinase in actin disrupted G2/M delay of IMR90 cells,
p90RSK kinase-dead form (DN- p90RSK) was overexpressed in actin disrupted cells. Consistent
with the sustained activation of p90RSK in actin disrupted G2/M delay, the cells entered mitosis
without delay when p90RSK kinase-dead form (DN- p90RSK) was overexpressed. We also
observed the reduced inhibitory phosphorylation of Cdc25C (S216) and Cdc2 (Y15) in kinasedead form (DN-p90RSK) p90RSK transfected IMR90 cells with actin dysfunction. However, when
we examined the phosphorylation of two major kinases, Chk1 and Chk2 in presence of actin
defect IMR90 cells, their phosphorylation were not observed, suggesting that DNA Damage
Checkpoint pathway is not involved in monitoring actin disruption at G2/M. Moreover, MEK
kinase phosphorylation was similar in cytochalasin D treated and untreated primary IMR90 cells.
This indicates that MEK kinase may not be the upstream kinase as in regular MEK-ERK-RSK
pathway in delaying cell cycle by actin defects at G2/M. Thus we suggest that there is an actin
checkpoint at G2/M and p90RSK is a mediator kinase that regulates mitotic entry through
inhibitory phosphorylation of Cdc25C (S216) in presence of actin defect IMR90 cells.
2730
CAP2 and its contribution to 6p22 Syndrome.
D. Ye1, M. Shinde1, G. Vizcarrondo1, J. Field1; 1Department of Pharmacology, University of
Pennsylvania, Perelman School of Medicine, Philadelphia, PA
Patients with 6p22 syndrome have deletions in this region of the chromosome and have varying
degrees of the following phenotypes: autism spectrum disorders, short stature, eye
abnormalities, ear anomalies, short neck, and mild heart defects. Little is known about most of
the genes in the region. One of them is CAP2, a member of the Cyclase-associated proteins
(CAP), a family of proteins that bind actin monomers (G-actin) and regulate the balance
between G-actin and filamentous actin (F-actin). There are two homologs of CAP in mammals,
CAP1 and CAP2. CAP1 is expressed in most tissues, while CAP2 is expressed primarily in
heart, skeletal muscle and brain.
To determine the function of CAP2 in vivo we generated
CAP2 knockout (KO) mice and found a number of phenotypes, all of which are more penetrant
in males. Both male and female mice are viable but by weaning are found below Mendelian
ratios (17% females and 5 % males). The surviving mice continue to have reduced survival
rates and are smaller. Treadmill experiments showed that males ran shorter distances and
exhausted faster than the controls while there were no significant differences in females. Male
CAP2 KO mice have increased heart rates, diastolic and blood pressure. An analysis of hearts
by ECHO and histology revealed that they were enlarged. Together the cardiovascular
MONDAY-LATE POSTER PRESENTATIONS
phenotypes are consistent with dilated cardiomyopathy, which would account for the sudden
death of most animals. In addition to the cardiac phenotypes, we also observed frequent eye
infections and microphthalmia in the CAP2 KO mice, suggesting an eye developmental problem
as also seen in Drosophila homolog mutants. Taken together, our results show that CAP2 is
required for growth, maintaining cardiac homeostasis and eye development. The similarities of
these phenotypes with the patients who have deletions of 6p22 suggest that CAP2 contributes
to some of their clinical phenotypes.
2731
Functions of tropomyosin in vivo.
A. Cho1, D. Montell1; 1Department of Biological Chemistry, The Johns Hopkins School of
Medicine, Baltimore, MD
Cytoskeletal tropomyosins (Tm) are F-actin binding proteins that stabilize contractile actin
filaments in the lamellum. Tms are also thought to inhibit lamellipodium formation by modulating
the actions of Arp2/3 and cofilin. However there are more than 40 different mammalian Tm
isoforms having different subcellular localizations and possibly different functions. Little if
anything is known regarding the functions of cytoskeletal Tm isoforms in vivo.
Drosophila egg chambers provide a genetically tractable in vivo model for studying cytoskeleton
dynamics in both migrating and stationary epithelial cells. Drosophila has one gene that
encodes cytoskeletal Tm isoforms. Here, we report that of the six predicted non-muscle
isoforms of Tm1, only two, Tm1-A and Tm1-I, are expressed detectably in follicle cells of the
Drosophila egg chamber. When these two isoforms are overexpressed in S2 cells, they show
mutually exclusive localizations. Moreover, these isoforms also exhibit distinct subcellular
localizations in vivo. Since previous studies report that different Tms localize to different actin
structures and some play a role in recruiting myosin to the actin fibers, we looked at the outer
follicle cells, where distinct stress fibers can be observed near their basal surfaces. The
organization of F-actin was disturbed and the recruitment of myosin was reduced in Tm1 mutant
follicle cells. Furthermore, the cell diameter appeared to be affected. Overexpression of the two
isoforms together, but not the individual isoforms, causes the opposite effect on cell size and a
change in the overall size of egg chambers. We are also investigating the role of Tm1 in the
collective migration of the border cells where Tm1-A and Tm1-I are the only two isoforms
detectably expressed. Using two different mutant alleles of Tm1, we have shown that reduced
Tm in border cells results in migration defects. Isoform-specific functions in these cells are
currently under investigation. The fruit fly egg chamber serves as an excellent in vivo model in
which to study Tm function at the subcellular, cellular and whole organ levels.
2732
Insights into the structure and function of the RING ubiquitin ligase AO7 (RNF25).
D. K. Stringer1, Y. C. Tsai1, J. Mariano1, S. Li1, Y. Liang2, R. Das3, A. Byrd3, X. Ji2, A.
Weissman1; 1Laboratory of Protein Dynamics and Signaling, National Cancer Institute,
Frederick, MD, 2Macromolecular Crystallography Laboratory, National Cancer Institute,
Frederick, MD, 3Structural Biophysics Laboratory, National Cancer Institute, Frederick, MD
The discovery that the RING finger protein AO7 (RNF25) binds tightly to ubiquitin-conjugating
enzymes (E2s) and has ubiquitin ligase (E3) activity helped lead to the determination that RING
finger proteins are, in general, ubiquitin ligases. This high affinity E2 binding is atypical for RING
fingers, which generally bind to E2s with relatively low affinity. We have solved the structure of a
complex of AO7 RING finger and the E2, UbcH5B (Ube2d2). This structure reveals that, in
addition to the well-characterized interaction between RING finger domains and E2s, AO7 also
contains a second discrete alpha-helical UbcH5B binding region (U5BR), providing a molecular
MONDAY-LATE POSTER PRESENTATIONS
explanation for the high affinity interaction between these two molecules. The function of AO7 in
cells is not clear. We found that transfection of AO7 induced formation of long protrusions in
cells. We are currently investigating the molecular effects of AO7 overexpression in cells.
2733
Cortactin phosphorylation recruits Vav2 to regulate cell edge protrusion.
B. J. Rosenberg1, C. Mader1, A. Koleske1; 1Yale School of Medicine, New Haven, CT
Tight spatiotemporal control of the actin cytoskeleton is vital for motile cells that undergo
directed cell migration, including fibroblasts during wound healing and cancer cells during
metastasis. For example, cell edge protrusion is necessary to form a leading edge in fibroblast
chemotaxis.
In previous work, we have shown that cortactin, an actin nucleation promoting factor, is
phosphorylated by Abl family kinases downstream of growth factor receptor activation, and that
this interaction is important in promoting invadopodia formation in breast cancer cells and cell
edge protrusion in fibroblasts. Nevertheless, the biochemical and functional downstream
consequences of cortactin phosphorylation are as yet poorly understood. In an effort to explore
these downstream events, we screened a library of SH2 domains in the human proteome for
specific interaction with phosphorylated cortactin. Of the 10 SH2 domains identified in that
screen, we focused on that belonging to the Rho-family guanine nucleotide exchange factor
Vav2. Vav2 is a member of a small family of RhoGEFs that are unique in that they contain both
a Dbl homology domain and SH2/SH3 motifs, which are likely important in regulating the
spatiotemporal activation of their catalytic activity.
We show that Vav2 binds specifically to phosphorylated cortactin, but not to a nonphosphorylatable mutant both by in vitro binding assays and in cells by co-immunoprecipitation.
We also show that Vav2 and cortactin co-localize in fibroblasts spreading on fibronectin at cell
edge protrusions, and that disruption of the vav2:cortactin interaction interferes with this colocalization, as well as fibroblast dorsal ruffle formation downstream of PDGF stimulation.
2734
wdr1 (AIP1) is required for myeloid development in zebrafish.
M. J. Redd1, M. Yousfi2, E. Carlot-Muryama2, W. Horsley1, M. Tauzin2, P. Herbomel2, N. Trede1;
1
Oncological Sciences, University of Utah, Salt Lake City, UT, 2Institute Pasteur, Paris, France
In a genetic screen for deficiencies in neutrophil development in zebrafish, we recovered carmin
(car), a mutant that is characterized by the loss of neutrophils and macrophages at three days
post fertilization (dpf). The car mutants eventually succumb with severe edema between six and
ten dpf. Mapping led to the identification of a mutation in the wdr1 gene on chromosome 14.
Since WDR1 is known to act together with cofilin to dissassemble actin filaments, we stained
car mutants with phalloidin and found that F-actin accumulates in myeloid cells prior to their
disappearance as well as in the epidermis. Time-lapse microscopy revealed that the few
surviving neutrophils in car mutants fail to chemotax normally towards small wounds in the fins.
In addition we find examples of car neutrophils failing to complete cytokinesis followed by
apoptosis. The car mutant phenocopies activating alleles of WASp that lead to severe
congenital neutropenia in humans.
MONDAY-LATE POSTER PRESENTATIONS
2735
Characterization of a novel player in myosin independent cytokinesis.
J. Xu1, A. Nayeri2, W. Cande2, A. R. Paredez1; 1Biology, University of Washington, Seattle, WA,
2
MCB, UC Berkeley, Berkeley, CA
Giardia is a highly divergent eukaryote that is responsible for over 100 million parasitic
infections annually. While Giardia lacks all canonical actin binding proteins (ABPs), the actin
cytoskeleton has an elaborate organization that is dynamically regulated throughout the
lifecycle. Moreover, the giardial actin cytoskeleton is required for essential cellular processes
including trafficking, cytokinesis, and cellular organization. Thus defining the mechanisms of
actin regulation in these essential processes will provide both evolutionary insight as well as
novel chemotherapeutic targets for treating this parasite. Like in other eukaryotes, the actin
cytoskeleton of Giardia is in part regulated by Rho family G-protein signaling. Perturbing this
signaling system leads to abnormal cytokinesis as well as reduced rates of proliferation and
attachment. How this signaling system is linked to the cytoskeleton remains unresolved. We
are currently focused on understanding the mechanism of cytokinesis as Giardia lacks both the
contractile ring and midbody structures normally associated with cytokinesis. Given that other
parasitic (and presumably deep branching) eukaryotes such as Trichomonas and
Trypanosomes also lack these structures, studying cytokinesis in Giardia may lead to a broader
understanding of this process. Through a combination of biochemical and bioinformatic
approaches we have identified novel ABPs. One of these proteins, although novel, contains a
Bro1 domain which is thought to bind actin and recruit the ESCRT membrane remodeling
complex. Interestingly this protein is enriched in the ingressing cleavage furrow and its
depletion leads to cytokinesis defects. This protein represents a starting point for defining the
mechanism of cytokinesis in Giardia.
2736
Mechanisms underlying the organization of polarized cytoskeletal structures in the
Drosophila embryo.
A. K. Spencer1, S. Yamamoto2, V. Bayat2, M. Jaiswal2, N. Haelterman2, H. J. Bellen2, J. A.
Zallen1; 1Developmental Biology, HHMI and Sloan-Kettering Institute, New York, NY, 2Baylor
College of Medicine, Houston, TX
Epithelial cells are capable of producing a wide variety of specialized structures with unique
shapes, from hairs to sensory bristles to stereocilia. Ventral epidermal cells of the late
Drosophila embryo generate an array of actin and microtubule-based apical protrusions called
denticles. Each group of denticle cells undergoes directional cell-shape changes and cell
rearrangements to form six distinct columns of highly aligned and elongated cells that elaborate
denticles at their posterior margin. Each cell column produces denticles that display unique
morphological properties, including denticle size, shape, and the directionality of the denticle tip.
These differences, coupled with the regularity of the overall denticle array, makes this an ideal
system for identifying new regulators of actin and microtubule organization, planar cell polarity,
and cell-cell communication. We have used forward genetic screening of the X chromosome to
identify mutants that fail to generate the normal organization of denticles in the cuticle. Of
approximately 750 lines screened, we identified 17 mutants with specific defects in denticle
organization, polarity, or morphology. We are currently examining the effects of these mutations
on the planar polarized organization of the actin cytoskeleton, microtubules, and the cell cortex.
In one mutant that displayed denticle disorganization in the initial screen, confocal images
stained for cell junctions and F-actin revealed that this phenotype is due to both the incorrect
placement of denticle structures within cells as well as the failure of denticle cells to correctly
change shape, align and elongate. We are currently using live imaging to identify cell behaviors
MONDAY-LATE POSTER PRESENTATIONS
in the denticle field that may be responsible for these defects. Several other mutants had
defects in denticle orientation in the cuticle, but cell shape and denticle placement appeared
normal by confocal imaging. We are now examining the role of localized cytoskeletal regulators
involved in directional actin organization at the denticle tip. The goal of these studies is to
identify new mechanisms and processes that are involved in the generation, maintenance, and
large-scale organization of cytoskeletal structures during tissue development.
2737
Rnd3 controls osteoclast bone resorption by regulating podosome organization.
D. H. Georgess1, M. Mazzorana1, J. Terrado2, I. Perez-Roger3, I. Machuca-Gayet1, P. Jurdic1;
1
Cell Biology and Bone Physiopathology, Ecole Normale Supérieure de Lyon, Institute of
Functional Genomics of Lyon, Lyon, France, 2School of Veterinary Sciences, Department of
Animal Medicine and Surgery, Universidad CEU Cardenal Herrera, Valencia, Spain, 3School of
Health Sciences, Department of Chemistry, Biochemistry and Molecular Biology, Universidad
CEU Cardenal Herrera, Valencia, Spain
Osteoclasts (OCs) are giant multinucleated cells of the monocytic lineage that are responsible
for bone resorption. On a non-mineralized substrate, differentiating OCs form actin-rich
adhesive structural units called podosomes that organize into clusters or rings. In mature OCs,
rings fuse into a peripheral belt. On bone matrix, however, podosomes condense in a thick
central belt called the sealing zone (SZ). The latter isolates the resorption pit from the rest of the
extracellular milieu thus locally maintaining a high concentration of actively secreted protons
and enzymes. The molecular mechanisms involved in the spatiotemporal substrate-dependent
regulation of podosomes are still poorly understood.
In vitro, OCs differentiate from monocytes or dendritic cells (DCs). Recently, a new population of
human multinucleated giant cells (MGCs) resulting from DC differentiation has been described.
These MGCs share several common markers with OC but are, however, unable to resorb bone.
By immunofluorescent co-labeling of actin with cortactin, vinculin or paxillin, we showed that
MGCs form bona fide podosomes. Additionally, when seeded on a bone mimicking substrate,
MGC podosomes organize into circular structures reminiscent of podosome rings in OCs. We
decided to compare OCs and MGCs to identify novel proteins essential for OC-mediated bone
resorption. Therefore, we performed differential transcriptomic microarrays to identify genes that
are highly expressed only in OCs but not in DC-derived MGCs. Their expression was confirmed
by Taqman Low Density array. One of the shortlisted genes was Rnd3. By culture assays using
primary murine Rnd3-/- OCs, we evinced that Rnd3 is not important for OC differentiation but
essential for bone resorption. Interestingly, we observed that clustered podosomes are fewer
per cell, but larger in diameter in Rnd3-/- OCs compared to Rnd3+/+ OCs. FRAP analysis
revealed, in Rnd3 deficient osteoclasts, a decreased turnover rate and increased mobile fraction
of actin in podosome clusters. In contrast, only actin turnover rate was reduced in podosome
rings. The imbalance in actin recruitment and kinetics in podosomes was also correlated with a
higher number of OCs with rings.
Altogether, these results demonstrate an essential role of Rnd3 in OC-mediated bone resorption
by tightly balancing podosome remodeling. We may thus speculate that ring formation is an
essential checkpoint for podosome maturation and bone resorption
MONDAY-LATE POSTER PRESENTATIONS
2738
Force-regulated actin filament assembly and disassembly during formation of stress
fibers.
S. S. Tojkander1, G. Gateva1, P. Lappalainen1; 1University of Helsinki, Institute of Biotechnology,
Helsinki, Finland
Stress fibers are central determinants of adhesion and motility of non-muscle cells. Based on
their protein compositions and associations with the focal adhesions (FAs), stress fibers can be
divided into three different groups: dorsal stress fibers, transverse arcs and ventral stress fibers.
Dorsal stress fibers are anchored to FAs at their distal ends and elongate towards cell center
through actin polymerization at FAs. Transverse arcs, which are actomyosin bundles connected
to dorsal stress fibers, are generated through annealing of Arp2/3- and mDia2-nucleated actin
filament nodes parallel to the leading edge of the cell. During cell migration, arcs flow towards
the cell center along with the elongating dorsal stress fibers. Contractile ventral stress fibers,
which are anchored to focal adhesions at each end, are then derived from the pre-existing arc –
dorsal stress fiber network. However, the mechanisms regulating the assembly and
disassembly of actin filaments in the arc – dorsal stress fiber network that ensure proper
formation and contractility of ventral stress fibers, have remained elusive. Here we show that
during retrograde flow, transverse arcs fuse together to form thicker actomyosin bundles.
Coinciding with the fusion, arcs become more contractile and consequently affect the angles of
FAs residing in the ends of arc-associated dorsal stress fibers. Importantly, along with the
formation of contractile ventral stress fibers, actin polymerization at FAs in the ends of these
actomyosin bundles halts. Inhibition of actin polymerization at FAs appears to be indeed
dependent on contractility, because disruption of myosin II containing arcs led to persistent actin
polymerization at FAs. Also actin filament disassembly during ventral stress fiber formation is
dependent on contractility, because cofilin-1 localized only to non-contractile dorsal stress fiber
regions. Furthermore, cofilin-1 depletion by siRNA led to defects in arc fusions as well as
formation of an abnormal ventral stress fiber network. Together, these data provide evidence
that contractility controls both actin filament assembly and disassembly in cells to ensure the
proper formation of ventral stress fibers.
2739
A nuclear function for formins in MRTF-A regulation.
C. Baarlink1, H. Wang1, R. Grosse1; 1University of Marburg, Marburg, Germany
Formins are defined by the presence of the formin homology 2 (FH2) domain, which directly
promotes asssembly of actin filaments for cellular processes such as motility, cytokinesis and
morphogenesis. One consequence of formin induced actin dynamics is the regulation of the
actin binding protein and myocardin-related transcription factor MRTF-A (MAL). Signal-induced
depletion of actin monomers promote release of actin from MRTF-A in both the cytoplasm and
the nucleus resulting in transcriptional co-activation of its partner protein SRF. Interestingly, it
has been previously noted that certain formins such as mDia1 and mDia2 passage through the
nuclear compartment but the functional relevance of this is entirely unclear. Here we show that
formins specifically control actin dynamics inside the mammalian nucleus to drive serumdependent MAL/SRF activity. Nuclear mDia-dependent actin turnover appears to be necessary
and sufficient to efficiently regulate MAL/SRF. Our data indicate that the function of nuclear
formins is part of the serum response to induce rapid assembly of endogenous actin filaments.
MONDAY-LATE POSTER PRESENTATIONS
2740
ROBO1's Role in Mediating Tensional Homeostasis.
L-N. T. Le1, A. Moran2, H. Macias3, P. Keely4, L. Hinck2; 1Molecular, Cell, and Developmental
Biology and Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, CA,
2
Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, Santa Cruz,
CA, 3Diabetes Center, University of California, San Francisco, San Francisco, CA,
4
Pharmacology, Biomedical Engineering, University of Wisconsin, Madison, WI
The extracellular matrix (ECM) plays a critical role in mediating cellular processes such as
adhesion, differentiation, and proliferation by modulating signaling and structure molecules.
Matrix rigidity is capable of altering biological processes, thereby increasing the risk of tumor
formation.
Many studies have demonstrated a critical role for transmembrane receptors in
communicating extracellular cues and mediating biological activity.However, little is known
about how transmembrane receptors convert physical stimuli from the ECM into chemical
signals that remodel the cytoskeleton and maintain tensional equilibrium. We have identified the
transmembrane Roundabout receptor, ROBO1, as upregulated in several types of tumors and
in
breast cells plated in a high-tension environment. This, combined with its ability to induce
remodeling of the cytoskeleton, leads us to hypothesize a role for ROBO1 in mediating tensional
homeostasis by balancing forces in and outside the cell.
We use the mammary gland as a model system to study cellular dynamics. The
mammary gland is a tubular, tree-like structure, composed of an outer layer of myoepithelial
cells (MECs), encircling an inner layer of luminal epithelial cells (LECs). During development,
ROBO1 is expressed in the MEC layer that is adjacent to the ECM and stroma. Our preliminary
data show decreased stress fiber formation in Robo1-/- tissue as compared to wild-type.
Concordantly, treatment of mammary MECs with SLIT activates Rac1 and increases the
thickness of stress fibers. Our co-immunoprecipitation (co-IP) data show that ROBO1 interacts
with membrane cytoskeletal proteins paxillin and beta-catenin, suggesting a potential role in
linking focal adhesions to adherens junctions. Together, these data suggest ROBO1’s
regulatory role on actin dynamics could take place at cellular adhesion sites.
Studies are currently underway to address ROBO1 regulation on cellular dynamics in
response to tension by using 3D collagen assays. Furthermore, we intend to model ROBO1
intracellular interactions in response to ECM rigidity by constructing deletion mutants of
conserved protein binding domains and probing for changes in protein association through coIP. We propose to elucidate how ROBO1 regulates internal and external cellular forces through
mechanotransduction that ultimately leads to the remodeling the ECM and actin cytoskeleton.
2741
COTL1 is required for lamellipodial protrusion at the immune synapse (IS) during T cell
activation.
J. Kim1, V. Shapiro2, H. Higgs3, D. D. Billadeau1; 1Biochemistry and Molecular Biology, Mayo
Clinic Graduate School, Rochester, MN, 2Immunology, Mayo Clinic Graduate School,
Rochester, MN, 3Biochemistry, Dartmouth Medical School, Hanover, NH
For T cell activation, immunological synapse (IS), specialized interface between T cells and
antigen-presenting cells (APCs) or target cells forms and spatiotemporal multiple processes
take place after T cell recognition of APCs. TCR engagement with peptide-MHC complex elicits
rapid rearrangement of cytoskeletal network to the IS which is critical for molecular signaling
pathways as well as molecular structure, namely, signaling molecules cluster at the central
supramolecular activation complex. It is well studied that actin is polymerized and polarized at
MONDAY-LATE POSTER PRESENTATIONS
the IS for T cell activation, and here we suggest that Coactosin like 1 (COTL1), a member of
actin depolymerizing factor homology (ADF-H) family regulates filamentous (F)-actin dynamics
by knocking down COTL1 using shRNA. We demonstrate that COTL1 depletion does not have
effects on TCR-mediated signaling processes, IL-2 secretion, adhesion between T cells and
APCs, cSMAC formation, and MTOC polarization to the IS. However, we observed that COTL1
and F-actin are recruited to the IS in response to TCR ligation as well as disruption of binding to
F-actin abrogates its polarization. Confocal live images show that lammellipodial protrusions by
TCR-ligation by anti-CD3 antibody coated on coverslip as well as by conjugation with Raji B
(APCs) are impaired by COTL1 depletion and mutation in F-actin binding.
Cell Migration and Motility
2742
Lysosomal activity is required for fast migration of mature dendritic cells.
M. Bretou1, P. Vargas1, P. Pierobon1, E. Terriac2, M. Piel2, A-M. Lennon-Dumenil1;
1
U932, Institut Curie, Paris, France, 2UMR 144, Institut Curie, Paris, France
Migration of Dendritic Cells (DC) to the lymph nodes is an essential step for the initiation of
immune responses. At the steady state DC reside at the periphery of the body, where they
sense for the presence of danger signals, such as bacterial particles. This key function of DC
relies on their ability to internalize important amounts of extracellular material -a process
referred to as macropinocytosis- while they migrate across the tissue. In case of infection DC
recognize and internalize the pathogenic antigens and initiate a maturation program. This
process is characterized by an increase in lysosomal activity (to degrade internalized antigens)
and the cell ability to migrate towards the lymph nodes, to interact and activate specific T
lymphocytes.
To study in-vitro the mechanism controlling DC migration after activation, we developed microfabricated channels that mimic the natural constrained environment of tissues. Using LPS as an
activator, we quantified changes in motility induced during DC maturation, and found that LPS
triggered a faster and more persistent migration. This increased motility correlated with changes
in the actin cytoskeleton. In non-activated DC polymerized actin was mainly localized at the
front of the cells, around vesicular structures formed during migration (macropinosomes). After
LPS activation actin was relocalized at the cell rear, in a cortical patch located behind the
nucleus. Further analysis revealed that this patch was in close apposition to lysosomes. Since
lysosomal activity is increased after DC activation, we hypothesized that it is required for fast
DC migration. To test this idea we inhibited lysosomal activity using both pharmacology and
genetics. Such inhibition indeed impaired LPS induced increase in motility, without altering the
activation of DC. We propose that lysosomal activity couples antigen degradation to fast cell
motility to favors the migration of the cells in which antigenic processing has occurred.
2743
FIGNL2 is a negative regulator of cell motility in vitro and in vivo.
R. A. Charafeddine1, J. Makdisi1, P. Nacharaju1, D. Schairer1, A. Friedman1, J. Friedman1, J. D.
Nosanchuk1, D. Sharp1; 1Physiology and Biophysics, Albert Einstein College of Medicine, Bronx,
NY
Fidgetin-like 2 (FIGNL2) is a previously uncharacterized member of the AAA protein superfamily
so named because of its similarity to the known microtubule severing enzyme, Fidgetin. Here
MONDAY-LATE POSTER PRESENTATIONS
we show that FIGNL2 is a strong negative regulator of cell motility both in vitro and in vivo.
Depletion of FIGNL2 from human tissue culture cells results in a significant elevation of cell
migration in standard wound healing assays. Immunofluorescence analysis shows that FIGNL2
knockdown affects both microtubule density and organization, especially, the more stable,
acetylated microtubules. These changes are accompanied by an increase in actin filament
density. Consistent with these in vitro observations, topical application of nanoparticle
encapsulated FIGNL2 siRNA to full thickness biopsy wounds made in the back of living mice
significantly enhances the rate of wound closure relative untreated and mock treated controls.
Studies are currently underway to characterize the impacts FIGNL2 on various aspects of
cytoskeletal dynamics as well as wound healing kinetics.
2744
SCAR/WAVE-mediated processing of engulfed apoptotic corpses is essential for
effective macrophage migration in Drosophila.
I. R. Evans1, P. Ghai1, K-L. Tan1, W. Wood1; 1Department of Biology and Biochemistry,
University of Bath, Bath, UK
In vitro studies have shown that SCAR/WAVE activates the Arp2/3 complex to generate actin
filaments, which in many cell types are organised into lamellipodia that are thought to play an
important role in cell migration. Here we demonstrate that SCAR is utilised by Drosophila
macrophages to drive their developmental and inflammatory migrations and that it is regulated
via the Hem/Kette/Nap1-containing SCAR/WAVE complex. SCAR is also important in
protecting against bacterial pathogens and in wound repair as SCAR mutant embryos succumb
more readily to both sterile and infected wounds. However, in addition to driving the formation
of lamellipodia in macrophages, SCAR is required cell autonomously for the correct processing
of phagocytosed apoptotic corpses by these professional phagocytes.
Removal of this
phagocytic burden by preventing apoptosis rescues macrophage lamellipodia formation and
partially restores motility. Our results demonstrate that efficient processing of phagosomes is
critical for effective macrophage migration in vivo. These findings have important implications
for the resolution of macrophages from chronic wounds and the behaviour of those associated
with tumours, since phagocytosis of debris may serve to prolong the presence of these cells at
these sites of pathology.
2745
Calcium flashes orchestrate the wound inflammatory response through DUOX activation
and hydrogen peroxide release.
W. Razzell1, I. R. Evans2, P. Martin1, W. Wood2; 1University of Bristol, Bristol, United Kingdom,
2
University of Bath, Bath, United Kingdom
Hydrogen peroxide (H2O2) is a crucial early signal that leads to immune cell recruitment to
wounds in model organisms ranging from Drosophila to Zebrafish. The H2O2 signal is in part
generated by activation of DUOX, an NADPH oxidase, however to this date little is known as to
how wounding activates DUOX. Using a fluorescent reporter of calcium, GCaMP3, we show that
laser wounding of the Drosophila embryo epidermis induces an instantaneous calcium wave,
which extends several cell rows back from the wound edge. We observe this wave as it travels
intracellularly and resolves after about 15 mins which is long before the wound has fully healed.
Using pharmacological agents, thapsigargin and EGTA, we blocked the calcium wave and
showed that Drosophila embryonic immune cells, hemocytes, now exhibit much reduced
capacity to detect and migrate to wounds, suggesting that the calcium flash could indeed be
involved in initiating attraction to the wound for immune cells. We then used a fluorescent
reporter of hydrogen peroxide, Amplex Red, to show that expression of DUOX RNAi results in
MONDAY-LATE POSTER PRESENTATIONS
the knockdown of the hydrogen peroxide signal. This can be rescued by co-expression of full
length DUOX but not DUOX lacking the calcium sensing EF domain of DUOX indicating that this
domain is important for detecting the calcium flash and triggering hydrogen peroxide synthesis,
which in turn, draws immune cells to wounds. We have also investigated the role of a DUOX cochaperone, DUOXA/ NIP, previously unstudied in the wound scenario and found NIP mutant
embryos have reduced wound hydrogen peroxide production and a retarded inflammatory
response. Overall our data shows how calcium functions as the earliest known signal in the
wound inflammatory response.
2746
Spatial modeling of myosin, actin, and adhesion in the migrating cell.
S. Pulford1, A. Mogilner2,3; 1Mechanical Engineering, UC Davis, Davis, CA, 2Mathematics, UC
Davis, Davis, CA, 3Neurobiology, Physiology, and Behavior, UC Davis, Davis, CA
Cell migration is a fundamentally important phenomenon that is a part of morphogenesis, wound
healing and immune response. A cell migrates on a two-dimensional substrate by protruding at
its leading edge, adhering to the substrate, and then contracting in order to pull the cell rear
forward. Usually, the cell achieves these migratory actions by using a system of three
cytoskeletal agents: actin forms a protrusive viscoelastic network by polymerizing at the cell’s
edge; adhesions anchor the actin network to the extracellular substrate; and myosin locally
contracts the actin network. Cell shape and movements evolve as a result of a balance between
these cytoskeletal agents and their three respective forces (actin-based viscoelastic shear,
adhesion-based drag and myosin-based contractile stress). Together, these forces determine
the flow of the actin network, which redistributes actin, myosin and adhesions; but then the
actin, myosin and adhesion densities cause local forces that determine the actin flow. Thus the
effects of these three forces on the cell are highly interdependent. Many questions remain about
how respective mechanical feedbacks self-organize the cytoskeleton of the motile cell.
Quantitative modeling has been instrumental in answering these questions.
The objective of this work is to further advance our understanding of the cytoskeletal motility
system of the cell through spatial modeling. In this poster, we present a minimal computational
model of this actin-myosin-adhesion system that encompasses the interdependence of actin
flow, myosin concentration, adhesive drag, and cell shape. This model reproduces characteristic
cytoskeletal patterns that have been experimentally observed in migrating cells. The resulting
simulations allow us to see distinct regimes of cytoskeletal interactions, and also show us that
this system of cytoskeletal agents is capable of the spontaneous symmetry breaking required
for cell polarization. The conclusion of this work is that small differences in a cell’s shape,
velocity, and adhesion can belie vastly different regimes of cytoskeletal dynamics within
migrating cells.
2747
MVB-associated clathrin drives lymphocyte migration.
G. Ramirez-Santiago1,2, M. Torres-Torresano1, J. Robles-Valero2, E. Veiga1; 1Cellular and
Molecular Biology, CNB-CSIC, Madrid, Spain, 2Instituto Investigación Sanitaria Hospital
Princesa, Madrid, Spain
Clathrin is a eukaryotic ubicuous protein which has many different functions. Its role during
clathrin-mediated endocytosis has been extensively studied, but clathrin is involved in many
other cellular processes such as, bacterial uptake viral infections or mitotic division. Some of
these processes require massive local actin rearrangements, for example at bacterial entry foci.
In agreement with these observations, recently, it has been demonstrated that clathrin drives
MONDAY-LATE POSTER PRESENTATIONS
actin polymerization at the immunological synapse during T cell activation. In this work we
analyze whether clathrin supports actin polymerization at the leading edge of migrating cells,
playing therefore a major role in cellular migration. Using lymphocytes (cells lines and primary
human lymphoblasts), we show that clathrin is necessary for cellular migration and it promotes
actin accumulation at the leading edge of migrating cells. Moreover, we also show that
multivesicular bodies (MVB) accumulates together with clathrin at the leading edge and the
clathrin adaptor for these MVBs, Hrs, is also required for actin accumulation and cellular
migration. Together, our data indicate that clathrin associated with MVB drive actin
polymerization at the cellular leading edge of migrating cells and thus it appears as a major
player in cell migration.
2748
The N-BAR domain protein, Bin3, regulates skeletal muscle cell migration and muscle
regeneration.
A. Simionescu1, Y. Wang2, P. Pham1, A. Ramalingam3, J. Duhadaway3, V. Faundez4, G.
Prendergast3, G. Pavlath1; 1Pharmacology, Emory University, Atlanta, GA, 2Cell and Molecular
Physiology, Loyola University Chicago, Maywood, IL, 3Lankenau Institute for Medical Research,
Wynnewood, PA, 4Cell Biology, Emory University, Atlanta, GA
During muscle repair, satellite cells migrate and fuse to give rise to regenerated myofibers.
Myofiber formation requires both membrane and actin dynamics. BAR domain proteins regulate
both of these processes in various cell types, by virtue of their ability to bend membranes and
bind small GTPases, but have been little studied in skeletal muscle. We analyzed the role of the
N-BAR domain protein, Bridging Integrator 3 (Bin3), during myogenesis in vitro and in vivo. Bin3
steady state levels are upregulated during muscle regeneration in vivo when small myofibers
form. Moreover, Bin3-/- mice exhibited muscle regeneration defects, characterized by both
decreased myofiber cross-sectional area, and increased myofiber branching compared to wildtype mice. Studies of myogenesis in vitro revealed that Bin3 protein was expressed by primary
mouse muscle cells throughout differentiation, and that Bin3-/- muscle cells exhibited smaller
myotubes compared to wild-type. To determine the cause for reduced myotube size in Bin3-/cells, we examined the possible contribution of endocytosis, F-actin localization and cell-cell
fusion. Bin3-/- myocytes did not show a defect in receptor-mediated endocytosis, but displayed
reduced migration compared to wild-type cells, reduced numbers of lamellipodia, as well as
reduced F-actin localization at the leading edge of lamellipodia. Moreover, Bin3 colocalized with
F-actin at the leading edge of lamellipodia in myocytes, suggesting a role for Bin3 in regulating
actin-dependent processes in myocytes. We are currently examining if and how Bin3 regulates
F-actin localization in myocytes via small GTPases. Interestingly, Bin3 is also required in
myotubes for later stages of fusion during myogenesis, when migration is minimal, suggesting a
potential role for Bin3 specifically in the fusion process. Overall, these data indicate potentially
differential functions for Bin3 during early and late myogenesis. These data open new avenues
for studying the role of BAR domain proteins in regulating muscle cell migration and muscle
regeneration both in skeletal muscle and other tissues.
2749
Ca2+ pulses control local retraction and adhesion in migrating cells.
F-C. Tsai1,2, T. Meyer1; 1Department of Chemical and Systems Biology, Stanford University
School of Medicine, Stanford, CA, 2Program of Cancer Biology, Stanford University School of
Medicine, Stanford, CA
Ca2+ signals are important for the polarity and turning of migrating cells. However, it is not yet
understood how Ca2+ signals exerts its role in regulating cell movement. By studying sheet
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migration using endothelial cells loaded with Fura-2/AM, we identified pulsatile local Ca2+
signals near the cell front that each control a local retraction and local adhesion of locally
extending lamellipodia. Eliminating endogenous Ca2+ pulses decreases lamellipodial retraction,
while photo-release of Ca2+ from NP-EGTA induced front retraction and adhesion similar to
endogenous Ca2+ pulses, indicating these small amplitude Ca2+ signals are both necessary
and sufficient for cyclic lamellipodia activity. We further demonstrate that these local Ca2+
pulses act through myosin light-chain kinase and myosin II to mediate actin cable retraction and
to strengthen focal adhesion.
Removing extracellular Ca2+ did not eliminate local Ca2+ pulses, suggesting that these local
signals are generated from the activation of inositol 3 phosphate receptors (IP3R) in the
endoplasmic reticulum (ER). Interestingly, inhibition of receptor tyrosin kinases (RTK) eliminates
local Ca2+ pulses, confirming that RTK-induced IP3R activation is the major source of these
Ca2+ signals. Further experiments show that leader cells in migrating sheets have, in the front,
more phospho-tyrosine, active phospholipase C and diacylglycerol, which likely contributes to
generating cell polarization. We also demonstrate that cytosolic Ca2+ is pumped out of the
plasma membrane more efficiently in the front of migrating cells, enabling the cells to locally
lower basal Ca2+ level and sensitize the effect of local Ca2+ pulses near the lamellipodia
region. Last but not the least, Ca2+ in the front ER is lowered after local Ca2+ pulse release
from ER and the enhanced pump activity in the front plasma membrane. This Ca2+ depletion is
restored by the accumulation and activation of stromal interaction molecule I (STIM1) to
increase store-operated Ca2+ influx locally. STIM1 is brought to the front ER through its affinity
with the plus ends of microtubules. Therefore, temporal-spatial synchronization of Ca2+
signaling control directed migration by regulating local adhesion, turning, and polarization of
migrating cells.
2750
Hypoxia induces targeted migration and enhances activation of RhoA in human
mesenchymal stem cells (hMSCs)
G. K. Vertelov1, G. Sanati1, L. I. Kharazi1, M. G. Muralidhar1, A. I. Kharazi1;
1
R&D, Stemedica, San Diego, CA
Background. Human mesenchymal stem cells (hMSC) have attracted increasing interest due
to their great therapeutic potential. The efficacy of MSC in treatment of ischemic injury in various
animal models has been sufficiently demonstrated in the literature. MSC migration to the site of
injury is believed to be one of the key factors in overall successful tissue/organ recovery. The
ability to migrate is a critical feature of stem cells that weakens upon differentiation of cells.
Long term culture of MSCs in hypoxia promotes a genetic program maintaining their
undifferentiated and multipotent status. Whilst most studies address the effect of transient
hypoxia on MSC in regards of MSCs migration, long term culture in hypoxia was not thoroughly
investigated, though the latter should better mimic the physiological conditions and prevent
differentiation, thus favoring better migration of MSCs. We studied the involvement of Rho A in
migration of MSC, the molecule is believed to play an essential role in reorganization of
cytoskeleton during all stages of migration.
Materials and Methods. Human bone marrow derived hMSC were expanded under normal
(21%) or “normoxic” and/or low oxygen (5%) or “hypoxic” conditions up to passage 4. A series
of 17 cytokines was used to induce chemotaxis.. Migration efficiency was assessed in vitro in
Corning Costar transwell chambers towards the gradient of the chemokines. We also compared
the level of GTP-bound RhoA in the cell extracts of Calpeptin activated Hypoxic and Normoxic
hMSC. MSCs cultured in hypoxia and normoxia were assayed for migration both in hypoxic and
normoxic environments.
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Results. In our study we show that hypoxic hMSC significantly outperform analogous normoxic
hMSC in vitro in transmigration assay towards most of the cytokines studied in this work. We
also demonstrated for the first time that hypoxic MSC are dramatically more sensitive to
activation of RhoA.
Conclusions. “Stemness” of MSCs undoubtedly depends on cell culturing conditions. Long
term hypoxia preserves MSCs in their most primitive, undifferentiated state; allowing them to
retain high migratory capacity, the key attribute of the stem cells. The results of this study
indicate that high directional migration of hMSCs permanently grown in hypoxia is associated
with the enhanced activation of RhoA. The enhanced migratory capacity of hypoxic hMSC
would further suggest their potential advantages for clinical applications.
2751
The role of Arp2/3 complex in fibroblast cell directional migration.
P. K. Suraneni1, B. Rubinstein1, R. Li1; 1Stowers Institute for Medical Research, Kansas city, MO
Arp2/3 complex is the key component of the actin polymerization engine that drives amoeboid
cell motility. Although the biochemical activity of the Arp2/3 complex has been well
characterized, it remains unclear how the Arp2/3 generated actin network is involved to drive
the cell motility in vivo. Previously we established a knockout mice model for ARPC3, a crucial
component of the Arp2/3 complex. To further investigate the function of the Arp2/3 complex in
different motile cells, we developed an approach to derive ARPC3+/+, +/- and -/- ES cells from
blastocysts of ARPC3+/- crosses and differentiate them into fibroblasts cells. Using these cells
with different genotypes, we recently addressed the question whether the Arp2/3 complex is
necessary for lamellipodia extension and cell motility? The ARPC3 deficient fibroblast cells
generate leading edge composed of filopodia-like structures which makes them morphologically
different from the wt type. Our result indicated that the Arp2/3 complex is required for
lamellipodia formation in differentiated mouse fibroblasts. We further examined the migration
behavior of these cells in 2D cultures. Surprisingly, compared with wild-type fibroblasts, the
Arp2/3 null fibroblasts cells are capable of rapid spreading and highly motile using filopodia-like
structures. However they failed to sustain directional movement towards the EGF
chemoattractant signal. While this observation is interesting, we further asked whether these
cells behave similarly in response to other growth factors. Experiments designed to address this
question is under way.
2752
Towards the understanding of cell migration in a 3D environment.
C. Guetta1, H. Long2, E. Yim1, S. Chew2, B. Ladoux1,3, N. Gauthier1; 1Mechanobiology Institute,
Singapore, Singapore, 2Nanyang Technological Institute, Singapore, Singapore, 3Laboratoire
Matière et Systèmes Complexes, Centre National de la Recherche Scientifique Unité Mixte de
Recherche, Paris, France
Cell locomotion is one of the most easily visible but yet one of the most complex coordinated
process exhibited by a living cell. Cell migration is essential for all organisms needing to search
for energy sources and it’s involved in many processes key to eukaryotic animal life, such as
embryonic development, wound healing and inflammatory responses. In contrast, a
deregulation of cell movement causes several pathological states including developmental
defects, healing abnormalities and cancer metastases.
In-vitro studies of cell migration have been primarily conducted on flat, two-dimensional
substrates. These studies have helped to elucidate the mechanisms by which cells migrate,
interact with the substrates and sense their environment. The simplicity of 2D culture has
enabled reductionist approaches, but this model might not faithfully capture the physiological
MONDAY-LATE POSTER PRESENTATIONS
behavior of cells in vivo. However, in-vivo, cells are embedded within complex and informationrich environments. Unsurprisingly, cells show large dissimilarities when migrating on flat
surfaces as compared to topographically complex three-dimensional environments (Yamada et
al. 2009). Furthermore, recent observations have led to the notion that the dimension in which
cells are cultured is a crucial fate determinant.
Several approaches have been used to embed cells into 3D environments. In particular,
extracellular matrices such as collagen gels are commonly used as simplified models to study
cell migration. It has also been proposed that one-dimensional topography functionally mimics
migration in a 3D fibrillar environment (Yamada et al. 2009). With the development of
bioengineering, electrospinning techniques have been used to produce in vitro fibers with
dimension and properties that can be tuned to resemble in vivo fibers.
In this poster we present our preliminary results in our attempt to develop a powerful fibrillar 3Dlike microenvironement that combines together a reductionist approach, an easy imaging and a
customization of the fibers properties (coating, geometry like diameter…). Our main goal is to
reconstruct an accurate model of 3D cell motility.
2753
Orientation-Specific Responses to Sustained Uniaxial Stretching in Focal Adhesion
Turnover.
Y. Chen1, A. M. Pasapera1, A. P. Koretsky1, C. M. Waterman1; 1National Institutes of Health,
Bethesda, MD
Cells are known to be mechanosensitive in response to extracellular matrix (ECM) deformation,
which, for example, can be caused by muscle contraction or changes in hydrostatic pressure in
the body. Focal adhesions (FAs) are protein complexes mediating the linkage between the cell
and the ECM, which initiate signaling events triggered by mechanical stimuli. We developed a
stretching apparatus in which cells growing on fibronectin-coated elastic substrates can be
stretched and imaged live to study how FAs dynamically respond to ECM deformation. U2OS
cells were transfected with GFP-paxillin as FA marker and subjected to 5% sustained uniaxial
stretching. Two responses at different timescales were observed: rapid FA growth within
seconds after stretching, and delayed changes in steady-state FA disassembly/assembly rate
and changes in cell polarity that occurred tens of minutes. Rapid FA growth occurred in all
cells, however steady-state FA disassembly/assembly and cell polarity changed in an
orientation-specific manner: significant FA disassembly and cell depolarization was observed in
cells with their long axes perpendicular to the stretching direction, but not in cells with their long
axes parallel to stretching. Pharmacological treatments demonstrated that FAK promotes but
Src inhibits rapid FA growth, while FAK, Src, and calpain 2 all contribute to delayed FA
disassembly and cell depolarization in cells perpendicular to stretching. Immunostaining for
phospho-FAK at specific times after stretching revealed that FAK activation was maximal at 5
seconds after stretching specifically in FAs oriented perpendicular to stretch. We hypothesize
that putative mechanosensitive proteins in FAs upstream to FAK and Src align with the long
axes of FAs, while a majority of FAs alig­­ns with cellular long axis, resulting in orientationspecific responses.
MONDAY-LATE POSTER PRESENTATIONS
2754
Quantitative metrics to assess the resolution and sensitivity of traction force
microscopy.
W. R. Legant1, C. Choi2, L. Shao1, L. Gao1, E. Betzig1, C. Chen2; 1HHMI Janelia Farm Research
Campus, Ashburn, VA, 2Bioengineering, University of Pennsylvania, Philadelphia, PA
Measurements of cell generated tractions, collectively termed traction force microscopy (TFM),
have emerged as an indispensible tool to study cell mechanics, migration and
mechanotransduction. The general approach of these methods involves culturing cells upon
flexible elastic substrates coated with adhesive ligands. As the cells adhere, spread and
migrate, they exert forces on the surface of the substrate. These forces combine to induce
deformations within the elastic substrate which can be monitored by tracking embedded fiducial
markers. This displacement field can then be converted to quantitatively measure the tractions
exerted by cells on the surface. Although numerous advances have been made to date, many
TFM varieties utilize different experimental parameters and underlying algorithms, and this
makes comparing traction stress maps across different studies difficult. To resolve this, we
present a general approach utilizing simulated data to fully probe the spatial resolution and
traction sensitivity, parameters which are inherently interdependent in TFM measurements.
This approach is readily compatible with all current methods of TFM and should serve as a
useful metric for characterizing and comparing future measurements of cell generated traction
forces. As a demonstration, we apply this method to characterize both the three-dimensional
(3D) tractions exerted by cells cultured on top of planer half-spaces (2.5D TFM) and the 3D
tractions exerted by cells fully surrounded by a 3D matrix (fully-3D TFM). Within these contexts,
we assess the effects of finite displacement sampling (bead density), measurement noise, and
regularization on the recovered surface tractions.
2755
Nitrosyl-cobinamide promotes wound healing through enhanced cell migration.
R. Spitler1, R. Schwappacher2, T. Wu3, R. Pilz2, G. Boss2, M. Berns1,2; 1University of California
Irvine, Irvine, CA, 2University of California San Diego, San Diego, CA, 3Harvard Medical School,
Massachusetts General Hospital, Boston, MA
Currently, there is no effective treatment for smoke-induced respiratory epithelial damage, which
represents a combination of burn-related injury and toxic injury due to the components carried
within the smoke itself. Previous studies have demonstrated the use of nitric oxide donors and
chelation agents to enhance wound healing and mitigate smoke-induced toxicity, but there is
still an unmet need for effective regimens to address both of these serious problems. The main
goal of this research was to develop a high-throughput in vitro wound healing assay. With the
use of a novel photonics robotic imaging microscope and software, we were able to monitor the
closure of a mechanically created scratch wound in a cell monolayer, and examine the effects of
pre-treating cells with nitrosyl-cobinamide (NO-Cbi), a nitrosylated derivate of vitamin B12. We
have found that NO-Cbi is able to enhance cell migration into the wound in multiple cell types
[rat kangaroo kidney epithelial (PtK2), human osteosarcoma (U2OS), adenocarcinomic human
alveolar basal epithelial cells (A-549) and normal human lung fibroblast (NHLF)]. In particular,
NO-Cbi treated A-549 cells showed an enhanced cell migration rate, generally 20% faster than
untreated control cells. Using pharmacological inhibitors and activators of the NO/cGMP/Protein
kinase G (PKG) signaling pathway as well as specific siRNAs targeting PKG, we determined
that NO-Cbi enhanced wound closure proceeds at least in part through a cGMP- and PKGdependent mechanism. These studies revealed that our optically based real-time quantitative
system can be used as a translational research tool to enhance the fundamental understanding
MONDAY-LATE POSTER PRESENTATIONS
of the wound healing process and determine the mechanisms of action of wound closureenhancing agents.
2756
The Drosophila large Maf transcription factor Traffic Jam regulates border cell migration.
F. Gunawan1, M. Arandjelovic1, D. Godt1; 1Cell and Systems Biology, University of Toronto,
Toronto, ON, Canada
Border cell cluster (BCC) migration in the Drosophila ovary is an excellent system to study
regulatory networks that govern migratory cell behavior in vivo. The BCC, which contains 6-8
migratory cells, invades the germline cyst and migrates toward the oocyte during midoogenesis.
An essential regulatory cascade that enables border cell motility includes the Jak/Stat pathway
and its downstream target Slow border cells (Slbo). Slbo activates expression of downstream
effectors that enable cell migration including the adhesion molecule DE-cadherin (DEcad).
We found that the large Maf transcription factor Traffic Jam (Tj) has an important function in
keeping this regulatory cascade in balance, thereby enabling normal border cell migration. Tj
expression gradually decreases in border cells during migration, but never disappears. Tj
overexpression in the border cells causes a strong delay in migration, suggesting that the
natural reduction of Tj expression is needed for border cell motility. Our analysis of interactions
between Tj and the components of the motility-enabling cascade revealed that Tj
overexpression affects the cascade at all three levels: it causes loss of Slbo, prevents DEcad
upregulation, and inhibits the Jak/Stat pathway by enhancing the expression of its inhibitor
Socs36E. Surprisingly, loss of Tj function also causes a significant delay in migration. Similar to
Tj overexpression, loss of Tj causes a decrease in Slbo expression, suggesting that the correct
amount of Tj is needed to maintain Slbo expression. Our data indicate that Tj works with Slbo,
DEcad and the Jak/Stat pathway in a regulatory network, keeping these factors in balance and
ensuring proper cell motility.
Recent data suggest that Tj might have additional functions in the BCC that are important for
the formation of normal actin-based cellular protrusions during migration. Increased Tj
expression leads to a severe reduction of actin protrusions and a significant shortening and
rounding of the cluster. In contrast, Tj loss-of-function causes BCCs to abnormally stretch and
extend unusually long actin protrusions. These morphological changes within the cluster are
likely to contribute to the migration delays observed when Tj expression is modulated. To further
understand the function of Tj in regulating migratory cell behavior, we are currently investigating
other downstream targets of Tj, focusing on those that directly regulate the actin cytoskeleton
and cell-cell adhesion.
2757
Interactions between actin and its effectors yields both polarized and dynamic
phenotypes: a modeling study.
W. R. Holmes1,2; 1Mathematics, University of California, Irvine, Irvine, CA, 2Mathematics,
University of British Columbia, Vancouver, BC, Canada
Patterns of intracellular waves, patches, and peaks of actin are observed experimentally in
many living cells. A typical view of cell function is that regulation (mediated by GTPases for
example) leads to reorganization (i.e. growth and contraction of the actin cytoskeleton).
However, some form of feedback is usually necessary to produce dynamic behavior of this sort.
We thus discuss an alternative view where these levels of function mutually influence each
other. A model of the interplay between filamentous actin (F-actin) and its nucleation promoting
MONDAY-LATE POSTER PRESENTATIONS
factors (NPF) is presented. Assumptions on NPF kinetics are based on properties of GTPases
(such as Rac or Cdc42), known effectors of actin nucleation. These NPF’s are assumed to both
up regulate F-actin growth and at the same time be down regulated by F-actin. We find that the
feedback inherent in this NPF-actin model generates a rich set of behaviors. Weak actin
feedback yields static patterning akin to polarization, moderate feedback yields dynamical
behavior such as travelling waves, and strong feedback can lead to wave trains or total
suppression of dynamics. The scope of these dynamics, and in particular the transition
between static polarization and dynamic behavior is discussed.
2758
Directional migration of polarized cells by polarization of store-operated Ca2+ entry.
G-W. Huang1, W-T. Chiu1, M-R. Shen1, M-J. Tang1; 1National Cheng Kung University, Tainan,
Taiwan
Formation of cell polarity is essential for directional migration and plays an important role in
physiological and pathological processes of organism. Previous studies indicated that Ca2+ and
its associated molecules, such as calpain and calcineurin, concentrated on the rear end of
polarized cells. Store-operated Ca2+ entry (SOCE) is the major form of extracellular Ca2+ influx
in non-excitable cells. The major molecular components in regulation of SOCE are the ER Ca2+
sensor stromal interaction molecule1 (STIM1) and two plasma membrane Ca2+ channels
transient receptor potential channel 1 (TRPC1) and Orai1. Activated SOCE molecular
complexes are present in caveolin-1 (Cav-1)-rich lipid rafts/caveolae where the channelosome
is formed. In order to test the role of SOCE in formation of cell polarity, we employed human
bone osteosarcoma U2OS cells that exhibited distinct morphological polarity during directional
migration. Our studies showed that extracellular Ca2+ influx was important for cell polarization.
Moreover, Ca2+ levels, Cav-1 and STIM1/TRPC1/Orai1 channelosomal complexes were
concentrated in the rear end. Inhibition of SOCE by specific inhibitors SKF96365, YM58483,
Gd3+ and La3+ disrupted cell polarization in a dose dependent manner. Non-selective blocker of
the TRPC family, flufenamic acid (FFA), also reduced cell polarity formation. Furthermore,
disruption of lipid rafts and overexpression of Cav-1 downregulated cell polarity. Finally, we
examined whether substrate rigidity affected formation of cell polarity. Our data showed that cell
polarization was markedly decreased by low substratum rigidity. Taken together, we conclude
that polarized distribution of SOCE plays important roles in formation of cell polarity. The
underlying mechanism of uneven distribution of SOCE remains to be investigated.
2759
High Temperature Requirement A4 (HtrA4) Suppresses the Fusogenic Activity of
Syncytin-1 and Promotes Trophoblast Invasion.
L-J. Wang1, H. Chen1; 1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
Cell-cell fusion and cell invasion are essential for placental development. Human
cytotrophoblasts in the chorionic villi may undergo cell-cell fusion to form syncytiotrophoblast to
facilitate nutrient-gas exchange or differentiate into extravillous trophoblasts (EVTs) to facilitate
maternal-fetal circulation. The placental transcription factor, glial cell missing 1 (GCM1),
regulates syncytin-1 and -2 expression to mediate trophoblast fusion. Interestingly, GCM1 and
syncytin-1 are also expressed in EVTs with unknown physiological functions. In this study, we
perform ChIP-chip analysis and identify high temperature requirement A4 (HtrA4) as a GCM1
target gene, which encodes a serine protease facilitating cleavage of fibronectin and invasion of
placental cells. Importantly, HtrA4 is immunolocalized in EVTs at the maternal-fetal interface
and its expression is decreased by hypoxia and in preeclampsia, a pregnancy complication
associated with placental hypoxia and shallow trophoblast invasion. We further demonstrate
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that HtrA4 interacts with syncytin-1 and suppresses cell-cell fusion. Therefore, HtrA4 may be
crucial for EVT differentiation by playing a dual role in prevention of cell-cell fusion of EVTs and
promotion of their invasion into the uterus. Our study reveals a novel function of GCM1 and
HtrA4 in regulation of trophoblast invasion and that abnormal HrtA4 expression may contribute
to shallow trophoblast invasion in preeclampsia.
2760
FilGAP, a Rho/ROCK-regulated GAP for Rac controls tumor cell migration.
K. Saito1, Y. Ozawa1, K. Hibino1, Y. Ohta1; 1Department of Biosciences, Kitasato University,
Sagamihara, Japan
Tumor cells exhibit two interconvertible modes of cell motility referred to as mesenchymal and
amoeboid migration. Mesenchymal mode is characterized by elongated morphology that
requires high GTPase Rac activation whereas amoeboid mode is dependent on actomyosin
contractility induced by Rho/ROCK signaling. While elongated morphology is driven by Racinduced protrusion at the leading edge, how Rho/ROCK signaling controls amoeboid movement
is not well understood. We identified FilGAP, a Rac GAP, as a mediator of Rho/ROCKdependent amoeboid movement of carcinoma cells. We show that depletion of endogenous
FilGAP in carcinoma cells induced highly elongated mesenchymal morphology. Conversely,
forced expression of FilGAP induced a round/amoeboid morphology that requires Rho/ROCKdependent phosphorylation of FilGAP. Moreover, depletion of FilGAP impaired breast cancer
cell invasion through extracellular matrices and reduced tumor cell extravasation in vivo. Thus,
phosphorylation of FilGAP by ROCK appears to promote amoeboid morphology of carcinoma
cells and FilGAP contributes to tumor invasion and metastasis.
2761
A Role for IQGAP1 in Cell Edge Retraction.
S. Foroutannejad1, N. Rohner1, J. M. Schober1; 1Pharmaceutical Sciences, Southern Illinois
University, Edwardsville, IL
Cell migration is characterized by restricted temporal-spatial distribution of intracellular proteins
that regulate actin protrusion and trailing edge retraction. IQGAP1 has emerged as a key
component in the regulation of cytoskeleton dynamics during cell migration, maintenance of
adherens junctions, microbial pathogenesis and intracellular trafficking. Several reports indicate
a role for IQGAP1 in regulation of actin protrusion at the leading edge of lamellipodia during
migration. IQGAP1 is known to localize to the protruding edge of lamellipodia in a variety of cell
types and interact with regulators of actin dynamics. Here, we provide evidence suggesting a
novel role of IQGAP1 in cell motility. We classified a number of common cell lines according to
their degree of WAVE co-localization with IQGAP1 near the cell edge. Interestingly, in some of
the cell lines IQGAP1 was distinctly separated from WAVE localization, suggesting IQGAP1
may localize to retracting edges instead of protruding edges. Of these cell lines, B16F10 cells
had the most restricted separation of IQGAP1 from WAVE. We co-transfected B16F10 cells
with GFP-IQGAP1 and mCherry-Arp3 to examine localization dynamics during cell edge
movement. The disappearance of Arp3 correlated with the appearance of IQGAP1 as the cell
edge transitioned from protrusion to retraction. Furthermore, the appearance of IQGAP1
preceded the formation of retraction fibers. These results demonstrate that in some cell types
IQGAP1 may function to promote cell retraction not lamellipodium edge protrusion. In addition,
we examined co-localization of adhesion site markers and myosin activity with GFP-IQGAP1 in
B16F10 cells. In areas rich in IQGAP1, there was decreased immunofluorescence staining of
vinculin, paxillin and phosphorylated-tyrosine indicating adhesion site disassembly.
Phosphorylated myosin light chain localized alongside IQGAP1 toward the cell nucleus.
MONDAY-LATE POSTER PRESENTATIONS
Overall, these results point to a new role of IQGAP1 in cell migration, namely retraction of the
cell edge potentially through regulation of myosin contractility and downregulation of adhesion
sites.
2762
A PIP5kinase required for transmission of Ras signalling is crucial for chemotactic
gradient sensing
L. V. Fets1, R. Kay1; 1Cell Biology Division, MRC Laboratory of Molecular Biology, Cambridge,
United Kingdom
Phosphoinositides are actively regulated during chemotaxis, and while it is now known that
PI(3,4,5)P3 signalling is non-essential for gradient sensing, the role of PI(4,5)P2 is little
understood. We describe a PIP5kinase, PikI, which produces PI(4,5)P2 and is essential for
efficient chemotaxis of Dictyostelium cells. Without PikI, PI(4,5)P2 levels are reduced by 90%,
and while pikI- cells move at normal speeds, they are highly disorientated in cAMP gradients.
This appears to be independent of the PI(3,4,5)P3 and PLC pathways, since a PI3Kinase-PLCmutant is essentially wild-type. Following chemotactic stimulation, Ras is efficiently activated in
pikI- cells, yet a range of Ras-dependent responses (including the activation of PKB) are
severely impaired. PikI is known to be phosphorylated by PKB. A phospho-mimic mutant
suggests that this phosphorylation increases PikI activity, thus creating a positive-feedback
loop. We propose that adequate PI(4,5)P2 levels are required to couple activated Ras to its
downstream effectors and that these levels are maintained by PikI, providing a novel method of
modulating the intensity of Ras signalling.
2763
p27(Kip1) Is a Microtubule-Associated Protein that Promotes Microtubule Polymerization
during Neuron Migration.
J. Godin1; 1GIGA neuroscience, Liege, Belgium
The migration of cortical interneurons is characterized by extensive morphological changes that
result from successive cycles of nucleokinesis and neurite branching. Their molecular bases
remain elusive, and the present work describes how p27(Kip1) controls cell-cycle-unrelated
signaling pathways to regulate these morphological remodelings. Live imaging reveals that
interneurons lacking p27(Kip1) show delayed tangential migration resulting from defects in both
nucleokinesis and dynamic branching of the leading process. At the molecular level, p27(Kip1)
is a microtubule-associated protein that promotes polymerization of microtubules in extending
neurites, thereby contributing to tangential migration. Furthermore, we show that p27(Kip1)
controls actomyosin contractions that drive both forward translocation of the nucleus and growth
cone splitting. Thus, p27(Kip1) cell-autonomously controls nucleokinesis and neurite branching
by regulating both actin and microtubule cytoskeletons.
2764
The scaffolding adapter Gab1 mediates the interaction of p120 Catenin with VAV2 and
Cortactin to promote VEGF-induced Rac activation and endothelial cell invasion.
C. Caron1, K. Spring1, I. Royal1; 1Department of Medicine, University of Montreal, Montreal, QC,
Canada
Angiogenesis is the formation of new blood vessels from pre-existing vasculature, and VEGF is
one of the most important factors regulating this process. Our previous work demonstrated the
promoting role of the scaffolding adapter Gab1 on VEGF signalling and biological functions
including cell survival, cell migration, and capillary formation. We further demonstrated that
MONDAY-LATE POSTER PRESENTATIONS
Gab1 is also involved in the reorganization of the actin cytoskeleton and mediates lamellipodia
formation upon VEGF stimulation. However, the molecular mechanisms involved are still not
well known.
Here we show that RNAi-mediated knockdown of Gab1 expression in human umbilical vein
endothelial cells (HUVECs) results in the attenuation of Rac activation upon VEGF stimulation.
We also find that knockdown or overexpression of Gab1 modulates the activation of VAV2, a
guanine nucleotide exchange factor (GEF), as well as its association with p120 Catenin
previously reported to promote Rac activation. In addition, we observed that Gab1 modulates
the phosphorylation of Cortactin, which correlates with its increased actin polymerizing activity,
as well as Cortactin’s capacity to associate with p120 Catenin, a protein known to redirect its
localization to membrane ruffles. Consistent with these observations, defective association of
Cortactin to the membrane of Gab1-depleted cells is observed upon VEGF stimulation. Gab1 is
reported to interact in a direct and constitutive manner with Cortactin, and we observed its
induced association with VAV2 and p120 Catenin in VEGF-stimulated cells. Interestingly, the
deletion of Gab1’s PH domain, which mediates its association to the membrane, results in the
disruption of the VAV2/p120 Catenin complex, and in the block of Rac activation. As Rac,
Cortactin and p120 Catenin are critical for invasion, we further validate Gab1 as a positive
mediator of endothelial cell lamellipodia formation and invasion in response of VEGF, an
essential step of angiogenesis. Our work thus bring novel insights into the Gab1-dependent
pathways mediating the biological responses of endothelial cells to VEGF, and shed light on the
molecular mechanisms underlying the reported promotion of in vivo angiogenesis via Gab1.
2765
Protein kinase cα influences on focal adhesions.
B. S. Fogh1, H. A. Multhaupt1, C. Larsson2, J. R. Couchman1; 1Biomedical Sciences,
Copenhagen University, Copenhagen, Denmark, 2Center for Molecular Pathology, Lund
University, Malmö, Sweden
Protein kinase C alpha (PKCα) is the only classical PKC to localize to fibroblast focal adhesions
(FAs). PKCα priming phosphorylation of T638 at the turn motif and S657 at the hydrophobic
motif is believed to be important for PKCα localization at membranes and for its activity.
However, it has also been shown that kinase dead and T638A/S657A PKCα mutants display
persistent translocation to membranes when activated.
Our aim was to investigate whether phosphorylation of the two priming sites is essential for
PKCα translocation to FAs, as well as their impact on FA morphology, dynamics and cell
behavior. WT PKCα-EGFP, its non-phosphorylatable (T638A/S657A) or phosphomimetic
(T638E/S657E) mutants were transfected into primary fibroblasts and evaluated by total internal
reflection fluorescence (TIRF) microscopy. mCherry-Zyxin was co-expressed as an FA marker.
PKCα was visible at FAs irrespective of phosphate at the two priming sites.
Analysis of FA dynamics showed that FAs of cells over-expressing PKCα(T638A/S657A) had a
longer half-life. In addition, immuno-fluorescence staining showed that sizes of those FAs were
increased and more elongated. No statistically significant changes in FA morphology or lifetime
were noted in cells expressing WT or phosphomimetic PKCα.
Individual cell tracking revealed that cells stably overexpressing the WT kinase or the
phosphomimetic mutant migrated with increased speed compared to control cells.
Therefore, phosphorylation of the priming sites (T638/S657) of PKCα is not required for PKCα
to localize at FAs. FA morphology and dynamics are, however, changed by expression of PKCα
that is not phosphorylated at these two residues.
MONDAY-LATE POSTER PRESENTATIONS
Regulation and Organization of the Genome
2766
Transcriptional Regulation of the ATP2A3 gene during differentiation of human gastric
and colon cancer cell lines.
L. Flores-Peredo1, G. Rodriguez1, A. Zarain-Herzberg1; 1Biochemistry Department, National
Autonomous University of Mexico, Mexico City, Mexico
The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) regulates calcium concentration
inside the endoplasmic reticulum and cytosol in most cell types. Normal colon and gastric
epithelial cells express high levels of the SERCA3 isoform, which is selectively lost in cancer
cells. Previously, Gelebart et al. found that SERCA3 protein expression is induced when human
gastric and colon cancer cells undergo in vitro differentiation.
In this work, we induced differentiation of KATO-III gastric cancer cells and Caco-2 colon
adenocarcinoma cells in culture using butyrate and confluence, respectively. We found a large
increase in mRNA expression of SERCA3 (up to 50-fold), suggesting that a transcriptional
mechanism regulates ATP2A3 gene expression. We measured transcriptional activity of pGL3hATP2A3-gene-promoter constructs in both cancer cell lines and we found a marked increase in
the activity up to 30-fold after induced differentiation. We narrowed the responsive elements to
the region -135 to +142 bp of the 5’-flankig-region of the human ATP2A3 promoter. This region
is inside a CpG island and contains 8 putative Sp factors binding sites. Sp is a family of
transcription factors including Sp1, Sp3, Sp4 and KLF1 to 17. Previously, in mouse endothelium
and human lymphocytes it has been shown an important role of Sp factors in transcriptional
regulation of the ATP2A3 gene. High level of KLF4 is expressed in intestinal epithelium where is
essential for terminal differentiation of normal tissue. Besides, it has been reported that both the
expression of KLF4 and SERCA3 is decreased in gastric and colon cancer, and that both
proteins and mRNAs increases after induced differentiation of these cancer cells in culture.
Therefore, to demonstrate a direct interaction of KLF4 and/or Sp with DNA elements within the
ATP2A3 promoter in gastric and colon cancer cell lines during in vitro differentiation, we
performed DNA-protein binding assays. Through EMSA and ChIP assays we identified a
specific and direct interaction of KLF4, Sp1 and Sp3 with their target sequences in the promoter
of the human ATP2A3 gene. We found that KLF4 binding increases in differentiated cancer cell
lines, suggesting that might function as an activator of ATP2A3 gene expression. In contrast,
the results suggest that Sp3 functions as a repressor in tumor cells keeping low ATP2A3
expression. Previously it has been shown that KLF4 could function as a transcriptional activator
or repressor depending on other protein interactions. By ChIP assays we found that KLF4 binds
to the proximal ATP2A3 promoter as part of a co-activator complex with the histone
acetyltransferase p300. We conclude that ATP2A3 expression is regulated at the transcriptional
level, positively by KLF4 and negatively by Sp3, which binds to the proximal promoter.
Supported by grants PAPIIT IN213613 and CONACyT 164413.
2767
Genome architecture and combinatorial use of transcription factors determine the timing
of cell cycle-dependent transcription.
U. Eser1, J. M. Skotheim2; 1Applied Physics, Stanford University, Stanford, CA, 2Biology,
Stanford University, Stanford, CA
The Start checkpoint regulates cell cycle commitment and associated transcription in budding
yeast. We previously showed that commitment to cell division corresponds to activating the
positive feedback loop of G1 cyclins controlled by the transcription factors SBF and MBF.
MONDAY-LATE POSTER PRESENTATIONS
Although this pair of transcription factors has over 200 targets, genes within this regulon have a
well-defined distribution of transcriptional activation times. We found that activation of the G1
cyclins precedes the activation of the bulk of the G1/S regulon. This feedback-first regulation
ensures that commitment to cell division occurs before large-scale changes in transcription and
is conserved in the related yeast S. bayanus and human cells. However, the molecular
mechanisms underlying the precise temporal order at the basis of feedback-first regulation
remained unclear. Here, we show that timing is partially explained by the combinatorial use of
SBF and MBF transcription factors, which implement a logical OR function for gene activation.
This conclusion is supported by both statistical analysis of genome-wide activation times as well
as single cell analysis of transcription from promoters lacking either SBF or MBF binding sites.
Additionally, we analyze genome-wide chromosome conformation capture data to examine the
potential link between the timing of gene expression and 3-D genome architecture. The early
activated genes of the ~300 gene G1/S regulon are significantly enriched for the number of
physical interactions. Further analysis revealed two main clusters, whose interactions covary
and whose activation time distributions are distinct. Taken together, our work explains a
significant amount of timing variation within cell cycle-dependent gene expression. Thus, the cell
utilizes both genome architecture and logical regulation to implement feedback-first regulation at
the start of the cell division cycle.
2768
Low-intensity infrared laser on action gene expression in skin and muscle tissue.
A. S. Fonseca1, A. L. Mencalha1, V. M. Campos1, S. De Paoli2, S. C. Ferreira-Machado1, A. A.
Peregrino1, M. Geller3, F. De Paoli4; 1UERJ, Rio de Janeiro, Brazil, 2Unesa, Rio de Janeiro,
Brazil, 3Unifeso, Teresópolis, Brazil, 4UFJF, Juiz de Fora, Brazil
Background and objective: Biostimulative effect of low-intensity lasers is the base for treatment
of diseases in soft tissues. However, data about influence of biostimulative lasers on gene
expression are scarce yet. The aim of this work was to evaluate effects of low-intensity infrared
laser on expression of actin mRNA in skin and muscle tissue.
Material and methods: Skin and muscle tissue of Wistar rats were exposed to low-intensity
infrared laser at different fluences and frequencies. One and twenty four hours after laser
exposition, tissue samples were withdrawn for total RNA extraction, cDNA synthesis and
evaluation of actin gene expression by quantitative polymerase chain reaction.
Results: Data obtained show that laser radiation alters the expression of actin mRNA differently
in skin and muscle tissue of Wistar rats depending of the fluence, frequency and time after
exposure.
Conclusions: The results could be used to laser dosimetry, as well as to justification of
therapeutic protocols for treatment of diseases of skin and muscle tissues based on lowintensity infrared laser.
2769
Intragenic transcription and the regulation of protein function.
K. McKnight1, H. Liu2, Y. Wang1; 1Biomedical Science, Florida State University College of
Medicine, Tallahassee, FL, 2Pharmacology, University of Texas Southwestern Medical Center,
Dallas, TX
The function of a given gene can be regulated at transcriptional, translational and posttranslational levels. The goal of this project is to demonstrate that under stressful conditions
transcription can be initiated within the coding region of a group of genes through intragenic
transcription initiation in order to produce smaller protein isoforms. Using western blotting we
have identified S-phase checkpoint-dependent shorter protein isoforms of the conserved spindle
MONDAY-LATE POSTER PRESENTATIONS
midzone protein Ase1 when yeast cells are treated with Hydroxyurea (HU), a drug that blocks
DNA synthesis and arrests cells in S-phase. Northern blotting was used to confirm the presence
of a smaller mRNA transcript upon HU treatment. 5’ Rapid Amplification of cDNA Ends
(5’RACE) was used to identify the transcriptional start site for both full length ASE1 mRNA and
the short form of ASE1 mRNA. From the data we hypothesized that ASE1 is subjected to
intragenic transcription after HU treatment and the shorter protein isoforms are produced to
counteract the function of the full length protein and maintain the proper response to DNA
replication block. By using live-cell fluorescence microscopy to examine spindle dynamics and
full length Ase1 localization upon overexpression of the short isoform of Ase1, we observed a
defect in spindle elongation and restricted spindle midzone localization of Ase1 during the
normal cell cycle. We conclude that under stressful DNA replication conditions, intragenic
transcription of ASE1 occurs in an S-phase checkpoint dependent manner. This intragenic
transcription produces smaller protein isoforms of Ase1 that act in a dominant negative manner
to prevent mitosis until DNA replication is completed successfully. Next generation RNA
sequencing was performed and data analysis is expected to identify other genes subjected to
similar regulation. Further analysis will be performed to confirm genes identified from RNA
sequencing and elucidate the mechanism by which the S-phase checkpoint induces intragenic
transcription.
2770
Connections between pre-mRNA splicing and the SWR complex in S. pombe.
K. E. Nissen1, K. Patrick1, C. Guthrie1; 1University of California, San Francisco, San Francisco,
CA
We are using a genetic approach to investigate the interplay between splicing factors and other
cellular pathways in fission yeast. Gene expression in Schizosaccharomyces pombe is highly
reliant on splicing, with ≈60% of all genes contain at least one intron. Using EMAPs (Epistatic
Mini-Array Profiles) to globally quantify genetic interactions between pairs of mutants, we have
recently shown multiple connections between RNA processing and a large number of chromatin
modification and remodeling factors in S. pombe1. In particular, this high-throughput double
mutant analysis revealed that many splicing factors had a significant enrichment of synthetic
sick/lethal genetic interactions with components of the SWR complex. To probe the functional
interactions between SWR complex components and the splicing machinery, we performed a
splicing-sensitive microarray to screen for pre-mRNA accumulation in Δpht1 and Δswr1 strains.
Consistent with the EMAP results, we observed that both the Δpht1 and Δswr1 strains exhibit
pronounced splicing defects in an intron-specific manner, the profile(s) of which are complex
and not completely overlapping. We are confirming and further characterizing these splicing
defects by qRT-PCR. One explanation for the splicing defect in the Δpht1 and Δswr1 strains is
that the SWR complex facilitates the recruitment of the splicing machinery; to test this
hypothesis we are preparing to perform chromatin immunoprecipitation-qPCR of splicing
factors. To date, our work indicates that efficient splicing is highly sensitive to mutations in both
the splicing and chromatin machineries.
1. Ryan et al. 2012 Mol Cell 46(5) 691-704
2771
Exploring the role of an hnRNP-related protein at the yeast telomere.
J. Jones1, A. Ito1, S. Spencer1, J. Y. Lee-Soety1; 1Saint Joseph's University, Philadelphia, PA
The maintenance of linear chromosome ends, or telomeres, is an important aspect of
chromosomal stability and telomeric replication. The TG-rich telomeric sequences end in a
MONDAY-LATE POSTER PRESENTATIONS
single stranded overhang causing telomeres to shorten with each round of DNA replication. The
progressive shortening causes growth arrest, also known as cell senescence. The telomerase
enzyme complex may relengthen telomeres and help solve the end replication problem.
Telomeres may also be maintained by other proteins that serve to cap chromosome ends or
prevent the activation of DNA damage signals. Saccharomyces cerevisiae constitutively express
telomerase but may be genetically manipulated to study telomere maintenance mechanisms.
Recently, proteins involved in RNA processing have been associated with telomere
maintenance, including heterogeneous nuclear ribonuclear protein (hnRNP) related proteins in
mammals. The yeast hnRNP-related protein, Npl3, is very similar to mammalian hnRNP A1. We
have found that yeast cells with an NPL3 deletion increased the rate of senescence in
telomerase-null cells. Furthermore, we have demonstrated that the conserved RNA recognition
motif in Npl3 was critical in preventing this accelerated senescence. Npl3 was able to bind
telomeric DNA, either double or single stranded, and telomeric RNA in vitro. However, Npl3 did
not appear to bind to telomeres in vivo in chromatin immunoprecipitation (ChIP) experiments. In
addition to processing and exporting mRNA, the multi-faceted Npl3 has also been implicated in
regulating transcription and translation. Our data suggest a role for Npl3 in regulating
transcription at subtelomeric regions, by which non-coding telomeric repeat-containing RNA
(TERRA) is made. Investigation into the function of Npl3 at telomeres will aid in understanding
the complex dynamics of telomere maintenance.
2772
Identification and localization of candidate transcription factors for the regulation of rat
ventral prostate gland response to androgen deprivation
R. Rosa-Ribeiro1, R. O. Vidal2, H. F. Carvalho1;
1
State University of Campinas, Campinas, Brazil, 2Brazilian Synchrotron Light Laboratory,
Campinas, Brazil
Androgens are key regulators of prostate physiology and exert their effects through the
androgen receptor (AR). AR function in transcription regulation is better characterized as an
enhancer and is modulated by hundreds of co-activators and co-repressors co-opted to activate
or repress a large number of genes. Estrogen acts not only locally in the prostate gland via
estrogen receptors (ER), but also as a strong systemic anti-androgen. This work was
undertaken to identify candidate transcription factors (TF) working in concert with AR to regulate
gene expression after androgen deprivation in rat ventral prostate by castration for three days
(Cas), one high dose of 17β-estradiol (E2) and a combination of both (Cas+E2). We approached
this by identifying gene expression profile utilizing DNA microarray (Rat Gene ST1.0,
Affymetrix). Gene ontologies and term enrichment pertaining to treatment-exclusive and shared
gene subgroups were determined and TF within the regulatory pathways were found. We have
also performed a search for putative TF binding sites in the proximal promoter (3,000 pb) of the
10 most up- or down-regulated genes in each experimental group and common to the three
groups by in silico analysis using Match1 program. The results revealed forty-one TF and
transcription regulators recovered by both approaches. Among them, Evi-1, NF-Y, HNF-4, Elk-1,
GATA-2, c-Rel, v-Myb and NFκB showed differences with respect to the promoter regions of the
regulated genes GAPDH and TBP-7. GATA-1 and GATA-2 were characteristic of the Cas group
and NFκB of the E2 group. Validation of these results showed that all these factors but HNF4
were expressed in the ventral prostate. Immunohistochemistry revealed that these TF are
differentially distributed in epithelial and stromal cells. In conclusion, a series of TF was
identified as candidates to regulate gene expression profiles in the rat ventral prostate gland in
an androgen deprived environment and/or in response to high dose estrogen and are likely
involved in the regulation of the different phenomena resulting in organ shrinkage, including
ECM remodeling.
MONDAY-LATE POSTER PRESENTATIONS
Financial support from FAPESP 2012/07512-4.
2773
Biochemical insights into the mechanism of Mot1.
R. Viswanathan1, D. Auble1; 1Biochemistry, Molecular Biology and Genetics, University of
Virginia, Charlottesville, VA
Mot1, an essential protein in S. cerevisiae, regulates the dynamics of TATA-binding protein
(TBP) by displacing it from promoters in an ATP-dependent manner. Mot1 also belongs to the
Swi2/Snf2 enzyme family, whose members broadly regulate various processes like
transcription, replication and repair. While many of the Swi2/Snf2 ATPases are subunits of large
complexes, Mot1 and its human homolog, BTAF1, act as single polypeptides on TBP. In order
to gain insight into the mechanism by which these enzymes disrupt protein-DNA interactions,
we used a FeBABE-mediated hydroxyl radical cleavage assay to map the domains of Mot1 that
interact with promoter DNA. Using this assay, we found first that the Mot1 ATPase domain
interacts with DNA upstream of the TATA box. Using the ATP analog, ADP.AlF4, we trapped a
putative transition-state complex in which the ATPase domain has undergone a conformational
change in which an additional domain is engaged with DNA and the complex is primed for TBPDNA dissociation. The C-terminal Mot1 ATPase domain is tethered to TBP by a spring-like
array of HEAT repeats. We propose that the HEAT array might not function as a passive tether
and instead may store conformational energy in the spring so that it can be used to facilitate the
dissociation of TBP from DNA.
2774
ATP-independent role for DEAD-box ATPase Prp28 in spliceosome assembly.
A. Price1, C. Guthrie1; 1Biochemistry and Biophysics, University of California, San Francisco,
San Francisco, CA
The spliceosome is a macromolecular machine that must identify and excise introns with single
nucleotide precision. This identification and excision is accomplished by a cascade of
recognition and rearrangement events, catalyzed by members of the DExD/H-box family of
ATPases. DEAD-box protein Prp28 promotes release of the U1 snRNP during a critical step of
spliceosome assembly. This step results in the hand-off of the 5’ splice site from the U1 snRNP
to the U6 snRNP in the catalytic core of the spliceosome. Previous work also implicated Prp28
in association of the U4/U6-U5 triple snRNP, which takes place prior to or in coordination with
U1 release. However, the mechanism of this involvement was unknown.
We sought to investigate the role of Prp28 in triple-snRNP association by monitoring kinetics of
spliceosome assembly extracts from a cold-sensitive prp28-1 strain. We also used native gels to
monitor the steps in spliceosome assembly that occur prior to triple-snRNP association and
prior to U1 release. Surprisingly, we found that prp28-1 extracts form less of commitment
complex 2, which is one of the first complexes formed during spliceosome assembly, well before
tri-snRNP association. Formation of commitment complex 2 is ATP-independent. We therefore
propose that the DEAD-box ATPase Prp28 plays two roles in spliceosome assembly: one ATPindependent role in formation of commitment complex 2 and a subsequent ATP-dependent role
in U1 release after triple-snRNP joining.
MONDAY-LATE POSTER PRESENTATIONS
2775
Closure of the RNA polymerase active center - too much fidelity.
A. Parshin1, J. Lee2, S. Borukhov2; 1Cell Biology, UMDNJ-SOM, GSBS, Stratford, NJ, 2Cell
Biology, UMDNJ-SOM, Stratford, NJ
Accurate transcription ensures correct transfer of genetic information form DNA to proteins.
Given the structural and mechanistic similarities between all classes of multi-subunit RNA
polymerases (RNAP), the mechanism of transcription fidelity is likely to be conserved in nature.
According to a current model, fidelity of RNAP is controlled at two key steps of nucleotide
addition cycle by the trigger loop (TL), an essential mobile structural element of RNAP β’
subunit. At the first step, NTP binds to a pre-insertion site of the catalytic center with the “open”
TL conformation. Correct NTP is recognized through Watson-Crick base-pairing with +1 DNA
base. At the second step, TL refolds into helix, “closes” the active center and delivers the NTP
to the insertion site. “Closed” TL verifies the NTP’s steric fit and catalyzes incorporation of only
correct NTP into RNA. To examine this model, we fixed the TL of E. coli RNAP in the “closed”
conformation by engineered covalent disulfide bridge. The resulting RNAP was subjected to
biochemical and in vitro transcriptional assays. We show that the “closed TL”-RNAP is
catalytically active, displaying the NTP-binding affinity and the rates of substrate incorporation
comparable to those of the wild type enzyme. Remarkably, the “closed TL”-RNAP possesses
dramatically increased fidelity of transcription (by 10-100 fold) which makes it a nearly
“errorless” enzyme. As we have shown previously, a negative outcome of TL “closing” was a
decreased maximal rate of transcription elongation due to limited diffusion of NTPs to the active
site. Perhaps, RNAP with flexible rather than fixed TL has an evolutionary advantage as it finds
an optimal balance between the accuracy and efficiency of transcription.
2776
Splicing in Yeast is Sensitive to RNAPII Elongation Rate.
E. Moehle1, H. Braberg1, C. Kaplan2, N. Krogan1, C. Guthrie1; 1Biochemistry, UCSF, San
Francisco, CA, 2Texas A&M University, College Station, TX
From diverse experimental systems, is becoming clear that the rate of RNA polymerase can
affect the efficiency of splicing, as well as the resulting isoform of an mRNA. The factors that
affect polymerase speed, as well as the generality of this coupling, remain poorly understood in
S. cerevisiae. To better study the coupling of splicing and transcription, we employed a series
of point mutations of RNA Polymerase II that affect in vitro transcription rates over a 40-fold
range. We used genome-wide splicing-sensitive microarrays to look for changes in splicing
efficiency in this series of point mutants. As predicted by the kinetic coupling model, increasing
the rate of transcription adversely affects the splicing of many genes, and a slower polymerase
exhibits a number of genes with depleted intron levels. Additionally, a number of transcripts
display a splicing defect when polymerase rate varies in either direction from the wild-type rate.
Furthermore, results from high throughput genetic screens show that these point mutants
genetically interact with a number of chromatin. These factors are intriguing candidates for
further studying how splicing may be affected by the chromatin structure of a spliced gene's
template.
MONDAY-LATE POSTER PRESENTATIONS
2777
Looking at chromatin structure with super-resolution microscopy
D. A. Cisneros1,2, M. Molodtsov2, A. Tedeschi1, J-M. Peters1, A. Vaziri1,2; 1Research Institute of
Molecular Pathology, Vienna, Austria, 2Max F. Perutz Laboratories, Vienna, Austria
The higher-order structure of chromatin is poorly understood, but it has been proposed that the
cohesin complex controls chromatin structure by contributing to the formation of chromatin
loops. Several proteins regulate the interaction of cohesin with chromatin, including the cohesin
associated protein Wapl which is thought to release cohesin from DNA. Here we have used
photo activated localization (PALM) super-resolution microscopy to study the structure of
chromatin in mouse embryonic fibroblasts in which the Wapl gene can be deleted and in which
cohesin stably accumulates on chromatin following Wapl depletion (Tedeschi, et al.,
unpublished). PALM supersedes conventional microscopy by localizing single fluorescent
proteins with 30-50 nm precission, which is better to the typical diffraction limit of ~200 nm. We
find that in control cells cohesin complexes are located in axial chromosomal domains which
become more prominent in Wapl depleted cells. Our data indicate that these chromosomal
domains are formed by clustering of cohesin complexes. We discuss how stabilization of
cohesin on DNA following Wapl depletion might lead to changes in cohesin-cohesin interactions
and changes in chromatin structure.
2778
Analysis of TIN2 isoform specificity.
B. Cimini1, E. Blackburn1; 1Department of Biochemistry and Biophysics, University of CaliforniaSan Francisco, San Francisco, CA
Mammalian telomeres are protected by the shelterin complex, made up of TRF1, TRF2, POT1,
TPP1, RAP1, and TIN2. Recent studies by Kaminker et al have shown that TIN2 is actually
expressed as two separate isoforms, TIN2S and TIN2L. The differences between these two
isoforms have not been characterized. We have created a system that allows us to knock down
the endogenous TIN2 and replace a single isoform, allowing us to examine the functional
differences of these two isoforms. We will show the isoform-specific differences in telomeres'
DNA damage protein recruitment, shelterin recruitment, and telomerase recruitment in multiple
cell lines expressing exogenous TIN2S vs TIN2L.
2779
The Role of Sir3-Sir4 Interactions in the Spreading of Heterochromatin in Saccharomyces
cerevisiae.
K. Harding1, R. Gerson1, C. Dore1, A. Rudner1; 1University of Ottawa, Ottawa, ON, Canada
Heterochromatin in eukaryotes regulates gene expression during development and formation of
specialized chromatin structures at telomeres and centromeres. In budding yeast, the SIR
(Silent Information Regulator) complex creates silenced chromatin regions at the mating loci,
telomeres and rDNA repeats. The SIR complex consists of histone deacetylase Sir2, and
histone binding proteins Sir3 and Sir4. Sir4 recruits Sir2 to silencer elements, where
deacetylation of H4K16 by Sir2 results in high affinity binding of Sir3 to nucleosomes. Current
models propose a polymerization model of SIR complex spreading involving reiterated histone
deacetylation and recruitment of Sir3 and Sir4.
The polymerization model of heterochromatin formation assumes a dependence of Sir3
binding to histones modified by Sir2. However, overexpression of Sir3 alone can extend
heterochromatin into euchromatic regions and oligomeric complexes of Sir3 have been
described in vitro, suggesting that Sir3 alone can polymerize and spread independent of specific
MONDAY-LATE POSTER PRESENTATIONS
histone modifications. We therefore wanted to test if Sir2 and Sir4 are required for Sir3mediated heterochromatin spreading.
Recently, the crystal structure of a C-terminal region of Sir3 was solved and a cluster of
lysine residues was proposed to form a binding site for Sir4. We have created alleles of SIR3
(sir3-2A, sir3-3A and sir3-4A) that mutate these residues and confirmed that they are unable to
bind to Sir4 through co-immunoprecipitation assays. Additionally, we have shown that this
region on Sir3 binds to the C-terminal Sir4 coiled-coil, which was previously mapped as a Sir3binding domain. These mutants cannot assemble silent chromatin in the absence of wild type
Sir3, showing that the Sir3-Sir4 interaction is required to nucleate heterochromatin. However,
the mutants are not dominant negative when expressed in a wild type cell, suggesting that they
do not interfere with the nucleation of silent chromatin.
Using a quantitative reporter assay, we show that overexpression of sir3-4A results in
silencing of subtelomeric regions without Sir4. When wild-type Sir3 is overexpressed, silencing
of a reporter gene is proportional to its distance from the telomere. Our data suggest that
silencing by sir3-4A is less dependent on this relationship, and this may reflect a property of
Sir4-independent silencing. We are currently testing if we can completely bypass the
requirement for Sir4 for both nucleation and spreading of silent chromatin.
2780
Identification and characterization of synthetic interactions between cdc13-1 and yKU80
mutant alleles on yeast telomeres.
J. Lorquet1, S-A. Flores1, W. Azaizeh1, L. Sanchez1, M. Jean1, C. J. Hengartner1, L. R. Vega1;
1
Biology, Barry University, Miami Shores, FL
Telomeres are the physical ends of eukaryotic chromosomes that function to protect DNA ends
from degradation and from end to end fusion. Telomeres consist of stretches of repeated C/Grich DNA ending with 3’ single stranded G-rich overhangs. Telomere maintenance and function
are facilitated by the enzyme telomerase and by accessory proteins such as Ku and Cdc13p. In
S. cerevisiae, the essential telosome protein Cdc13p binds to single-stranded G-tails and is
required to protect telomeric DNA from degradation. cdc13-1 is a temperature sensitive allele of
CDC13, that is defective for telomere end protection. Another yeast telosome component is the
heterodimeric Ku complex, composed of Ku70 and Ku80. Ku plays multiple roles in DNA
metabolism including: non-homologous end joining and recombination and protects telomeres
from degradation and inappropriate recombination. Yeast cells limited for Cdc13p or for the Ku
complex have uncapped telomeres and exhibit longer single-stranded G-tails. Ku also interacts
with TLC1, the RNA template of the telomerase enzyme and has been shown to exhibit endbinding activity. This study examines the effects of mutations in yKU80 on cdc13-1 strains.
Using a genetic library of yku80 mutations generated by A. Bertuch’s laboratory, we have
introduced 125 mutant yku80 alleles into the cdc13-1 background by plasmid shuffle and
determined the effects on viability and telomere end protection of the various yku80 mutant
alleles in cdc13-1 strains. We found that 22 out of 125 yku80 alleles tested increased the
temperature sensitive phenotype of cdc13-1 strains, suggesting a telomeric/end protection role
for these mutant yku80 alleles. We are currently characterizing the telomere phenotypes of
double mutant strains.
Supported by nih-nigms mbrs rise: R25 GM059244, Barry University, and nih-nigms/Nci mbrs
Score grant, 5sc 2ca 138567
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2781
Exploring C. elegans heterochromatin through the HP1 homolog HPL-2.
J. Garrigues1, S. Strome1; 1Molecular, Cell, and Developmental Biology, UC Santa Cruz, Santa
Cruz, CA
Formation of heterochromatin is important for organization of the genome and regulation of
gene expression. Heterochromatin protein 1 (HP1) and methylation of histone H3 at lysine 9
(H3K9me) are hallmarks of heterochromatic regions; HP1 can directly bind H3K9me via its
chromodomain.
The nematode C. elegans does not have obvious densely stained
heterochromatin by electron microscopy. To infer the possible distribution of heterochromatin in
worms, we performed chromatin immunoprecipitation followed by microarray analysis (ChIPchip) using antibodies specific for the worm HP1 homolog, Heterochromatin Protein 1-Like 2
(HPL-2) and compared its distribution to the distributions of H3K9me1, H3K9me2, and
H3K9me3. We observed that HPL-2, like H3K9me, is enriched on autosomal “arms” and
depleted from the X chromosome. Consistent with the autosomal arms being worm
heterochromatin, they contain more repetitive sequences and have fewer genes than
chromosome interiors. Interestingly, HPL-2 shows the highest correlation with H3K9me2 and
the lowest correlation with H3K9me3. To test whether HPL-2 association with chromosomes
requires H3K9me, we performed ChIP-chip analysis of HPL-2 in met-2 set-25 double mutants,
which the Gasser lab has shown to completely lack H3K9me (Towbin et al. Cell 150: 934-947,
2012). Surprisingly, the distribution of HPL-2 in met-2 set-25 mutants remains largely
unchanged compared to wild type. This finding suggests that HPL-2 can localize to and persist
on chromatin independently of H3K9me. We also observed that hpl-2 mutants display
significantly stronger transgene desilencing in the germline and sterility than met-2 set-25
double mutants. Taken together, these results suggest that HPL-2 has functions independent of
H3K9me, and that H3K9me may not be an essential component of worm heterochromatin.
2782
Genome-wide studies of replication origins in Candida albicans reveal both sequence
and chromatin features.
H-J. Tsai1, J. Baller1, L. Burrack1, J. Berman1; 1Genetics, Cell Biology and Development,
University of Minnesota, Minneapolis, MN
Faithful DNA replication is required for genome inheritance during cell division. All eukaryotes
have conserved replication machinery; however, the determinants of replication initiation remain
elusive. Origin recognition complex (ORC) binds replication origins, but no sequence-specific
DNA binding of ORC has been found in most eukaryotes. Importantly, ORC is necessary but not
sufficient to form the pre-replication complex. Our goal is to identify active replication origins
and characterize their features in the genome of the pathogenic yeast Candida albicans. We
used genome-wide approaches to specify ORC binding sites and compared ORC binding
regions with the patterns of phased nucleosomes to predict the locations of potential active
origins. A distinct class of constitutive origin is found in all of the C. albicans regional
centromeres, and origin firing at the centromere requires the presence of a functional
kinetochore (Koren and Tsai et al. PLoS Genetics 2010). The centromeric origins are active in
each cell cycle as revealed by a nucleotide skew pattern. Furthermore, aligning ORC binding
sites with positioned nucleosomes throughout the genome revealed the locations of potential
non-centromeric (chromosomal) origins. 139 chromosomal origins have been identified. These
origins do not appear to be constitutive, and the origin firing efficiency varies, depending on the
local genetic features. Interestingly, a novel 21 bp sequence motif was found as a putative ARS
consensus sequence (ACS). In C. albicans, circular plasmids rapidly integrate into the genome
and are not maintained autonomously. We have designed a plasmid system mimicking the
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linear chromosome structure to test our hypothesis that this novel motif can serve as an ACS in
C. albicans genome to facilitate efficient replication origin firing during the cell cycle. In addition
to the sequence motif, histone methylation and acetylation appear to have a strong effect on
ORC binding affinity at a subset of chromosomal ORC binding sites. Thus, local histone
modifications may regulate non-constitutive origins. Taken together, we conclude that C.
albicans has two different classes of replication origins: constitutive origins at the centromeres
and stochastically firing origins, which may be associated with an ACS motif at many loci across
the chromosome arms.
2783
Reducing lysyl hydroxylase 2 expression in a pro-fibrotic environment by introducing
DNA methylation in the plod2 promoter.
R. A. Gjaltema1, C. Huisman1, H. G. Kazemier1, M. G. Rots1, R. A. Bank1; 1Pathology & Medical
Biology, University Medical Center Groningen, Groningen, Netherlands
Lysyl hydroxylase 2 (LH2), encoded by plod2, initiates pyridinoline (pyr) cross-linking of
collagen. These hard-to-degrade cross-links are increased in fibrotic collagen, resulting in poorly
digestible collagen and subsequently altered biomechanical support of tissues. To date, no
successful therapeutics have been developed to reduce pyr cross-linking in fibrotic diseases.
Therefore we aim to reduce LH2 expression by targeted DNA methylation of important
transcription factor (TF) binding sites, which can be inherited by daughter cells and withstand
activation of fibroblast by the pro-fibrotic cytokine TGFβ1. To identify LH2 expression related
TFs and their binding to the plod2 promoter, we performed promoter-reporter assays, low
molecular weight compound (LMWC) inhibition of TFs, and chromatin immunoprecipitations
(ChIPs) in primary human skin fibroblasts. Influence of DNA methylation on LH2 expression was
assessed by bisulfite sequencing and in vitro methylation of promoter-luciferase constructs.
Currently we are targeting zinc-finger proteins fused to DNA methyltransferases to the plod2
promoter to introduce targeted DNA methylation. The -1000 ATG deletion fragment showed the
highest reporter activity after TGFβ1 stimulation. This region contains putative SP1 and Smad3
binding sites, and LMWC inhibition of either TF reduced LH2 expression of TGFβ1 stimulated
fibroblasts. ChIP indicated binding of both TFs to the plod2 promoter. No differences in DNA
methylation was observed after TGFβ1 stimulation. However, in vitro methylation of the plod2
promoter by M.SssI prevented reporter activity during TGFβ1 stimulation. To conclude, our data
indicates that introducing DNA methylation at indicated TF binding sites to be a potent tool in
reducing fibrosis-related LH2 expression.
2784
A Chemical Genetic Strategy for Studying Substrate Specificity of the PCAF
Acetyltransferase Domain.
T. Zekovic1, D. Lloyd1, H. Luecke1; 1NIDDK, NIH, Bethesda, MD
Specific amino acids within the histone tails are sites of regulatory post-translational
modifications (PTM). Various PTMs, including acetylation, methylation, phosphorylation, ADPribosylation and ubiquitination, are recognized to play pivotal roles in the regulation of gene
expression and functions of many eukaryotic proteins. Acetylation of lysine side chains, carried
out by histone acetyltransferases (HATs) is now recognized to be one of the most important
epigenetic modifications. Even though lysine acetylation was initially identified in histones, many
nonhistone proteins are subject to acetylation, raising an important question about crosstalk
between PTMs in cells. So, with the increase in complex regulatory processes at the cellular
level, it is necessary to design new experimental tools to solve the conundrum of acetylation as
a dynamic protein PTM. We are interested in identifying specific sites of histone
MONDAY-LATE POSTER PRESENTATIONS
acetyltransferase P300/CBP-associated factor (PCAF) function in mammalian cells. Our
strategy is a chemical genetic approach based upon engineering the HAT domain to selectively
transfer ‘clickable’ acyl groups to substrate proteins. Using this approach, we were able to
identify known and novel substrates of various HAT domains, in human cell lysates and intact S.
cerevisiae cells. We will present our progress toward the identification of novel substrates of the
PCAF HAT domain, with an emphasis on comparing PCAF substrates with those of related HAT
proteins. Taken together, these approaches will facilitate deconvolution of HAT signaling
mechanisms in diverse eukaryotic cells.
2785
HACking the centromere: epigenetic engineering on Human Artificial Chromosome
reveals plasticity of centrochromatin.
N. M. Martins1, J. H. Bergmann2, A. Kagansky1, W. C. Earnshaw1; 1Wellcome Trust Centre for
Cell Biology, Edinburgh, United Kingdom, 2Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY
Centromeres are loci in eukaryotic chromosomes that form the foundation of kinetochores,
allowing chromosome attachment to the mitotic spindle. Flaws in kinetochore stability lead to
segregation defects, resulting in aneuploidy, genomic instability and eventually cell death or
cancer. Centromeres are determined epigenetically, in part by the incorporation of the histone
H3 variant CENP-A, forming a specialized chromatin landscape dubbed ‘centrochromatin’.
Trancription-associated factors and centromeric transcripts have been reported to play a role in
centromere maintenance, suggesting that specific processes are at work within
centrochromatin. The fact that centromeres do not have strict dependencies on DNA sequence
for localization, and can change to other locations on a chromosome over the course of
evolution, makes them a mysterious piece of the chromatin regulation puzzle.
To further dissect the nature of centrochromatin, we have developed a synthetic Human
Artificial Chromosome (HAC), to which protein constructs of interest can be targeted.
By tethering chromatin modifiers to the HAC centromere, we have previously discovered that
centromeres require active chromatin marks to be maintained, and are sensitive to KAP-1 and
HP1 heterochromatin silencing.
To better understand the properties of centrochromatin, we have manipulated both transcription
activation and repression.
By tethering p65 and VP16, two different kinds of transcription activators, we found that
centromeres are compatible with open, acetylated chromatin. However, strong transcriptional
induction by VP16 (unlike p65) disrupts the centromere, by evicting pre-existing CENP-A.
We also found that, unlike with heterochromatin repression, centromeres are resistant to
Polycomb silencing: after tethering EZH2, kinetochore proteins and active chromatin marks are
reduced, but the HAC suffers no segregation defects or loss of transcription.
These results suggest that active centromeres can be surprisingly plastic in their chromatin
activity: they can be hyperacetylated or repressed with Polycomb while still maintaining normal
kinetochore functions, but are very sensitive to histone turnover processes, such as high levels
of transcription.
MONDAY-LATE POSTER PRESENTATIONS
2786
Epigenetic regulation of the sperm specific alpha4 Na,K-ATPase and sodium hydrogen
exchanger 10 isoform genes.
D. L. Kumar1, P. L. Kumar1, P. F. James 1; 1Zoology, Miami University, oxford, OH
The α4 Na,K-ATPase and the sodium hydrogen exchanger 10 (NHE10) are sperm-specific
transport proteins important for normal sperm function. Previously, it has been shown that some
germ cell-specific genes are regulated by DNA methylation. DNA methylation is an epigenetic
mechanism that involves addition of a methyl group to a cytosine residue, which is linked by a
phosphodiester bond to guanine residue in the DNA molecule, i.e. a CpG dinucleotide.
Hypermethylation of CpG dinucleotides in CpG islands (regions rich is CpG dinucleotides)
associated with gene promoters is known to result in transcriptional repression and
hypomethylation of these CpG islands is known to result in transcriptional activation of these
genes. Differential methylation of intragenic CpG islands has been shown to be associated with
both up- and down-regulation of different genes. We have identified intragenic CpG islands in
the α4 Na,K-ATPase and NHE10 genes (Atp1a4 and Slc9a10 respectively). Here, we present
evidence of differential methylation in the CpG island of Atp1a4 with reduced DNA methylation
in sperm cells compared to somatic cells. Further, we show up-regulation of Atp1a4 and
Slc9a10 expression following treatment of male germ cell line GC-1spg cells with 5-Aza-2deoxycytidine, an inhibitor of DNA methyltransferase enzymes (DNMT’s) demonstrating the
direct involvement of DNA methylation in the regulation of Atp1a4 and Slc9a10 expression. We
also have preliminary data from mouse embryonic stem cells (ES cells) lacking DNMT1 that
demonstrates up regulation of Atp1a4 expression. Further studies will involve looking at Atp1a4
expression levels in DNMT3A and 3B and determine the functional significance of the intragenic
CpG island as an alternative promoter using luciferase reporter constructs. To our knowledge,
this is the first study addressing the role of DNA methylation in regulation of the Atp1a4 and
Slc9a10 expression.
2787
Quantitative single-cell analysis of uORF-regulated translation in the integrated stress
response.
G. Dey1, K. Han1, T. Meyer 1; 1Chemical and Systems Biology, Stanford University, Stanford, CA
Ribosomes frequently engage at and translate through upstream open reading frames (uORFs)
located in the 5’ untranslated regions (UTRs) of a significant fraction of mammalian messenger
RNAs, a surprising observation supported by a large number of recent ribosome profiling
studies. The functional role of these uORFs or their translated peptides is almost completely
unknown. The stress-regulated transcription factor ATF4 provides one interesting counterexample. When cells are subjected to stress, multiple signals converge to inactivate the ratelimiting initiation factor eIF2-α, attenuating translation globally but increasing the translation of
ATF4. The ATF4 5’ UTR contains two conserved functional uORFs: a short activating uORF1
that promotes ribosome re-initiation positioned upstream of a longer inhibitory uORF2. The
prevailing hypothesis holds that depletion of the active pool of eIF2-α delays post-uORF1
reinitiation, allowing the ribosome to bypass uORF2 and translate the coding sequence. This
simple kinetic scheme has never been quantitatively explored as a general mechanism for
inducible translation. Here we describe the development of a novel genetically encoded dualcolor fluorescent reporter to monitor translation rates in real-time in single cells. We are
currently using this sensor to obtain quantitative measurements of translational induction
regulated by the human ATF4 5’ UTR. We have encoded the working model for ATF4 regulation
in a simple computational framework, allowing us to predict the effects of varying the (highly
conserved) length and relative positions of uORF1 and uORF2 on ATF4 induction. These
MONDAY-LATE POSTER PRESENTATIONS
predictions will be quantitatively tested using the translation sensor in order to validate the
model. In addition, we will attempt to extract generalized constraints on eIF2α-regulated
translational induction, allowing us to filter the human genome for similar motifs and identify
novel stress-regulated genes. We hope that this study will serve as a starting point for the
quantitative investigation of functional regulation by uORFs, and we expect the translation
reporter to be a widely useful tool for investigating the dynamics and regulation of translation.
Signal Transduction and Signaling Networks I
2788
Vasopressin induces mTOR activation in vascular smooth muscle cells cultured in high
extracellular glucose concentration.
D. K. Montes1, V. C. Muñoz1, P. V. Burgos1, C. B. Gonzalez1; 1Department of Physiology,
Universidad Austral de Chile, Valdivia, Chile
Vascular smooth muscle cells (VSMCs) proliferation and remodeling are associated with many
vascular diseases. Hyperglycemia contributes to the development of vascular disease by
promoting cell growth and proliferation. Mammalian target of rapamycin complex (mTOR) is a
key regulator of autophagy, cell growth and proliferation. Here, we investigated the effects of
high glucose on mTOR activation induced by AVP in VSMCs. Vascular smooth muscle cells (A10), which endogenously expresses the V1 vasopressin receptor, were cultured in DMEM
containing 25 mM glucose for 10 days and then were stimulated with AVP (50 nM). We studied
the mTOR activation by Western blotting using phospho-specific antibodies. To determine the
pathway involved in mTOR activation, cells were pre-incubated with inhibitors prior to AVP
stimulation. Autophagy was analyzed using the ratio LC3 I/II proteins by Western blotting.
Incubation with AVP of VSMCs, which were cultured in high extracellular glucose (25 mM),
induced the mTOR phosphorylation, showing a maximum at 4 hours, in contrast to the AVP
stimulation of cells cultured in normal glucose (5.5 mM), wherein, only basal phosphorylation
was observed. When cells were pretreated with the a PI3K specific inhibitor or with the PKC
inhibitor the AVP-induced mTOR activation was greatly attenuated; whereas, AG1478,
PD98059 or 1-butanol did not inhibit the AVP-induced mTOR activation, ruling out the
involvement of PKD, ERK and the EFGR transactivation. Analysis of the autophagy markers
LC3I and LC3II by Western blotting showed an inhibition of the autophagy with a similar kinetic
as the mTOR increases. Our results suggest that AVP is able to stimulate mTOR and
consequently to inhibit autophagy in VSMCs by activating a PKC and the PI3K/AKT pathway in
a high extracellular glucose concentration. These results might explain why hyperglycemia is a
risk factor in cardiovascular disease.
Supported by FONDECYT 1100871 and DIDUACH.
2789
Analysis of the role of the amino acid receptor T1R1-T1R3 and amino acid deprivation on
insulin synthesis and secretion.
A. Lorente-Rodriguez1, E. M. Wauson1, M. H. Cobb1; 1Pharmacology, UT Southwestern Medical
Center, Dallas, TX
Hormones, growth factors and nutrients (including glucose, fatty acids and amino acids)
regulate steps in insulin synthesis and secretion. These signals, along with pathways that
respond to the metabolic state of the cell are coordinated by the mammalian target of rapamycin
complex 1 (mTORC1) and the adenosine monophosphate (AMP)-activated protein kinase
MONDAY-LATE POSTER PRESENTATIONS
(AMPK).
Under nutrient replete conditions AMPK is inactive and active mTORC1
phosphorylates Ulk1 to suppress autophagy. Starvation decreases ATP and increases ADP
and AMP which activate AMPK. AMPK suppresses mTORC1 and activates the autophagy
inducing kinase Ulk1. In addition, amino acid starvation inactivates mTORC1 independent of
AMPK signaling.
We find that the G-protein-coupled receptor complex T1R1-T1R3 senses amino acids, activates
mTORC1 and supports insulin secretion. Notably, knockdown of either T1R1 or T1R3 induces
autophagy in a broad set of cell lines including cardiac, lung and beta cells. In receptor
knockdown cells grown in nutrient replete conditions, the major mTORC1 and AMPK sites on
Ulk1 are under phosphorylated suggesting that AMPK is not responsible for inducing
autophagy. Receptor knockdown also causes a catastrophic loss of beta cell insulin content
that is not caused by a reduction of pre-proinsulin mRNA levels. This suggests that cellular
insulin content is regulated post-transcriptionally in receptor knockdown cells: either there is
decreased message translation, increased insulin degradation, or both. Because autophagy is
upregulated in receptor knockdown cells, we are now addressing whether this reduced insulin
content is mediated through autophagic degradation of secretory granules. In addition, we are
addressing whether autophagy is responsible for secretory granule turnover in beta cells, and
whether amino acid starvation of untransfected beta cells has any effect on insulin content and
secretion.
2790
TORC2 Signaling is Antagonized by PP2A and the Far Complex in Saccharomyces
cerevisiae.
T. Pracheil1, J. Thornton2, Z. Liu1; 1University of New Orleans, New Orleans, LA, 2Stowers
Institute for Medical Research, Kansas City, MO
The target of rapamycin (TOR) kinase is a central regulator of eukaryotic cell growth, and exists
in two essential, yet distinct, TOR kinase complexes: rapamycin-sensitive TORC1 and
rapamycin-insensitive TORC2. Lst8, a component of both TOR complexes, is essential for cell
viability. However, whether the essential function of Lst8 is linked to TORC1, TORC2, or both
remains unclear. To address this question, we conducted a genetic screen in the budding yeast
Saccharomyces cerevisiae to isolate lst8 deletion suppressor mutants. Here we report that
mutations in SAC7 and FAR11 suppress lethality of lst8 deletion and TORC2-deficient (tor2-21)
mutations but not TORC1 inactivation by rapamycin treatment. Furthermore, an lst8 deletion
results in mislocalization of TORC2 components Bit61 and Avo3 but not TORC1 component
Kog1 and has little effect on the expression of RPL3 and RPS6A, genes encoding ribosomal
proteins, which are positively regulated by TORC1 suggesting that the essential function of Lst8
is linked only to TORC2. Interestingly, characterization of lst8 deletion bypass mutants revealed
a role for protein phosphatase 2A (PP2A) in the regulation of TORC2 signaling. We show that
Far11, a member of the Far3-7-8-9-10-11 complex (Far Complex) involved in pheromoneinduced cell cycle arrest, interacts with Tpd3 and Pph21, conserved components of PP2A, and
deletions of components of the Far complex and PP2A rescue growth defects in lst8 deletion
and tor2-21 mutants. In addition, loss of Rts1, the regulatory B’ subunit of PP2A or Far11
restores phosphorylation to the TORC2 substrate Slm1 in a tor2-21 mutant. Mammalian Far11
orthologs FAM40A/B exist in a complex with PP2A known as STRIPAK, suggesting a conserved
functional association of PP2A and Far11. Other components of the STRIPAK complex, SLMAP
and the striatins, have been shown to have some limited sequence similarity with other
members of the Far complex, Far9/10 and Far8 respectively. The STRIPAK complex contains
the cerebral cavernous malformation 3 (CCM3) protein, mutations of which have been
implicated in familial cerebral cavernous malformations. The PP2A-Far complex is likely the
MONDAY-LATE POSTER PRESENTATIONS
yeast counterpart of the mammalian STRIPAK complex. Antagonism of TORC2 signaling by the
PP2A-Far complex represents a novel regulatory mechanism for controlling spatial cell growth
of yeast. Future studies on PP2A and the Far complex in yeast may provide important insights
into regulation mechanisms of the mammalian STRIPAK complex.
2791
Cellular responses to LAT1-mediated leucine uptake revealed by comprehensive
phosphoproteomics.
P. Wiriyasermkul1, S. Nagamori1, S. Serada2, M. Matsumoto3, K. I. Nakayama3, T. Naka2, Y.
Kanai1; 1Biosystem Pharmacology, Department of Pharmacology, Graduate School of Medicine,
Osaka University, Osaka, Japan, 2Laboratory for Immune Signals, National Institute of
Biomedical Innovation, Osaka, Japan, 3Department of Molecular and Cellular Biology, Medical
Institute of Bioregulation, Kyushu University, Kyushu, Japan
Amino acids are important not only for protein synthesis but also for cellular signaling regulating
cell growth and proliferation. To promote cell growth, amino acids mobilize mammalian target of
rapamycin (mTOR) signaling pathway. mTOR is a serine/threonine kinase forming two distinct
complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), to control diversely
cellular responses. mTORC1 accelerates translation by phosphorylating downstream molecules
such as p70 ribosomal S6 kinase 1 (p70 S6K) and initiation factor 4E binding protein 1 (4EBP1). Among amino acids, leucine activates mTORC1 most prominently. However, the
mechanisms of leucine-mediated mTORC1 activation are largely unknown. We have shown that
many types of cancer cell lines highly expressed L-type amino acid transporter 1 (LAT1). LAT1
is a large neutral amino acid transporter which transports several essential amino acids
including leucine for cancer cell growth. In our cell model, leucine was transported into the cells
by LAT1 and further activated mTORC1. A LAT1-inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2carboxylic acid (BCH), suppressed leucine-induced phosphorylations of mTOR, p70S6K and
4EBP1. To analyze comprehensively the cellular responses to leucine transported by LAT1, we
took a phosphoproteomics approach. The cancer cell lines were stimulated by leucine in the
presence or absence of BCH. Phosphoproteins obtained on the whole cell lysate were analyzed
quantitatively and classified based on the responsiveness to leucine and/or BCH. In the
analysis, the phosphorylation induced by leucine and suppressed by BCH was considered as
the phosphorylation caused by LAT1-mediated leucine uptake. Our results confirmed that
leucine uptake by LAT1 regulates signaling molecules involved in the translation process.
Additionally, we found that leucine also induces phosphorylation of the proteins responsible for
the other cellular responses such as transcription, cell cycle regulation and cell structure. These
data have revealed the roles of leucine in multiple cellular responses and opened up the door to
study the missing link between LAT1-mediated leucine uptake and mTORC1 stimulation.
2792
A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino
acid sufficiency to mTORC1.
L. Bar-Peled1, L. Chantranupong1, A. D. Cherniack2, W. W. Chen1, E. D. Spear3, M. Meyerson4,
D. Sabatini1; 1Whitehead Institute for Biomedical Research, Cambridge, MA, 2Broad,
Cambridge, MA, 3John Hopkins School of Medicine, Baltimore, MD, 4Dana-Farber Cancer
Institute, Boston, MA
The mTOR Complex 1 (mTORC1) pathway promotes cell growth in response to many cues,
including amino acids, which act through the Rag GTPases to promote mTORC1 translocation
to the lysosomal surface, its site of activation. Although progress has been made in identifying
positive regulators of the Rags, it is unknown if negative factors also exist and whether they are
MONDAY-LATE POSTER PRESENTATIONS
mutated in cancer. Here, we identify GATOR as a complex that interacts with the Rags. GATOR
is composed of two subcomplexes we call GATOR1 and 2. Its GATOR1 subunits have been
implicated as tumor suppressors and their inhibition makes mTORC1 signaling insensitive to
amino acid deprivation. In contrast, inhibition of GATOR2 subunits suppresses mTORC1
signaling, and epistasis analysis reveals that GATOR2 is an upstream negative regulator of
GATOR1. Furthermore, the GATOR1 subcomplex has GTPase activating protein (GAP) activity
for RagA and RagB, consistent with its inhibitory role in mTORC1 signaling. In cancer cell lines
containing GATOR1 inactivating mutations, mTORC1 is hyperactive and resistant to amino acid
regulation, and these cells are hypersensitive to rapamycin, an FDA-approved mTORC1
inhibitor. Thus, we identify the GATOR complex as a critical regulator of the pathway that
signals amino acid sufficiency to mTORC1 and reveal that, like other mTORC1 regulators, the
Rags can be deregulated in cancer.
2793
Identification of Wnt/Ryk signaling target genes in regulating Xenopus gastrulation.
Y. Hong1, E. C. Park1, E-Y. Shin1, G-H. Kim1; 1Division of Life Science, Korea Basic Science
Institute, Daejeon, Korea
Ryk is a member of atypical receptor tyrosine kinase family that consists of an extracellular WIF
(Wnt inhibitory factor) damain, an intracellular atypical kinase domain, and a PDZ binding motif
at C-terminus. Ryk has previously been shown to regulate canonical Wnt/β-catenin signaling by
directly binding to Wnt ligands and Dishevelled. A recent study showed that Ryk also regulates
noncanonical Wnt pathway in convergent extension (CE) movements by promoting Wnt11mediated endocytosis. However, downstream of Wnt/Ryk signaling during Xenopus
embryogenesis is less well characterized. In this study, we report possible target genes of
Wnt/Ryk signaling that may regulate Xenopus gastulation. To identify target genes of Ryk
signaling pathway, microarray analysis was performed with (1) uninjected, (2) Ryk mRNA
overexpressed and (3) Ryk MO injected dorsal marginal zone tissues on Affimatrix GeneChip
Xenopus laevis Genome 2.0 Array. We found 245 genes were upregulated in XRyk
overexpressed tissues and downregulated in XRyk depleted tissues at 1.5-fold or above. GO
annotation analysis showed that these genes are mainly associated with cell adhesion and
movements. It strongly suggests that these genes may contribute to the regulation of Xenopus
gastrulation. (Y. Hong and E.C. Park contributed equally to this work.)
2794
Cdo inhibits canonical Wnt signaling via interaction with LRP6 during neuronal
differentiation.
S-M. Ho1, J-S. Kang1; 1Department of Molecular Cell Biology, Sungkyunkwan University School
of Medicine, Suwon, Korea
Neurogenesis involves consecutive steps of specification, proliferation and terminal
differentiation of stem cells into mature neurons. The precise control of this process is critical to
ensure the generation of an adequate number of neurons required for brain development.
Several signaling pathways including canonical Wnt signaling are implicated in regulation of this
process. Previously, we have shown that a receptor protein Cdo promotes neuronal
differentiation. In this study, we investigated the interaction between canonical Wnt signaling
and Cdo in neurogenesis. Upon neuronal induction of P19 EC cells by retinoic acid, canonical
Wnt signaling components, a Wnt coreceptor LRP6 and Wnt ligands, and Cdo are
simultaneously upregulated. Cdo depletion and deficiency caused the upregulation of canonical
Wnt ligands and their signaling activities during neuronal differentiation of P19 EC and C17.2
MONDAY-LATE POSTER PRESENTATIONS
neural progenitor cells, resulting in impaired neurogenesis. The defective neurogenesis of Cdo
depleted cells was recovered by the treatment of Wnt inhibitor. Moreover, Cdo forms complexes
with LRP6 via its 2nd immunoglobulin domain, thereby inhibiting Wnt signaling. These data
suggest that Cdo promotes neuronal differentiation by suppression of canonical Wnt signaling.
2795
Activation of wnt/β-cate.nin signaling pathway by a phytoestrogen diarylheptanoid in
preosteoblastic cells.
A. Chairoungdua1,2, K. Bhukhai1,2, K. Suksen1, A. Suksamrarn3, P. Piyachaturawat1,2;
1
Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand, 2Research Center of
Transport Proteins for Medical Innovation, Faculty of Science, Mahidol University, Bangkok,
Thailand, 3Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok, Thailand
Wnt/β-catenin signaling pathway plays crucial roles in bone cell differentiation. Previous study
reported that the effect of estrogen on bone formation was mediated through the activation of
Wnt/β-catenin signaling pathway. Curcuma comosa Roxb. (C. comosa) has long been used in
Thai traditional medicine for treatments of illness in the uterus and ovarian hormone deficiency.
ASPP 049 is the major diarylheptanoids isolated from C. comosa, which exhibits high estrogenic
activity. However, the estrogenic activity of ASPP 049 on bone cell differentiation has not yet
been investigated. In the present study, we aim to investigate the mechanistic effect of ASPP
049, on Wnt/β-catenin signaling pathway in preosteoblastic (MC3T3-E1) cells. By using a
TOPFlash luciferase assay in MC3T3-E1 cells, ASPP 049 rapidly activated β-catenin activity
and induced a significant increase in β-catenin protein levels similar to those of estradiol.
Treatment with LiCl, the specific inhibitor of GSK-3β, ASPP 049 and E2 failed to increase the
expression of β-catenin protein, suggesting that the activation of Wnt/β-catenin signaling
induced by ASPP 049 and E2 is dependent on GSK-3β activity. Indeed, ASPP 049 inhibited
GSK-3β activity by induction the GSK-3β phosphorylation at serine 9 residue. In addition, the
induction of β-catenin protein expression by ASPP 049 and E2 is required estrogen receptor
because it was inhibited by ICI, an ER blocker. The effect of ASPP 049 in promoting bone cell
differentiation was determined by expression of ALP, a bone differentiation marker. We found
that ASPP 049 induced ALP expression in concentration-dependent manner. The induction of
ALP activity by ASPP 049 was inhibited by wnt signaling antagonist, DKK-1. Collectively, our
results suggest that ASPP 049 induces preosteoblastic cell differentiation through the activation
of Wnt/β-catenin signaling pathway. Therefore, our results provide important information
required for further development of this natural compound as supplement for postmenopausal
women.
2796
Selective Small Molecule Targeting β-C.atenin Function Discovered by an In Vivo
Chemical Genetic Screen.
J. Rickmyre1, J. Hao1, A. Ao1, A. Frist1, C. Hong1,2,3; 1Cardiovascular Medicine, Vanderbilt
University School of Medicine, Nashville, TN, 2Pharmacology, Vanderbilt University School of
Medicine, Nashville, TN, 3Cell & Developmental Biology, Vanderbilt University School of
Medicine, Nashville, TN
The canonical Wnt signaling pathway, mediated by the transcription factor β-catenin, plays
critical roles in embryonic development, and represents an important therapeutic target. In the
zebrafish, two β-catenins function in early developmental patterning. β-catenin 2 is required for
dorsal organizer formation, a region equivalent to the Spemann-Mangold organizer in Xenopus.
After formation of the dorsal organizer, zygotic contributions of β-catenin 1 are thought to
promote ventral and posterior cell fates. In a zebrafish-based in vivo screen for small molecules
MONDAY-LATE POSTER PRESENTATIONS
that specifically perturb embryonic dorsoventral patterning, we discovered a novel compound,
named windorphen, which selectively blocks the Wnt signal required for ventral development.
Windorphen exhibits remarkable specificity toward the c-terminal transactivation domain of
zebrafish β-catenin 1, disrupting its association with the co-activator p300. Thus, our chemical
genetic study indicates that the two zebrafish β-catenin proteins are not functionally redundant,
with β-catenin 1 required selectively for ventral, but not dorsal, development of the embryo.
Lastly, windorphen robustly kills cancer cells that harbor Wnt-activating mutations, supporting
the therapeutic potential of this novel Wnt inhibitor that engages a nontraditional pharmaceutical
target.
2797
Role of PI3K-Akt in regulating the TGF-beta receptor levels at the plasma membrane.
E. H. Budi1, R. Derynck2; 1Department of Cell and Tissue Biology, UCSF, San Francisco, CA,
2
The Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, UCSF,
San Francisco, CA
Cells rely on their receptors at the plasma membrane to bind extracellular molecules and trigger
intracellular responses. In response to TGF-β, the TGF-β receptors relay signals into
transcriptional responses that affect many cellular processes, including differentiation, cell
growth, cell death, homeostasis to diseases. TGF-β signals through binding to type II and type I
serine/threonine kinase receptors, which leads to rapid activation and hetero-oligomerization of
Smad2 and Smad3 which subsequently can activate Smad signaling, and non-Smad signaling
through kinase pathways. Insulin, a potent inducer of the PI3K-Akt pathway, activates TGF-β
signaling. We hypothesize that insulin regulates TGF-β signaling by inducing an enhancement
of TGF-β receptor levels at the plasma membrane through activation of PI3K-Akt pathway.
Indeed, we found that insulin upregulates TGF-β receptors at the plasma membrane. Our data
suggest that the increased TGF-β receptor presentation at the cell surface is mediated through
the PI3K-Akt pathway. Inhibition of this pathway using a pharmacological inhibitor or RNAi
showed lower levels of TGF-β receptors at the plasma membrane. We also determined whether
increased cell surface TGF-β receptor levels are affecting cell’s sensitivity and responsiveness
to TGF-β signal. We found that, at low TGF-β levels, insulin-treated cells showed higher levels
of phospho-Smad2/3 and higher Smad-mediated transcription activity than cells in the absence
of insulin. This effect was blocked by SB431542, a specific of TGF-β type I receptor kinase
inhibitor. Our results support the hypothesis that activation of PI3K-Akt signaling in response to
insulin initiates the rapid transport of the TGF-β receptors to the cell surface, which leads to
increased cell sensitivity and responsiveness to TGF-β signal. Studies are ongoing to define the
mechanism through which PI3K-Akt regulates TGF-β receptor cell surface levels.
2798
ILK modulates TGF-β receptor signalling in dermal fibroblasts.
L. Dagnino1, S. Boo1, L. Vi1; 1Physiology and Pharmacology, University of Western Ontario,
London, ON, Canada
Transforming growth factor-beta (TGF-β) signalling pathway is one of the fundamental cellular
signalling pathways described in many cell types. It is involved in many biological and
pathological processes, including pathogenesis of many abnormal wound healings and
tumourigenesis. In dermis, TGF-β1 modulates skin homeostasis, and participates in wound
healing through several mechanisms, including induction of proliferation and migration of dermal
fibroblasts, as well as differentiation of dermal fibroblasts into contractile myofibroblasts. We
recently established that normal induction of dermal myofibroblast differentiation by TGF-β1
requires integrin-linked kinase (ILK), a scaffolding protein, which also has been associated with
MONDAY-LATE POSTER PRESENTATIONS
various human cancers. ILK interacts with TGF-β1 receptor type II (TβRII) in various cell types
and ILK-deficient fibroblasts display decreased levels of receptor complexes containing TGF-β
receptor type I (TβRI) and TβRII. This decrease is accompanied by reduced Smad2
phosphorylation and alpha-smooth muscle actin expression in response to TGF-β1. This
decrease in Smad2 phosphorylation is partly rescued by exogenous ILK. In ILK-deficient cells,
increased levels of TβRII ubiquitination and degradation through the proteasomal pathway are
observed, suggesting a modulatory role of ILK in TβR trafficking. Inhibition of TβRII turnover
with a proteasomal inhibitor in ILK-deficient fibroblasts partially restored Smad2 phosphorylation
in response to TGF-β1. Similarly, disruption of lipid-raft formation by nystatin, a drug that
interferes with proteasomal TβRII degradation, partly restored levels of Smad2 phosphorylation
induced by TGF-β1 in ILK-deficient cells. This suggests a role of ILK in modulation of TβRII halflife, through modulation of its trafficking to lipid-rafts, thus reducing its proteasomal degradation
and increasing the receptors available to traffic to endosomes and signal through Smad2.
Supported with funds from the Natural Sciences and Engineering Research Council of Canada.
2799
PAWS1: a new player in the BMP pathway.
J. Vogt1, K. Dingwell2, T. Macartney1, J. Smith2, G. Sapkota1; 1MRC Protein Phosphorylation
and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom, 2MRC National Institute
for Medical Research, London, UK
Bone morphogenetic proteins (BMP) play critical roles in key cellular processes and during
embryogenesis. BMPs signal through receptor-mediated tail-phosphorylation of SMAD
transcription factors 1, 5 & 8 (SMADs1/5/8). In the canonical pathway, phosphorylated SMADs
associate with SMAD4 and translocate to the nucleus, where they regulate the expression of
multiple target genes. However, even in the absence of SMAD4, BMP ligands still control
multiple cellular responses. How these are achieved is poorly understood. Through a proteomic
approach, we identified a novel protein associated with SMAD1 (PAWS1). We demonstrate that
endogenous PAWS1 interacts preferentially with SMAD1 and forms a complex in SMAD4independent manner. Furthermore, we observed that BMP induces phosphorylation of PAWS1
and this is inhibited by a relatively non-specific ALK3-inhibitor, LDN-193189. In vitro active BMP
type I receptors ALK2, 3 and 6 also phosphorylate PAWS1. Interestingly, BMP-induced
phosphorylation of PAWS1 is critical in mediating the transcription of some non-canonical BMPtarget genes. We further investigated the role of PAWS1 during early Xenopus embryogenesis.
Overexpression of PAWS1 in X. laevis embryos leads to duplication of axis, a phenotype that is
associated with the inhibition of BMP signalling. Collectively, these observations imply that
PAWS1 is a new player in the BMP signalling pathway. We are deciphering the precise
molecular mechanisms by which PAWS1 modulates the BMP-SMAD1 pathway.
2800
Arginine methylation initiates BMP-induced Smad signaling.
J. Xu1, J. Liu1, H. A. Wang1, J. Oses-Prieto1, M. Pei1, A. Burlingame1, K. Bruckner1, R. Derynck1;
1
University of California, San Francisco, San Francisco, CA
Kinase activation and substrate phosphorylation commonly form the backbone of signaling
cascades. Bone morphogenetic proteins (BMPs), a subclass of TGF-β family ligands, induce
activation of their signaling effectors, the Smads, through C-terminal phosphorylation by
transmembrane receptor kinases following type I and type II receptor complex formation.
However, the slow kinetics of Smad activation in response to BMP suggests a preceding step in
the initiation of BMP signaling. We now show that arginine methylation, which regulates gene
expression, yet also modifies some signaling mediators, initiates BMP-induced Smad signaling.
MONDAY-LATE POSTER PRESENTATIONS
BMP-induced receptor complex formation promotes presentation of the methyltransferase
PRMT1 to the inhibitory Smad6, resulting in Smad6 methylation and relocalization at the BMP
receptors. BMP-induced Smad6 methylation by PRMT1 occurs at a specific arginine, arginine
74, as revealed by mass spectrometry analysis and confirmed with a mutagenesis approach.
Antibodies specific for mono or asymmetric di-methylated arginine 74 further confirmed Smad6
methylation and Smad6 recruitment to the cell membrane upon BMP stimulation. BMP-induced
and PRMT1-mediated Smad6 methylation leads to Smad6 dissociation from BMP type I
receptors, allowing activation of effector Smads through phosphorylation. This requirement for
PRMT1 in BMP-induced biological responses is conserved across species, as apparent by the
role of the PRMT1 ortholog Dart1 in BMP signaling during Drosophila wing development.
2801
Signaling pathways involved in AVP-mediated ERK1/2 activation and cell proliferation in
vascular smooth muscle cells.
M. Brenet1, C. I. Villanueva1, P. L. Carmona1, C. B. Gonzalez1; 1Department of Physiology,
Universidad Austral de Chile, Valdivia, Chile
Vascular smooth muscle cell (VSMC) proliferation and remodeling are associated with many
vascular diseases. Arginine vasopressin (AVP) induces proliferation of VSMC; however, the
pathway by which vasopressin receptor actives ERKs (Extracellular Regulated Kinases) and its
contribution to cell proliferation are poorly understood. We investigated the signaling event
triggered by AVP and its participation in the proliferation of VSMCs. To determine proliferation
and ERKs phosphorylation, A-10 cells, derived from rat vascular smooth muscle cells, were
stimulated with AVP and ERKs phosphorylation was determined by Western blotting using
phospho-specific antibodies and proliferation was studied using BrdU labeling assay. AVP
activates ERK1/2 in A-10 cells with a biphasic kinetic; an early and a late phase. The early
phase (2.5-15 min) depends upon EGFR transactivation and involves the activation of PKC and
Src; whereas, the late phase (60-120 min) was EGFR-transactivation independent. Similarly,
AVP-stimulated cell proliferation was blocked by inhibition of PKC and Src activities and also by
preventing EGFR transactivation. ERK1/2 were co-immunoprecipitated with ELK-1, a nuclear
substrate of ERKs, from cell extracts stimulated for 2.5-15 min with AVP but not at longer times.
Moreover, V1a cytoplasmic C-terminus contains several potential PKC and GRK
phosphorylation sites. To study the role of these sites in AVP-induced ERK1/2 phosphorylation
signals we generated two mutants, one truncated at the first potential phosphorylation site
V1a387stop; and the other in which Ser-388 was mutated to Gly-388. The AVP-induced early
ERK1/2 phosphorylation peak was significantly attenuated in the truncated or mutated V1a
receptors. Interestingly, only the truncated receptor did not proliferate after AVP stimulation.
Taken together, these results suggest that the AVP induces the ERK phosphorylation by EGFRtransactivation dependent and independent mechanisms. Moreover, Src and PKC appear to be
involved in both mechanisms. The EGFR-dependent mechanism is essential for AVP-induced
VSMCs proliferation.
Supported by CONICYT fellowship and FONDECYT 1100871 and DIDUACH.
2802
Mechanisms of fibroblast growth factor 9-induced steroidogenesis in mouse testis.
M. S. Lai1, P. R. Chen1, B. S. Pan1, Y. M. Lin1, S. J. Tsai 1, B. M. Huang1; 1National Chen Kung
university, Tainan, Taiwan
Fibroblast growth factor 9 (FGF9) is a multifunctional poly-peptide belonging to the FGF family,
which has been demonstrated with biological functions related to bone formation, lens fiber
MONDAY-LATE POSTER PRESENTATIONS
differentiation, nerve development, gap junction formation and sex determination. In previous
study, we demonstrated that FGF9 could stimulate the production of testosterone in mouse
Leydig cells. In the present study, we used both primary mouse Leydig cells and MA-10 mouse
Leydig tumor cells to further investigate the detail cellular mechanism of FGF9 stimulating
steroidogenesis. Results showed that the FGF9 could significantly activate steroidogenesis both
in mouse primary and tumor Leydig cells (p<0.05). Further investigation showed that FGF9
significantly induced the level of phospho-AKT at 0.5 and 24 hr; phospho-JNK at 0.25, 0.5 and
24 hr; phospho-p38 at 0.5 hr; and phospho-ERK1/2 from 0.25 to 24 hr in primary Leydig cells,
respectively (p<0.05). Also, FGF9 significantly up-regulated the level of phospho-AKT at 3 and
24 hr; phospho-JNK at 0.25 and 1 hr; and phospho-ERK1/2 at 0.5 to 1 hr in tumor Leydig cells,
respectively (p<0.05). By using specific inhibitor of AKT, JNK, p38 and ERK1/2, we further
demonstrated that the inhibitor of AKT and ERK1/2 could significantly repress the stimulatory
effect of FGF9 on mouse Leydig cell steroidogenesis. According to these findings, we
concluded that FGF9 induced testosterone production by activating JNK, ERK1/2 and p38 of
MAPK pathway and AKT of PI3K pathway.
2803
The Role of Pseudophosphatase MK-STYX in Neuronal Differentiation.
B. M. Flowers1, K. Wong1, S. D. Hinton1; 1Biology, College of William and Mary, Williamsburg,
VA
The rat pheochromocytoma PC12 cell line is a widely used system to study neuronal
differentiation. It is well established that sustained activation of the extracellular signaling related
kinase (ERK) pathway is required for neuronal differentiation in these cells. Here, we investigate
MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding
protein] role in neuronal differentiation. MK-STYX is a member of the MAPK phosphatase
(MKP) family, which is responsible for dephosphorylating the ERKs. However, MK-STYX is a
pseudophosphatase that doesn’t have such catalytic activity, because its signature motif lacks
the essential nucleophilic cysteine for catalysis. Here we show that PC12 cells endogenously
express MK-STYX. To investigate the role of MK-STYX in neuronal differentiation further, PC12
cells were transfected with pMT2 or MK-STYX-FLAG and pEGFP vectors. Cells overexpressing MK-STYX showed neurite outgrowth. Furthermore, branching was observed in cells
over-expressing MK-STYX stimulated with nerve growth factor. We inhibited neuronal growth
factor stimulatory effects with the MEK inhibitor PD98059. However, MK-STYX induced
neuronal differentiation even in the presence of this inhibitor, suggesting that MK-STYX doesn’t
affect the ERK pathway but is involved in another pathway such as the Rho pathway whose
inactivation leads to neuronal differentiation. Our RhoA activity assays show that within twelve
minutes MK-STYX decreased RhoA activation. These results show that MK-STYX can induce
neuronal differentiation through inactivation of RhoA and apparently independently of the MAPK
pathway, strongly supporting our hypothesis that the pseudophosphatase MK-STYX has a role
in neuronal differentiation.
2804
The Pseudophosphatase MK-STYX Modulates RhoA Activity.
L. Rusnak1, S. D. Hinton1; 1Biology, College of William & Mary, Williamsburg, VA
The protein tyrosine phosphatase (PTP) family has members that display the structural features
of the enzymes, but lack the critical nucleophilic cysteine in the active site for catalysis. Thus,
these proteins are catalytically dead and referred to as pseudophosphatases. The
pseudophosphatase MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein], a member of the dual specificity phosphatase
MONDAY-LATE POSTER PRESENTATIONS
subfamily of PTPs, is catalytically inactive due to the fact that it has a phenylalanine and serine
in its signature motif where the expected histidine and cysteine would be. Our previous studies
showed that MK-STYX interacts with G3BP-1 [Ras-GAP (GTPase-activating protein) SH3 (Src
homology 3) domain-binding protein-1], and inhibits stress granules. However, MK-STYX’s
mode of action has not been determined. To understand its mode of action we focus on the
pseudophosphatase’s role in various signaling pathways. This study investigates the role of MKSTYX in the Rho pathway, which regulates the stress granule and apoptotic pathways. Rho
activity pull-down assays with cells over-expressing MK-STYX showed that MK-STYX increases
RhoA activation early after stimulation. Furthermore, Coomassie stain showed that other
proteins were pulled down with active Rho in the presence of MK-STYX. These results show
that MK-STYX modulates the Rho pathway, strongly supporting our hypothesis that the
pseudophosphatase has a role in regulating signaling pathways. Future goals of this study are
to identify these proteins, and to determine whether MK-STYX affects any downstream effectors
of Rho.
2805
The pseudophosphatase MK-STYX increases Hsp70 expression.
C. J. Jackson1, L. E. Rusnak2, J. E. Barr2, S. D. Hinton2; 1Hampton University, Williamsburg, VA,
2
The College of William and Mary, Williamsburg, VA
MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding
protein] is a pseudophosphatase, a member of the dual-specificity family subfamily of protein
tyrosine phosphatases that lacks the essential nucleophilic cysteine in its signature motif
required for catalytic activity. Previously, we reported that MK-STYX interacts with G3BP-1
[Ras-GAP (GTPAse-activating protein) SH3 (Src homology 3) domain binding protein-1], and
inhibits stress granule formation. Stress granules, cytoplasmic storage sites for mRNA, form as
a protective mechanism against stress caused by UV irradiation, hypoxia, and heat shock.
Stress induces stress granules, and activates many mRNAs encoding heat shock proteins
included in stress granules, and a few excluded from granules. For example, Hsp70 and Hsp90
are excluded from stress granules, but are translated preferentially during stress conditions.
Furthermore, Hsp70 expression increases as stress granules disappear, leading to translational
recovery, which requires protective proteins such as chaperones. Since Hsp 70 has been
implicated as an interacting partner of MK-STYX, and MK-STYX drastically decreases stress
granules, mapping out the Hsp70 and MK-STYX connection could greatly expand the
understanding of stress granule disassembly. To determine MK-STYX affects on Hsp 70
expression at the transcriptional and translational level, we over-expressed MK-STYX, stressed
the cells, and performed reverse transcriptase PCR, and immunoblotting, respectively. Here, we
report that the MK-STYX increases the Hsp70 at both the transcript and protein levels. Future
studies will include real-time PCR, and characterizing MK-STYX’s affects on other components
involved in Hsp70 regulation.
2806
The atypical C1 domain of RAF mediates membrane translocation.
M. Seo1, R. Kolesnick1; 1Weill Cornell Medical graduate school, New York, NY
Activation of Raf by extracellular signals is initiated by association with intracellular membranes.
However, membrane recruitment of Raf by RAS-GTP binding is not sufficient to stimulate Raf
kinase activity and membrane lipid acts as a co-factor in conjunction with RAS in Raf activation.
In this regard, previous studies showed that C-Raf binds ceramide, a lipid second messenger,
but it is not clear which domain of C-Raf mediates ceramide-binding and what biologic function
ensues. All Raf isoforms contain cysteine-rich domains classified as atypical C1 domains.
MONDAY-LATE POSTER PRESENTATIONS
Contrary to typical C1 domains, which bind to diacylglycerol to mediate membrane
translocation, atypical C1 domains do not bind diacylglycerol and there is no consensus as to
which lipid mediates its translocation.
We postulate that Raf binds ceramide and translocates to the plasma membrane via its atypical
C1 domain by a mechanism analogous to diacylglycerol binding by typical PKCs. To address
this hypothesis, we have developed biochemical and imaging assays to examine whether
ceramide mediates plasma membrane recruitment of Raf. Our ELISA binding assay shows the
atypical C1 domains of A-, B-, C-Raf bind ceramide in a dose-dependent manner and that
binding is decreased with C1 domain structure-disrupting mutants. Our imaging data also
indicate that c-Raf-1 translocates to plasma membrane co-localizing with ceramide by confocal
microscopy.
These studies will determine a potential role of ceramide in regulation of translocation and
subsequent activation of Raf.
2807
SH2B1 interacts with STAT3 and regulates FGF1 responsive genes during neuronal
differentiation.
Y-J. Chang1, L. Chen1; 1National Tsing Hua University, Hsinchu, Taiwan
Genetic studies have established the crucial roles of fibroblast growth factors (FGFs) in
embryogenesis. We have previously shown that overexpression of a signaling adaptor proteins
SH2B1 enhances FGF1-induced neurite outgrowth mainly through MEK-ERK1/2-STAT3
pathway. SH2B1 also up-regulates the expression of FGF1-induced expression of Egr1. While
SH2B1 is able to shuttle between the cytoplasm and the nucleus, its nuclear role remains
unknown. In this study, we provided evidence showing that SH2B1 interacted with STAT3
transcription factor through its SH2 domain to regulate FGF1-induced expression of Egr1 and Ncadherin. These findings may underlie the mechanism of how SH2B1 regulates gene
expression through binding to transcription factors and thus promotes neuronal differentiation.
2808
Epidermal growth factor (EGF) triggers localized calcium release and protein kinase C
activation within the nucleus of liver cells.
A. C. Campos1,2, M. A. Rodrigues1, M. H. Nathanson2, D. A. Gomes1; 1Biochemistry and
Immunology, Federal University of Minas Gerais, Belo Horizonte, Brazil, 2Internal Medicine,
Yale University, New Haven, CT
Nuclear Ca2+ signals are important for transcriptional activation and cell proliferation. However
little is known about how this process is regulated. Recent studies on the novel nuclear mode of
EGF Receptor (EGFR) signaling have shown that EGFR acts as a transcriptional regulator and
is involved in biological functions such as cell proliferation, tumor progression and chemo- and
radiotherapy-resistance. This work aimed to investigate whether nuclear calcium signaling is
triggered by EGFR. EGF-induced translocation of EGFR to the nucleus was observed in
hepatocytes isolated from rats and in SKHep-1 liver cells by confocal microscopy and
subcellular fractionation. InsP3 buffers selectively targeted to the nucleus or cytosol were
expressed in SKHep-1 cells that were stimulated with 200 ng/mL EGF, and changes in cytosolic
or nuclear calcium were followed by FLUO-4 fluorescence using a confocal microscope. EGF
increased calcium both in the nucleus and cytosol, and the nuclear but not the cytosolic InsP3
buffer prevented calcium signals in response to EGF. Moreover, cells treated with clathrin
siRNA, which blocks translocation of EGFR to the nucleus, also inhibited increases in calcium.
The amount of phosphatidylinositol 4,5-bisphosphate (PIP2) in nuclei isolated from resting or
stimulated cells was assessed by ELISA assay and showed that nuclear PIP2 decreased
MONDAY-LATE POSTER PRESENTATIONS
dramatically 5 min after EGF treatment. To ascertain the activation of downstream effectors we
assessed protein kinase C (PKC) activity by the FRET-based sensors, CytoCKAR and
NucCKAR, targeted to cytoplasm and nucleus, respectively. NucCKAR but not CytoCKAR
CFP/YFP emission ratio increased after EGF addition. These data collectively provide evidence
that activation of EGFR induces PIP2 hydrolysis in the nucleus, which in turn promotes calcium
release through intra-nuclear InsP3 receptors and leads to nuclear-localized PKC activation.
Inhibition of EGF-induced nuclear calcium signals by clathrin knockdown suggests that EGFR
must translocate to the nucleus to activate this pathway.
2809
Regulation of adipocyte differentiation by phosphoinositide-3 kinase enhancer-A.
M. C-L. Tse1, X. Liu1, K. Ye1, C-B. Chan1; 1Department of Pathology and Laboratory Medicine,
Emory University, Atlanta, GA
Phosphoinositide-3 kinase enhancer-A (PIKE-A) is a GTPase that promotes tumor
transformation and invasion. We have previously reported that PIKE-A expression was elevated
in the adipose tissue of obese mice. However, its role in this tissue remains unexplored. We
here investigate if PIKE-A participates in adipogenesis. Both mRNA and protein amount of
PIKE-A are progressively increased when 3T3-L1 cells are committing adipogenic
differentiation. Moreover, dexamethasone/isobutylmethylxanthine/insulin-induced adipocyte
differentiation is augmented when PIKE-A is overexpressed in 3T3-L1 preadipocytes. On the
other hand, PIKE-ablated (PIKE -/-) mouse embryonic fibroblasts (MEF) are refractory to
hormone-induced lipid accumulation. Interestingly, the activity of Signal Transducer and
Activator of Transcription 5a (STAT5a), a well-known adipogenic transcription factor, is reduced
in PIKE -/- MEF and white adipose tissue. It is also found that PIKE-A interacts directly with
STAT5a to facilitate its activation. Thus, our data demonstrate a novel role of PIKE-A in
promoting adipocyte maturation through regulating the activity of STAT5a.
2810
A structure function analysis of the Cdc42-specific guanine nucleotide exchange factor
Fgd1 in the process of invadopodia formation.
P. Ciufici1, M. Capestrano2, T. Daubon1, R. Buccione2, E. M. Genot1; 1inserm, Pessac, France,
2
Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy
Background: Invadopodia are proteolytically active protrusions that extend from the ventral
surface of invasive tumour cells when cultured on an extracellular matrix (ECM). Cdc42 is a
master regulator of invadopodia formation but the molecular mechanisms operating upstream of
this GTPase are ill-defined. The guanine exchange factor (GEF) Fgd1 is a novel invadopodia
component. It is required for invadopodia formation and function in carcinoma cells but how
Fgd1 is regulated is still unknown. A structure-function analysis was performed to characterize
the molecular mechanisms regulating Fgd1 in the process of invadopodia formation.
Rationale: Fgd1 contains a Dbl homology (DH) domain adjacent to a pleckstrin homology (PH)
domain which catalyzes the exchange of bound GDP for GTP on Cdc42, an N-terminal prolinerich domain (PRD) upstream of the DH/PH domain, a cysteine-rich zinc-finger FYVE domain
and a second C-terminal PH domain (PH2) downstream of it. The PRD negatively regulates
GEF activity and contains two putative Src-homology3 (SH3)-binding domains. It also contains a
binding sequence (CBD) for cortactin and mammalian actin-binding protein 1 (Abp1) .
Results: GFP tagged constructs encoding the full length protein (GFP-FL-Fgd1), Fgd1 lacking
either the N-terminal (ΔPRD), the C-terminal (ΔFYVE-PH2) or both regions (DH/PH1 only) were
transiently expressed in MDA-MD-231 human breast cancer cells and analyzed for subcellular
localization and consequences on cell’s ECM degradation potential.
MONDAY-LATE POSTER PRESENTATIONS
Compared to FL-Fgd1, expression of the DH/PH1 domain alone enhanced invadopodia
formation and associated ECM degradation whereas neither Fgd1ΔPRD nor Fgd1ΔFYVE-PH2
had an effect. Localization at invadopodia was lost for DH/PH1 or Fgd1ΔPRD mutants but
retained for Fgd1ΔFYVE-PH2 mutant. Upon knockdown of endogenous Fgd1, only expression
of the DH/PH1 mutant was able to rescue the ECM degradation function.
Cortactin and mAbp1 are involved in invadopodia formation and are also known as Fgd1
interacting partners, we therefore focused on the PRD domain which contains the binding
sequence for these two proteins. Deletion of this region (Fgd1ΔCBD mutant) relocalised Fgd1
from invadopodia to the perinuclear area. In Fgd1 depleted cells, in contrast to FLFgd1,
Fgd1ΔCBD was unable to elicit invadopodia formation. This mutant exerted a dominant
negative effect as it significantly reduced invadopodia formation and associated ECM
degradation when the endogenous Fgd1 was present.
Collectively, our data indicate that the PRD domain is essential for Fgd1 localization at
invadopodia sites and involves cortactine binding, thus providing a potential link between Fgd1
binding partners and Fgd1 specific functions
2811
IL-1b regulates osteoclastic resorbing activity by increasing autophagy.
E-J. Chang1, Y-H. Chung1, D-H. Song1, H-S. Jeon1, B. Choi1; 1Department of Medicine,
University of Ulsan College of Medicine, Seoul, Korea
Bone-resorbing activity of osteoclast (OC) is enhanced by inflammatory signals. Recently it had
been defined that osteoclastic resorbing activity requires LC3-conatinging autophagosome for
cathepsin K release; however, the critical mediator involved in this event remains to be defined.
We found that IL-1b leads to an increase in the conversion of soluble LC3-I to lipid-bound LC3-II
in OCs during RANKL-induced osteoclastogenesis was correlated with cathepsin K release and
following OC activity. IL-1b increased the formation of LC3-containing autophagosome, which
co-localized with cathepsin K in OCs. Knockdown (KD) of LC3 using small interfering RNA
suppressed autophagosome formation, actin ring formation, and cathepsin K release. Taken
together, it decreased bone resorbing activity of OCs by IL-1b and that might be through
inhibiting cdc42 activity. More importantly, lysosomal pH was increased by the IL-1b treatment,
and this led to an increase in cathepsin K release. In contrast, IL-1R antagonist failed to
increase lysosomal pH in OCs as well as cathepsin K release. The results demonstrate that IL1b contributes to osteoclast activity through regulating autophagy flux, providing a cellular
mechanism for the role of inflammatory cytokine IL-1b in accelerating osteoclastic function.
2812
The Rho-GEF Trio is crucial for laminar flow-induced endothelial cell alignment.
J. Kroon1, F. P.J. van Alphen1, J. van Rijssel1, J. van Buul1; 1Department of Molecular Cell
Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University
of Amsterdam, Amsterdam, Netherlands
Endothelial cells that line the lumen of arteries are constantly exposed to pulsatile laminar flow.
This results in high shear forces that play an important role in vascular remodeling and are
atheroprotective. Turbulent flow on the other hand is associated with low shear stress and
correlates with the onset of diseases, such as atherosclerosis. It has been shown by us and
others that endothelial cells, exposed to high shear, remodel their actin cytoskeleton by inducing
actin stress fiber formation. This results in cell elongation and alignment in the direction of flow.
However, it is not clear how the signals that are induced by laminar flow are translated into
endothelial cell alignment.
MONDAY-LATE POSTER PRESENTATIONS
Remodeling of the actin cytoskeleton is mediated by small GTPases that in turn are activated by
Rho-guanine nucleotide exchange factors (GEFs). The GEF Trio activates Rac1, RhoA and
RhoG, key regulators of the actin cytoskeleton.
Upon induction of flow, Trio protein and mRNA expression levels are increased, whereas Vav2
expression levels are unaltered. Therefore, our current focus is to elucidate the role of Trio in
primary human endothelial cell functioning and actin remodeling under shear stress conditions.
Our data show that laminar flow increased Trio localization at VE-cadherin-mediated cell-cell
contacts. Using shRNA to reduce Trio protein expression, we found that flow-induced
endothelial cell alignment was abrogated, whereas Vav2 silencing did not have any effect. In
addition, flow-induced Golgi polarization is hampered in Trio-deficient endothelial cells.
Furthermore, laminar flow increased the transendothelial electrical resistance, indicative for
more stable cell-cell contacts, which did not occur in Trio-shRNA-transduced endothelial cells.
Conversely, subsequent expression of a shRNA-resistant Trio construct rescued the loss in
flow-induced resistance in Trio-deficient endothelial cells.
These data show that flow-induced cell alignment and increased endothelial barrier function is
mediated by the Rho-GEF Trio. This work thus identifies Trio as an important novel component
of a mechanosensory signalling pathway.
2813
Rapid and comprehensive analysis of protein-protein interactions with combinatorial use
of bio-nanoparticles in living cells.
T. Kyung1, S. Lee1, H. Lee1, W. D. Heo1; 1Biology, KAIST, Daejeon, Korea
In living cells, many kinds of protein molecules dynamically interact with each other to transfer
their signaling information and ultimately induce biological outputs. Therefore, identifying
molecular interactions is an essential step for understanding signaling networks involved in
many biological functions and human diseases including cancer and neuronal diseases.
Although several kinds of technologies have been developed to screen and identify molecular
interactions, these methods suffer from their intrinsic problems such as limited dynamic range
and indirect readout. To overcome these limitations, we present a screening platform analyzing
protein-protein interactions in live mammalian cells with high sensitivity and selectivity. This
method is based on supramolecular assemblies of nanoclusters with genetically engineered bionanoparticles, which is triggered by specific protein-protein interactions. With this technology,
we screened interactions of small GTPases and effector proteins and visualized signaling
complexes in living cells. Also, we identified essential region of effector protein for binding small
GTPases. This highly sensitive and convenient method provides a novel platform for rapid and
comprehensive analysis of signaling networks in live cells.
2814
Regulation of transcriptional activity of PPARγ by lipin1.
H. Kim1, E. Koh1, E. Bae1, D-Y. Jeong1, K. Kim1; 1College of Medicine, Yonsei University, Seoul,
Korea
Peroxisome proliferator-activated receptorγ(PPARγ) is a key transcription factor involved
inadipogenesis and high expression levels of adipocyte-specific genes in adipocytes. In the
absence of ligand, PPARγ interacts with corepressors, acting as repressor to its target genes.
PPARγ ligands replace the corepressors with coactivators, converting PPARγ to transcriptional
activator. The present study demonstrates that lipin1 activates PPARγ activity even in absence
MONDAY-LATE POSTER PRESENTATIONS
of ligands by replacing the corepressors with coactivators as does artificial PPARγ ligand, such
as rosiglitazone. The transcriptional activation domain (TAD) in lipin1 resides between residues
217 to 399, where any conserved motifis not found and there is no homology with other lipin
isoforms. TAD in lipin1 plays a critical role only in PPARγ activation, but not in PPARα
activation. Activity of lipin1 TAD is enhanced by p300, and SRC-1 but not by PGC-1α and
P/CAF. The physical interaction with PPARγ occurs at C-terminal region from residue 825 to
926. VXXLL motif at residue 885 is more critical in coactivation function for PPARγ rather than
LXXIL motif at residue 725 which is critical in PPARα activation. In summary, lipin1 is key
modulator to activate PPARγ activity via differential mechanism from that in PPARα activation.
2815
Insulin stimulates Akt activation via peroxisome proliferator activated receptor-mediated
phosphatidylinositol 3 kinase.
Y-J. Kim1, S. Kim1; 1Kyung Hee University, Seoul, Korea
Phosphatidylinositol 3-kinase (PI3K) plays an important role in the metabolic actions of insulin.
The PIK3R1/p85α regulatory subunit is a critical component of the PI3K signaling pathway.
Peroxisome proliferator-activated receptor γ (PPARγ) as a transcription factor is known as a key
regulator for adipogenesis. However, the transcriptional regulation of PIK3R1 in adipocytes
remains unknown. In the present study, we investigated that PIK3R1 is a direct transcriptional
target of PPARγ. The level of PIK3R1 expression was increased in 3T3-L1 cells after induction
of adipocyte differentiation and was also induced in preadipocyte by overexpression of PPARγ.
Two putative peroxisome proliferator response elements (PPREs) in PIK3R1 promoter were
identified as PPARγ binding sites. By chromatin immunoprecipitation assay, we observed that
PPARγ interacts to the two PPREs region of PIK3R1 promoter. In addition, luciferase reporter
assays were showed that the -1183/-1161 and -573/-551 region of the PIK3R1 promoter
contains essential elements for binding PPARγ. Furthermore, up-regulation of PPARγ-mediated
PIK3R1 expression enhanced the insulin-stimulated Akt activation in 3T3-L1 cells. Taken
together, these results suggest that PPARγ is essential for transcriptional activity of PIK3R1
during adipogenesis.
Proteostasis, Cell Stress, and Aging
2816
Identification of E3 ubiquitin-ligase SCF1(FBXO25) substrates using protein microarrays
(Protoarray).
M. D. Gomes1, F. R. Teixeira1, A. O. Manfiolli1, C. S. Soares1; 1Biochemistry and Immunology,
Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Brazil
FBXO25 is one of 69 known human F-box proteins (FBPs) that serve as specificity factors for a
family of ubiquitin ligases composed of Skp1, Rbx1, Cullin1 and F-box protein (SCF1). FBXO25
localizes to the nucleus as a component of the nuclear body named FANDs (FBXO25Associated Nuclear Domains), partially colocalizes with Cullin1, Skp1, ubiquitinated proteins
and the proteasome. Interestingly, the inhibition of RNA polymerase I disrupts FANDs, and
FBXO25 antibodies interferes with RNA polymerase II transcription in vitro, indicating the role of
FBXO25 in the transcriptional process. In the present study, we have applied a powerful and
high-throughput proteomic approach, known as Protoarray, to identify ubiquitinated substrates
of FBXO25. The scanning of protoarrays showed 75 differentially ubiquitinated proteins. We
have already results indicating the degradation of two these targets mediated by FBXO25 and in
MONDAY-LATE POSTER PRESENTATIONS
cellulo ubiquitination and functional assays have been performed to relate FBXO25 with these
substrates.
2817
FBXO11 targets BCL6 for degradation and is inactivated in diffuse large B-cell
lymphomas.
S. Duan1, L. Cermak1, J. K. Pagan1, M. Rossi1, C. Martinengo2, P. Francia di Celle3, B. Chapuy4,
M. Shipp4, R. Chiarle2, M. Pagano1; 1New York University Medical Center, New York City, NY,
2
Department of Biomedical Sciences and Human Oncology, CERMS, University of Torino,
Torino, Italy, 3San Giovanni Battista Hospital, Torino, Italy, 4Medical Oncology, Dana-Farber
Cancer Institute, Boston, MA
BCL6 is the product of a proto-oncogene implicated in the pathogenesis of human B-cell
lymphomas. By binding specific DNA sequences, BCL6 controls the transcription of a variety of
genes involved in B-cell development, differentiation and activation. BCL6 is overexpressed in
the majority of patients with aggressive diffuse large B-cell lymphoma (DLBCL), the most
common lymphoma in adulthood, and transgenic mice constitutively expressing BCL6 in B cells
develop DLBCLs similar to the human disease. In many DLBCL patients, BCL6 overexpression
is achieved through translocation (~40%) or hypermutation of its promoter (~15%). However,
many other DLBCLs overexpress BCL6 through an unknown mechanism. Here we show that
BCL6 is targeted for ubiquitylation and proteasomal degradation by a SKP1–CUL1–F-box
protein (SCF) ubiquitin ligase complex that contains the orphan F-box protein FBXO11. The
gene encoding FBXO11 was found to be deleted or mutated in multiple DLBCL cell lines, and
this inactivation of FBXO11 correlated with increased levels and stability of BCL6. Similarly,
FBXO11 was either deleted or mutated in primary DLBCLs. Notably, tumour-derived FBXO11
mutants displayed an impaired ability to induce BCL6 degradation. Reconstitution of FBXO11
expression in FBXO11-deleted DLBCL cells promoted BCL6 ubiquitylation and degradation,
inhibited cell proliferation, and induced cell death. FBXO11-deleted DLBCL cells generated
tumours in immunodeficient mice, and the tumorigenicity was suppressed by FBXO11
reconstitution. We reveal a molecular mechanism controlling BCL6 stability and propose that
mutations and deletions in FBXO11 contribute to lymphomagenesis through BCL6 stabilization.
The deletions/mutations found in DLBCLs are largely monoallelic, indicating that FBXO11 is a
haplo-insufficient tumour suppressor gene.
2818
The Novel Interaction Between Microspherule Protein Msp58 and Ubiquitin E3 Ligase
EDD Regulates Cell Cycle Progression.
M. F. Benavides1,2, L-F. Chow-Tsanga1, J. Zhang3, H. Zhong1,2; 1Biological Sciences, Hunter
College-CUNY, New York, NY, 2Biological Sciences, The Graduate Center-CUNY, New York,
NY, 3College of Medicine, University of Cincinnati, Cincinnati, OH
Microspherule protein Msp58 (or MCRS1) plays a role in numerous cellular processes including
transcriptional regulation and cell proliferation. It is not well understood either how Msp58
mediates its myriad functions or how it is itself regulated. Here, by immunoprecipitation, we
identify EDD (E3 identified by differential display) as a novel Msp58-interacting protein. EDD,
also called UBR5, is a HECT-domain (homologous to E6-AP carboxy-terminus) containing
ubiquitin ligase that plays a role in cell proliferation, differentiation and DNA damage response.
Both in vitro and in vivo binding assays show that Msp58 directly interacts with EDD.
Microscopy studies reveal that these two proteins co-localize in the nucleus. We have also
found that depletion of EDD leads to an increase of Msp58 protein level and extends the half-life
of Msp58, demonstrating that EDD negatively regulates Msp58’s protein stability. Furthermore,
MONDAY-LATE POSTER PRESENTATIONS
we show that Msp58 is up-regulated in different cell lines upon the treatment with proteasome
inhibitor MG132 and exogenously expressed Msp58 is ubiquitinated, suggesting that Msp58 is
degraded by the ubiquitin-proteasome pathway. Finally, knockdown of either Msp58 or EDD in
human lung fibroblast WI-38 cells affects the levels of cyclins B, D and E, as well as cell cycle
progression. Together, these results suggest a role for the Msp58/EDD interaction in controlling
cell cycle progression. Given that both Msp58 and EDD are often aberrantly expressed in
various human cancers, our findings open a new direction to elucidate Msp58 and EDD’s roles
in cell proliferation and tumorigenesis.
2819
The Budding Yeast STUbL Subunit Slx5 Functionally Interacts with the SUMO Ligase
Siz1.
E. P. Szymanski1, M. L. Guillotte1, N. Pasupala1, J. W. Westerbeck1, B. C. Matson1, O.
Kerscher; 1Biology, The College of William and Mary, Williamsburg, VA
The budding yeast proteins Slx5 and Slx8 constitute the two subunits of a SUMO-targeted
ubiquitin ligase (STUbL). Slx5 is the targeting subunit of the Slx5/Slx8 complex, and both
subunits are required for the ubiquitin ligase function. STUbLs are essential for the DNA
damage response and prevention of gross chromosomal rearrangements, but currently only a
few STUbL substrates are known. Here we describe the interaction of the yeast STUbL
Slx5/Slx8 with a new target, the SUMO ligase Siz1. In mitosis, Siz1 is phosphorylated and
exported from the nucleus to sumoylate proteins in the cytosol, including septins. Several lines
of evidence suggest that Slx5 functionally interacts with nuclear localized Siz1. First, Slx5 and
Siz1 interact in two-hybrid and in pull-down assays. Second, Slx5 modulates the steady state
levels and phosphorylation of Siz1. Third, Siz1 is a substrate for ubiquitylation by Slx5 both in
vitro and in vivo. We propose a model for the functional cross-talk between sumoylation and
SUMO-targeted degradation.
2820
Investigating the Effect of SUMO-Dependent Degradation on Huntingtin Toxicity and
Aggregation in Yeast.
G. Driessnack1, O. Kerscher1; 1Biology, College of William and Mary, Williamsburg, VA
The causative agent of Huntington’s disease is a mutant protein called huntingtin (mHtt) that
accumulates and forms aggregates in neuronal cells. Aggregates form as a result of
polyglutamine (poly-Q) expansion in mHtt, with repeats over 36 causing toxicity in neuronal
cells. mHtt can be covalently modified with ubiquitin and SUMO (small ubiquitin-like modifier).
Ubiquitin modified mHtt can be targeted for degradation by the ubiquitin-proteasome system
(UPS) while sumoylation may cause mHtt proteins to become more soluble resulting in
enhanced toxicity. Both the aggregation and toxicity of mHtt can be studied in the genetically
tractable budding yeast cell model system. SUMO-targeted ubiquitin ligases (STUbL), such as
yeast Slx5/Slx8 STUbL, have emerged as an important component of a regulatory system for
the homeostasis of SUMO conjugates in the cell. Here we show that yeast strains with SLX5 or
SLX8 deletions exhibit a severe growth defect and enhanced lethality due to expression of a
toxic mHtt construct with 103 Glutamine residues (103Q). In contrast, a non-toxic mHtt construct
with 25 Glutamine residues (25Q) showed little or no effect. High-molecular weight aggregates
of 103Q mHtt were greatly enhanced in a SUMO mutant, smt3-331, that also shows an altered
distribution of Slx5 in the nucleus. These findings suggest that STUbLs may be involved in the
mediation of toxic huntingtin protein aggregates.
MONDAY-LATE POSTER PRESENTATIONS
2821
Conjugation of polyubiquitin chains to lipid droplet proteins by cytosolic ubiquitination
machinery.
C. M. Richter1, J. A. Olzmann1, T. Shaler2, R. R. Kopito1; 1Stanford University, Stanford, CA,
2
SRI International, Menlo Park, CA
Lipid droplets (LDs) are cellular organelles that act as reservoirs for triacylglycerol (TAG) and
cholesterol esters (CE) and, thus, are critical for regulation of cellular metabolism. The TAG
and CE core of the LD is surrounded by a phospholipid monolayer that is decorated with a
variety of proteins, including proteins that regulate lipid metabolism, signaling, and vesicular
trafficking. Inhibition of cellular proteasomes results in the accumulation of polyubiquitin
conjugates in LD fractions, suggesting that the ubiquitin-proteasome pathway may regulate the
turnover of LD-associated proteins or that LDs are sequestration sites for non-LD proteasome
substrates. However, the identity of these polyubiquitinated proteins is unknown, as is the
machinery responsible for their ubiquitination. Using mass spectrometry, we have identified a
protein complex in LDs composed of a ubiquitin (Ub) conjugation enzyme (Ube2G2), the AAA+
ATPase p97/VCP and several cofactors (AUP1, UBXD8, Ufd1, Npl4) that link this versatile
ATPase to Ub-dependent processes. Although these data suggest that LD contain machinery
required for Ub conjugation, neither the substrates of ubiquitination nor the E3 ligases that
provide substrate specificity have been identified in LDs. To identify LD ubiquitination machinery
and substrates, we conducted in vitro ubiquitination assays using LD fractions isolated from
human cell lines. LDs themselves were incapable of supporting polyubiquitin conjugate
formation in the presence of recombinant E1 enzyme, ATP, and FLAG-Ub, consistent with the
absence of candidate E3s from the LD proteome. We tested if AUP1, which lacks a canonical
E3 ligase catalytic domain but binds and recruits Ube2G2 to LDs, functioned as a non-canonical
E3 ligase. However, AUP1 was not sufficient to support the polyubiquitination of LD proteins in
vitro, even when recombinant AUP1 was added in excess to LD fractions. By contrast,
polyubiquitin chains were conjugated to LD proteins upon the addition of cytosol to LD fractions.
These data indicate that at least a subset of the machinery that mediates the polyubiquitination
of LD proteins is not stably associated with LDs.
2822
Anti-α1-Antitrypsin Single Chain Variable Fragment (scFv) Prevents Z-AAT Toxicity and
Aggregation.
K. Xiao1, Y. Levites2, R. L. Wang1, R. Oshins1, F. Rouhani1, S. Song3, M. Brantly1; 1Medicine,
University of Florida, Gainesville, FL, 22. Center for Translational Research in
Neurodegenerative Disease, University of Florida, Gainesville, FL, 3Pharmaceutics, University of
Florida, Gainesville, FL
Alpha-1 antitrypsin (AAT) is a neutrophil elastase inhibitor. The Z mutation (E342K) of AAT
causes retention of the mutant protein as an inclusion body within the ER of hepatocytes, which
may cause severe liver diseases such as fibrosis and cirrhosis. Current methods of blocking the
loop-β-sheet polymerization of Z-AAT with ligands or small peptides have been proven to be
feasible in vitro, but have limitations in the delivery method to the cell. We designed a new
strategy using a single chain variable fragment (scFv) derived from a monoclonal antibody to
inhibit AAT polymerization; we can further improve the scFv-ZAAT complex degradation by
adding a tag recognized by proteasome machinery. Anti-AAT scFv was constructed and
validated based on a parent hybridoma. The mRFP-Z-AAT stable-expression CHO cells were
transfected with scFv expression constructs or a GFP expression vector as a control and Z-AAT
accumulation levels and cell stress were evaluated. The ability of the scFv to bind AAT was
validated in vitro and ex vivo. To facilitate degradation of the complex, Hsp70 binding motif,
MONDAY-LATE POSTER PRESENTATIONS
FKBP12, or KDEL sequence was attached to the C-terminus of the scFv; of these, only scFvFKBP12 treated cells showed significant reduction of polymers within the ER. In addition, the
scFv-FKBP12-Z-AAT complexes were degraded by the proteasome pathway and reduction of
Z-AAT polymerization lead to decreased cell stress levels. Furthermore, although the scFv can
recognize both M and Z AAT, the expression of scFv-FKBP12 does not interfere with the
secretion or activity of normal M-AAT in the cells.
Conclusion: ScFv-FKBP12 can bind and direct α-1 antitrypsin towards proteasome degradation,
which reduces ER stress.
2823
Inhibition of hepatic Cyp4a reduces ER stress and improves insulin resistance in type 2
diabetes.
E. C. Park1, Y. Hong1, G-H. Kim1; 1Division of Life Science, Korea Basic Science Institute,
Daejeon, Korea
Abnormal activation of ER stress signaling is responsible for hepatic insulin resistance and can
result in type 2 diabetes (T2DM). However, the underpinnings of mechanisms that regulate ER
stress and T2DM are poorly understood. Here we show the biochemical and physiological
characterizations of cytochrome P450 4A (Cyp4a) as a novel regulator of ER stress-induced
hepatic insulin resistance and apoptosis. We found that Cyp4a is highly up-regulated in db/db
diabetic mice liver. Inhibition of Cyp4a activity in db/db and high fat diet (HFD) mice using its
specific inhibitor HET0016 improved the diabetic physiological phenomena, such as glucose
tolerance, insulin secretion and hepatic steatosis. The insulin resistance and apoptosis were
also rescued by loss of Cyp4a function in the db/db and HFD mice liver. We revealed that the
amelioration of diabetes was due to the reduction of ER stress which was up-regulated in
diabetic mice liver. On the contrary, induction of Cyp4a in db/db mice accelerated ER stress,
insulin resistance and apoptosis. These results show the importance of Cyp4a in the remission
of diabetes and of ER stress-induced insulin resistance and apoptosis and suggest that the
reduction of Cyp4a activity would be a therapeutic target against T2DM.
2824
Transcriptional repression and the Unfolded Protein Response: The role of C/EBPβ in
regulating Il4ra expression during endoplasmic reticulum stress.
A. M. Arensdorf1, D. T. Rutkowski1; 1Anatomy and Cell Biology, University of Iowa, Iowa City, IA
The Unfolded Protein Response (UPR) is an intracellular signaling cascade initiated by
physiological or pathophysiological endoplasmic reticulum (ER) stress. Activation of the UPR
culminates in the production of three transcription factors (ATF4, ATF6 and XBP1) and
comprehensive regulation of gene expression. While gene activation following ER stress has
been extensively studied, the mechanisms of gene suppression are poorly understood. To begin
to elucidate these mechanisms, we followed the expression of the sentinel mRNA Il4ra, which
was suppressed by ER stress in multiple cell types. Il4ra mRNA suppression occurred through a
mechanism distinct from regulated IRE1-dependent decay (RIDD) and dependent upon
PERK/eIF2α signaling. Analysis of the Il4ra promoter suggested a role for the transcriptional
regulator C/EBPβ, which was confirmed in C/EBPβ-deficient cells. eIF2α phosphorylation, along
with a concomitant eIF2α-independent ER stress signal, was necessary for production of the
transcriptionally repressive isoform of C/EBPβ (LIP). Thus, ER stress signaling pathways
converge on transcriptional repression of Il4ra through C/EBPβ and consequently impair
signaling through IL-4/IL-13. Our results reveal a novel mechanism for gene repression during
ER stress, and highlight the ability of the UPR to influence inflammatory signaling by impinging
upon a parallel gene regulatory pathway.
MONDAY-LATE POSTER PRESENTATIONS
2825
The ATF6 pathway of the UPR response is important in regulating lipid accumulation in
the kidney caused during ER stress.
D. D. McCabe1, D. T. Rutkowski1; 1University of Iowa, Iowa City, IA
Obesity has developed into a global epidemic affecting over a billion adults worldwide and is
linked to many serious health problems, including chronic renal failure and fatty kidney. Fatty
kidneys have also been connected to increased hypertension and generation of chronic kidney
disease in humans. The pathways regulating lipid metabolism in the kidney are not wellunderstood, nor are the mechanisms by which these pathways become dysregulated during
obesity. However, It is well-established that obesity leads to stress in the endoplasmic reticulum
(ER) of several metabolically active cell types in vivo including the liver and adipose, and that
this stress is accompanied by dysregulation of lipid metabolic pathways. These observations
suggest that ER stress in the kidney might upset lipid metabolism, leading to the development of
fatty kidneys.
ER stress leads to activation of the unfolded protein response (UPR), which culminates in broad
changes in gene transcription. In previous work, we have found that exposure to ER stress
causes mice lacking the ER stress sensing protein ATF6 to display profound fatty liver, which is
a consequence of transcriptional alteration in the expression of genes that regulate lipid
metabolism. Strikingly, Atf6-/- animals also showed persistent ER stress in the kidneys, as well
as a dramatic accumulation of renal fat. These results suggest that, as in the liver, direct
pathways lead from ER stress and UPR activation to lipid metabolism. They also raise the
exciting possibility that Atf6-/- mice can serve as a novel model system for understanding how
renal lipid metabolism becomes dysregulated during obesity and chronic kidney disease. Thus,
persistent ER stress may cause metabolic dysregulation via gene expression changes in the
kidney, which leads to fatty kidney and further downstream consequences.
2826
ER stress response to type I procollagen misfolding in osteogenesis imperfecta
E. Makareeva1, L. Mirigian1, B. Ashinsky1,2, S. Leikin1;
1
Section on Physical Biochemistry, Eunice Kennedy Shriver National Institute of Child health
and Human Development, National Institutes of Health, Bethesda, MD, 2Carleton College,
Northfield, MN
Type I collagen is a triple helical protein that assembles into fibers, forming the scaffold of
extracellular matrix. It is by far the most abundant protein in vertebrates, yet folding of its
procollagen precursor does not conform to conventional protein folding paradigms. Procollagen
misfolding caused by mutations in the molecule or by deficiencies in its chaperones leads to
severe bone fragility and skeletal deformities (osteogenesis imperfecta, OI). Endoplasmic
Reticulum (ER) stress due to this misfolding and the resulting osteoblast malfunction appear to
play a major role in OI, but the underlying molecular mechanisms are still poorly understood. ER
stress response to procollagen misfolding caused by Gly substitutions in the triple helical region,
which are responsible for over 80% of severe OI cases, is particularly puzzling. Our
immunofluorescence studies and biochemical analysis of secreted and intracellular collagens
suggest that retention of misfolded procollagen molecules in the ER is a common consequence
of such mutations. Nevertheless, we find that this retention does not cause upregulation of BiP
or activation of conventional unfolded protein response (UPR) signaling, consistent with
previous reports. Instead, the ER stress response seems to involve the NF-κB pathway
reminiscent of the termed ER-overload response in serpinopathies. Better understanding of this
unconventional ER stress response mechanism might help identifying novel targets for
therapeutic intervention.
MONDAY-LATE POSTER PRESENTATIONS
2828
A direct role for Hrd3p in ERAD.
N. Vashistha1, S. Caroll2, R. Hampton1; 1Section of Cell and Developmental Biology, University
of California, San Diego, La Jolla, CA, 2University of Washington, Seattle, WA
The HMG-CoA Reductase Degradation (HRD) pathway is a conserved ubiquitin-dependent
route of endoplasmic reticulum associated degradation (ERAD), used to degrade numerous
misfolded ER proteins. The HRD complex contains the Hrd1 RING-H2 E3 ubiquitin ligase and
other proteins including Usa1p and Hrd3p. Usa1 and Hrd3 tightly regulate the Hrd1 stability and
levels. In the absence of Hrd3, Hrd1 undergoes rapid self-catalyzed ubiquitination, and Hrd1
self-degradation is completely dependent on Usa1. We have exploited this Usa1 requirement to
test Hrd3 for a direct role in ERAD.
Hrd3 was one of the original HRD proteins. It is required for ERAD of both lumenal
(ERAD-L) and membrane-bound (ERAD-M) substrates. It is hypothesized that Hrd3 plays a
central role in recognition of ERAD substrates, since it recruits lumenal factors required for
ERAD-L, and it has also been shown to directly cross-link the Hmg2 ERAD-M substrate.
However, the profound drop in Hrd1 that occurs in a hrd3Δ null has left Hrd3’s role in ERAD an
open question: it is not clear to what extent Hrd3’s importance in ERAD is due to support of
Hrd1 levels, rather than direct action as a ligase component. This is an especially salient
question since ERAD defects in a hrd3Δ null mutation are suppressed by over expression of
Hrd1.
We have shown that the UBL domain of Usa1p is responsible for Usa1-dependendent
self-degradation of Hrd1p: in a strain with Usa1-ΔUBL, Hrd1 is fully stable in a hrd3Δ null. Thus,
in the Usa1-ΔUBL background, Hrd1 stability is no longer dependent on the presence of Hrd3,
allowing for the first time examination of the direct role of Hrd3 in ERAD independent of Hrd1
stability effects. Degradation of prototype ERAD-M substrates Hmg2, Sec61-2 and PDR5 was
strongly dependent on Hrd3 in Usa1-ΔUBL where Hrd1 is stable. Similarly, degradation of
classic ERAD-L substrates was also totally Hrd3 dependent in the Usa1-ΔUBL background.
These results indicate for the first time a direct role of Hrd3p in both ERAD-M and ERAD-L.
Although a direct role of Hrd3 in ERAD-L is consistent with its engagement of several lumenal
mediators of this ERAD branch, the importance of Hrd3 in ERAD-M may indicate novel modes
of membrane substrate recognition mediated by this key component of the HRD pathway. We
are currently exploring the mechanistic details of Hrd3’s direct ERAD function for both substrate
classes.
MONDAY-LATE POSTER PRESENTATIONS
2829
The effect of HMX1 deletion on spore viability in Saccharomyces cerevisiae
K. T. Kern1, S. W. Gorsich1; 1Biology, Central Michigan University, Mount Pleasant, MI
HMX1 has been shown to contribute to hydrogen peroxide (H2O2) tolerance in Saccharomyces
cerevisiae by the regulation of glutathione (GSH1), glutathione peroxidase (GPX1), and
catalase (CTT1) genes. In addition, the deletion of HMX1 in haploid cells increases cell
sensitivity to H2O2. When cells are experiencing oxidative stress mitochondria will change from
an evenly distributed tubular network to clumped aggregates. This could have drastic effects on
sporulation as mitochondrial health and function is essential for spore formation during meiosis.
This is in line with studies demonstrating that mutations impacting mitochondrial morphology
have decreased spore viability and overall fitness. Our preliminary results demonstrate that the
deletion of HMX1 in Saccharomyces cerevisiae shows no difference in sporulation and spore
viability compared to the wild type under non-stress conditions, but when incubated in the
presence of H2O2 spore viability decreases in comparison to the wild type. Further studies
showing the effects of overexpressing HMX1 and other stress tolerance genes are ongoing.
2830
Aging of yeast Saccharomyces cerevisiae RAS2val19 mutant
A. Pichova1; 1Institute of Microbiology, ASCR, Praha, Czech Republic
Yeast, especially S. cerevisiae, is suitable single cell model organism for studies of aging and
apoptosis, multifactorial processes involving a complex network of regulations, because it
exhibits mother cell specific aging, where the cell divides asymmetrically, giving rise to a nextgeneration of larger mother cell and a smaller daughter cell that resets the clock to zero while
mother cell accumulates all aging-connected changes. This asymmetry is disturbed in the protooncogene mutant strain RAS2val19, known as a short-lived mutant. Micromanipulations revealed
a high percentage of virgins unable to form micro- and macro-colonies. Young cells, mostly
virgins, separated by centrifugal elutriation show markers of apoptosis, such as having a high
load of ROS (reactive oxygen species) detected by DHR (dihydrorhodamine); many cells
appeared with DNA breaks stained by the TUNEL test and their nuclei also appeared with
aberrant morphology. Quantitative comparison of extrachromosomal DNA rings (ECR) in
separated young and old cells of the wild type, RAS2val19 mutant and a strain with delayed aging
together with other data point to the premature aging being connected with symmetric aging,
where the daughter cell is already inheriting some features of aging.
2831
Effects of soybean peptide intake on inhibition of muscle atrophy in mice.
A. Ozeki1, E. Kizaki1, A. Ohashi1, S. Ymada1; 1Human Life Science, Graduate School of Jissen
Women's University, hino city in Tokyo, Japan
There are some reports that the atrophy of skeletal muscles is expected to be improved by the
nourishment and the movement. Soybean peptide is effective to inhibit muscle protein
degradation in vitro. However, this effect hasn ft been clear in vivo. Recently we demonstrated
the soybean peptide(AM) intake inhibited muscle atrophy by tail suspension in mice. Then we
examined the mechanism of inhibition muscle atrophy by AM intake.
Mice were purchased from SLC Japan. @They were individually housed in stainless steel
cages in a temperature, humidity and light controlled room(24 Ž, 60%,12 light /dark cycle). All
mice were fed the MF for 7days. The mice were divided into four groups consisting of given a
control(water) group, suspension(water+suspension) group, an AM drink(175 mg/l in water)
group and an AM drink (175 mg/l in water+suspension) group. Body weight was measured
MONDAY-LATE POSTER PRESENTATIONS
before and after tail suspension. After the experiment, all mice were sacrificed by under
anesthesia. Soleus was collected and weighted on the Electronic balance and stored at 80 Žuntil analysis. The area of a muscle cell was measured by using the preparation stained
by HE with a digital microscope. Myofibrillar protein content was determined spectrophotometric
ally based on the Bradford method at a wavelength of 700nm. A standard curve was generated
using bovine serum albumin. Total RNA was extracted with miRNA easy Mini Kit. 1ƒÊg RNA
was retro-transcribed using the cDNA Archive kit following manufacturer fs instructions. Gene
expression was measured by quantitative real-time PCR using ABI 7000 Sequence Detector
System. Specific assays on demand for atrogin-1 were from Applied Biosystems. The level of
each transcript was measured with the threshold cycle (Ct) method using as endogenous
controls ƒÀ-actin mRNA form mouse tissues.
The AM intake inhibited the atrophy of skeletal muscles by tail suspension. In particular, the
intake of the AM was effective in reducing slow muscle atrophy. We examined the effect of soy
peptide on inhibition of muscle cell atrophy. The result showed the soy peptide reduced the
muscle cell atrophy significantly. In this experiment, the intake of the AM also controlled a
decrease of the muscle protein. Atrogin-1 gene expression was significantly reduced by AM
intake. These results suggested AM intake inhibit protein degradation process in soleus muscle.
2832
Late effects of heavy-ion irradiation on ex vivo osteoblastogenesis and cancellous bone
microarchitecture.
L. H. Tran1, J. S. Alwood1, A. Kumar1, C. L. Limoli2, R. K. Globus1;
1
NASA Ames Research Center, Moffett Field, CA, 2UC Irvine, Irvine, CA
Prolonged spaceflight causes degeneration of skeletal tissue with incomplete recovery even
after return to Earth. We hypothesize that heavy-ion irradiation, a component of Galactic Cosmic
Radiation, damages osteoblast progenitors and may contribute to bone loss during long
duration space travel beyond the protection of the Earth’s magnetosphere. Male, 16 week-old
C57BL6/J mice were exposed to high-LET (56-Fe, 600MeV) radiation using either low (5 or
10cGy) or high (50 or 200cGy) doses at the NASA Space Radiation Lab and were euthanized 34, 7, or 35 days later. Bone structure was quantified by microcomputed tomography (6.8 µm
pixel size) and marrow cell redox assessed using membrane-permeable, free radical-sensitive
fluorogenic dyes. To assess osteoblastogenesis, adherent marrow cells were cultured ex vivo,
then mineralized nodule formation quantified by imaging and gene expression analyzed by RTPCR. Interestingly, 3-4 days post-exposure, fluorogenic dyes that reflect cytoplasmic generation
of reactive nitrogen/oxygen species (DAF-FM Diacetate or CM-H2DCFDA) revealed irradiation
(50cGy) reduced free radical generation (20-45%) compared to sham-irradiated controls.
Alternatively, use of a dye showing relative specificity for mitochondrial superoxide generation
(MitoSOX) revealed an 88% increase compared to controls. One week after exposure, reactive
oxygen/nitrogen levels remained lower(24%) relative to sham-irradiated controls. After one
month, high dose irradiation (200 cGy) caused an 86% decrement in ex vivo nodule formation
and a 16-31% decrement in bone volume to total volume and trabecular number (50, 200cGy)
compared to controls. High dose irradiation (200cGy) up-regulated expression of a late
osteoblast marker (BGLAP) and select genes related to oxidative metabolism (Catalase) and
DNA damage repair (Gadd45). In contrast, lower doses (5, 10cGy) did not affect bone structure
or ex vivo nodule formation, but did down-regulate iNOS by 0.54-0.58 fold. Thus, both low- and
high-doses of heavy-ion irradiation cause time-dependent, adaptive changes in redox state
within marrow cells but only high doses (50, 200cGy) inhibit osteoblastogenesis and cause
cancellous bone loss. We conclude space radiation has the potential to cause persistent
damage to bone marrow-derived stem and progenitor cells for osteoblasts despite adaptive
changes in cellular redox state.
MONDAY-LATE POSTER PRESENTATIONS
2833
Investigating the bioenergetic regulation of parkin-induced mitophagy.
T. MacVicar1, J. Lane1; 1University of Bristol, Bristol, UK
Our lab studies the role of autophagy and mitochondrial quality control in cellular homeostasis.
The mitochondrial network is a finely tuned and adaptable organelle system that must remain
healthy in order to generate sufficient ATP for cellular function. Damaged mitochondria must be
removed and replaced with healthy ones in order to maintain the energy supply. Recent
research has highlighted the importance of mitochondrial quality control in human disease. For
example, the PINK1-parkin mediated removal of dysfunctional mitochondria by mitophagy has
received much attention and has been genetically implicated in Parkinson’s disease. We are
particularly interested in how cellular bioenergetics affects parkin-mediated mitophagy. To this
end we have employed a range of imaging and biochemical techniques to explore the
bioenergetic regulation of mitophagy and mitochondrial dynamics.
We have generated human cell lines stably expressing YFP-parkin allowing these cells to
activate mitophagy in response to treatment with the mitochondrial depolarizing agent, CCCP.
Remarkably, we find that close to 100% of RPE1 cells expressing YFP-parkin comprehensively
remove their mitochondria as detected by fixed antibody labeling and live cell imaging.
However, it is understood that transformed cell lines are predominantly glycolytic and may not
depend on their mitochondria for ATP production. To encourage a greater dependence on
mitochondrial respiration we changed the energy substrate from glucose to galactose.
Subsequent bioenergetic experiments confirmed increased oxygen consumption and
mitochondrial ATP production in galactose fed cells. Interestingly, galactose fed YFP-parkin
cells do not undergo mitophagy after CCCP treatment, despite robust translocation of parkin.
This suggests an inhibition of mitophagy in conditions that require increased oxidative
phosphorylation.
Whilst the autophagy pathway itself does not appear to be inhibited in galactose fed cells, we
have found intriguing differences in the control of mitochondrial dynamics between glycolytic
and mitochondrial respiring cells. Mitochondria normally fragment following CCCP treatment,
however our data show that galactose fed cells maintain a more interconnected network.
Intriguingly, we find that CCCP induced cleavage of the key inner mitochondrial membrane
fusion protein OPA-1 is diminished in galactose fed cells. We have also observed a PKA
dependent inhibition of Drp1 mediated fission in these cells. We therefore question whether
cells that require their mitochondria to maintain the energy supply are able to protect their
mitochondrial network from mitophagy via alterations in the regulation of mitochondrial
dynamics. Further research into the bioenergetic regulation of mitophagy will especially help us
to understand mitochondrial homeostasis in disease vulnerable, high ATP demanding human
tissue such as dopaminergic neurons.
2834
Different types of autophagy pathways compete for the common autophagic machinery.
A-Y. Liu1, K-H. Ho1, W-P. Huang1,2; 1Institute of Zoology, National Taiwan University, Taipei,
Taiwan, 2Department of Life Science, National Taiwan University, Taipei, Taiwan
Cells are sensitive to rapid changes of external environment, which induce quick adjustment to
cellular pathways for survival. Autophagy is one of the stress-induced pathways, which breaks
down cytoplasmic proteins/organelles to increase cell survival when facing environmental
stresses. It has been reported that ER stress induced specific autophagy called reticulophagy,
which selectively targets expanded ER for degradation and serves a protective role during
strong unfolded-protein response (UPR) activity. Here, we showed that reticulophagy is a
specific type of autophagy distinct from the other selective autophagy pathways, the Cvt
MONDAY-LATE POSTER PRESENTATIONS
pathway and macroautophagy. Using reticulophagy as a model, we found competition between
different types of autophagy occurring under growing condition, Dithiothreitol treatment, and
nitrogen starvation. Additionally, activation of reticulophagy delayed endosomal trafficking to the
vacuole, suggesting that reticulophagic machinery has altered the fusion dynamic at the
vacuole. We propose that different types of autophagy pathways compete for the common
autophagic machinery to best benefit cells facing specific environment conditions.
2835
Small molecule inhibitors and endogenous interactions that modulate ATG7 activity.
J. Nabhan1, W. Zhang1, J. Minissale1, P. LeRoy1, J. Gavin1, X. Yang1, H. Liao1, H-K. Loke1, A.
Lubinsky1, N. D’Amore1, L. Dick1, J. Brownell1; 1Millennium Takeda, Cambridge, MA
Macroautophagy (autophagy) is a conserved pathway required for cellular homeostasis.
Autophagy is initiated through formation of double-membrane vesicles, called autophagosomes,
around cytoplasmic proteins and/or organelles that eventually fuse with lysosomes to degrade
vesicular contents and yield recycled cellular nutrients. Aside from homeostasis, a role for
autophagy has been demonstrated in cancer, neurodegenerative diseases, and in microbial and
viral infections. The E1-like enzyme ATG7 is an essential component of the autophagic pathway
required for activation of ubiquitin-like substrates and is indispensable for autophagosome
formation. To better understand the function of ATG7 and autophagy, we sought to identify
chemical inhibitors, as well as endogenous regulators of ATG7 activity. We recently generated
small molecule inhibitors that perturb the ATG7 enzymatic activity. The mechanism of inhibition,
termed “substrate-assisted inhibition”, is similar to that previously reported for MLN4924, a
Nedd8 -activating enzyme inhibitor. Treatment of cultured cells or purified ATG7 with specific
inhibitors diminished ATG7 thioester formation with its known ubiquitin-like (Ubl) substrates and
led to formation of tightly bound inhibitor-substrate adducts. In cells, inhibition of ATG7 blocked
the autophagy pathway through diminished lipidation of the Ubl substrates. Furthermore, we
purified and identified these inhibitor-adducts as Gabarap and Gabarap-L1. Lastly, we utilized
an in situ cross-linking protocol combined with immunoprecipitation to identify novel ATG7
interaction partners, which may serve as endogenous regulators. This approach unveiled new
associations with several proteins of known function in oncogenesis. Ongoing studies aim to
delineate the functional relevance of these interactions and to ultimately determine if they may
serve as additional targets for cancer chemotherapy.
2836
Autophagy Positively Regulates Primary Ciliogenesis by Removing OFD1 from Centriolar
Satellites.
M. G. Lin1, Z. Tang1, Q. Zhong 1; 1Molecular and Cell Biology, UC Berkeley, Berkeley, CA
The primary cilium is a microtubule-based organelle that functions in sensory and signaling
pathways. It originates from a centriole-derived basal body, which is surrounded by granular
structures called centriolar satellites. Regulation of primary cilia biogenesis and the role of
centriolar satellites in ciliary function remain poorly understood. Here, we show that autophagic
degradation of OFD1 (oral-facial-digital syndrome 1) at centriolar satellites promotes primary
cilia biogenesis. Autophagy is a catabolic pathway in which cytosol, damaged organelles, and
protein aggregates are engulfed in autophagosomes and delivered to lysosomes for destruction.
We show that the population of OFD1 at the centriolar satellites is selectively degraded upon
serum starvation. The degradation of OFD1 is mediated by the autophagy-lysosome pathway
via its interaction with autophagy-specific protein LC3. In autophagy-deficient Atg5 null mouse
embryonic fibroblasts and human retina-pigmented epithelial cells treated with lysosome
inhibitors, OFD1 accumulates at centriolar satellites and blocks BBS4 (Bardet-Biedl syndrome
MONDAY-LATE POSTER PRESENTATIONS
4) recruitment to cilia, leading to fewer and shorter primary cilia. Notably, the primary
ciliogenesis defect in Atg5 null cells is fully rescued by OFD1 knockdown. Strikingly, OFD1
depletion promotes cilia formation in both cycling ciliary cells and “non-ciliary” breast cancer and
osteosarcoma cells. Our results demonstrate that removal of OFD1 by autophagy at centriolar
satellites represents a general mechanism to promote ciliogenesis. This discovery may further
our understanding of human ciliary diseases and provide new therapeutic tools for their
treatment.
2837
Salt as an oxidative stress in gastric mucosal cells.
H. Matsui1, M. Tamura1, Y. Nagano1, T. Kaneko1, T. Tomita2; 1Division of Gastroenterology,
University of Tsukuba, Tsukuba, Japan, 2Timelapsevision Inc., Asaka, Japan
[Background and Aims] Sodium chloride (NaCl) has been reported to involve acute gastric
mucosal lesions. Moreover, NaCl- rich food has been reported to be one of carcinogenic factors
for the stomach. However, there are a few reports to clear the pathogenesis for NaCl-induced
the gastric lesions and neoplasms. Recently, we have reported that gastric acid is an oxidative
stress which involves cellular apoptosis via mitochondrial reactive oxygen species (ROS),
particular superoxide anion production (Matsui et al. J Gastro. 2011). ROS have been reported
to play very important role for the involvement of the gastric lesions and carcinogenesis.
Hyperosmotic condition derived by NaCl may inhibit mitochondrial electron transfer thereby
producing ROS. To elucidate this possibility, we investigated the relations between NaCl and
ROS production in gastric epithelial cells.
[Methods] A gastric epithelial cell-line RGM-1 and RGM-MnSOD which is a MnSOD overexpressing stable clone after a gene transfection treatment were used. These cells were
exposed 300, 400, 600, 800 and 1300mOsm. Cellular cytotoxicity after each treatments was
investigated by TC-1 method. Electron paramagnetic resonance spectra were detected with a
spin trapping reagent CYPMPO. Cellular apoptosis was investigated by Cell-death detection kit.
[Result] NaCl treatment involved ROS, particular supeoxide anion production. Mild
hyperosmotic condition involved not only necrosis but also apoptosis. Overexpression of
MnSOD restrained NaCl-derived ROS production and cell death.
[Conclusions] NaCl is one of oxidative stress for gastric epithelial cells.
2838
Identification of human sequences that regulate autophagic-like Programmed Cell Death
in yeast.
S. Sheibani1,2, J. F. Lapointe2, R. Eid1, A. Horowitz2, C. Clapp1, L. Portt1, N. Gharib1, H. Vali2, C.
A. Mandato2, M. T. Greenwood1; 1Department of Chemistry and Chemical Engineering, Royal
Military College of Canada, Kingston, ON, Canada, 2Department of Anatomy and Cell Biology,
McGill University, Montreal, QC, Canada
Expression of human Bax, a critical regulator of mitochondrial membrane permeabilisation,
causes cell death in yeast. By screening a human cardiac cDNA library in yeast cells
conditionally expressing mammalian Bax, we previously isolated several clones capable of
preventing the deleterious effects of Bax in yeast. We found that one clone, human 14-3-3β/α, a
protein whose paralogs have been reported to have numerous chaperone-like functions, was
able to prevent cell death in response to traditional apoptotic inducing stresses including
cadmium and cycloheximide (Clapp et al. 2012 Cell Death Dis. 3:e348). The expression of 14-33β/α also conferred resistance to cell death induced by the TOR inhibitor rapamycin and by
starvation for the amino acid leucine, conditions that serve to induce autophagy. This represents
one of the first sequences that can prevent cell death not by inhibiting the initiation of autophagy
MONDAY-LATE POSTER PRESENTATIONS
but instead by preventing cell death at the end of autophagy. To ascertain if other regulators of
autophagy exists in the clones identified as Bax suppressors, the viability of yeast cells
expressing the different Bax suppressors was determined after 6 days of leucine starvation. We
thus identified three other potential regulators of autophagy named Bh34, Bh80 and Bh109. The
three Bax suppressors were able to delay cell death in response to apoptotic inducing stimuli
indicating that they are strong candidates to be anti-apoptotic sequences. One clone, Bh80,
behaved like human 14-3-3β/α and significantly delayed leucine starvation mediated PCD. The
two other clones, Bh34 and Bh109, gave rather surprising results in that they promoted and
increased the lethal effects of leucine starvation. All three clones nevertheless conferred
resistance to the autophagic inducing drug rapamycin. These results indicate that a subset of
anti-apoptotic genes may also represent anti-autophagic genes. Such genes allow for the
possibility to identify similarities and differences between apoptotic and autophagic cell death.
Analysis of these autophagic regulators is continuing in mutants that are defective in apoptotic,
autophagic and necrotic PCD.
2839
The Role of Progranulin Deficiency in TDP43-Mediated Neurotoxicity.
A. R. Mason1,2, S. Barmada2,3, L. Herl-Martens4,5, X. Xu4, A. Daub2,6, R. Farese4,7, S.
Finkbeiner2,3,8; 1Graduate Program in Developmental and Stem Cell Biology, University of
California San Francisco, San Francisco, CA, 2Institute of Neurological Disease, Gladstone
Institutes, San Francisco, CA, 3Department of Neurology, University of California San Francisco,
San Francisco, CA, 4Institute of Cardiovascular Disease, Gladstone Institutes, San Francisco,
CA, 5Graduate Program in Neuroscience, University of California San Francisco, San Francisco,
CA, 6Graduate Program in Bioengineering, University of California San Francisco, San
Francisco, CA, 7Departments of Medicine, Biochemistry and Biophysics, University of California
San Francisco, San Francisco, CA, 8Department of Physiology, University of California San
Francisco, San Francisco, CA
Frontotemporal Dementia (FTD) is the most common dementia in patients younger than 60.
FTD patients exhibit massive neuronal loss in the frontal and temporal lobes, leading to
behavioral changes or language impairment and death in 6-9 years. No effective treatments
exist. Among the known genetic causes of FTD, Progranulin (PGRN) haploinsufficiency is one
of the most common. Patients with mutations in PGRN exhibit accumulation of TDP43, a
normally nuclear protein, in the cytoplasm of neurons in affected brain regions. Previous studies
have demonstrated that cytoplasmic TDP43 is neurotoxic. The goal of this study is to elucidate
the link between PGRN and TDP43. We show preliminary data indicating that PGRN rescues
from TDP43 neurotoxicity and decreases TDP43 levels in vitro. Future studies will elucidate the
mechanism of this effect in both primary rodent neurons and human neurons derived from FTD
patient iPS cells.
2840
Prechordate conservation of glia-type specific expression and function of Neuropathy
Target Esterase.
S. Dutta1, J. McFerrin1, B. L. Patton1, D. Kretzschmar1; 1C.R.O.E.T., Oregon Health & Science
University, Portland, OR
Human loss-of-function mutations in the Neuropathy Target Esterase (NTE) gene cause
inherited spastic paraplegia; NTE inactivation by organophosphate pesticide exposures and
nerve gases cause delayed neuropathy (OPIDN). However, cellular mechanisms remain
uncertain. In Drosophila, loss of the NTE homologue Swiss Cheese (SWS) causes progressive
degeneration including glia as well as neurons. Previously, we showed cell-specific expression
MONDAY-LATE POSTER PRESENTATIONS
of SWS in neurons or glia in SWS-null flies separately rescued axonal and overensheathment
defects, suggesting SWS is independently required in neurons and at least one glia type to
maintain neural function. Here, using UAS/GAL4 conditional expression and cell-type specific
drivers for RNAi constructs to knockdown SWS/NTE levels in wildtype Drosophila, we show
directly that neuronal degeneration independently results from loss of SWS in neurons (by panneuronal driver APPL), and hyperensheathment from loss in glia (pan-glial drivers LOCO,
REPO). Surprisingly, brain vacuolization resulted from loss of SWS expression in glia, not
neurons. Further, glial-type drivers mz0709 and alrm showed SWS expression is specifically
required in Ensheathing glia, and not Astrocytic and Barrier glia. Fly behavior assays revealed
severe neural deficits result from both neuronal and glial deficiency; e.g. Climbing Index for both
were reduced 85-90% compared to controls expressing UAS-LacZ. However, progressive loss
of function was differentially sourced: loss of glial SWS caused activity deficits by day 2
posteclosion, while neuronal loss caused delayed onset deficits after day 14. Transgenic
expression of mouse and human NTE fully compensated Drosophila lacking SWS, including
both glial and neuronal degenerative phenotypes. In mice, pan-specific antibodies to NTE
showed little distinct reactivity in embryonic nerves, but sharply increased levels from P8-14
when axonal sorting and Schwann Cell (SC) differentiation end. In adult mice, NTE was present
at high levels in nonmyelinating SC, colocalized with GFAP and CD44, and low levels in
myelinating SC and axons. SC cultured from PND 0 nerves upregulated NTE over 36 hours,
along with nuclear differentiation factor Oct-6. NTE also accumulated to high levels in
denervated Schwann cells following nerve injury in vivo (4 days post-axotomy) and remained
high at 10 days, beyond initial axon reinnervation. Conversely, NTE accumulated in unsorted
axon bundles in nerves of adult laminin alpha2 and alpha4 deficient mice. Thus, NTE is
independently expressed in PNS axons and glia, in mice as in flies. Combined results suggest a
critical NTE target function conserved in ensheathing glia since prechordate divergence, which
loss may induce delayed pathogeneses in human OPIDN and spastic paraplegia.
2841
Stress and apoptosis modify the expression of Ro60, PCNA, p53 and HSP70 with a
similar pattern at the observed in Systematic Lupus Erythematosus.
A. W. Hernández-Rodríguez1, E. M. Sánchez-Sierra1, R. M. Ramírez-Santoyo1, E. E. LópezRobles1, M. A. López-Luna1, S. H. Sánchez-Rodríguez1; 1Laboratorio de Biología Celular, UAZ,
Zacatecas, Mexico
Systemic Lupus Erythematosus is a polyclonal autoimmune disease of connective tissue, with
skin affections; there is a relationship between sun exposure and skin manifestations. This
study was to analyze the effect of environmental stressors on skin, through the expression of
Ro60, p53, PCNA and HSP70. Balb/c mice were subjected to UV, heat, HgCl2, ethanol, H2O2,
camptothecin and cycloheximide in the absence or presence of an inhibitor of caspase 3.
Subsequently, an extract was prepared with the skin and then characterized the Ro60, p53,
PNCA and HSP70 proteins by Western blot-ECL. There was a change in the antigenic pattern
of Ro60 with heat, HgCl2, camptothecin and cycloheximide. Also, there was an increase in the
expression of Ro60 antigen and band, which decreased by administer an inhibitor of caspase.
The PCNA protein reduced expression due to UV light, heat, camptothecin and cicloheximide.
When given the inhibitor of caspase 3, before subjecting mice to stress, the decrease in the
expression of PCNA was not very strong. p53, showed an increase in expression with heat,
HgCl2, camptothecin and cycloheximide. By using the inhibitor of caspase 3 was not observed
expression of this protein in any condition. Todos los factores físicos y químicos inducen estrés
celular censado a través de la HSP70. Conclusion: stressors induce increase of HSP70 and
changes in the expression of Ro60. Stressors heat and mercuric chloride, as apoptotic drug
MONDAY-LATE POSTER PRESENTATIONS
camptothecin and cicloheximide increase the expression of p53 and decreased PCNA
expression.
2842
Novel assays for imaging mitophagy and aggregated protein removal.
N. J. Dolman1, K. Chambers1, W. Zhou1, T. Huang1, R. Smith1, K. Gee1, C. Bartel1, U. Singh1,
M. S. Janes1; 1Molecular Probes, Life Technologies, Eugene, OR
Autophagy functions as a predominately pro survival mechanism removing damaged or
aggregated cellular components from the cytosol. This is achieved by the encompassment of
cargo, via an emerging family of receptors, by the isolation membrane that then fuses to form
the double-membrane autophagosome. The autophagosome itself carries no degradative
capability. To degrade these components, the autophagosome needs to deliver them to the
lysosome thereby forming the autolysosome. Here we show imaging assays that allow
discrimination of autophagosome to autolysosome progression through multi-color imaging. As
stated above autophagy functions as a mechanism by which a cell can remove damaged
organelles or aggregated proteins. We also describe novel imaging assays for specific forms of
autophagy such as mitophagy or of aggregated proteins. For mitophagy we describe the use of
environmentally sensitive and insensitive mitochondrial probes in conjunction with autophagy
specific markers in multi-color time-lapse imaging. Through the use of metabolic labeling of
proteins we show imaging assays to asses the formation, localization and clearance of protein
aggregates. Data relevant to clearance of both short- and long-lived proteins by the
Proteasomal or lysosomal pathways respectively will be shown.
Cancer Cell Biology I
2843
Identification of a cleavage product of BRCA2 in cancer cell lines and its formative
mechanism.
N. Wali1, K. Hosokawa1, H. Saito2, S. Ohmi3, A. Nakanishi1, Y. Miki1,2; 1Molecular Genetics,
Tokyo Medical and Dental University, Tokyo, Japan, 2Molecular Diagnosis, Janpanese
Foundation for Cancer Research, Tokyo, Japan, 3Med.Sci.Inst,Tokyo University, Tokyo, Japan
Identification of a cleavage product of BRCA2 in cancer cell lines and its formative mechanism.
Background: BRCA2 is a multifunctional tumor suppressor protein with critical roles in DNA
repair, centrosome amplification, and cytokinesis. Loss of BRCA2 expression has been linked to
several cancer types. We previously performed immunoblotting for BRCA2 in the MCF7 breast
cancer cell line using two antibodies with different epitopes, i.e., recognizing the central region
(a.a. 1651-1821) and C terminus (a.a. 2959-3418). The specific band for wild-type BRCA2
(molecular weight; 384-kDa) was detected at 239-kDa using the antibody that recognizes the
central region. The 239-kDa band was not detected using the antibody that recognizes the C
terminus. However, the mechanisms governing BRCA2 cleavage are not clearly understood.
We hypothesized that cleavage of BRCA2 is achieved by proteolytic processing of wild-type
BRCA2.
Methods: To identify the candidate protease, we immunoprecipitated whole cell extracts
prepared from HeLa S3 cells using anti-BRCA2 antibody (central region). Captured BRCA2associated protease was separated by gel electrophoresis and subjected to mass spectrometry.
Mass spectrometry was also used to determine the mass of cleavage products and the
MONDAY-LATE POSTER PRESENTATIONS
localization of the scissile bond. To examine the role of the cleavage BRCA2 fragments, we
generated cleavage-site-directed antibodies for immunoblotting, which specifically recognize the
two fragments of BRCA2 but do not cross-react with the unproteolyzed wild-type BRCA2 (384kDa).
Results: Analysis of immunoprecipitated protein complexes and mass spectrometry revealed
membrane type-1 matrix metalloproteinase (MT1-MMP) protein complexed with BRCA2. We
also found that the sequence of the BRCA2 cleavage site was at 2135N-L2136 by mass
spectrometry. Furthermore, following transfection of BRCA2-FLAG into HeLa cells, we observed
differences in intercellular localization of cleaved BRCA2 and BRCA2-FLAG under
immunofluorescence microscopy using cleavage-site-directed antibodies and FLAG antibody.
Conclusions: We suggest that BRCA2 is a cleavage target of MT1-MMP and that MT1-MMP
possesses an intercellular proteolytic function, which may be importantly involved in malignant
transformation.
Keywords: BRCA2, MT1-MMp, LC-MSMS
2844
DNA Polymerase θ (POLQ) and defense against DNA damage.
M. Yousefzadeh1, R. Wood1; 1Molecular Carcinogenesis, University of Texas MD Anderson
Cancer Center -- Science Park, Smithville, TX
The mammalian genome encodes 15 different DNA polymerases that function in DNA
replication, DNA repair, immunological diversity, and the bypass/tolerance of DNA damage.
DNA polymerase theta (POLQ) is an A-family nuclear DNA polymerase found in vertebrate
genomes but its function is not yet certain. It is intriguing that breast cancers with elevated
levels of POLQ expression correlate with poorer outcomes. Multiple functions of POLQ have
been proposed including lesion bypass, antibody class switching and hypermutation, and a
backup role in base excision repair. Recombinant full length POLQ has low fidelity, can bypass
thymine glycols and can efficiently bypass and extend from abasic sites in vitro. POLQ-depleted
cancer cells have an altered response to ionizing radiation. Bone marrow stromal cells
(BMSCs) from mice lacking Polq are hypersensitive to ionizing radiation and bleomycin, a
radiomimetic agent. To investigate potential pathways in which POLQ may participate, we
analyzed the sensitivity of Polq-deficient BMSCs and controls to commonly used DNA
damaging agents. Using colony formation assays, we found Polq-/- BMSCs are more sensitive
than wild type cells to DNA damaging agents such as IR and topoisomerase inhibitors such as
camptothecin, etoposide, and ICRF-193, but not interstrand crosslinking agent mitomycin c. Our
data supports a role for POLQ in the defense against DNA strand break damage possibly
through an alternative end joining mechanism, as proposed for the Drosophila POLQ homolog
Mus308. Current efforts in the lab are focused on using complementation of POLQ in
mammalian cells to rescue the damage sensitivity phenotypes.
MONDAY-LATE POSTER PRESENTATIONS
2845
Live imaging of tumor incipient cells with Eisuke mice expressing FRET biosensor for
ERK.
T. Hiratsuka1, T. Nonaka2, K. Kinoshita3, M. Matsuda1; 1Department of Pathology and Biology of
Diseases, Graduate School of Medicine, Kyoto University, Kyoto, Japan, 2Department of
Immunology and Cell Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan,
3
Department of Evolutionary Medicine, Shiga Medical Center Research Institute, Shiga, Japan
Mouse skin tumor model with two carcinogens, DMBA and TPA, has long been utilized to reveal
multistep characteristics of tumorigenesis. DMBA is involved in the initiation step inducing an
activating mutation of HRas, and the following repetitive applications help the initiated cells
preferentially proliferate. In this process, initiated cells are thought to acquire additional genetic
or epigenetic changes as they grow into a full grown tumor. However, dissecting the cellular and
molecular events of incipient cells in the tumorigenesis has been extremely difficult lacking the
methods to detect them despite their significance to address problems such as which cell
lineage is tumor origin, how tumor-candidate cells are deemed to survive or die, and how they
can interact with their surrounding microenvironment.
We previously developed Eisuke mice, which express a FRET biosensor for ERK. Eisuke mice
enable us to monitor ERK activity in living tissues under a two-photon excitation microscope.
We topically treated Eisuke mice with DMBA and TPA, and microscopically observed the ERK
activity in the wide ear skin area on various days after DMBA treatment. The estimated number
of imaged keratinocytes is 3 ~105 cells per one mouse. We expected incipient tumor cells
could be detected by their outstanding ERK activity among the majority of other cells with
normal ERK activity.
Approximately one month after the initial DMBA treatment, we found cell clusters with
extremely-high ERK activity. Such ERKhigh clusters never appeared in mice treated with DMBA
alone or acetone (solvent). We followed the number of ERKhigh cells in each cluster and
classified the fate of each cluster into the following patterns: (1) Decrease and disappearance
(2) Stable state (3) Exponential increase. Most ERKhigh clusters exhibited gDecrease and
disappearance h pattern. In a gStable state h, the number of ERKhigh cells remained 20 to 40
for approximately 70 days. Intriguingly, the period of gStable state h was sometimes followed
by sudden transition to gExponential increase h, suggesting the acquisition of additional
genetic or epigenetic changes to help the incipient cells proceed to more advanced stage.
Genetic analysis of the ERKhigh cells is now undergoing. In conclusion, the Eisuke mice provide
an excellent method to identify the tumor incipient cells in DMBA/TPA-induced skin
tumorigenesis.
2846
Dysregulation of O-GlcNAcylation contributes to colitis and colitis-associated
tumoregenesis.
Y. Yang1, D. Kim1, H. Jang1, D. Park2, S. Choi1, J. Kim1, H. Moon1, P. Suh1; 1Nano-Bioscience
and Chemical Engineering(NBC), UNIST, Ulsan Metropolitan City, Korea, 2Postech, Pohang,
Korea
O-GlcNAcylation is dynamic and essential post-translational modification. Although
dysregulation of O-GlcNAc modification catalyzed by O-GlcNAc transferase (OGT) and OGlcNAcase (OGA) contributes to the etiology of chronic diseases of aging, including cancer,
cardiovascular disease, type 2 diabetes, and Alzheimer¡¯s disease, its pathphysiological role of
gastrointestinal tract is unknown. Here we address this tissue using a dextran sodium sulfate
(DSS) - colitis and colitis-associated cancer model. We observe that O-GlcNAcylation was
increased in colonic tissues of WT mice given DSS, concomitant with induction of O-GlcNAc
MONDAY-LATE POSTER PRESENTATIONS
cycling enzymes. Importantly, increased O-GlcNAc levels enhanced TNF-a-induced activation
of NF-kB signaling in colon epithelial cell line, suggesting that O-GlcNAcylation regulates NF-kB
signaling. In DSS-induced colitis, OGA+/- mice exhibited increased susceptibility to DSS
compared with OGA+/+ mice, with significantly higher clinical and histological colitis scores, and
myeloperoxidase activity. Inflammatory cytokines and chemokines were markedly elevated in
OGA+/- mice. Correlatively, enhanced activation of the nuclear factor-kB (NF-kB) and signal
transducer and activator of transcription 3 (STAT3) pathways were observed in colonic tissues
of OGA+/- mice. In mouse models of colitis-associated cancer (CAC) model, OGA+/- mice
exhibited a higher incidence of colon tumors than OGA+/+ mice. Based on these results, we
suggest that increased O-GlcNAc level in colon tissues aggravates intestinal inflammation in
mouse model of DSS-induced colitis and CAC model by modulating NF-kB signaling.
2847
Repression of Cyclin G2 Blunts the Cell Cycle Arrest Response of MCF7 Cells to
Antagonists of Estrogen Receptor Signaling and to the AMPK Activator Metformin.
M. Zimmermann1, A. Arachchige Don 2, M. Donaldson1, M. C. Horne1; 1Pharmacology,
University of California, Davis, CA, 2Pharmacology, UMDNJ-Robert Wood Johnson Medical
School, Piscataway, NJ
Acquired tumor cell resistance to endocrine-based therapeutics poses a significant challenge for
long-term abatement of estrogen receptor (ER)-positive breast cancers (BCs). Signaling crosstalk between activated ER and peptide growth factor receptor pathways (e.g. HER2 and IGF1R) is one adaptive mechanism promoting ER positive BC tumor cell resistance to drugs
inhibiting estrogen (E2) signaling. Identification of the gene products regulated by these
pathways that influence resistance to the ER-antagonists tamoxifen and fulvestrant could
improve future therapeutic approaches for control of BC. Transcript levels of the CCNG2 gene
encoding the unconventional cell cycle arrest response protein, cyclin G2 (CycG2), are reduced
upon activation of ER, HER2, insulin and insulin-like growth factor-1 receptor (IGF-1R)
signaling. Our previous work showed that blockade of HER2, PI3K and mTOR signaling
upregulates CycG2 expression in HER2 overexpressing BC cells, and that ectopic expression of
CycG2 induces cell cycle arrest of BC cell lines. Here we show that E2 depletion and
pharmacological blockade of ER signaling in E2-dependent BC cells enhances CycG2
expression and nuclear localization. Using shRNA-mediated RNAi we determined that blunting
CycG2 upregulation promotes proliferation of E2-deprived and fulvestrant-treated MCF7 cells.
Evidence suggests that loss of CycG2 increases phopho-activation of MEK1 and inhibitory
phosphorylation of RB. Our work also indicates that CycG2 can form complexes with CDK10, a
recently identified determinant of tamoxifen resistance linked to inhibition of the
RAF/MEK/MAPK signaling pathway. Recent studies suggest that the anti-diabetic drug
metformin (MTFN) inhibits BC cell growth by promoting AMPK–mediated suppression of growth
factor receptor activation of mTOR. Patients taking MTFN exhibit a dose-dependent reduction in
cancer risk and clinical trials indicate that BC therapies including MTFN improve response rates.
We found that MTFN treatment stimulates CycG2 expression and potentiates both fulvestrantmediated upregulation of CycG2 expression and growth-inhibition of MCF7 cells. Moreover,
knockdown of CycG2 expression blunts the enhanced anti-proliferative effect of MTFN on
fulvestrant treated cells. Importantly, analysis of BC tumor cDNA microarray databases
indicates that CCNG2 transcripts are reduced in aggressive, poor-prognosis BCs.
MONDAY-LATE POSTER PRESENTATIONS
2848
The expression and cellular localization of DLC family proteins in hepatocellular
carcinoma.
D. Lukasik1, E. Wilczek1, A. Walczak2, G. Szparecki1, G. M. Wilczynski2, B. Gornicka1;
1
Pathology, Medical University of Warsaw, Warsaw, Poland, 2Laboratory of Molecular and
Systemic Neuromorphology, Nencki Insitute of Experimental Biology, Warsaw, Poland
Deleted in liver cancer (DLC) protein family gathers proteins that function mainly as a Rho
GTPase activating proteins and by the regulation of small GTPases. It was found that the first
identified gene from this family, DLC1, is often lost in hepatocellular carcinoma and thus may
function as a liver tumor suppressor. Following DLC1, two other forms of DLC protein family,
DLC2 and DLC3 were identified. To date, there are only few published data concerning these
proteins role in malignant transformation. The aim of the present study was to determine
whether the loss of DLC1, DLC2 and/or DLC3 is a common change in HCC development.
We assessed by the means of the immunohistochemical approach DLC immunoreactivities in
tissue sections, as well as the gene status (by the fluorescence in situ hybridization) of DLC1
and DLC2 in different types of hepatocellular carcinoma - typical form and fibrolamellar variant.
We found that DLC1 gene is deleted in high percentage of both types of HCC. Additionally, by
the methylation-specific PCR we found DLC1 promoter to be epigenetically silenced in
tumorous tissues. DLC2 gene was deleted only in minority of cases. Surprisingly, in a subset of
tumors, we found DLC2 protein to be more abundantly expressed in cancer cells when
compared to normal hepatocytes. For both gene and protein status we did not find differences
between HCC variants. There were no differences between the normal and malignant tissue
regarding DLC3 immunoreactivity. The localization of all three protein forms in tumorous tissue
was distinct. DLC1 was seen predominantly in tumor stroma, whereas DLC2 and DLC3 in the
cytoplasm of cancer cells.
Our results indicate, that the loss of the DLC1, but not DLC2 and DLC3, is a common step
during hepatocarcinogenesis. It appears that members of the DLC family may function in HCC
development in a differential manner.
2849
Identifying a regulatory role for the tumor metastasis suppressor gene KAI1/CD82 in
metastatic prostate cancer cell lines.
P. Dodla1, M. Durston1, J. Kaminski2, S. Singh1, C. K. Miranti3, S. Sridhar2; 1Cell and Molecular
Biology, Grand Valley State University, Allendale, MI, 2Biomedical and Health Sciences, Grand
Valley State University, Allendale, MI, 3Lab of Integrin signaling and tumorigenesis, VanAndel
Research Institute, Grand Rapids, MI
KAI1/CD82, a metastasis prostate tumor suppressor gene expression is lost when the cancer
progresses from a primary to a metastatic stage. CD82 has also been shown to be downregulated in cancers of the gastrointestinal tract, colon, cervix, breast, lung, pancreas, skin,
thyroid and liver etc. As a member of the tetraspanin family of proteins, CD82 interacts with
proteins and may act as a master regulator of membrane organization at the cell surface. Even
though some of the interacting proteins have been identified, the significance of these
associations and its role in metastasis prevention is unclear. By reintroducing CD82 into highly
metatstatic prostate cells (PC3), we have shown CD82 to regulate c-Met (phosphorylation) and
activation. Currently we are focused on studying the exact mechanism by which CD82 regulates
c-Met. CD82 does not seem to associate with c-Met nor does it seem to down-regulate c-Met.
Preliminary indications are that as a tetraspanin and thus as a molecular organizer it may be
redistributing c-Met on the cell surface. It is also highly possible that it may bring a c-Met
MONDAY-LATE POSTER PRESENTATIONS
specific phosphatase (such as DEP-1) to the surface to dephosphorylate and deactivate c-Met.
We are currently exploring both possibilities.
Even though we have identified c-Met protein to be regulated by CD82, we have reason
to believe that there may be more proteins regulated by CD82. Microarray studies done on
CD82 (+/-), on both tumor and normal prostate cells suggests that CD82 may be regulating
genes involved in cell cycle, growth, and metastatic suppression. To validate the results, we
have utilized Q-PCR assays, investigating genes specifically involved in metastasis suppression
and growth. These genes include: CXCR4, CXCR7, RUNX3, TFF-3, and MMP10. CXCR4 and
CXCR7 are chemokine receptors, RUNX3, a tumor suppressor gene, and MMP10, which
encodes the matrix metalloproteinase 10 needed for invasion and continuation of metastasis.
Two of the genes involved in metastasis (TFF-3, RUNX-3) have been quantified and the data
correlates with the microarray data. MMP10, CXCR4, and CXCR7 are currently being validated.
Identifying the proteins regulated by CD82 and deciphering the downstream signaling
mechanisms involved in this regulation is the focus of our future studies.
2850
Effects of HSET (KIFC1) and Eg5 (KIF11) gene silencing on cell viability and centrosome
features during mitosis in human breast cancer cell lines.
K. Hanestad1, M. Y. Zhang1, W. C. Wayne1, A. Coxon1, R. L. Kendall2, M. N. Payton1; 1Oncology
Research, Amgen Inc., Thousand Oaks, CA, 2Therapeutic Innovation Unit, Amgen Inc.,
Thousand Oaks, CA
Centrosome amplification frequently occurs in human cancer cells and is associated with
increased chromosome instability and poor prognosis. Cancer cells with extra centrosomes can
form a bipolar spindle by clustering extra centrosomes. Recently, kinesin motor protein, HSET,
has been proposed to be required for efficient centrosome clustering in cell lines with extra
centrosomes (Kwon et al. Genes & Dev. 2008). The investigators showed that depletion of
HSET with pooled siRNAs was lethal to cancer cell lines that contain extra centrosomes. This
suggests that inhibition of the centrosome clustering pathway may selectively kill cancer cells
with extra centrosomes with minimal impact on normal dividing cells.
The aim of the current study was to evaluate the phenotypic effects of HSET and Eg5 gene
knockdown using individual and pooled siRNAs in human breast cancer cell lines. We
evaluated BT-549 and MDA-MB-231 cells which contain extra centrosomes, and MCF-7 cells
were included as a control cell line, containing a normal centrosome number. Cells were
transfected with siRNAs targeting HSET (10 individual, 1 pool), Eg5 (4 individual, 1 pool), or
non-targeting control siRNAs (10 individual, 1 pool). Efficient gene silencing of HSET and Eg5
at the RNA and protein level was demonstrated in all 3 cell lines. We confirmed the HSET
siRNA pool reported by Kwon et al. increased multipolarity and induced cell killing in cell lines
with extra centrosomes. In contrast, the majority of individual siRNAs targeting HSET showed
only a modest effect on cell viability in cell lines with extra centrosomes, with considerable
phenotype variability (e.g. nuclear morphology, effects on cell-cycle). We determined that only
1 of 4 individual siRNAs from the HSET siRNA pool significantly killed cancer cells; however all
4 siRNAs increased multipolarity. Individual and pool siRNAs targeting Eg5 induced a dramatic
loss of cell viability in all 3 cell lines, irrespective of centrosome number, and displayed
phenotypes consistent with literature findings.
Together these data show that individual siRNAs targeting HSET increase multipolarity, but fail
to induce robust cell killing. Furthermore, our data suggest the cell killing observed with pooled
siRNAs targeting HSET may be due to off-target effects and independent of centrosome
declustering.
MONDAY-LATE POSTER PRESENTATIONS
2851
The Role of TFG-TEC Fusion Gene in Human Extraskeletal Myxoid Chondrosarcomas.
B. Lim1, A-Y. Kim1, J. Choi1, J. Kim1; 1Department of Life science, Laboratory of Molecular and
Cellular Biology, seoul 121-743, Korea
The t(3;9)(q11–q12;q22) translocation associated with humanextraskeletal myxoid
chondrosarcomas results in a chimeric moleculein which the N-terminal domain (NTD) of the
TFG (TRK fusedgene) is fused to the TEC gene. The results showed that the chimeric gene
encoded a nuclear protein that bound DNA with the same sequence specificity as the parental
TEC protein. Comparison of the transactivation properties of TFG-TEC and TEC indicated that
the former has higher transactivation activity for a known target reporter containing TEC-binding
sites. Finally, deletion analysis of thefunctional domains of TFG (NTD) indicated that the
PB1(Phox and Bem1p) and SPYGQ-rich region of TFG (NTD) were capableof activating
transcription and that full integrity of TFG (NTD) was necessary for full transactivation. These
results suggest that the oncogenic effect of the t(3;9) translocation may be due to theTFG-TEC
chimeric protein and that fusion of the TFG (NTD) to the TEC protein produces a gain-offunction chimeric product.
2852
Segregated protection of pro-apoptotic macrophage inhibitory cytokine 1 (MIC-1)
transcript induced by endoplasmic reticulum stress in cancer cells.
S. Park1, H. Choi1, Y. Moon1; 1Lab of Mucosal Exposome and Biomodulation, Department of
Microbiology & Immunology, Pusan National University School of Medicine, Yangsan, Korea
Endoplasmic reticulum (ER) stress-induced insults lead to apoptotic cell death that is implicated
in a range of chronic inflammatory diseases and cancers. Macrophage inhibitory cytokine 1
(MIC-1), a member of the transforming growth factor beta superfamily, is variously associated
with the pathogenesis of cancer. To investigate the molecular mechanisms of MIC-1 gene
regulation, ER stress and its related signals were studied in human colon cancer cells.
Functionally, C/EBP homologous protein (CHOP), a well-known apoptotic mediator induced by
ER stress, was a positive regulator of MIC-1 gene expression. ER stress-induced CHOP
enhanced MIC-1 mRNA stability by triggering protein kinase C-mediated cytosolic translocation
of HuR/ELAVL1 RNA binding protein, which bound to and stabilized the MIC-1 transcript as a
first critical mechanism for MIC-1 mRNA stabilization. Another critical mechanism for MIC-1
mRNA was due to ER stress-activated ERK1/2 signals contributing to avoiding from destabilization of MIC-1 transcript by controlling the extended holding of the nucleated mRNA in
the stress granules fusing with the mRNA-decaying processing body. We propose these two
mRNA modulations across the compartment can account for stabilized transcription and
subsequent translation of pro-apoptotic MIC-1 gene in human cancer cells during ER stress
(This work was supported by the Basic Science Research Program through the National
Research Foundation of Korea, funded by Ministry of Education, Science, and Technology
Grant 2012R1A1A2005837).
MONDAY-LATE POSTER PRESENTATIONS
2853
C/EBPβ Mediates Inflammation-associated PAI-2 Gene Expression: Implications for
Ovarian Cancer.
E. A. Udofa1,2, B. W. Stringer3, P. Gade1,4, D. Mahony3, D. V. Kalvakolanu1,4, T. M. Antalis1,2,4;
1
University of Maryland, Baltimore, Baltimore, MD, 2Center for Vascular and Inflammatory
Diseases, Baltimore, MD, 3Queensland Institute of Medical Research, Herston, Queensland,
Australia, 4University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore,
MD
Plasminogen activator inhibitor type 2 (PAI-2) is a multifunctional serine protease inhibitor that is
highly upregulated in response to cellular stress in many cell types, including macrophages and
tumor cells. Of particular interest is the finding that PAI-2 expression is deregulated in several
cancers and has metastasis suppressor activity. Clinically, PAI-2 expression in ovarian cancer
metastases is indicative of longer disease-free and overall patient survival. PAI-2 expression
has been suggested as a biomarker to distinguish ovarian cancer from uterine cancer since it
was the most upregulated gene identified in a microarray study between these two cancer
types. This is important considering that high-grade ovarian cancer and uterine cancer
represent two histologically similar malignancies characterized by markedly different biological
behavior and response to chemotherapy, thus suggesting that the status of tumor-associated
PAI-2 expression may be used to direct therapeutic intervention. Given the potential importance
of PAI-2 expression to cancer as well as a host of other inflammatory diseases, we are
investigating molecular mechanisms that regulate inflammation-associated PAI-2 gene
expression. Our results demonstrate that the PAI-2 proximal promoter confers the greatest
response to lipopolysaccharide (LPS), a hallmark inflammatory agent, in a mouse macrophage
cell model. Further, we find that the PAI-2 proximal promoter is critically dependent on a CCAAT
enhancer binding (C/EBP) element at -203/- 192 and that C/EBPβ binds the C/EBP element in
an LPS-inducible manner, both in vitro and in vivo. Functional studies reveal that PAI-2
induction in response to LPS challenge is severely abrogated in C/EBPβ-KO mouse embryonic
fibroblasts. Together, these data indicate that C/EBPβ mediates inflammation-associated PAI-2
gene expression. Given that both C/EBPβ and PAI-2 are important for cellular differentiation,
stable ovarian cancer cell lines overexpressing PAI-2 and its catalytic mutants have been
generated to test the hypothesis that PAI-2 expression sensitizes ovarian cancer cells to
differentiation-inducing agents.
2854
Ell3: novel oncogene to regulate cancer stem cell population.
K-S. Park1, H-J. Ahn1; 1Department of Biomedical Science, CHA University, Seong-Nam Si,
Korea
Ell3 is a testis-specific RNA polymerase II elongation factor whose cellular function is not clear.
In previous study, we reported that Ell3 is activated during the differentiation of mouse
embryonic stem cells (mESCs) and stimulating lineage differentiation by promoting epithelialmesenchymal transition (EMT) and suppressing apoptosis. In this study, we suggest that Ell3
functions as a oncogene in human breast cancer cells. Over-expression of Ell3 in breast cancer
cell lines increases cell growth, mammosphere forming ability and resistance for anti-cancer
drug (5-FU). Ell3 over-expressing breast cancer cells have enhanced cancer stem cell
populations. Interestingly, overexpression of Ell3 reduces phospho-smad2 level in breast cancer
cell line. It is under investigation whether oncogenicity of Ell3 in breast cancer cell is induced by
modulation of TGF-beta signal pathway.
MONDAY-LATE POSTER PRESENTATIONS
2855
p53 response to energy stress depends on p73.
Y. Adamovich1, Y. Shaul1; 1Department of Molecular Genetics, Weizmann Institute of Science,
Rehovot, Israel
p53 cooperates with p73 and p63 paralogs in augmenting tumor suppression. p73 is involved
in the maintenance of genomic stability in normal cells, and in radio- and chemosensitivity of
human cancer cells. In response to DNA damage stress, p73 is sufficient to activate
transcription of p53-responsive genes that inhibit proliferation and induce apoptosis. In this
study our goal was to examine the role of p73 in cell response also to energy stress. The AMPactivated protein kinase (AMPK) is a central energy sensor that is activated in response to
decrease in intracellular ATP levels. Chronic stimulation of AMPK results in either growth arrest
or apoptosis depending on cell type. We found that pharmacologic stimulation of AMPK led to
p73 accumulation, which was mediated by direct phosphorylation of p73 by AMPK on a novel
Ser426 residue. The phosphorylated p73 was stabilized and accumulated to higher levels by
escaping the E3 ligase Itch and ubiquitine-dependent proteasomal degradation. We further
show that pharmacologic stimulation of AMPK resulted in cell cycle arrest and apoptosis that
were fully depended on the presence of p53. Surprisingly, and in contrary to the response to
DNA damage, p73 could not replace p53 in the apoptotic response to the energy stress caused
by chronic stimulation of AMPK. However, activation of p73 was important in achieving a more
rapid apoptotic response. Remarkably, we found that p73 was important for the stabilization and
accumulation of p53 under AMPK stimulation but not under DNA damage, suggesting that p53
and p73 may cooperate distinctively under different cellular stresses. Thus, our findings
establish a new coherent type 1 feed forward loop (C1-FFL) between p73 and p53, which is
different from the diamond-shaped loop that is described under DNA damage.
2856
Identification of Target Genes of the miRNA-148 Family Intronically Encoded by the
COPZ Genes.
D. J. Oliver1, A. Chaparala1, D. Altomare1, H. Ji1, K. Creek1, M. Shtutman1; 1Drug Discovery and
Biomedical Sciences, University of South Carolina, Columbia, SC
Coatomer complex I (COPI) is an essential component of intracellular membrane traffic involved
in Golgi-ER trafficking, early endosome transportation, and autophagic vesicle maturation. Each
protein in the COPI complex is coded by a single gene except the gamma and zeta subunits
that are encoded by two genes - COPG1, COPG2 and COPZ1, COPZ2 respectively. It is
currently unknown whether these isoforms play different functional roles in the COPI complex.
COPZ1 and COPZ2 genes both host a single microRNA in their intronic region miR-148b and
miR-152, respectively. These microRNAs are members of the miR-148 family of microRNAs
which include miR-148a, miR-148b, and miR-152. While some targets of these miRNAs have
been described, it remains to be seen how these miRNAs function as a family, especially
considering that miR-152 and miR-148a are selectively downregulated in various cancers while
miR-148b is not. The sequences of miR-148 family of miRNAs are highly conserved and their
seed regions are almost identical. Therefore all miRNA target prediction algorithms identify
highly overlapping target subsets for each miRNAs. To determine differentially regulated target
genes, we prepared prostate and breast carcinoma cells, PC3 and MDA-MB-231,
overexpressing miR-148b and 152. Using microarray analysis, we identified common and
differentially regulated genes. We validated several target genes including tumor-specific
Epithelial Adhesion molecule (EPCAM) by QPCR and western blotting. Broad effects of miR148 family miRNAs on signal transduction network will be discussed.
MONDAY-LATE POSTER PRESENTATIONS
2857
Mxd3 regulation of cell cycle progression and apoptosis defined by time of activation in
medulloblastoma cells.
T. T. Ngo1, G. A. Barisone1, E. Díaz1; 1Pharmacology, University of California Davis School of
Medicine, Davis, CA
Medulloblastoma, the most common brain tumor in children, develops due to uncontrolled
growth of cerebellar granule neuron precursors (GNPs). The regulatory networks involved in
medulloblastoma formation and progression have yet to be completely characterized.
Previously, we identify Mxd3 as a critical regulator of GNP proliferation during normal
development (Yun et al., 2007). Mxd3 is a basic-helix-loop-helix-leucine-zipper (BHLHZ)
transcription factor that is part of the Myc/Max/Mxd network in which Myc and Mxd form
heterodimers with Max to invoke opposite cellular responses. Specifically, Myc and Max
heterodimers are associated with the progression of proliferation where as Mxd and Max
heterodimers are associated with differentiation. Mxd3, however, is an atypical member of the
Mxd family as it has been shown to promote proliferation and Nmyc expression in GNPs (Yun et
al. 2007). Moreover, recently we showed that Mxd3 is upregulated in medulloblastomas and
knockdown of Mxd3 resulted in reduced proliferation (Barisone et al., 2012). To study Mxd3 in
the context of medulloblastomas in a time-dependent manner, we engineered the DAOY human
medulloblastoma cell line to stably express a fusion protein between truncated Estrogen
Receptor and full-length Mxd3 (ER-Mxd3). The fusion protein effectively migrates into the
nucleus upon treatment of 4-hydroxytamoxifen allowing for the timed activation of Mxd3. We
unexpectedly observe a decrease in cell counts 24 hours after Mxd3 activation. Pulse-chase
experiments with BrdU indicate that prior to this effect a transient increase in BrdU incorporation
occurs at 12 hours of treatment, indicating that ER-Mxd3 translocation to the nucleus initially
promotes cell cycle progression but that sustained activation ultimately leads to reduction in cell
number. These results provide an explanation for our previous surprising findings in
medulloblastoma cells whereby persistent expression of Mxd3 lead to reduced cell number due
to activation of apoptotic pathways (Barisone et al., 2012). To identify genes regulated upon
Mxd3 activation, we conducted expression profiling microarray experiments on samples taken at
key time-points after ER-Mxd3 translocation. In agreement with our previous findings (Barisone
et al., 2012), preliminary functional clustering analysis of these data suggest that apoptotic
genes are activated as early as 12 hours and remain activated as long as 72 hours in response
to Mxd3. In summary, these results suggest that Mxd3 promotes cell cycle progression but
ultimately level/or duration of Mxd3 expression ultimately leads to cell death. We hypothesize
that the pro-apoptotic activity of Mxd3 serves as a safe-guard mechanism for Mxd3
overexpression that might lead to uncontrolled proliferation.
2858
Regulation of p57kip2 and tumorigenesis by the prolyl hydroxylase PHD3 in renal cell
carcinoma.
E. Koh1, J-S. Moon1, W. Han1, H. Kim1, E. Bae1, D-Y. Jeong1, K-S. Kim1; 1College of Medicine,
Yonsei University, Seoul, Korea
Prolyl hydroxylase domain-containing protein3 (PHD3) is one of enzymes catalyzing prolinehydroxylation of hypoxia inducible factor(HIF)¥á- subunits, which is key regulatory event of HIF
activity. In renal cell carcinoma (RCC) tissues, PHD3 is commonly expressed at markedly
higher level than surrounding normal tissue. PHD3 is one of the target genes, of which the
expressions are induced by HIFs. Because high level of HIF activities is known to play a crucial
role in tumorigenesis of RCC, the control of PHD3 by HIFs is simply expected to be negative
MONDAY-LATE POSTER PRESENTATIONS
feedback loop. However, PHD3 shows strong tumorigenic effects in RCC by itself. The forced
expression of PHD3 in Caki-1, where PDH3 is expressed at low level, stimulated the
proliferation and led to tumor formation in xenographed nude mice. The knocking-down of PHD3
in A498 and 768-O RCC cell lines, where PHD3 is highly expressed, severely inhibited the cellcycle progression and the proliferation. A498 tumors formed in xenographed nude mice were
effectively shrinked by injection of lentivirus expression shRNA for PHD3. When the conditional
expression system of shRNA for PHD3 was introduced to A498 cells, the xenographed tumors
are completed disappeared by administration of doxycycline. The expression of p57KIP2 is
characteristically reduced in RCC tissues.
2859
Awake the sleeping: Gene specific re-expression of epigenetically silenced candidate
tumor suppressor genes in cervical cancer by artificial transcription factors.
C. Huisman1, F. Falahi1, G. B. Wisman2, M. Schokker1, I. Kazemier1, E. Schuuring1, A. G. van
der Zee2, M. G. Rots1; 1Pathology and Medical Biology, University Medical Center Groningen,
Groningen, Netherlands, 2Gynaecological Oncology, University Medical Center Groningen,
Groningen, Netherlands
Epigenetic silencing of tumour suppressor genes (TSGs) is a significant event in the
progression of cancer. In cervical cancer, we and others found TFPI-2, C13ORF18, CCNA1 and
EPB41L3 among those genes which are often hypermethylated. In this study, we aim to
specifically re-express these candidate TSGs using self-designed Artificial Transcription Factors
(ATFs), as this may lead to a decrease in tumour growth.
A total of 25 engineered ATFs, targeting either TFPI-2, C13ORF18, CCNA1 or EPB41L3, were
retrovirally delivered into (primary) cervical cancer cell lines. Gene inductions were quantified
using qRT-PCR, also after epi-drug treatment (5-Aza-2'-Deoxycytidine and Trichostatin A).
Functional effects of re-expression were measured using growth/apoptosis assays. Repressive
epigenetic marks were identified using chromatin IP and bisulfite sequencing, before and after
ATF treatment.
Gene silencing of all 4 genes in cervical cancer was associated with DNA hypermethylation and
repressive H3K9me3 and H3K27me3 marks could be identified. ATF induced re-expression
could be achieved in (primary) cell lines for all genes to similar levels as epi-drugs treatment.
Re-expression was associated with reduced cell growth (all genes) and induction of apoptosis
(EPB41L3, C13ORF18). Co-treatment of ATFs with epi-drugs further enhanced the ATF
induced effects. Interestingly, the forced re-expression by ATFs led to partial demethylation and
a decrease in histone methylation. These data demonstrate the potency of ATFs to re-express
and potentially demethylate hypermethylated silenced genes. Furthermore, this is the first time
that C13ORF18 and EPB41L3 are reported as functional TSGs in cervical cancer and may thus
serve as new therapeutic anti-cancer target.
Financed by NWO-VIDI to MR.
2860
Disparity of HSP60 (heat shock protein 60) mRNA Expression Level in colon cancer.
L. Yong Hyun1, P. Young Hoon1, K. Sua1, L. Seul Ki Na1, K. Dae Young1, Y. Hwang1;
1
Department of Biological Science, College of BioNano Technology, Gachon University,
Incheon, Korea
As eating habit has been changed, incidence rate of colon cancer is increasing. Thus, studies
about colon cancer are performed and focused on its early detection, diagnosis and treatment.
Especially, researches about interactions between colon cancer and HSP60 (heat shock protein
MONDAY-LATE POSTER PRESENTATIONS
60) are performed in some laboratories. HSP60, a mitochondrion protein, is featured to function
protein folding in cancer cell. Furthermore, HSP60 is located in mitochondria, cytoplasm, and
cell surface, taking part in reaction to stress response, apoptosis and cancer growth. Our study
focused on relationship between HSP60 and cancer growth. Other researches showed that
HSP60 is overexpressed in particular cancers. Our study focused on colon cancer in order to
find variation of HSP60 mRNA. we extracted mRNA samples of colon cancer tissue and made
cDNA through RT-PCR (Reverse Transcription PCR) and performed Real-time PCR. Through
electrophoresis of these cDNAs, we contrasted expression levels of HSP60 cDNA of colon
cancer tissue and normal tissue. Expression level of colon cancer HSP60 mRNA was higher
than that of normal tissue. Different expression of HSP60 mRNA between colon cancer and
normal tissue will be a good tool for early detection or diagnosis of colon cancer. And this is
important role to identify potential cancer biomarker.
2861
Elucidating LEF/TCF dynamics with ChIP-Seq and Gro-Seq.
N. P. Hoverter1, M. Zeller1, A. Garibaldi1, P. Baldi1, K. Hertel1, M. Waterman1; 1UC Irvine, Irvine,
CA
LEF/TCF transcription factors are downstream DNA binding proteins in the WNT signaling
pathway, which drives intestinal tumorigenesis. LEF/TCFs have a sequence specific HMG box
that binds Wnt Response Elements (WREs). The “E tail” isoforms of LEF/TCFs are alternatively
spliced to include a second DNA binding domain called the C-clamp, which binds short RCCG
elements adjacent to WREs. To elucidate C-clamp function in colon cancer cells, we
engineered DLD1 cell lines that can induce the expression of wild-type TCF1E (dnTCF1EWT) or
a mutant that lacks C-clamp DNA binding activity (dnTCF1Emut) with the addition of
doxycycline. Microarray analysis indicated that a WNT/p21 circuit is directed by C-clamp-driven
WNT target gene selection.
However, a problem with microarrays is they are relatively insensitive to rapid changes in
transcription after treatment of the cell (growth factor addition, overexpression of a protein, etc.).
RNA species that have long half-lives or show compensatory changes at the level of stability will
appear to be unchanged, even if the transcription rate has changed. In addition, long time
intervals after treatment are used, resulting in a cascade of gene expression changes, most of
which are indirectly related to the initial treatment. To identify rapid response WNT target
genes, we will perform GRO (Global Run On)-Seq after induction of dnTCF1E. GRO-Seq
utilizes a pulse of the Thymidine analogue 4-Thiouridine to label all RNA that is being actively
transcribed. 4-thiouridine transcripts are then biotinylated and pulled down by streptavidin
columns. Preliminary GRO-RTPCR reveals that a strong reduction in Wnt target gene
transcription can be detected at 2hrs post doxycycline addition (dnTCF1E induction). Thus
GRO-Seq will allow the identification of genes that are particularly dependent on
WNT/LEF/TCFs for their expression.
To understand the mechanism behind dnTCF1EWT induction of p21 and determine the
importance of C-clamp DNA binding activity to global WRE selection, we will perform ChIP-Seq
in tandem with GRO-Seq. Our preliminary ChIP-Seq identified 3765 peaks bound by
dnTCF1EWT. These peaks were highly enriched near known Wnt target genes and were
strongly associated with colon cancer in an unbiased KEGG test. Motif analysis revealed
enrichment of the canonical WRE (CTTTGAT), but the presence of adjacent RCCG elements
were not predictive of binding. However, the degeneracy and shortness of the RCCG element
makes statistical analyses difficult. Therefore additional ChIP-seq analysis with dnTCF1Emut is
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currently being performed. This will allow us to determine the contribution of the C-clamp to the
global DNA binding of dnTCF1E.
2862
Phenotypic screening for modulators of oncogene addiction pathways by label-free
assessment of cell morphology.
J. Irelan1, W. Tang2, B. Xi1, L. Zhao1, J. Wu2, P. Ye2, J. Zhu2, W. Zhang1, L. Mao1, Y. Abassi1,
W. Xu2, X. Xu1; 1ACEA Biosciences Inc., San Diego, CA, 2Hangzhou High-throughput Drug
Screening Center, Zhejiang, China
Oncogene addiction may be defined as the dependence of cancer cells on a single oncogenic
pathway for sustained proliferation and/or survival. This phenomenon has been described as
the “Achilles heel” of cancer and may represent a promising therapeutic opportunity. We have
evaluated the utility of an impedance-based label-free system for screening of modulators of
receptor tyrosine kinase mediated signaling in the context of oncogene addiction. Label-free
approaches to phenotypic screening allow for real-time dynamic monitoring of cellular
responses. Cell morphology, attachment, and proliferation phenotypes can be altered in
response to modulators of many disease-relevant pathways. For many targets, direct
measurement of these characteristics allows for evaluation of endogenously expressed targets
and pathways in a therapeutically relevant cellular context, and is thus a perfect fit for
phenotypic approaches to drug discovery. We evaluated several oncogene addicted cell lines
comprising known pathways for phenotypic screening using the impedance readout to measure
cell morphology and viability. These cell lines showed robust and sensitive responses to ontarget or on-pathway tool compounds with relatively high specificity. One cell line was chosen to
execute a large scale screen using the impedance readout as both the primary and secondary
screening tool, leading to identification and development of a novel inhibitor. These inhibitors
were further characterized by in vitro kinase assays as well as immunoblotting using phosphospecific antibodies directed against the oncogene of interest.
2863
CD24 regulates tumor cell behaviour in a c-Src/STAT3 dependent fashion.
P. Altevogt1, N. P. Bretz1, A. V. Salnikov1, G. Moldenhauer1; 1D015, German Cancer Research
Center (DKFZ), Heidelberg, Germany
Membrane lipid rafts have been identified as important signaling platforms. CD24 is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that is exclusively localized in lipid rafts.
CD24 is frequently over-expressed in human carcinomas and is associated with poor prognosis.
Previous work has shown that CD24 expression affects cellular properties such as proliferation
and apoptosis in vitro and tumor growth in vivo. However, little is known how CD24 mediates
these effects. The objective of the present study was to analyze the functional consequences of
CD24 knock-down or over-expression using human cancer cell lines of different tumor entities
(ovarian, lung, glioma). We used western blot, animal experiments and RT-PCR for the
analysis.
We observed that the presence or absence of CD24 is crucial to regulate the amount and
activity of the tyrosine kinase c-Src in lipid rafts. Altered activity of c-Src mediated by CD24
knock-down was responsible for reduced levels of phosphorylated “signal transducer and
activator of transcription 3” - STAT3 (Y705). Diminished activity of STAT3 was confirmed by
luciferase-reporter assays. Silencing of c-Src, similar to CD24, reduced the expression of
prototype STAT3-regulated genes (MCL-1, Cyclin D1 and Survivin). Likewise, the overexpression of CD24 augmented c-Src activity, the recruitment of c-Src into lipid rafts, levels of pSTAT3 and the expression of STAT3-dependent target genes. Functionally, depletion of CD24
MONDAY-LATE POSTER PRESENTATIONS
reduced cell proliferation and increased apoptosis, suggesting a pivotal role of regulated target
genes for these functional effects.
We observed that antibodies to CD24 showed significant therapeutic effects in mouse xenograft
models of lung- or pancreatic cancers. Antibody treatment affected the levels of c-Src-activity in
the treated tumors and altered expression of STAT3 target genes. We conclude that CD24
promotes raft mediated signaling by regulating STAT3 activity and that c-Src has an important
role in this process.
2864
Involvement of cyclooxygenase-2 in prostate cancer cell migration and invasion.
T-S. Cheng1, Y-C. Lu1, J-R. Ko1, B-F. Lai1, M-S. Lee1; 1Department of Biochemistry and
Molecular Biology, National Taiwan University College of Medicine, Taipei, Taiwan
The high mortality of prostate cancer is mainly due to high cancer metastasis. Recent
investigations have shown that cyclooxygenases-2 (COX-2) modulates cell migration and
invasion in several types of cancers, including colon, lung, breast, pancreas and prostate
cancers. However, the molecular mechanism how COX-2 promotes prostate cancer cell
invasion is not understood. In this study, using an invasion progression model (parental and
highly invasive M2I2 PC-3 cells), we found that M2I2 PC-3 cells had high COX-2 and matriptase
expression, compared to parental PC-3 cells. To further explore the role of COX-2 in prostate
cancer cell migration and invasion, we examined the effect of a COX-2 inducer, IL1-£], on
prostate cancer cell migration and invasion. Our results showed that IL1-£] could promote
prostate cancer cell migration and invasion. Moreover, using a COX-2 product, PGE2, also
could induce matriptase activation via down-regulating matriptase cognate inhibitors, hepatocyte
growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2). Furthermore, we examined
the effect of a COX-2 specific inhibitor, celebrex, on prostate cancer cell migration and invasion,
and found celebrex could suppress cell migration and invasion, that is concurrent with reducing
matriptase activation and up-regulating HAI-1 and HAI-2 expression. To further address whether
HAI-1 and HAI-2 expression could reduce the expression or activation of matriptase in prostate
cancer cells, we established a stable clone of PC-3 cells with HAI-1 or HAI-2 over-expression
and found that HAI-1 and HAI-2 could reduce the matriptase activation, prostate cancer cell
migration and invasion. In addition, celebrex also could suppress the prostate cancer cell
migration and invasion promoted by matriptase-overexpression. Taken together, these data
indicate that COX-2 signaling can promote prostate cancer cell migration and invasion, partly
due to down-regulating HAI-1 and HAI-2 and up-regulating matriptase function. In addition,
celebrex exhibits a suppressive effect on prostate cancer cell migration and invasion.
2865
The role of CADM1 in suppression of cancer cell metastasis as a new type of
dependence receptor (DR)
H. Kogai1, M. Sakurai-Yageta1, Y. Murakami1;
1
Division of Molecular Pathology, Institute of Medical Science, The University of Tokyo, Tokyo,
Japan
CADM1(cell adhesion molecule 1) is a tumor suppressor involved in cell-cell adhesion.
Inactivation of CADM1 was observed in various cancers and its restoration suppressed in vivo
metastasis. It suggests that trans-interaction of CADM1 has two distinct functions in cell
adhesion and in the elimination of dissociated cancer cells similarly to dependence receptors
(DRs). DRs activate cell survival and differentiation signals in the presence of ligands while they
induce apoptosis in the absence of ligands by activating caspase pathway. Here, we
MONDAY-LATE POSTER PRESENTATIONS
investigated apoptosis induction by the loss of CADM1-mediated cell-cell adhesion. The
cytoplasmic region of CADM1 contains a consensus recognition site by caspase-3 (DAAD), and
wild type (WT) CADM1 but not CADM1 mutant (NAAN) was cleaved by caspase-3 in vitro.
When breast cancer cells, MCF-7, were cultured in single cell suspension, CADM1-WTexpressing cells showed enhanced apoptosis in comparison with CADM1-NAAN clones.
Furthermore, the apoptosis induction was correlated with the fragmentation of the cytoplasmic
domain of CADM1 by caspase, and the C-terminal fragment was sufficient for apoptosis
induction. These results suggest that dissociation of CADM1-mediated interaction may trigger
the cleavage of the cytoplasmic domain of CADM1 by caspase-3, and that the C-terminal
fragment could induce apoptosis. Taken together, CADM1 has a potential to prevent cancer
metastasis as a new type of DR.
2866
Mechanobiology of epithelial-mesenchymal-transition in breast cancer cell line: role of
vimentin.
C-Y. Liu1, Y-K. Wang2, H-H. Lin1, H-C. Harn1, M-J. Tang1; 1Department of Physiology, National
Cheng Kung University, Tainan, Taiwan, 2Graduate Institute of Biomedical Materials and Tissue
Engineering, Taipei Medical University, Taipei, Taiwan
Disruption of actin filaments and the decrease in focal adhesions are common features of
transformed cells displaying anchorage-independent growth and cellular tumorigenicity.
However, some cancer cells undergo epithelial-mesenchymal-transition (EMT) and become
more malignant, a process accompanied by the lost of cell-cell junction and gain of
mesenchymal markers such as vimentin. In this study, we hypothesized that intermediate
filament vimentin may function as a force transmission linker between actin filament and
microtubules in EMT cancer cells. To test this hypothesis, we first evaluated the biophysical
properties of three breast cancer cell lines, MCF7, MDA-MB 468, and MDA-MB 231 by
employing atomic force microscopy (AFM) to measure cell stiffness. The results revealed that
among these breast cancer cell lines, MDA-MB 231 showed the highest cell stiffness. Western
blot analysis showed that MCF7 expressed the lowest levels of β1 integrin and FAK, whereas
MDA-MB 231 displayed the highest levels of EMT-related proteins, such as β1 integrin, FAK,
vimentin, and fibronectin. With the vimentin si-RNA application in MDA-MB 231 cells, these cells
become larger and the cell stiffness was decreased. The immunofluorescence staining also
showed that actin filament and microtubule were reorganized toward cell edge when vimentin
knockdown. Further, the AFM indentation revealed that the reorganized cytoskeletons increased
cell stiffness at cell edge but decreased the nucleus stiffness. Taken together, our studies
showed that vimentin may not only function as a cell tension transmitter, but also a regulator for
the maintenance of cytoskeleton architecture and the balance of cell force generation.
2867
Protein Kinase G-Iα Hyperactivation and Potential Role of VASP Phosphorylation in
Promoting Ovarian Cancer Cell Proliferation, Cell Adhesion and Cell Migration.
J. C. Wong1,2, R. Coffman1, H. Rosenberg1, R. R. Fiscus1; 1Center for Diabetes & Obesity
Prevention, Treatment, Research & Education, Cancer Research Program, and College of
Pharmacy (Pharmaceutical Sciences), Roseman University of Health Sciences, Las Vegas, NV,
2
Department of Chemistry, University of Nevada, Las Vegas, Las Vegas, NV
Platinum-based drugs such as cisplatin have dominated the drug therapy of ovarian cancer
during the past three decades. Our recent studies have shown that cisplatin regulates the
endogenous expression of nitric oxide synthases (NOSs) in human ovarian cancer cells, and
that the chemoresistance/cytoprotective effects of endogenous endothelial NOS (eNOS) involve
MONDAY-LATE POSTER PRESENTATIONS
hyperactivation of PKG-Iα. We have identified that PKG-Iα is the predominant isoform of PKG in
ovarian cancer cells, as determined by Western blot analysis as well as using the new,
ultrasensitive NanoPro100 capillary electrophoresis-based-nano-fluidic protein analysis system.
The chemoresistance and exaggerated cell proliferation are likely mediated by the constitutive
hyperactivation of PKG-Iα (reflected in the high levels of VASP phosphorylation at Ser239).
Using small interfering RNA (siRNA) PKG-Iα and VASP gene knockdown, we showed that the
inhibition of endogenous PKG-Iα kinase activity reduced VASP Ser239 phosphorylation,
proliferation rate, migration and adhesion in three cisplatin-resistant ovarian cancer cell lines,
SKOV3, OVCAR3 and A2780cp. In our recent study, epidermal growth factor (EGF)-induced
activation of Src family kinase (SFK) was found to tyrosine-phosphorylates PKG-Iα, increasing
its serine/threonine kinase activity in ovarian cancer cells. siRNA knockdown of PKG-Iα
expression inhibits the EGF-stimulated increases in VASP Ser239 phosphorylation and Src/SFK
autophosphorylation at the equivalent of Tyr416 (the phosphorylation site for activating the
tyrosine kinase activity) in A2780cp cells. Moreover, PKG-Iα-siRNA dramatically decreases cell
migration both with and without EGF-induced stimulation. Furthermore, both PKG-Iα and VASP
siRNA knockdown significantly decrease proliferation and adhesion in three ovarian cancer cell
lines (SKOV3, OVCAR3 and A2780cp). These data confirm the role of endogenous PKG-Iα
activity, potentially via VASP Ser239 phosphorylation, in promoting cell proliferation, adhesion
and migration/invasion in ovarian cancer. Data from our laboratory has shown that PKG-Iα
phosphorylates several downstream proteins, including c-Src, Bad, CREB and VASP, leading to
enhanced cell proliferation and cytoprotection, contributing to chemoresistance in ovarian
cancer cells. The unique features of this signaling pathway in ovarian cancer cells may provide
a novel therapeutic target for disrupting metastasis and secondary tumor formation during
ovarian cancer progression.
2868
A structure function analysis of the Cdc42-specific guanine nucleotide exchange factor
Fgd1 in the process of invadopodia formation.
P. Ciufici1, M. Capestrano2, T. Daubon1, R. Buccione2, E. Genot1; 1IECB, Pessac, Bordeaux,
France, 2Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy
Background: Invadopodia are proteolytically active protrusions that extend from the ventral
surface of invasive tumour cells when cultured on an extracellular matrix (ECM). Cdc42 is a
master regulator of invadopodia formation but the molecular mechanisms operating upstream of
this GTPase are ill-defined. The guanine exchange factor (GEF) Fgd1 is a novel invadopodia
component. It is required for invadopodia formation and function in carcinoma cells but how
Fgd1 is regulated is still unknown. A structure-function analysis was performed to characterize
the molecular mechanisms regulating Fgd1 in the process of invadopodia formation.
Rationale: Fgd1 contains a Dbl homology (DH) domain adjacent to a pleckstrin homology (PH)
domain which catalyzes the exchange of bound GDP for GTP on Cdc42, an N-terminal prolinerich domain (PRD) upstream of the DH/PH domain, a cysteine-rich zinc-finger FYVE domain
and a second C-terminal PH domain (PH2) downstream of it. The PRD negatively regulates
GEF activity and contains two putative Src-homology3 (SH3)-binding domains. It also contains a
binding sequence (CBD) for cortactin and mammalian actin-binding protein 1 (Abp1) .
Results: GFP tagged constructs encoding the full length protein (GFP-FL-Fgd1), Fgd1 lacking
either the N-terminal (ΔPRD), the C-terminal (ΔFYVE-PH2) or both regions (DH/PH1 only) were
transiently expressed in MDA-MD-231 human breast cancer cells and analyzed for subcellular
localization and consequences on cell’s ECM degradation potential. Compared to FL-Fgd1,
expression of the DH/PH1 domain alone enhanced invadopodia formation and associated ECM
degradation whereas neither Fgd1ΔPRD nor Fgd1ΔFYVE-PH2 had an effect. Localization at
invadopodia was lost for DH/PH1 or Fgd1ΔPRD mutants but retained for Fgd1ΔFYVE-PH2
MONDAY-LATE POSTER PRESENTATIONS
mutant. Upon knockdown of endogenous Fgd1, only expression of the DH/PH1 mutant was able
to rescue the ECM degradation function.
Cortactin and mAbp1 are involved in invadopodia formation and are also known as Fgd1
interacting partners, we therefore focused on the PRD domain which contains the binding
sequence for these two proteins. Deletion of this region (Fgd1ΔCBD mutant) relocalised Fgd1
from invadopodia to the perinuclear area. In Fgd1 depleted cells, in contrast to FL-Fgd1,
Fgd1ΔCBD was unable to elicit invadopodia formation. This mutant exerted a dominant
negative effect as it significantly reduced invadopodia formation and associated ECM
degradation when the endogenous Fgd1 was present.
Collectively, our data indicate that the PRD domain is essential for Fgd1 localization at
invadopodia sites and involves cortactine binding, thus providing a potential link between Fgd1
binding partners and Fgd1 specific functions.
2869
Depletion of pro-metastatic protein NEDD9 promotes caveolae formation though
activation of caveolin-1 and inhibits invasion and migration of breast cancer cells.
P. Kozyulina1,2, Y. Loskutov1, J. Liu3, E. Pugacheva1; 1Department of Biochemistry, West
Virginia University and Mary Babb Randolph Cancer Center WVU School of Medicine,
Morgantown, WV, 2Department of Inracellular Singaling and Transport, Institute of Cytology
RAS, St.-Petersburg, Russia, 3Department of Physiology and Pharmacology, West Virginia
University, Morgantown, WV
NEDD9 is a member of a of non-catalytic scaffolding protein family with a wide number of
functions including regulation of cell cycle, apoptosis and cell motility. Upregulation of this
protein can promote tumor cell migration, invasion and metastasis in multiple cancer cell types
(Singh et al., 2010). In our previous study, we demonstrated that depletion of NEDD9 in breast
cancer cells leads to a significant decrease in cell migration and invasion concomitant with
accumulation of integrin β1, β4 and α5 in MDA-MB-231LN cells. However, the total level of
these receptors did not change, which implies that NEDD9 does not impact integrin synthesis or
degradation but trafficking. Integrin trafficking is known to be mediated by caveolae
internalization (Echarri and Del Pozo, 2006). Caveolin-1 is a major scaffold protein of caveolae;
it regulates multiple signaling pathways, contributes to membrane homeostasis, and can
promote cell migration (Urra et al., 2012). Upon NEDD9 depletion, we discovered a two fold
increase in the volume of caveolin-1 positive vesicles and a significant increase in tyrosine
phosphorylation of caveolin-1. Immunofluorescence staining of MDA-MB-231LN
cells
transfected with GFP-tagged NEDD9 showed partial colocalization of caveolin-1 and NEDD9.
Mechanistically, we found that NEDD9 directly binds to caveolin-1 and inhibits caveolin-1
activation and thus, induces a decrease in caveolae formation. FACS analysis indicated that
reduction of caveolin-1 in NEDD9 depleted cells brings back to normal levels the amount of
integrin β4 on the cell surface, thus providing evidence of a connection between the
accumulation of caveolin-1 positive vesicles and surface integrins in NEDD9 knockdown cells.
2870
ATM suppresses SATB1-induced malignant progression in breast epithelial cells.
E. Ordinario1, H-J. Han1, S. Furuta1, L. Heiser1, L. Jakkula1, F. Rodier1, P. Spellman2, J.
Campisi1, J. Gray2, M. Bissell1, Y. Kohwi1, T. Kohwi-Shigematsu1; 1Lawrence Berkeley National
Laboratory, Berkeley, CA, 2Oregon Health And Science University, Portland, OR
SATB1 drives metastasis in breast cancer cells by radically reprogramming gene expression.
We show that SATB1 also has an oncogenic activity in certain non-malignant breast epithelial
cell lines. We studied the non-malignant MCF10A cell line, which is used widely in the literature.
MONDAY-LATE POSTER PRESENTATIONS
We obtained aliquots from two different sources (referred to here as MCF10A-1 and MCF10A2), but found them to be surprisingly dissimilar in their responses to oncogenic activity of
SATB1. Ectopic expression of SATB1 in MCF10A-1 induced tumor-like morphology in threedimensional cultures, led to tumor formation in immunocompromised mice, and when injected
into tail veins, led to lung metastasis, whereas SATB1 had no such effects in MCF10A-2. DNA
copy-number analysis revealed that MCF10A-1 is indistinguishable genetically from MCF10A-2;
however, gene expression profiling analysis revealed that these cell lines have significantly
divergent signatures for the expression of genes involved in tumorigenesis, including cell cycle
regulation and signal transduction. The early DNA damage-response kinase, ATM, was greatly
reduced in MCF10A-1 cells compared to MCF10A-2 cells, and we found the reason for
reduction to be phenotypic drift due to long-term cultivation of MCF10A. ATM knockdown in
MCF10A-2 and two other non-malignant breast epithelial cell lines, 184A1 and 184B4, enabled
SATB1 to induce tumors similar to that observed in MCF10A-1. These data indicate a novel role
for ATM as a suppressor of SATB1-induced malignancy in breast epithelial cells, and also raise
a cautionary note that phenotypic drift could give rise to dramatically different functional
outcomes.
2871
H3 lysine 9 methylation promotes cell motility in vitro and drives tumor formation in vivo;
a possible explanation for the cancer-associated up-regulation of H3K9 methylation.
M. Hieda1, Y. Yokoyama1, Y. Nishioka1, S. Matsuura1, N. Matsuura1; 1School of Medicine and
Helth Science, Osaka University, Suita, Japan
Global histone modification patterns correlate with tumor phenotypes and prognostic factors in
multiple tumor types. Recent studies suggest that aberrant histone modifications play an
important role in cancer. However, the effects of global epigenetic rearrangements on cell
functions remain poorly understood. In this study, we demonstrate that the histone H3 lysine 9
(H3K9) methyltransferase SUV39H1 is clearly involved in regulating cell migration in vitro.
Overexpression of wild-type SUV39H1, but not enzymatically inactive SUV39H1, activated
migration in breast and colorectal cancer cells. Inversely, migration was reduced by knockdown
of SUV39H1 or chemical inhibition by chaetocin. In addition, H3K9 tri-methylation (H3K9me3)
was specifically increased in invasive region of colorectal cancer tissues. Moreover, the
presence of H3K9me3 positively correlated with lymph-node invasion in colorectal cancer
patients. Furthermore, overexpression of SUV39H1 drove tumorigenesis in mouse, resulting in
a considerable decrease in survival rate. These data demonstrate that H3K9 tri-methylation
plays an important role in human colorectal cancer progression, possibly by promoting collective
cell invasion.
2872
Rab27a controls MT1-MMP secretion, cell invasiveness and tumour growth in breast
cancer cells.
L. Iannone1, A. Hendrix2, G. Bolasco3, D. Tracy-White4, S-P. Wong4, T. Dubois5, P. Chavrier6,
M. Seabra4, C. Recchi4; 1Department of Experimental Medicine and Toxicology, Imperial
College London, London, United Kingdom, 2Ghent University Hospital, Gent, Belgium, 3EMBL
Mouse Biology Unit, EMBL, Monterotondo, Italy, 4NHLI, Imperial College London, London,
United Kingdom, 5Department of Translational Research, Institut Curie, Paris, France,
6
UMR144, Institut Curie, Paris, France
Rab proteins have been implicated in cancer progression but their exact function remains to be
established. Here, we have investigated the role of the secretory Rab Rab27a in the context of
breast cancer invasiveness and metastasis and we have found that Rab27a controls the
MONDAY-LATE POSTER PRESENTATIONS
secretion of the metalloprotease MT1-MMP. Invasive cancer cells adopt secreted
metalloproteases to degrade the extracellular matrix (ECM) and migrate into distant organs,
where they seed and form metastases. In particular, MT1-MMP is a pivotal enzyme for ECM
degradation and remodelling by cancerous cells. We initially observed by both fluorescent and
electron microscopy that Rab27a associates with MT1-MMP positive vesicles. Then, we
established that depletion of Rab27a impairs cell invasiveness in a variety of bi- and tridimensional assays where invasiveness depends on MT1-MMP activity. In a complementary
approach, the overexpression of Rab27a increases matrix degradation and MT1-MMP levels at
the cell surface, pointing at a role for Rab27a in MT1-MMP export. Though cell growth in vitro is
not altered by modified levels of Rab27a expression, we observed a strong decrease in tumour
growth in vivo in mice when cancer cells were stably depleted of Rab27a. Furthermore, analysis
of a cohort of samples from breast cancer patients showed higher levels of Rab27a in
cancerous tissue. These data thus highlight the importance of Rab27a in cancer
aggressiveness, possibly mediated by Rab27a control on MT1-MMP trafficking and activity.
2873
Knockdown of LAT1 suppresses cholangiocarcinoma cell proliferation, invasion and
migration.
K. Janjorn1,2, S. Borwornpinyo3, A. Chairoungdua1,2; 1Physiology, Faculty of Science, Mahidol
University, Bangkok, Thailand, 2Research Center of Transport Proteins for Medical Innovation,
Faculty of Science, Mahidol University, Bangkok, Thailand, 3Biotechnology, Faculty of Science,
Mahidol University, Bangkok, Thailand
Cholangiocarcinoma (CCA) is highly prevalent in Northeastern part of Thailand. The current
chemotherapeutic treatment for CCA is ineffective. Therefore, new molecular target for
treatment of CCA is required. Our preliminary result found that L-type amino acid transporter 1
(LAT1) and 4F2 heavy chain (4F2hc or CD98), a heterodimeric amino acid transporter, is highly
expressed in CCA cells however, role of this amino acid transporter on malignant phenotypes of
CCA cells is currently unknown. Therefore, we aim to investigate role of LAT1 on CCA cell
proliferation, invasion and migration and its underlying molecular mechanisms. By using specific
lentiviral shRNA-mediated knockdown of LAT1 in CCA cells (KKU-M213), and quantitative real
time PCR, we found that the expression of LAT2, LAT3, LAT4 and ASCT2 was not changed in
spite of the shRNA successfully reduced the expression of LAT1 approximately 70%,
suggesting the specificity of shRNA and there is no compensatory adaptation in KKU-M213
cells after reduction of LAT1 expression. Knockdown of LAT1 resulted in the reduction of Na+independent [14C]L-Leucine uptake and decreased in cell proliferation by 60%. Cell invasion
and migration was examined and found that cell invasion and cell migration was inhibited by
25% and 50 %, respectively compared to shRNA control. Furthermore, the activity of mTOR
was reduced as illustrated by the reduction of the level of p70 S6 Kinase phosphorylation, a
well-known downstream target of mTOR in LAT1 knockdown KKU-M213 cells, suggesting the
suppression of cell proliferation, invasion and migration in LAT1 knockdown CCA cells mediated
through inhibition of mTOR signaling pathway. Collectively, our results suggest that
LAT1/4F2hc, heterodimeric amino acid transporter plays an important role on the malignant
phenotypes of cholangiocarcinoma cells. Therefore, LAT1 appears to be one of an excellent
target for cholangiocarcinoma treatment.
MONDAY-LATE POSTER PRESENTATIONS
2874
Immunofluorescent analysis of EMT using a duplex antibody cocktail.
C. S. Gagen1, G. Innocenti1, C. Hoyt2, R. Wetzel1; 1Cytometry, Cell Signaling Technology,
Danvers, MA, 2Caliper Life Sciences, Hopkinton, MA
Epithelial-mesenchymal transition (EMT) is a biological process involved in cancer invasion and
metastasis in which cells undergo a series of biochemical changes that induce a morphological
transformation from an epithelial to mesenchymal phenotype. The concurrent examination of
epithelial and mesenchymal markers in cells or tissues is desirable to visualize morphological
changes, such as those seen in metastasis, but can be challenging, as it requires robust and
specific reagents that can be combined in a multiplex immunofluorescent panel. To meet this
need, we have developed a simple three-color panel that includes antibodies directed against Ecadherin (epithelial) and vimentin (mesenchymal), along with a DNA dye. This multiplex panel
was validated on control cells and tissues, and subsequently used to screen formalin-fixed
paraffin-embedded (FFPE) cancer tissue. Using image analysis software, we were able to
quantify the expression of the two markers in regions of tumor that appeared to be undergoing
EMT. These results show that an immunofluorescence antibody cocktail can be used for the
convenient, efficient, and simultaneous detection of the EMT targets E-cadherin and vimentin in
cell- and tissue-based assays. Furthermore, the ability to detect multiple analytes in single
tissue sections is advantageous for quantitative research.
2875
Involvement of Myosin II in MDA-MB-231 Cell Migration.
B. Hindman1, Z. M. Goeckeler2, R. Wysolmerski3; 1Mary Babb Randolph Cancer Center, West
Viriginia University, Morgantown, WV, 2Center for Cardiovascular and Respiratory Sciences,
West Viriginia University, Morgantown, WV, 3Department of Neurobiology and Anatomy, West
Viriginia University, Morgantown, WV
Cancer cell invasion and metastasis is an important question in the study and treatment of
cancer. Investigators have gained significant insight into the attachment molecules participating
in cancer cell metastasis, however our knowledge of the motor protein Myosin II involvement in
the process is limited. To determine the importance of Myosin II in regulating cell invasion and
metastasis, we depleted MDA-MB-231 cells, an aggressive and metastatic breast cancer cell
line, of specific Myosin II isoforms. shRNA’s targeting nonmuscle myosin IIA and IIB were used
to make stable myosin IIA and IIB knockdown (KD) MDA-MB-231 cell lines. Myosin IIA and B
protein levels were reduced by 90-95% of vector controls and protein levels were routinely
monitored to ensure myosin II KD was consistent between experiments. Loss of either myosin
IIA or IIB led to altered cell morphology in both 2-D and 3-D cultures. In 2-D cultures both
myosin IIA and IIB KD cells exhibited loss/reorganization of F-actin. In 3-D, myosin IIB KD cells
were elongated and appeared localized to one focal plane while IIA KD cells where highly
branched sending processes in multiple directions. To assess how loss of myosin II affects the
cells’ ability to organize their extra-cellular matrix (ECM), MDA-MB-231 cells were embedded in
3-D collagen constructs and gel compression/contraction assays performed. Loss of Myosin IIA
inhibited gel-compression by 80% while loss of IIB caused minimal reduction. This data
suggested myosin II has a critical role in MDA-MB-231 interaction with the ECM and may be
directly involved in MDA-MB-231 migration.
To measure MDA-MB-231 migration, we
developed a “mini tumor” 3-D migration assay. Cells were cast in a collagen matrix to form a
“mini tumor”; after 24 hours, this construct is re-embedded in a 3-D matrix microenvironment.
Cells migrating outward from the mini-tumors are followed by time lapse microscopy over a 24
hour period. Loss of myosin IIB causes a 50% reduction in migratory velocity while loss of IIA
results in a 25% reduction compared to parental controls. Track displacement and track total
MONDAY-LATE POSTER PRESENTATIONS
length were also altered in IIA and IIB knockdown cell lines. Taken together, these data indicate
that Myosin II isoforms play significant and non-redundant roles in cancer cell migration.
2876
The transcription factor ZNF217 is a prognostic biomarker and therapeutic target during
breast cancer progression.
L. E. Littlepage1,2,3, H. Kouros-Mehr2, A. S. Adler4, K. Qu4, G. Huang5, C. Collins6, H. Y. Chang4,
J. W. Gray7, Z. Werb2; 1Chemistry and Biochemistry, Harper Cancer Research Institute,
University of Notre Dame, Notre Dame, IN, 2Anatomy, University of California at San Francisco,
San Francisco, CA, 3Harper Cancer Research Institute, South Bend, IN, 4Program in Epithelial
Biology, Stanford University School of Medicine, Stanford, CA, 5Helen Diller Family
Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA,
6
The Prostate Centre at Vancouver General Hospital, Vancouver, BC, Canada, 7Oregon Health
and Science University, Portland, OR
Amplification of 20q13 occurs in many cancers including 20-30% of primary human breast
cancers and correlates with poor breast cancer prognosis. The transcription factor ZNF217 is a
candidate oncogene within this region. In patient cohorts we found that patients expressing high
ZNF217 in breast tumors had reduced survival and increased chemoresistance. We also
studied the consequences of Znf217 overexpression using both in vivo and in vitro culture
models. Znf217 overexpression drove aberrant differentiation and signaling events, increased
self-renewal capacity, mesenchymal marker expression, motility, and metastasis, and repressed
an adult tissue stem cell gene expression signature downregulated in cancers.
To identify an inhibitor of ZNF217 function, we correlated the IC50s for drugs from the
Developmental Therapeutics database with ZNF217 expression levels in NCI60 tumor cell lines
and identified the AKT inhibitor triciribine. Mammary epithelial cells (MECs) expressing reduced
levels of ZNF217 (shRNA-ZNF217) required higher concentrations of triciribine to kill cells in
culture. Interestingly, other AKT pathway inhibitors were not as effective as triciribine. Treatment
of our mouse mammary tumor models with triciribine decreased the Znf217-induced tumor
burden, inhibited AKT and MAPK activation in the tumors and inhibited progenitor cell selfrenewal by mammosphere assays. ZNF217 overexpression in MECs made them resistant to
the chemotherapy doxorubicin, while treatment with triciribine generated synthetic lethality with
doxorubicin.
Taken together, our mouse models demonstrate that Znf217 overexpression induces multiple
phenotypes required for tumor progression including increased progenitor cell phenotype,
increased motility and increased resistance to cytotoxic drugs, indicating that it is both an
oncogene and potential drug target. We also find that ZNF217 is both a prognostic indicator of
survival and metastasis in breast cancer patients as well as a predictive indicator of response to
treatment. With ZNF217 as a biomarker, triciribine may be an effective therapy for poor
prognostic patients.
2877
Anti-angiogenic effects of zoledronic acid on osteotropic breast cancer cells.
C. Contreras1, S. Morshedian1, W. Hamud1, B. Dhakal1, B. Khanal1, J. Bush1,2; 1Biology,
California State University, Fresno, Fresno, CA, 2Fresno Medical Education Program, UCSF School of Medicine, Fresno, CA
Emerging data suggest the bisphosphonate, zoledronic acid (ZOL), exerts both indirect and
direct anti-tumor effects by decreasing tumor cell proliferation, increasing apoptosis, and
MONDAY-LATE POSTER PRESENTATIONS
inhibiting angiogenesis. Recent data indicate that anti-angiogenic factors exert cellular effects
via suppression of mitochondrial oxidative phosphorylation/respiration and elevation of
intracellular ROS levels. This project intended to define proteins involved in response to ZOLtreatment of an osteotropic breast cancer cell line, MDA-231BO. Results demonstrate that
MDA-231BO cells are more sensitive to ZOL cytotoxicity and salutary effects are both mediaand calcium-dependent. To investigate pro-apoptotic mechanisms of ZOL, total RNA, whole cell
protein lysates, or mitochondrial-enriched fractions were isolated from MDA-231BO cells and
using a combination of RT-PCR, 1D/2D-PAGE separation and MALDI-MS/MS, we identified
several angiogenesis inhibitors that were upregulated and pro-angiogenic factors that were
downregulated after ZOL treatment. Functional studies demonstrate the improved efficacy of
ZOL on MDA-231BO compared to MDA-MB-231 controls. Taken together, these results further
support a model that ZOL tips the ‘angiogenic switch’ in favor of anti-angiogenic factors in
cancer cells with a propensity for bone metastases.
Computational, Physical, and Synthetic Cell Biology
2878
Membrane spatial organization at Eph/Ephrin clusters on breast cancer cells.
A. Tian1, M. Dong1, M. P. Coyle1, A. C. Greene1, C-H. Yu2, H. H. Huang1, J. T. Groves1; 1UC
Berkeley, Berkeley, CA, 2National University of Singapore, Singapore, Singapore
Eph receptors are mostly expressed in embryonic tissues during development and their primary
function is to regulate tissue organization by controlling the adhesion and repulsion between
cells. Additionally, Eph receptors are found to be over-expressed in numerous cancer cell lines.
The role of Eph receptors in cancer cells is complex and still under debate. Therefore, better
understanding the function of Eph receptors in cancer cells is important for cancer therapies.
The spatial regulation of cell membrane receptors has a crucial role to the downstream signaling
and cell behavior, especially for Eph receptors due to their unique role in controlling cell/tissue
organization during development. Different from other RTKs, supramolecular assemblies are
formed after Eph interacts with its membrane bound ligand Ephrin. To elucidate the physiology
of Eph/Ephrin clusters, we studied the spatial organization of membrane components at the
EphA2/EphrinA1 cluster sites on cancer cell membranes. EphA2-expressing cancer cells were
presented on an EphrinA1-displaying supported lipid bilayer, allowing EphA2 and EphrinA1 to
interact at the interface and form clusters on the membranes. Total internal reflection
microscopy and confocal microscopy are used to monitor the spatial localization of fluorescent
lipid analogs and membrane proteins at the EphA2/EphrinA1 cluster sites on cell membranes.
The results show that proteins on the cytoplasmic side of the plasma membrane are excluded
from the EphA2/EphrinA1 clusters and actin is concentrated at the peripheral of the clusters.
MONDAY-LATE POSTER PRESENTATIONS
2879
Establishment of a real-time evaluation system of the osteoconduction using fluorescent
proteins
H. Ohsugi1, J. Hatsukawa1, Y. Otsuka1, M. Aizawa2,3, N. Kanzawa1; 1Dept of Materials and Life
Sciences, Sophia university, Tokyo, Japan, 2Dept of applied Chemistry, Meiji University,
Kanagawa, Japan, 3Next-generation bioceramics project, Kanagawa Academy of Science and
Technology, Kanagawa, Japan
The aging of the Japanese population has led to a significant increase in the number of patients
with bone disease. Tissue engineering plays an important role in reconstructive orthopaedic
surgery for the patients. The utilization of pluripotent stem cells in tissue engineering is a key
technique for the development of bone substitute materials. To know the changes in cell
function in response to abiotic stimuli such as attachment to the bone substitute materials, cell
differentiation markers for osteoblasts are need to be analyzed. Standard methods for analyzing
bone differentiation are the gene expression analysis using RT-PCR, evaluation by immunostaining of cells with specific markers, and measurement of alkaline phosphatase activity. But
these methods are unsuited for the continuous observations for cell differentiation, and the
process is sometimes very sophisticated and time-consuming. Thus we aim to establish a
system that is able to quickly and easily evaluate bone conduction ability.
To visualize the expression levels of bone differentiation marker genes, red fluorescence
protein, mCherry, and green fluorescence protein, EGFP, were used in our study. For the realtime observation of cells, two plasmids to express fluorescent proteins using promoter sequence
of bone differentiation marker gene were constructed. Promoter sequences of Type I Collagen
(ColI), an early-stage maker gene and osteocalcin (OC), a late-stage maker gene for bone
differentiation were amplified by PCR using genomic DNA of MC3T3-E1 mouse osteoblastic
cells as the template. ColI promoter sequence was subclone into a mCherry expression vector.
OC was ligated into an EGFP expression vector. These two constructions were transfected into
MC3T3-E1 cells to observe the real-time osteoblastic differentiation in the cell. On day 1, cells of
red color were observed. Cells having both colors were observed on day 4. Number of cells
having green fluorescence increased and that having red fluorescence decreased along with the
culture periods. We analyzed gene expression using PCR. Our result showed that expression
patterns of mCherry and EGFP genes corresponded with those of ColI and OC genes,
respectively. Thus the expression profiles of marker genes and those of innate ColI and OC
coincided, we have successfully in constructions of transgenic cell line which express marker
genes. No obvious change was observed in the proliferation and calcification in transgenic cells
compared with MC3T3-E1 normal cells. We will use this construct in other pluripotent cells such
as C2C12 mouse myoblasts to study the cellular responses to abiotic stimuli.
2880
Interaction of Sustained Release Vitamin K2 Microspheres and MG-63 Cells.
S. Whu1,2, M-D. Chung2, S-J. Shieh2, C-M. Lu2; 1Department of Medical Device, Medical and
Pharmaceutical Industry Technology and Development Center, New Taipei City, Taiwan,
2
Department of Chemical and Materials Engineering, National Chin-Yi University of Technology,
Taichung, Taiwan
In this work, we prepared microspheres containing vitamin K2 (VK2) and poly(lactic¡Vco-glycolic
acid) (PLGA) biodegradable polymer with the oil in water (O/W) emulsion nonaqueous phase
separation method. First, 0%, 0.01%, 0.1%, and 1.0% (w/v) vitamin K2 were loaded into the
PLGA microspheres, signature as VK2MS, and calculated the yield of VK2MS. Then VK2MS
were observed by scanning electron microscope (SEM) to observe its morphology. The laser
scattering particle size distribution analyzer (LS), UV/Visible spectrometer (UV/Vis) and
MONDAY-LATE POSTER PRESENTATIONS
attenuated total reflectance fourier transform infrared spectrometry (ATR-FTIR) were used to
determine the effect of VK2 concentration on particle size distribution and encapsulation
efficiency. VK2MS were determined with drug release assay for 70 days in vitro. Finally, the cell
growth rate and cell number were estimated for 7 days with MG-63 human osteosarcoma cells
(MG-63 cells) by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay,
and detection cell of bone differentiation situation by the assay of alkaline phosphatase (ALP)
activity. Hematoxylin & Eosin (H&E) and Alizarin red S staining were utilized to evaluate the
effect of VK2MS on MG-63 cells. The yield of VK2MS was increased with VK2 content, 1.0%
VK2MS had the heights value of 80.8 ¡Ó 6.9%. All microspheres were spherical with a smooth
surface, and particle size distribution was in the range of 2~10 μm, their scale expanded with
VK2 content. We confirmed VK2 loaded into the PLGA microspheres, and PVA has been
cleaned by ATR-FTIR assay. The encapsulation efficiency of VK2MS were above 67.6 ± 7.1%,
0.1% VK2MS had the highest value of 92.8 ± 5.2%. In drug release assay, the 0.01% VK2MS
showed that the VK2 was almost complete release in 35 days, 0.1% and 1.0% VK2MS had
released of 36.6 ± 1.4% and 7.0 ± 0.4% in 70 days, respectively. It seems that drug release rate
was increased by decreasing VK2 loaded into the microspheres. In vitro of cell proliferation
assay, VK2MS inhibited the cell proliferation, 1.0% VK2MS by which MG-63 cells for 7 days was
mostly inhibited in cell growth rate of 44.5 ± 0.3%; VK2MS could promote cell differentiation,
enhanced the ALP activity, and 0.1% VK2MS treatment of MG-63 cells at 3 days had the heights
value of 11.2 U/L-30 min. This same situation was also found by histological staining for bone
mineralization. VK2MS enhanced bone formation was demonstrated and it is intended to apply
for bone regeneration in clinic in the future.
2881
High throughput imaging reveals genes affecting C. elegans locomotion.
A. Brown1, E. Yemini1, L. Grundy1, T. Jucikas1, W. Schafer1; 1MRC Lab of Molecular Biology,
Cambridge, UK
Visible phenotypes have played a critical role in understanding the molecular basis of behaviour
and development in the nematode worm C. elegans. However, many mutants have subtle
phenotypes that are difficult or impossible to see by eye. To broaden the applicability of this
powerful approach to genetics, we are using an automated system to record high-resolution
video of freely behaving worms and a computer vision system to identify and classify worms
based on features of their movement, morphology, and posture yielding a rich phenotypic
“fingerprint”. Analysis of this fingerprint provides the most detailed view to date of the
behavioural repertoire of C. elegans and defines a baseline for comparison to mutants. To this
end, we have compiled a database comprising video data from several hundred mutant strains
that reveal many previously undescribed phenotypes that can be clustered into related classes.
All video data as well as the derived feature data are available online at
http://wormbehavior.mrc-lmb.cam.ac.uk/. Since mutants in the same class may have related
functions, phenotypic clustering leads to new hypotheses for gene function.
2882
Optical control of protein activity by fluorescent protein domains.
X. X. Zhou1, H. K. Chung2, A. J. Lam1, M. Z. Lin1,3; 1Bioengineering, Stanford University,
Stanford, CA, 2Biology, Stanford University, Stanford, CA, 3Pediatrics, Stanford University,
Stanford, CA
Fluorescent proteins (FPs) are widely used as sensors of protein localization and activity, while
other classes of light-absorbing proteins have been used for optogenetic control of proteins with
light. Here we describe a previously unknown feature of a variant of the photochromic green FP
MONDAY-LATE POSTER PRESENTATIONS
Dronpa – the ability to undergo light-dependent dissociation and association, and use it to
control protein activities with light. We created a fluorescent light-inducible protein (FLIP) design
in which Dronpa domains are fused to both termini of an enzyme domain. In the dark, the
Dronpa domains tetramerize and cage the protein, but light induces Dronpa dissociation and
activates the protein. This method enabled optical control over guanine nucleotide exchange
factor and protease domains without extensive screening. To our knowledge, this is the first
case of a light-dependent interaction outside of natural light-responsive regulatory proteins, and
this method also uniquely has self-reporting abilities. In ongoing work, we are examining the
mechanism of caging by Dronpa domains in more detail and adapting the FLIP design to
photochromic red FPs. Our findings extend the applications of FPs from exclusively sensing
functions to also encompass optogenetic control.
2883
Tuning gene expression through RNA polymerase binding site design.
D. L. Jones1, R. Brewster1, R. Phillips1; 1Applied Physics, California Institute of Technology,
Pasadena, CA
One of the paramount goals of synthetic biology is the ability to tune transcriptional networks at
will. As a step in that direction, we present a set of 18 unique binding sites for E. coli RNA
Polymerase (RNAP) σ70 holoenzyme designed using a model of sequence dependent binding
energy combined with a thermodynamic model of transcription to produce a targeted level of
gene expression. This promoter set allows us to determine the correspondence between the
absolute numbers of mRNA molecules or protein products and the binding energies measured
in kB T energy units (where kB denotes the Boltzmann constant). Over 3 orders of magnitude in
expression, constitutive expression from the binding sites adheres on average to the predicted
level of gene expression to within a factor of 3 in both protein and mRNA copy number.
This level of predictive accuracy is maintained when the promoters are put under control of a
simple repression motif, in which transcription is repressed via a single repressor binding site
adjacent to the promoter. Our simple thermodynamic model of constitutive transcription can be
extended to account for the effect of repressor binding, yielding an expression that predicts
gene expression as a function of both RNAP binding site strength and repressor copy number.
We have measured gene expression at RNAP binding energies ranging from 1.5 to 7.0 kB T,
and repressor copy number ranging from 0 to 100 per cell, and obtained good agreement with
model predictions. This indicates that our designed promoters still function as expected when
transplanted into a different regulatory context. We believe that manipulating protein-DNA
interactions to predictively tune gene expression as we have done here is useful for both the
quantitative dissection of regulatory architectures and as an engineering tool for use in synthetic
biology.
2884
Celx: an automated software to study mitotic chromosome movement in C. elegans onecell embryo.
I. Filiatreault1, M. Cormier1, T. Phi1, J. Dorn1, P. Maddox1; 1Université de Montréal, Montréal,
QC, Canada
During mitosis, chromosomes must be aligned to the metaphase plate in order to be correctly
segregated into two identical sets of sister chromatids. Their movement is made possible by the
microtubules emanating from opposite spindle poles. Microtubules attach to chromosomes via a
complex protein structure called the kinetochore, which plays a vital role in the regulation of
chromosome movement. The outer layer of the kinetochore is mostly responsible of microtubule
attachment, while the inner layer interacts with the centromere. It is not surprising that the role
MONDAY-LATE POSTER PRESENTATIONS
of many of the > 100 kinetochore components in governing the mechanical forces required for
proper chromosome movement is yet to be discovered. The C. elegans one-cell embryo is an
attractive model system to study the role of kinetochore proteins in regulating microtubule-based
force production. Its chromosomes are holocentric, i.e. the centromere is distributed along the
entire length of the chromosome axis. Consequently, tracking both position and shape of the
chromosome provides a rich readout of the forces exerted by microtubules. Since manual
tracking of the 3D shape of chromosomes is both difficult and prohibitively time-consuming, we
have developed automated software that segments and tracks entire chromosomes in C.
elegans from condensation to alignment at the metaphase plate. Using this software, we find
that in Nuf2-depleted embryos, chromosome movement speeds are unaffected, but that the
metaphase plate is wider while pole-to-pole distances are shorter, suggesting that Nuf2 plays a
critical role in regulating force balance and spindle architecture.
2885
Trajectory of Conformational Changes in Ligand Activation of E. coli cAMP Receptor
Protein Elucidated by Small Angle X-Ray Scattering.
R. Chkheidze1, J. C. Lee1; 1Dept. of Biochemistry & Molecular Biology, University of Texas
Medical Branch, Galveston, TX
cAMP receptor protein, CRP, a well-studied transcription activator, is proposed to regulate more
than 400 genes in E. coli. CRP is a homo-dimer in which each subunit consists of two domains,
namely the N-terminal cAMP- and the C-terminal DNA-binding domain. One remaining unclear
feature in the activation mechanism is the correlation between occupancy of cAMP to the Nterminal ligand-binding sites and the trajectory of conformational changes during the activation
process. The thermodynamics of binding of the two cAMP molecules to CRP exhibit
distinguished characteristics, namely, exothermic and endothermic for binding of the first and
second cAMP, respectively. But it is not known whether conformational shift occurs after binding
of two cAMP molecules or there is an intermediate conformational state upon binding of the first
cAMP, although the thermodynamics indicate a major conformational change is associated with
the binding of the second cAMP. In recent studies, both apo- and holo-CRP structures have
been elucidated by NMR and X-ray crystallography. Both studies show a consistent picture of
major rotation of the DNA-binding domain with respect to the ligand-binding domain is involved
in the activation process, although there are substantial differences between the apo-CRP
structures defined by the two approaches.
We used Small Angle X-ray Scattering (SAXS) to define the conformational changes and
evaluate the effect of different cAMP concentrations on the shape of wild type and mutant CRPs
in solution. The advantage of SAXS is that it is a solution technique and the information
generated about the general shape of the molecule can be compared with the NMR and
crystallographic data. Our results showed almost perfect correlation with the simulated
scattering curve obtained from the published X-ray crystal structure. We also showed that the
shift to the conformation of holo-CRP is correlated to the binding of two cAMP molecules and
this process can be significantly affected by the mutations that change the cooperativity of
cAMP binding. These observed changes in mutant CRP can be explained by the fact that apomutant S62F- and D53H-CRP exhibit characteristics of shifting towards the apo- and holoconformations, respectively. Thus, the effects of mutations are shifting the distribution of the
ensemble of structures in equilibrium between the apo and holo states.
MONDAY-LATE POSTER PRESENTATIONS
2886
Physical mechanisms of cortical flow during maintenance phase in C. elegans.
W. M. McFadden1, J. Alberts2, E. Munro1; 1University of Chicago, Chicago, IL, 2University of
Washington, Friday Harbor, WA
Cortical flows driven by contractile actomyosin networks play key roles in cell polarization,
cytokinesis, motility and tissue deformation. At a macroscopic level, the cortex can be
characterized as an “active fluid” in which active stresses produced by local actomyosin
interactions work against an effectively viscous network resistance to drive long range flows. But
we still know very little about how macroscopic quantities like stress and effective viscosity
emerge from microscopic interactions among actin filaments, crosslinkers, and myosin motors.
We are addressing these questions in the context of polarity maintenance in one-cell C. elegans
embryos. During maintenance, a stably asymmetric distribution of myosin drives a steady flow
of cortical actin and myosin towards a convergence zone near the center of the embryo.
We used spinning disk confocal microscopy and particle image velocimetry (PIV) to measure
density and velocity profiles along the AP axis, then used standard methods to fit our data to the
simple continuum model for an active fluid. The fits yield an estimate of a hydrodynamic length
scale (the ratio of internal effective viscosity to friction) that is consistent across multiple
embryos. Interestingly, this length scale is similar in magnitude to that measured by Mayer and
Grill (Mayer et al. Nature. 2010 Sep 30, pp. 617-21) during polarity establishment despite
significant differences in network organization and dynamics between establishment and
maintenance phases. We found that different assumptions about the dependence of active
tension on myosin density provided equally good fits, suggesting that the simple model is underconstrained by wild type data and that additional data is needed to infer the exact nature of
these dependencies.
However, these different assumptions yield sharply different predictions about the dependence
of flow speed on myosin density and we are currently testing these through systematic
manipulation of Myosin II levels via depletion of MRCK-1.
In a complementary approach, we are using agent-based simulations of cross-linked
actomyosin networks to explore microscale determinants of cortical flow. We find that
simulations benchmarked against in vitro assays for actomyosin interactions and constrained by
experimental observations of actin and myosin density can reproduce the patterns of flow we
observe during maintenance, and we are now using these models to systematically explore how
macroscopic tension depends on interplay between myosin density, actin density and actin
turnover.
2887
Ca2+-decoupled contraction of single rat adult cardiomyocytes induced by microscopic
heat pulse.
K. Oyama1, A. Mizuno1, S. A. Shintani1, H. Itoh1, T. Serizawa1, N. Fukuda2, S. Ishiwata3,4, M.
Suzuki3,4; 1Department of Physics, Faculty of Science and Engineering, Waseda University,
Tokyo, Japan, 2Department of Cell Physiology, The Jikei University School of Medicine, Tokyo,
Japan, 3Organization for University Research Initiatives, Waseda University, Tokyo, Japan,
4
Waseda Bioscience Research Institute in Singapore, Waseda University, Singapore, Singapore
Laser irradiation is recently demonstrated as an effective method to stimulate moving tissues
such as heart muscles. Such a non-contact method can take effect without interrupting the
mechanical functions of a heart, but the mechanism that induces contraction of cardiomyocytes
is still under discussion. This is mainly because the laser irradiation triggers various reactions
such as photochemical and photothermal effects. Among these effects, here we focused on the
MONDAY-LATE POSTER PRESENTATIONS
temperature change which can alter physiological functions of biological systems. We generated
a microscopic heat pulse (ΔT < ~13°C for 0.2~0.5 sec) by focusing an infrared laser light under
an optical microscope, and we found that the microscopic heat pulse induces contraction of
single rat adult cardiomyocytes. Heat pulses applied at 2.5 Hz could induce repetitive
contractions as a heat-induced oscillation of 2.5 Hz. At 36°C, smaller ΔT can induce
contractions than at 25°C. Contrary to a usual muscle contraction, Ca2+ transients were not
detected by means of Ca2+ indicators during the contraction. We further confirmed that the
contraction was also inducible in skinned cardiomyocytes in Ca2+-free condition. These results
indicate that a heat pulse regulates the contraction without the involvement of Ca2+ dynamics;
i.e., the microheating stimulation can skip several steps relating to Ca2+ signaling in the
excitation-contraction coupling of muscle, and directly activates actomyosin interaction. This
non-invasive method has the potential to stimulate the beating of failing hearts without
exacerbating abnormal Ca2+ dynamics.
2888
Single molecule measurements of TCR:pMHC binding kinetics in living primary T cells.
R. M. Pielak1,2, G. P. O'Donoghue1,2, A. A. Smoligovets1,2, J. J. Lin1,2, J. T. Groves1,2;
1
Department of Chemistry, University of California, Berkeley, Berkeley, CA, 2Physical
Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA
The central question in immunology is how T cells distinguish self from foreign antigens. At the
center of this question is the T cell receptor (TCR), which is constantly challenged with the
daunting task of recognizing foreign antigens among countless self-peptides presented by MHC
molecules. We use live primary T cells and supported lipid bilayers (SLB) to investigate the
mechanism of TCR triggering and subsequent T cell activation. Using TIRF microscopy and
multi-timescale imaging strategy, individual molecules were tracked with temporal resolution
spanning from milliseconds to minutes. These observations allow direct readout of pMHC:TCR
binding kinetics in situ and are being used to map the physical signature of antigen.
2889
A Minimal Theoretical Model of the E. Coli Bacterium in Exponential Phase Growth.
A. Maitra1, K. Dill1;
1
Laufer Center for Phys. & Quant. Biol., Stony Brook University, Stony Brook, NY
We study the fundamental process of exponential cell growth in the E. Coli bacterium under
conditions of extracellular glucose limitation using a minimal model by accounting for energy
metabolism and protein synthesis. The cell model has three nodes: metabolites, ribosomal
proteins and non-ribosomal proteins. Their interdependencies and dynamics are wrapped in a
system of ordinary differential equations. The formulations for the fluxes capture the essence of
cellular physiology under various conditions of growth. Specifically, there are three interweaving
circuits. The synthesis of non-ribosomal proteins requires ribosomal proteins, ATP production
requires metabolic enzymes (a subset of non-ribosomal proteins) and ribosome synthesis
occurs via autocatalysis through ATP consumption. We simulate glucose uptake by interfacing
the model cell under varied extracellular glucose concentrations.
We solve the model numerically for different glucose concentrations, and, where possible,
explore the cell states analytically under steady state conditions. We verify the model
predictions with available experimental data. The model lets us quantify the coupling between
energy generation and biomass growth. An implication of this model is that it lets us compute
the fitness landscape in terms of the parameters of the cells, such as the protein translation
rates, to make hypotheses about possible routes the cell could evolve under glucose limitation.
MONDAY-LATE POSTER PRESENTATIONS
2890
Remote, In-Plane Mechanosensing by Cells on Floating Collagen Gels.
H. Mohammadi1, P. A. Janmey2, C. A. McCulloch1; 1University of Toronto, Toronto, ON,
Canada, 2University of Pennsylvania, Philadelphia, PA
The mechanical properties of the extracellular matrix impact many cellular functions but little is
known about the contribution of matrix deformations to cellular mechanosensing that extends
beyond the immediate cell-matrix interface. We examined remote mechanosensing by
developing a cell culture model that employs collagen gels circumferentially supported by nylon
mesh frames that float on culture medium. This approach obviates mechanical interference from
the underlying rigid foundation of tissue culture plastic and enables assessment of remote, inplane mechanosensing. With this model we found that 3T3 cells rapidly formed cellular
processes whose lengths and number per cell depended on the frame opening size. Cells that
did not express ß1 integrin or the focal adhesion kinase, or 3T3 cells treated with blebbistain,
did not form cell processes. Knockdown of filamin A did not affect process formation. When the
opening sizes were increased (from 200 µm to 1700 µm widths) mean cell extension length,
mean number of extensions per cell, and the sum of cell extension lengths significantly
decreased. Strain fields generated by cells were measured by displacement of fluorescent
beads embedded in collagen gels. Cells sensed the presence of nylon frames when cellgenerated strain fields in the matrix reached the frame boundaries. This new model
demonstrates the ability of cells to sense remotely, variations of matrix stiffness in the absence
of a rigid, underlying substrate.
TUESDAY-LATE POSTER PRESENTATIONS
TUESDAY, DECEMBER 18
Cell-Matrix and Cell-Cell Interactions
2891
Effect of OPN Silencing on ECM Mineralization of Osteoblasts Grown on a
Nanostructured Ti Surface.
P. L. Adachi1, F. S. Oliveira1, M. M. Beloti1, R. D. Coletta2, A. L. Rosa1, P. T. Oliveira1; 1Cell
Culture Laboratory, School of Dentistry of Ribeirao Preto, University of Sao Paulo, Ribeirao
Preto, Brazil, 2Department of Oral Diagnosis, Piracicaba Dental School, University of Campinas,
Piracicaba, Brazil
Molecular and cellular interactions between biomaterials and biological environments are
affected by the nature of the biomaterial, such as its surface chemistry/energy and topography.
Current strategies in dental implantology to accelerate/enhance osteogenesis adjacent to
implants include the modification of implant surface topography at the nano-scale level, which
may affect the expression of major proteins found at the bone-biomaterial interface, such as
osteopontin (OPN). OPN is a multifunctional protein that regulates cell adhesion and
extracellular matrix (ECM) mineralization. Initial studies demonstrated that nanotextured
titanium (Ti) surfaces enhance cytoplasmic and extracellular OPN immunoreactivity at early
periods of osteogenic cell cultures. Besides, these cultures showed accelerated and/or
enhanced bone-like nodule formation. To verify whether there is a correspondence between
these two findings we aimed to evaluate the effect of OPN gene silencing on the ECM
mineralization by cells grown on nanotextured Ti. Ti discs were chemically treated by a mixture
of H2SO4/H2O2 for 4 h. Non-etched machined Ti discs were used as control. Primary osteogenic
cells derived from newborn rat calvarial bone were plated on both Ti surfaces. On day 2, cells
were transfected with either a scrambled (negative control group, C1) or a specific sequence of
siRNA for OPN (siRNA group). Non-transfected cells were also cultured (C2). OPN silencing
was confirmed by Real Time PCR at day 4. Bone-like nodule formation was assessed at day 14
by Alizarin Red (AR) staining. Cells grown on nanotextured Ti showed enhanced mineralization
when compared to machined surfaces (Tukey test, p < 0.05). However, there were no
significant differences between siRNA and control groups. Therefore, silencing of OPN gene
expression at the initial period of osteogenic cell cultures appears not to affect ECM
mineralization on either nanotextured Ti or machined Ti. Further studies should also address the
influence of later OPN expression on ECM mineralization under our in vitro conditions.
2892
WFDC/PS20 affects prostatic endothelial cell behavior by modulating uPA.
S. Pimentel1, G. Barbosa1, H. Carvalho1; 1Unicamp, Campinas, Brazil
It has been shown that prostatic regression following androgen deprivation by castration is
largely dependent on the behavior of endothelial cells (EC). The understanding of the
interactions among different cell types regulating angiogenesis and blood vessel physiology in
the prostate, as well as their probable regulation by androgens, is important due to the use of
castration in prostate cancer therapy. One such factor is the product of the gene WFDC1/ps20
(prostatic stroma 20 KDa protein), which is known to be secreted by smooth muscle cells
regulating both epithelial cell behavior as well as angiogenesis in xenograft models. To
approach this, we tested the effects of smooth muscle cell (SMC) conditioned medium in the
prostatic endothelial cells differentiation into capillary-like structures on growth factor-reduced
TUESDAY-LATE POSTER PRESENTATIONS
Matrigel substrate. The results showed that the SMC conditioned medium stimulated endothelial
cell differentiation. We further analyzed the expression of urokinase-type plasminogen activator
(uPA) in endothelial cells and the behavior of the cytoskeleton in these cells growing on different
substrates. The activity of uPA in the EC conditioned medium after treatment with peptide 8 (a
synthetic peptide derived from ps20), was higher when cells where cultured on matrigel, as
compared with type I collagen and plastic. In these cells, nuclear staining was observed for uPA
24 hours after treatment while an intense cytoplasmic staining was observed at 72 hours. The
uPA system is believed to play a key role in tissue degradation, cell migration, angiogenesis,
cancer invasion and metastasis. These findings suggest that ps20 and uPA are part of a
mechanism affecting endothelial cell behavior controlling angiogenesis in the rat ventral prostate
that seems important to coordinate gland activity and androgen levels, ultimately adapting the
gland to reproductive behavior. Financial support from FAPESP, CNPq and CAPES.
2893
Mutation in OA/Gpnmb Inhibits Bone Formation in vivo and Osteoblast Differentiation in
vitro.
S. Abdelmagid1, F. Moussa1, S. Lababidi1, G. Sondag1, J. Belcher2, H. Stinnett1, F. Safadi1;
1
Anatomy and Neurobiology, Northeast Ohio Medical University, Rootstown, OH, 2Temple
University School of Medicine, Philidelphia, PA
We have previously identified osteoactivin (OA/Gpnmb) as an osteogenic factor with high
expression in long bones and calvaria. OA/gpnmb stimulates osteoblast differentiation and
matrix mineralization in vitro. To help us better understand the importance of OA/Gpnmb in
bone formation and during skeletal modeling, we studied OA/Gpnmb mutant mice (DBA/2J),
with nonsense mutation on OA gene that produce 150 a.a truncated OA/Gpnmb protein. Bone
micro-CT (μCT) analysis showed decreased bone volume, trabecular number, trabecular
thickness and increased trabecular space in DBA/2J compared to wild-type (DBA) male 8week-old mice. Serum ELISA showed decreased bone formation markers; bone alkaline
phosphatase (BALP) and osteocalcin (OC) in DBA/2J compared to DBA mice. Static and
dynamic histomorphometric analyses showed decreased bone area and bone perimeter,
osteoid maturation time, bone formation and mineral apposition rates in DBA/2J compared to
DBA mice. However, trabecular osteoblasts were increased in numbers and surface in DBA/2J
compared to DBA mice. In addition, adipocytes numbers were also increased in DBA/2J bone
marrow, compared to DBA. Next, we examined proliferation, survival and differentiation of
calvarial osteoblasts isolated from DBA/2J and DBA ex vivo. Proliferation of primary osteoblasts
determined by fluorescent-labeled-DNA and propedium iodide (PI) flow cytometry was
decreased in DBA/2J compared to DBA. In contrast, serum starvation of primary osteoblasts
showed no differences in cell survival between DBA/2J and DBA, evaluated by formazen
crystals, annexin V and PI flow cytometry. Next, we examined primary osteoblast differentiation
(determined by ALP staining and activity) and matrix mineralization (determined by von Kossa
and alizarin red staining) from DBA/2J and DBA mice. At day 14 in culture, ALP staining and
activity was decreased in DBA/2J compared to DBA cells. Moreover, at day 21 in culture,
number and size of mineralized nodules and total calcium measured in matrix, or culture lysate
were also decreased in DBA/2J compared to DBA. Quantitative PCR analyses of osteoblast
differentiation markers ALP, collagen type I, and OC were significantly less in DBA/2J compared
to DBA. Given that DBA/2J osteoblasts express truncated OA, we were interested to examine
whether this truncated protein leads to ER stress and toxicity. Immuno-fluorescent peri-nuclear
expression of OA/Gpnmb in DBA/2J cells was detected and co-localized with rough ER and
Golgi markers when compared to eccentric signals in DBA osteoblasts. Furthermore, ER stressinduced cell toxicity determined by Caspase-12 activity showed no significant difference
between DBA/2J and DBA osteoblasts. These data was confirmed by transmission electron
TUESDAY-LATE POSTER PRESENTATIONS
microscopy. Collectively, these data suggest that osteogenesis is defective in DBA/2J mice due
to osteoblast differentiation and matrix mineralization. Further studies are warranted to
investigate the mechanism(s) of action of OA/Gpnmb during osteoblastogenesis and bone
formation.
2894
Suppressive effects of human lactoferrin on TNF-α-induced vascular inflammation in
human endothelial cells.
T. Lee1, K. Park1, C. Kim1, J. Kim1; 1College of Life Science and Graduate School of
Biotechnology, Kyung Hee University, Yongin-si, Korea
Vascular inflammation requires recruitment of leukocytes and their adhesion to vascular
endothelium. One of the major cell adhesion molecules, Intercellular Adhesion Molecule-1
(ICAM-1) is expressed in endothelium in response to various cytokines. We investigated the
effects of human lactoferrin (Lf) on expression of ICAM-1 by human umbilical vein endothelial
cells (HUVECs) stimulated with TNF-α. Adhesion of human monocytoid U937 cells to Lf-treated
HUVECs were evaluated by co-culture experiments using FITC-labeled U937 cells. Exposure of
endothelial cells to Lf resulted in a significant decrease of ICAM-1 expression. We demonstrated
that Lf down-regulated expression of human ICAM-1 at transcriptional level in ECs. Adhesion of
U937 cells to TNF-α-stimulated endothelial cells was reduced by Lf in a dose-dependent
manner, indicating correlation with down-regulation of ICAM-1. Involvement of NFκB in the
transcriptional control of ICAM-1 protein was assessed by gel electromobility assays, showing
that Lf inhibited binding of NF-κB to ICAM-1 promoter. Furthermore we showed that Lf inhibited
the TNF-α-induced expression of ICAM-1 and inflammatory cytokines in ex vivo and in vivo
animal model. The results suggest that lactoferrin may be useful for prevention of inflammatory
diseases involving up-regulation of cell adhesion molecules.
2895
Knockout and Transgenic Approaches to Study Protocadherins Function in Zebrafish.
H. U. Ortiz-Medina1, S. Miller 1, J. D. Jontes1; 1Neuroscience, The Ohio State University,
Columbus, OH
Protocadherins are the largest subgroup within the cadherin superfamily of calcium-dependent
cell surface receptors. The interaction of these molecules is a crucial step in the development
of the connections in the nervous system, as several protocadherins have been implicated in
neurodevelopmental disorders. However, the perturbations caused by mutations in these genes
remain unknown. Our laboratory uses zebrafish as a model to investigate the roles of
protocadherins in nervous system development. To accomplish these goals, we are pursuing
two, parallel approaches. First, we are using transcription activator-like effector nucleases
(TALENs) to introduce heritable lesions in protocadherin genes of interest. Second, we are
using transposon mediated BAC transgenesis to drive useful transgenes under the control of
protocadherin regulatory elements. Together with in vivo 2-photon imaging, these approaches
will allow us to assess the specific cellular defects resulting from loss of protocadherin function.
Specifically, we are targeting two members of the δ-protocadherin family, pcdh19 and pcdh18b.
Mutations in human pcdh19 result in a female-limited form of infantile-onset epilepsy, while loss
of pcdh18 has been associated with intellectual disability. Thus, our studies in zebrafish could
allow a better understanding of the developmental perturbations that give rise to various
developmental disorders.
TUESDAY-LATE POSTER PRESENTATIONS
2896
ICAM-1-mediated leukocyte adhesion is critical for the activation of endothelial LSP1.
M. Hossain1, Y. Su1, S. M. Qadri1, L. Liu1; 1Pharmacology, University of Saskatchewan,
Sasaktoon, SK, Canada
Leukocyte-endothelial interactions during leukocyte recruitment trigger signaling events in
endothelial cells prior to transendothelial migration of leukocytes. Leukocyte-specific protein 1
(LSP1) expressed in endothelial cells plays a pivotal role in regulating subsequent recruitment
steps following leukocyte adhesion. In neutrophils, LSP1 is activated by phosphorylation of its
serine residues by molecules downstream of p38 mitogen-activated protein kinase (MAPK) and
protein kinase C. Whether leukocyte adhesion on endothelial cells is crucial for endothelial
LSP1 activation remains elusive. In addition, discrepancies prevail on the functions of
endothelial and leukocyte LSP1 in leukocyte adhesion. Using an in vitro assay, we demonstrate
that adhesion of wild-type (Lsp1+/+) neutrophils to LSP1-deficient (Lsp1−/−) endothelial cells
was significantly reduced comparing that to Lsp1+/+ endothelial cells. Immunoblotting revealed
increased phosphorylated endothelial LSP1 in the presence of adherent Lsp1−/− neutrophils
(stimulated by MIP-2/CXCL2, KC/CXCL1, TNF-α or thapsigargin) but not cytokine or chemokine
alone. Pharmacological inhibition of p38 MAPK by SB203580 significantly blunted the
phosphorylation of endothelial LSP1. Similarly, the engagement of endothelial ICAM-1 crosslinking that mimics leukocyte adhesion resulted in phosphorylation of endothelial LSP1. In
neutrophil-depleted Lsp1+/+ mice, administration of ICAM-1 cross-linking antibody resulted in
increased phosphorylation of LSP1 and p38 MAPK in TNF-α-stimulated cremaster muscle. In
conclusion, endothelial LSP1 participates in leukocyte adhesion in vitro, and adhesion of
leukocytes through ICAM-1 fosters the activation of endothelial LSP1, an effect at least partially
mediated by the activation of p38 MAPK. Endothelial LSP1, in contrast to neutrophil LSP1, is
not phosphorylated by cytokine or chemokine stimulation alone.
2897
Effect of Monocyte Adhesion on the Mechanical Properties of Endothelial Cells.
Y. Hwang1, D. Gonzalez-Rodriguez1, A. Babataheri1, C. Hivroz2,3, A. I. Barakat1, J. Husson1;
1
Department of Mechanics, LadHyX, CNRS-École Polytechnique, Palaiseau, France, 2Institut
Curie, Centre de Recherche, Paris, France, 3Inserm, U932, Paris, France
During early development of atherosclerosis, monocytes transmigrate across the endothelium at
abnormally high rates and accumulate in arterial walls. It is not clear why the incidence of
transmigration increases. Our goal is to understand whether biophysical factors are involved in
the regulation of transmigration.
Other groups have used atomic force microscopy to show that shortly after adhesion of
neutrophils to endothelium, the stiffness of endothelial cells is transiently modified before
returning to baseline within minutes. Others have used electric cell-substrate impedance
measurements to show that monocyte adhesion leads to the formation of endothelial cell-tosubstrate gaps.
We hypothesize that similar endothelial stiffness changes occur during the adhesion of
monocytes, and that gap formation translates into a decreased endothelial cell-to-substrate
adhesion energy. In order to test these hypotheses, we develop a micropipette-based device
that enables us to:
(1) gently manipulate a single monocyte, (2) control the timing of its adhesion to an endothelial
monolayer, (3) measure subsequent changes in endothelial cell viscoelastic properties, (4)
correlate mechanical changes with cytoskeletal reorganization and calcium signaling, and (5)
measure the detachment force of an endothelial cell from its substrate in order to measure
endothelial cell-to-substrate adhesion energy.
TUESDAY-LATE POSTER PRESENTATIONS
2898
Localization of Usher 1 proteins to the calycal processes of primate and amphibian, but
not rodent, photoreceptors.
C. Schietroma1,2, I. Sahly1, E. Dufour1, V. Michel2, A. Balhoul2, I. Perfettini2, E. Papermans2, A.
Estivalet1, D. Carette2, A. Aghaie1, I. Ebermann2, A. Lelli2, M. Iribarne2, J-P. Hardelin2, D. Weil2,
J-A. Sahel1,3, A. El-Amraoui2, C. Petit1,2,3,4; 1Syndrome de Usher et autres Atteintes RétinoCochléaires, Institut de la Vision, Paris, France, 2Unité de Génétique et Physiologie de
l’Audition, Institut Pasteur, Paris, France, 3Département de Génétique, Institut de la Vision,
Paris, France, 4Collège de France, Paris, France
The mechanisms underlying retinal dystrophy in Usher syndrome type I (USH1) remain
unknown, primarily because mice lacking any of the USH1 proteins (myosin VIIa, harmonin,
cadherin-23, protocadherin-15, sans) while replicating faithfully the human auditory and
vestibular impairment, do not display retinal degeneration. The absence of an animal model has
delayed the development of gene replacement strategies, which cannot be guided by a correct
understanding of the defective cell function in need to be restored.
We investigated here the reasons for the discrepancy between human pathology and the
phenotype of the mouse mutants, in order to elucidate the roles of USH1 proteins in the human
retina.
We developed specific antibodies against the five USH1 proteins and characterized their
localization in primate, rodent and amphibian retinas, by means of high resolution confocal and
electron microscopy. We found that, in macaque photoreceptor cells, all USH1 proteins
colocalized at membrane interfaces (i) between the inner and outer segments in rods and (ii)
between the microvillus-like calyceal processes and the outer segment basolateral region in
rods and cones. This pattern, conserved in humans and frogs, was mediated by the formation of
an USH1 protein network, which was associated with the calyceal processes from the early
embryonic stages of outer segment growth onwards. By contrast, mouse photoreceptors lacked
calyceal processes and had no USH1 proteins at the inner–outer segment interface. We
suggest that USH1 proteins form an adhesion belt around the basolateral region of the
photoreceptor outer segment in humans, and that defects in this structure probably cause the
retinal degeneration in USH1 patients.
2899
Regulation of vascular integrity by mechanosensing at endothelial cell-cell junctions.
S. Huveneers1,2, J. Oldenburg2, J. de Rooij2; 1Molecular Cell Biology, Sanquin Research and
Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, Netherlands,
2
Hubrecht Institute, Utrecht, Netherlands
Destabilization of the VE-cadherin complex, the central component of endothelial cell-cell
junctions, is a key event of vascular permeability induction in inflammatory responses.
Permeability-inducing factors, transmigrating leukocytes and disturbed blood flow alter local
mechanical forces at the vascular wall that impinge on the VE-cadherin complex. Such forces
are able to switch stable endothelial cell-cell junctions into dynamic junctions. Our goal is to find
the mechanotransduction mechanisms that explain the translation of mechanical force into
regulation of endothelial cell-cell junctions.
We recently demonstrated that force-induced cell-cell junction destabilization takes place at a
molecularly and phenotypically distinct subset of VE-cadherin adhesions, that we named Focal
Adherens Junctions (FAJ). In this study (JCB 196(5), 641-652, 2012) we showed that FAJs are
attached to radial F-actin bundles that generate pulling forces at the VE-cadherin complex and
that FAJs are specifically marked by the mechanosensory protein Vinculin. By enhancing
TUESDAY-LATE POSTER PRESENTATIONS
actomyosin contraction inflammatory permeability factors such as thrombin induce the
transformation of stable junctions into FAJs. And live imaging of normal FAJs and Vinculindevoid FAJs revealed that Vinculin protects VE-cadherin junctions from opening too far during
their force-dependent remodeling. These results implicate Vinculin-dependent VE-cadherin
mechanotransduction in endothelial permeability induction during inflammatory responses.
To identify novel components involved in mechanotransduction at FAJs we currently investigate
the regulation of proteins implicated in the association of junctions to the actin cytoskeleton.
These experiments show that recruitment of the actin binding proteins VASP and Zyxin to FAJs
is force dependent like Vinculin. Next, we imaged fluorescently-tagged VASP and Zyxin in αcatenin mutant cells that are unable to recruit Vinculin to cell-cell junctions, and we find that the
force-dependent localization of VASP and Zyxin occurs independently of Vinculin. These results
suggests that multiple mechanosensory mechanisms occur at VE-cadherin-based junctions. We
currently aim to reveal the contributions of these apparently independent mechanisms to forcedependent cell-cell junction remodeling, and intend to elucidate the functional consequences of
Vinculin recruitment to endothelial cell-cell junctions in vascular integrity.
2900
N-cadherin homotypic binding regulates the cell surface localization of neuronal
nicotinic acetylcholine receptors through RhoA and ROCK.
J. L. Bruses1;
1
Anatomy and Cell Biology, University of Kansas School of Medicine, Kansas City, KS
N-cadherin is expressed at a variety of synaptic contacts in both the central and peripheral
nervous system where it forms adherens-like junctions commonly localized adjacent to the
active zone and the postsynaptic density (PSD). To examine whether N-cadherin homotypic
binding regulates the distribution of neuronal nicotinic acetylcholine receptors (nAChR) on the
cell membrane, Chinese Hamster Ovary (CHO) K1 cells (which do not expressed type I
cadherins) were transfected with N-cadherin C-terminally fused to EGFP, and with α3 and β4
nAChR subunits C-terminally fused to a myc-tag epitope which localizes at the cell surface.
nAChR expressed at the cell surface were labeled with anti-myc antibodies. The expression
levels of nAChR at cell-cell contacts were analyzed in confocal images by measuring nAChR
pixel area within the cell contact and normalized to receptor density on contact-free cell
membranes. nAChR were found evenly distributed on the surface of cells expressing
membrane-bound EGFP and the level of nAChR expression at contacting membranes was
considered 100% (100 ± 16.3%). In contrast, N-cadherin-mediated cell contacts were devoid of
nAChR (3.8 ± 2.2%). Deletion of the β-catenin binding domain increased the expression levels
of nAChR within the N-cadherin-mediated cell contact (78.3 ± 13.0 %), while nAChR expression
levels were similar to controls (102.2 ± 15.2%) in cell contacts mediated by N-cadherin lacking
the entire cytoplasmic domain. Treatment with inhibitors of actin polymerization (latrunculin A
[10µM] and cytochalasin D [2µM]) did not affect nAChR expression within the N-cadherinmediated contact area, which remained devoid of nAChR (3.8 ± 2.7% and 8.2 ± 2.7%
respectively). In contrast, treatment with a cell permeable RhoA inhibitor (C3-transferase
[1µg/ml]) promote some expression of the nAChR within the N-cadherin-mediated contact (34.9
± 7.1%), while higher expression levels were observed in cells treated with the ROCK inhibitor
Y27632 [10µM] (81.5 ± 23.4%). These results indicate that N-cadherin homotypic binding
regulates the cell surface localization of nAChR through a mechanism that involves N-cadherin
cytoplasmic domain and requires the activity of RhoA and its downstream effector ROCK. The
results suggest that N-cadherin-mediated junctions localized at the synaptic complex may
regulate the localization of nAChR at the PSD or may promote the displacement of the
receptors to extrasynaptic cell membranes of the pre and postsynaptic cells.
TUESDAY-LATE POSTER PRESENTATIONS
2901
CD44 required for neuregulin-dependent axonal sorting and nonmyelinating Schwann
cell differentiation.
S. Miller1, J. McFerrin2, F. Banine3, W. Su3, L. Sherman3, B. L. Patton2; 1Stanford University,
Palo Alto, CA, 2C.R.O.E.T., Oregon Health & Science University, Portland, OR, 3Oregon
National Primate Research Center, Oregon Health & Science University, Portland, OR
How developing Schwann cells (SC) adopt myelinating (mSC) and nonmyelinating (nmSC)
phenotypes properly matched to local axonal composition is not well understood. Signaling from
neuronal neuregulin-1 (Nrg1) through SC ErbB2/3 receptors is implicated in regulating
phenotypic differentiation, as well as the mature extent of axon ensheathment of both
myelinated axons and unmyelinated axons. Previously, in primary cultures we showed SC
ErbB2/3 receptor complexes include the surface receptor CD44 and that CD44 knockdown
inhibited Nrg-dependent receptor activation. Here, we show CD44: is unique in marking onset of
nmSC differentiation; is required by nmSC throughout the PNS to match process arborization to
their local complement of unmyelinated axons; and particularly required in mixed nerves to set
the SC phenotype boundary between small unmyelinated and similarly small myelinated axons.
By immunostaining, CD44 was absent from immature SC in embryonic mouse and rat nerves
and from myelinating SC in postnatal and adult nerve. In mice, CD44 was first detected at low
levels at P3-P5, long after E18 onset of axonal sorting, specifically in activated Oct6(+) SC
along peripherin-rich axons destined for nonmyelinated Remak fibers. CD44 levels sharply
increased after P5 specifically on mature nonmyelinating SC (L1, GFAP, p75LNTR +ve) and
prior to integrin a1 on NMSC (after P14). Thus, NMSC differentiate from immature SC through a
first CD44+ stage and a later integrin a1+ maturation. At high resolution, CD44 was
concentrated in an array of clustered domains distributed throughout the Remak bundle
processes, polarized to adaxonal surfaces (not basal lamina) and distinct from p75LNTR
clusters. Adult mice lacking CD44 had limb posture defects. Large axons in CD44null sciatic
nerves were fully sorted and properly myelinated, but the NMSC failed to form sufficient
processes to separate and cover unmyelinated axons; 86% of Remak axons were incompletely
ensheathed in mutants, irrespective of bundle size and without deficit in NMSC number. This
defect mirrors those from loss of Nrg1, ErbB, and associated factors. In contrast to sciatic nerve,
CD44null vagus and sympathetic trunk nerves had significantly fewer myelinated fibers (71%
and 13% of WT values, respectively), and myelinating SC nuclei (70% and 14%). Particularly
absent was myelination of axons less than 1.2 microns, which comprised 40% of all myelinated
fibers in WT control but 10% in nulls, thereby accounting entirely for total myelinated fiber
deficits. It has been unclear how ErbB activation is distinguished for mature SC differentiation.
These results suggest CD44 is required to activate both myelinating and nonmyelinating ErbB
responses to low levels of Nrg1 in Oct6(+) activated SC and mature NMSC.
2902
A conserved site and low N-glycosylation potential are associated with the regulation of
the discoidin domain receptor family by glucose-sensitive N-glycosylation.
T. Phan1,2, E. Wong1,2, X. Sun1,2, K. Kim1, S. Jung1, C. Yoon1, B-S. Yang1,2; 1Korea Institute of
Science and Technology, Seoul, Korea, 2University of Science and Technology, Daejon, Korea
A growing body of evidence shows that N-glycosylation regulates cell surface receptor tyrosine
kinases. For discoidin domain receptor 2 (DDR2) expressed in HEK293 cells, we detected at
least 4 isoforms with distinct molecular weights during the N-glycosylation process, where a
higher molecular weight corresponded to a more complex N-glycan. Similarly, DDR1 showed at
least 3 isoforms. Of the isoforms, only the largest molecular weight form could interact with
collagen for autophosphorylation. The molecular weights of the largest DDR1 and DDR2
TUESDAY-LATE POSTER PRESENTATIONS
isoforms were reduced sensitively when the cells were cultured in glucose-depleted conditions,
whereas that of EGFR did not show any significant changes under the same conditions. In
silico analysis of the N-glycosylation potential among N-glycosylation sites by using artificial
neural networks predicted that the DDR family has distinctively low N-glycosylation potential
while that of EGFR is more than moderate. Site-directed mutational analysis of asparagine to
glutamine in the 5 N-glycosylation sequences of DDR2 revealed that mutation of asparagine
213 alone inhibited the generation of the isoform containing a complex N-glycan, suggesting
that N-glycosylation proceeds in a controlled and orderly manner among the N-glycosylation
sites. Mutation of asparagine 211 in DDR1, the homolog of asparagine 213 in DDR2, yielded
the same result. Taken together, these results suggest that the DDR family is subject to
regulation by a glucose-sensitive and orderly N-glycosylation process, with which its conserved
site and low potential for N-glycosylation are associated.
2903
Ceruloplasmin Binds and Inactivates Matrix Metalloproteinase-2.
M. W. Thompson1; 1Division of Natural Sciences and Mathematics, Jefferson Community &
Technical College, Louisville, KY
An interaction between the iron-binding protein lactoferrin and matrix metalloproteinase-2
(MMP-2) suggests that MMP-2 may interact with other metal binding proteins. To examine this
possibility, the copper binding protein ceruloplasmin was examined to determine its effect on
MMP-2 and pro-MMP-2. Apoceruloplasmin, and to a lesser extent, holoceruloplasmin,
physically interacted with pro-MMP-2 in an immunoprecipitation assay. Furthermore,
apoceruloplasmin inhibited the protease activity of active MMP-2, suggesting that ceruloplasmin
may regulate the activity of MMP-2 in vivo.
2904
Remodeling of adherens junctions in response to tension force during Drosophila
gastrulation.
M. Weng1, E. Wieschaus1; 1Princeton University, Princeton, NJ
Cell-cell junctions, especially Cadherin-based adherens junctions, are known to mediate
Intercellular tension forces which are important for many tissue morphogenic events. However,
the possibility that adherens junctions might also actively respond to tension has only recently
been supported by experiments using tissue culture systems. We use gastrulating Drosophila
embryos as the in vivo system to explore the role of mechanical forces in remodeling adherens
junctions. In this system, adherens junctions are initially formed at a subapical position and
show an apical-basally elongated morphology. Once myosin-mediated apical surface tension is
induced to drive the invagination of mesodermal cells, adherens junctions become concentrated
at the most apical edge of the lateral membrane. We show that morphological change in
adherens junctions temporally correlates with myosin activity that drives cell shape changes and
the maintenance of the spot-like apical adherens junctions is dependent on myosin. Live
imaging show that the remodeling of adherens junctions happens in two phases. During late
cellularization, the domain of adherens junctions remains elongated while its most apical edge
gradually move towards the apical surface. Upon the constriction of the apical surface,
junctional domain rapidly shrinks from the basal edge and become apically concentrated. Such
rapid change in apical-basal distribution immediately follows the onset of apical myosin
contraction and temporally correlates with the reduction in cell apical surfaces. To examine the
specific role of myosin-mediated tension, myosin regulatory light chain (spaghetti squash or
sqh) is knocked down by RNAi. In sqh RNAi embryos, myosin activity is depleted and
mesodermal cell invagination is abolished. Meanwhile, adherens junctions in mesodermal cells
TUESDAY-LATE POSTER PRESENTATIONS
are specifically weakened and diffusely localized around the apical surface. They can not
concentrate into a spot and quickly disappear during gastrulation, suggesting the apical spot-like
adherens junctions that connect super-cellular myosin network during mesodermal cell
invagination depend on myosin activity.
2905
Tight Junction-based construction of the apical structure.
T. Yano1, M. Ito2, T. Matsui3, A. Tamura1, M. Uji1, Y. Yamazaki1, S. Tsukita1; 1Osaka university,
Osaka, Japan, 2Dokkyo University, Tochigi, Japan, 3Kyoto University, Kyoto, Japan
Epithelial cell sheets cover the outer and inner surfaces of every compartment in the body, at
every level, from tiny blood vessels to the entire body surface, and at all of these levels, they
function as a permselective barrier. The paracellular barrier, which is established between
epithelial cells by the formation of tight junctions (TJs) in combination with adherens junctions,
has received considerable attention lately, because of new findings about the claudin-family
proteins and related tight-junctional proteins. Current evidence indicates that actin filaments are
associated with TJs and that the apical cytoskeletal network is well developed, with microvilli in
some cases. Here, we show, by advanced immunofluorescence microscopy, that the apical cell
cortex consists of a layered structure of actin filaments and microtubules. We identified a
microtubule-binding protein that was integrated with TJs (TJ-MAP), by using a gel overlay
method to examine TJ-enriched fractions. Suppression of TJ-MAP expression by RNAi
disturbed the arrangement of the microtubule network. We next measured the transepithelial
electrical resistance (TER), an index of TJ integrity, and found that the TER was decreased in
the TJ-MAP-knockdown cells. In 3 dimensional culture, TJ-MAP-knockdown cells formed ductlike structure. These findings suggest that the apical microtubular network is associated with
TJs, and thus may play a key role in regulating the TJs f cell-cell adhesion/barrier function and
epithelial morphology.
2906
Changes in Cardiac Formin Expression Modify Connexin43 Gap Junctions.
C. Maulion1, C. Vasquez1, G. Morley1, K. Maass1; 1NYU School of Medicine, New York, NY
Dynamic changes of the actin cytoskeleton are instrumental in morphogenetic processes
including changes in cell shape and adhesion. Formin proteins regulate actin microfilament
assembly and can specifically influence adherens junction formation. Previous studies in our lab
have demonstrated remarkable plasticity of formin isoforms during heart development and in
vitro cardiomyocyte differentiation. As gap junction stability is dependent on the presences of
mechanical junctions we were interested if modulation of cardiac formins influences expression
of Cx43 protein and gap junction function. Objective: To investigate the effect of cardiac formin
knockdown (KD) on cell-cell contact formation and functional coupling of cardiomyocytes.
Methods: Cardiomyocytes were isolated from neonatal rat hearts (NRCM) and cultured as
monolayers (d0); NRCM were treated with transfection agent only (TF), control siRNA (Ctr) or
formin specific rat siRNAs (Daam1; Fhod1; Fhod3; Dharmacon) (d1); cultures were subjected to
high resolution optical mapping or processed for immunofluorescence analysis (d4). Results:
KD of Fhod1 or Fhod3 lead to disruption of sarcomers, cell rounding and ultimately resulted in
complete dissociation of NRCM. In contrast, Daam1 KD resulted in significant cell elongation
without loss of cell-cell contacts (mean cell areas in μm2: 681.8 ± 99.1 (Daam1) vs. 594.9 ± 67.6
(TF), 564 ± 53.3 (Ctr), 455.9 ± 47.4 (Fhod1), 339.3 ± 14.3 (Fhod3); P: 0.01, ANOVA). As
expected, optical mapping data for discontinuous Fhod1 and Fhod3 monolayers were very
variable due to areas of complete block of conduction. Optical mapping analysis of Daam1
silenced NRCM demonstrated significant increase in conduction velocity (0.241 ± 0.004 m/s;
TUESDAY-LATE POSTER PRESENTATIONS
n=4) compared to NRCM treated with TF only (0.197 ± 0.010 m/s, n=3) or Ctr (0.207 ± 0.005
m/s, n=3; P: 0.003, ANOVA). Average gap junction diameter (0.24 ± 0.03 µm (n=489; Daam1)
vs. 0.41 ± 0.03 µm (n=550; DF), 0.38 ± 0.04 µm (n=574; Ctr) P: 0.009, ANOVA), and total gap
junction area per cardiomyocyte (0.79 ± 0.22% (Daam1) vs. 1.38 ± 0.16% (DF), 2.79 ± 0.66%
(Ctr); P: 0.009, ANOVA) were significantly decreased in Daam1 KD compared to controls.
Conclusion: Changes in cardiac formins influence cardiomyocyte morphology, gap junction
size and intercellular communication. Targeting formin expression or activity might very well
improve maturity of in vitro differentiated cardiomyocytes and engraftment properties.
2907
Loss of Claudins 2 and 15 from Mice Causes Defects in Paracellular Na+ Flow and
Nutrient Transport in Lumen and Leads to Death from Malnutrition.
M. Wada1, A. Tamura1, N. Takahashi1, S. Tsukita1; 1Graduate School of Medicine, Osaka
University, Osaka, Japan
The intestinal symport system for moving nutrients across membranes via transporters, and is
required for absorption of major nutrients such as glucose, amino acids, and bile acids (which
are required for fat absorption). Most of these transporters are regulated by Na+, but the
standard diet does not provide sufficient levels of this ion to the intestinal lumen to support this
system. Claudins form paracellular barriers between epithelial cells, and claudin-2 and -15
regulate paracellular ion flow in the intestine. We investigated how cell adherence, tight junction
barriers, and claudins regulate the supply of Na+ to the intestinal lumen in mice. We created
Cldn2-/-Cldn15-/- (double knockout) mice and analyzed intestinal tissues by reverse
transcription PCR, immunoblot, immunofluorescence, electron microscopy, and hematoxylin
and eosin analyses. We also measured paracellular Na+ flow, the luminal Na+ concentration,
and the absorption of glucose, amino acids, and fats, which were orally administered to the
mice. Paracellular flow of Na+ from the intestinal submucosa to the lumen, and therefore the
concentration of Na+ in the lumen, was greatly reduced in intestines of Cldn2-/-Cldn15-/- mice.
Absorption of glucose, amino acids, and fats also decreased in the mice, which died by
postnatal day 25 from malnutrition. The paracellular flow of Na+ from the intestinal submucosa
is regulated by tight junctions that contain claudin-2 and -15. This system is required for
absorption of glucose, amino acids, and fats; disruption of this system in mice leads to infant
death due to malabsorption.
2908
A Genome-Wide shRNA Screen Identifies Novel Gene Targets for Increased Survival of
Placenta-Derived Adherent Cells in Low Attachment.
V. Voskinarian-Berse1, A. Persak1, R. Youngblood2, K. Sosa3, M. O’Kane1, T. Brieva1, G.
Russotti1, S. Abbot1, R. Hariri1, X. Zhang 1; 1Celgene Cellular Therapeutics, Warren, NJ,
2
Princeton University, Princeton, NJ, 3University of the Sciences, Philadelphia, PA
Placenta-derived adherent cells (PDAC) are a novel culture-expanded cell population derived
from human placental tissues obtained at birth following normal full-term pregnancy. PDAC are
plastic-adherent during culture expansion and are currently being developed as a novel
therapeutic agent to treat chronic inflammatory disorders. In this study we evaluated PDAC
survival in environments that may fail to provide sufficient anchorage support, such as may be
encountered in circulation post systemic administration, and identified several genes/pathways
underlying PDAC interactions with extracellular matrix.
A medium-throughput anoikis assay protocol was established by assessing growth and viability
of PDAC cultured in attachment-resistant cell culture vessels (anoikis condition). Less than 5%
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of PDAC survived after culture for 48 hours in Nunc low cell binding plates, which prevent cell
attachment with a polymer presenting a phosphorylcholine moiety and result in anoikis. Viral
transduction of PDAC with the genome-wide Decode Lentiviral shRNA Screening Libraries
(Thermo Scientific) was performed to identify genes/pathways that could enhance PDAC
survival in the anoikis condition.
A primary screen resulted in identification of 73 Refseq IDs, which were deemed likely to
improve PDAC survival in the anoikis condition. Of 73 putative inhibitors, commercially available
siRNAs were identified for 51 fully annotated genes and applied in the secondary confirmation
study. RNA-silencing of selected targets led to significantly increased viability, compared to nontargeting siRNA-treated PDAC in the anoikis assay. Target genes included FH2 domain
containing 1 (FHDC1), guanine nucleotide binding protein alpha inhibiting 2 (GNAI2), kinase
non-catalytic C-lobe domain containing 1 (KNDC1), lysophosphatidic acid receptor 4 (LPAR4),
mitogen-activated protein kinase kinase kinase 5 (MAP3K5), solute carrier family 2, member 3
(SLC2A3), and staufen homolog 2 (STAU2). Stable SLC2A3 knockdown PDAC, established
using lentiviral shRNA vectors, could be propagated in suspension culture in the anoikis
condition while exhibiting normal growth rate with high cell viability and no significant cell
aggregates.
In summary, we have successfully identified and confirmed novel gene targets, the knockdown
of which can result in enhanced PDAC survival under anoikis conditions. This study has
provided more insight into understanding the consequences of specific genetic modification to
growth properties of PDAC. The modified cell lines with anchorage independence may have
improved therapeutic potential and may facilitate large-scale propagation as suspension
cultures; however, further studies will be required to assess potential safety and functional
implications of the modifications.
2909
A novel cell culture surface supports effective formation of 3D cell spheroids in
suspension.
S. M. Carter1, A. D. Sinor-Anderson1, L. Gaarn1, J. Granchelli1, T. K. Marwood1, C. Neeley1;
1
Thermo Fisher Scientific, Rochester, NY
The study of cell biology via monolayer cell culture systems is not entirely adequate for the
investigation of complex inter-cell and cell to extracellular matrix interactions in vitro. Three
dimensional (3D) cell culture systems better mimic more complex interactions and are extremely
useful in broad applications of cell biology. In human cancer biology, a 3D system is better able
to simulate an extracellular microenvironment to study tumor cell progression and sensitivity to
anticancer agents. Additionally, the formation of spheres by stem cells or progenitor cells in
suspension (e.g. embryoid bodies, neurospheres) is widely used for producing different cell
lineages. However, variability in forming spheres has been a persistent problem. This
variability has been linked to medium composition and volume, cell density, duration in culture,
and most importantly, the cellular interactions with the culture dish itself. Here, we hypothesize
that a hydrophilic polymer coating surface inhibits cell attachment to the culture dish by blocking
binding of the extracellular matrix (ECM). First we demonstrate that the adsorption of several
ECM proteins (e.g. Collagen I and Fibronectin) to the hydrophilic polymer is extremely low
compared to the standard cell culture-treated surface, implying minimal interactions between the
hydrophilic polymer surface and the ECM. We then examined sphere formation on the
hydrophilic polymer surface in comparison to a standard cell culture treated surface. We were
able to show that unlike the standard cell culture surface, the hydrophilic polymer surface was
able to support spheroid development in suspension with virtually no cell attachment by several
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cancer cell lines and pluripotent stem cells. In order to show that the hydrophilic polymer coated
surface has no deleterious effects on cell growth, we conducted spheroid culture in these dishes
at relatively low cell seeding densities. Over time, the spheres of all initial seeding densities
were able to grow in volume, indicating that the hydrophilic polymer coating has no adverse
effect on cell survival and proliferation. Overall, the data supports using the hydrophilic polymer
surface for a consistent culture system for non-adherent spheroid growth, aiding in three
dimensional cancer cell modeling and progenitor cell differentiation in vitro.
2910
Mimicking biochemistry and biomechanics of adipose tissue stimulates adipogenesis.
A. Young1, Y. Choi1, A. Engler1, K. Christman1; 1Bioengineering, UC San Diego, La Jolla, CA
Several studies have demonstrated that human adipose-derived adult stem cells (ASCs) will
induce more de novo adipogenesis in vivo when they are pre-differentiated toward an adipocyte
lineage prior to injection. However, the use of chemically-modified media formulations
containing a cocktail of steroids and synthetic inhibitory molecules remains the predominant
method for priming these cells for adipogenic differentiation. Biochemical and biomechanical
extracellular matrix (ECM) cues have recently been shown to play a role in multiple
differentiation pathways, however, they have yet to be investigated for adipogenesis. The
objective of this study was to recapitulate both the composition and mechanics of adipose tissue
in vitro in order to stimulate ASC adipogenesis without the need for synthetic additives. For this
study, ASCs were isolated from lipoaspirate that was collected from patients undergoing routine
lipoplasty. The leftover tissue was then decellularized to obtain adipose-specific ECM proteins
and proteoglycans. Polyacrylamide gels were made with an elastic modulus of 2 kPa, which is
similar to that of adipose tissue, or with stiffer moduli at 20 and 40 kPa, as confirmed by atomic
force microscopy. The gels were functionalized with either collagen or adipose matrix
components derived from the decellularized lipoaspirate and seeded with human ASCs. The
cells were cultured for one week in DMEM:F12 media with only 10% FBS and 1%
penicillin/streptomycin. Within just 24 hours of culture, the cells on 2 kPa gels exhibited a
compact, rounded shape, with cell spreading significantly increasing with increasing substrate
stiffness. After 7 days, these spherical cells on the 2 kPa gels stained positive for small lipid
vacuoles via Oil Red O, as opposed to no staining on the stiffer substrates. PCR analysis
confirmed these results, as gene expression of adipogenic factors peroxisome proliferatoractivated receptor gamma (PPARγ) and a fatty acid binding protein, aP2, were upregulated for
cells cultured on the 2 kPa gels compared to stiffer gels and compared to cells cultured on
tissue culture plastic. Furthermore, this upregulation of adipogenic genes was enhanced when
the gels were functionalized with adipose-specific components compared to collagenfunctionalized gels. These results suggest that in vitro culturing of ASCs on substrates that
mimic both the biochemical and biomechanical properties of adipose tissue may be an effective
way for priming ASCs for adipose formation in vivo, without the need for chemical additives.
2911
Dynamics of cell division and matrix interaction in three dimensions.
A. Lesman1, J. Notbohm2, D. A. Tirrell1, G. Ravichandran2; 1Chemical Engineering, California
Institute of Technology, Pasadena, CA, 2Engineering and Applied Science, California Institute of
Technology, Pasadena, CA
Dynamic reciprocal interactions between cells and the extracellular matrix mediate many
essential cellular processes, but their role in cell division is not yet well understood. Nearly all
previous in vitro studies of cell division have used two-dimensional (2D) flat substrates which do
not capture the three-dimensional (3D) nature of many extracellular matrix environments. Here
TUESDAY-LATE POSTER PRESENTATIONS
we report simultaneous observations of cell division dynamics and traction force microscopy for
cells embedded in a 3D environment. We show that during division in a 3D fibrin matrix,
fibroblasts remain anchored to the matrix at the tips of persistent thin protrusions while the cell
body rounds and disengages from the matrix. We quantitatively map cell-induced matrix
displacements for various stages of division, and we suggest that cells use energy stored in
matrix deformation to facilitate re-spreading of daughter cells along the anchored protrusions.
Membrane blebs are formed during cytokinesis, and may provide a mechanism for prompt reengagement of cell-matrix adhesive contacts. This work highlights the importance of system
dimensionality and cell-matrix interactions for understanding the biophysics of the cell division
process.
2912
Three dimensional polydimethylsiloxane (PDMS) scaffolds for in vitro study of human
fibroblasts.
A. Parillo1, G. Peck1, M. Stanton2,3, W. G. McGimpsey4, C. R. Lambert2,3, R. M. Bellin1; 1Biology,
College of the Holy Cross, Worcester, MA, 2Bioengineering Institute, Worcester Polytechnic
Institute, Worcester, MA, 3Chemistry and Biochemistry, Worcester Polytechnic Institute,
Worcester, MA, 4Chemistry and Biochemistry, Kent State University, Kent, OH
Traditionally, cells have been cultured and studied on flat surfaces. These surfaces do not
replicate in vivo conditions; rather, they only allow cells to grow in two dimensions. We have
created a rough surface with ~ 6 μm features by casting polydimethylsiloxane (PDMS) onto a
well defined, textured surface and characterized it by atomic force microscopy and contact
angle goniometry. The surface was chemically derivatized with an amine terminated silane.
This surface was used as a substrate to culture human fibroblasts acquired from normal
foreskin (American Type Culture Collection, CRL-2097). After two days, the substrates with cells
attached were fixed with paraformaldehyde and antibody tagged for vinculin or fibronectin.
Fibronectin staining was performed both with cells present and after cells had been extracted
from the surface with 2% Triton X-100 to determine the fibronectin arrangement on the PDMS
surfaces.
Significant differences were observed in cellular morphology between rough and flat PDMS
surfaces. Fibroblasts grown on rough surfaces exhibit smaller cross-sectional areas and thicker
Z sections overall. Extracellular matrix (ECM) deposition, as shown by fibronectin labeling, was
observed as flat regions showing the size and shape of cell footprints on flat PDMS in contrast
to smaller ECM islands observed on hills and valleys of the rough PDMS. Focal adhesion
complexes, as represented by vinculin staining, were observed around the perimeter of the cells
on flat PDMS, whereas the rough PDMS cells showed smaller adhesion complexes. Overall,
these findings indicate that fibroblasts grown on this rough PDMS are able to adhere to the
surface in ways that are more consistent with three dimensional growth environments than
typical flat cell culture dishes. This new rough cell growth surface shows potential as an
inexpensive scaffold for in vivo-like growth of cells for use in a wide-range of biomedical
applications.
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Nuclear Structure and Function
2913
Protein Kinase A is part of a mechanism that regulates nuclear re-import of the nuclear
tRNA export receptors Los1p and Msn5p in Saccharomyces cerevisiae.
J. B. Pierce1, G. van der Merwe1, D. Mangroo1; 1Molecular and Cellular Biology, University of
Guelph, Guelph, ON, Canada
The two main signal transduction pathways that allow eukaryotes to sense and respond to
changes in the availability of glucose in the environment are the cAMP/PKA and AMPK/Snf1
kinase-dependent pathways. Previous studies have shown that nuclear export of mature tRNAs
is inhibited in Saccharomyces cerevisiae deprived of glucose, and is thought to be caused by
cytoplasmic retention of the nuclear tRNA export receptors Los1p and Msn5p. Here, we provide
evidence that nuclear retention of tRNA in cells deprived of glucose is due to a block in nuclear
re-import of the nuclear tRNA export receptors. Retention of the receptors in the cytoplasm is
not caused by activation of Snf1p, but by the inactivation of PKA during glucose deprivation.
Regulation of nuclear re-import of the receptors is not due to phosphorylation of the tRNA export
receptors by PKA. However, PKA phosphorylates known components of the tRNA export
pathway in vitro. A model that is consistent with the data is that PKA and an unknown
mechanism regulate the activity of these components or an unidentified protein to control
nuclear re-import of the receptors in reponse to glucose availability.
2914
The Role of Nucleoporins in Baculovirus Nuclear Import
S. Au1, N. Panté1;
1
Univ British Columbia, Vancouver, BC, Canada
Baculovirus is an arthropod-specific, enveloped, rod-shaped virus with circular double-stranded
DNA genome that replicates in the nucleus of its host insect cells. Using electron microscopy we
demonstrated that baculovirus capsids remain fully intact while entering the nucleus through
nuclear pore complexes (NPCs). Here, we sought to delineate the necessity of different cellular
proteins for the nuclear import of this virus. NPCs house numerous proteins that help regulate
nucleocytoplasmic trafficking. Selective transport of large macromolecules involves the
phenylalanine glycine repeat nucleoporins (FG nups). Nup62 and Nup153, positioned within the
central channel and the nuclear basket of NPCs, respectively, are FG nups that commonly act
as binding sites to mediate nuclear import. To dissect the role of Nup62 and Nup153 in the
nuclear import of baculovirus, we used siRNA to transiently deplete these nups in HeLa cells,
followed by infection with the baculovirus Autographa californica multiple nucleopolyhedrovirus
(AcMNPV). Baculovirus capsids were able to enter the nucleus of HeLa cells depleted of Nup62
or Nup153, an event that is similarly observed in non-treated cells. Nuclear import of the
baculovirus capsid was also tested in digitonin-permeabilized cells (previously depleted of
Nup62 or Nup153 by RNAi), instead of infected cells. We first optimized this nuclear import
assay to detect nuclear localization of the AcMNPV capsid under different conditions (with and
without energy, with and without cytosolic components). Using this system, we observed
nuclear accumulation of AcMNPV capsids in HeLa cells in both the presence and absence of
energy and cytosolic components. Experiments with digitonin-permeabilized cells depleted of
Nup62 or Nup153 yielded similar AcMNPV capsid nuclear accumulation compared to cells nontreated with siRNA. Our results suggest that nuclear import of baculovirus AcMNPV follows a
non-classical import pathway, thereby cytosolic components and energy are not required, and
nuclear import of this capsid can occur in the absence of Nup62 and Nup153.
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2915
Reconstruction of the cell nucleus under spatial constraints using microfluidic devices.
Y. Hara1, C. Merten1; 1Genome Biology Unit, European Molecular Biology Laboratory (EMBL),
Heidelberg, Germany
The size of the cell nucleus is changed dynamically during development, especially in
embryogenesis and has been observed to correlate with the cell size over the last 100 years.
However, there are few reports evaluating systematically how the size of the cell affects to set
the nuclear size. We are currently establishing a method that combine the Xenopus laevis egg
cell-free system, which can reconstruct the cell nucleus in vitro, with microfluidic devices to
manipulate the spatial constraints. First, we changed the concentration of the sperm nuclei
which we incubated with the egg extract while keeping the volume of the egg extract and the
dimension of the incubating test tube constant. The maximum (plateau) size of the
reconstructed nuclei correlated negatively with the sperm nuclei concentration, suggesting that
the maximum size of the cell nucleus depends on the amount of components required for
nuclear formation available for each sperm in the extract. Next, using our established method
we incubated the egg extract and the sperm nuclei in narrow and shallow microfluidic channels
without changing the overall extract volume and the concentration of the sperm nuclei. As a
result, the size of the reconstructed nuclei was smaller than that reconstructed in the test tube
(without spatial constraint). These findings suggested that the nuclear size can be controlled by
the available space surrounding the nuclei rather than the overall amount of the components in
the cytoplasm.
2916
Lacandonia granules are present in three species of the division Bryophyta.
C. D. Alonso-Murillo1, L. T. Agredano-Moreno1, R. Lara-Martínez1, L. F. Jimenez-Garcia1; 1Cell
Biology, UNAM, Mexico City, Mexico
Lacandonia granules were first described in the nuclei of the plant Lacandonia schismatica.
They are 32 nm in diameter intranuclear ribonucleoprotein particles. Ultrastructural
characteristics revealed by inmunoelectron microscopy and in situ hybridization for the presence
of SR proteins and poly RNA suggested that they are similar to Balbiani ring granules of the
nuclei of salivary glands of insect larvae. Moreover, the presence of Lacandonia granules in the
nuclei of Gingko biloba, prompted us to search for their presence in other groups of plants as
bryophyte. In this work we analyzed the presence of Lacandonia granules in three species of
the unseeded plants Bryophyta (Anthoceros punctatus, Marchantia polymorpha and
Polytrychum sp. Samples were prepared for electron microscopy either by the standard
technique or by a method for RNPs. Pictures were also taken to generate stereopairs by using a
goniometer coupled to the transmission electron microscope. Results indicate the presence of
Lacandonia granules in the nuclei of the three species. Stereopairs show that Lacandonia
granules are connected to fibrous material surrounding compact chromatin clumps. We
conclude that Lacandonia granules are also present in the division Bryophyta [D. A-M is a
graduate student supported by Posgrado en Ciencias Biológicas-UNAM].
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2917
3-D Chromosome organization and genomic regulation during the cell cycle in WI38 lung
fibroblasts.
A. Fritz1, N. Sehgal1, J. Vecerova1, B. Stojkovic2, J. Xu2, S. Bhattacharya3, R. Berezney1;
1
Biological Sciences, University at Buffalo, Buffalo, NY, 2Computer Science, University at
Buffalo, Buffalo, NY, 3Fayetteville State University, Fayetteville, NC
There is growing evidence that chromosome territories (CTs) have a probabilistic non-random
arrangement within the cell nucleus of mammalian cells. Beyond radial positioning, our lab
previously determined that subsets of human chromosomes have preferred probabilistic
arrangements of interchromsomal associations that are cell-type specific. While it is generally
assumed that 3-D arrangement and the spatial positioning of genes within the CT are linked to
genomic regulation, the degree of non-random organization of individual CT remains unclear
and no overall model has been proposed. As a first step to elucidate the 3-D topology of
individual CT, we performed multi-color fluorescence in situ hybridization (FISH) using 6 probes
extending from one end of each CT to the other in human WI38 lung fibroblasts throughout the
cell cycle. 6 CT were selected ranging in size and gene density (1,4,12,17,18 and X). In-house
computational geometric algorithms were applied to measured the 3-D distances between every
combination of probes and to evaluate data-mined structural patterns. Our findings suggest a
non-random arrangement of individual CT that varies from chromosome to chromosome.
Significant differences in 3-D topology were found in comparing the G1 and S phases of the cell
cycle with the gene-rich CT 1 and 17 displaying greater differences than the gene poor CT 18.
Moreover the overall shape of CT varies widely with gene-rich CT showing a high degree of
irregular shapes while gene-poor CT have more regular ellipsoid-like shapes. We propose that
these differences in structural organization and changes during the cell cycle are related to the
global gene expression programs of the individual chromosomes.
2918
“IMPDH Rod" - a novel nuclear inclusion.
P. Juda1, J. Smigova1, D. Cmarko1, I. Raska1; 1Cellular biology and pathology, First Faculty of
Medicine, Prague, Czech Republic
Inosine-5-monophosphate dehydrogenase (IMPDH) catalyzes the critical, rate-limiting step
in the de novosynthesis of guanine nucleotide, the oxidation of inosine monophosphate to
xanthosine monophosphate. In humans, there are two types of IMPDH: constitutively expressed
IMPDH1 and inducible IMPDH2. Both types of IMPDH proteins are dispersed through the
cytoplasm of the cell. If inhibited by specific inhibitors like ribavirin or mycophenolic acid, IMPDH
forms cytoplasmic structures in cultured cells. According to their shape, these structures are
called “Rings and Rods” (RR). Importantly, inhibition of IMPDH protein is intensively studied,
also because of its clinical importance. IMPDH inhibitors are used as a antiviral drugs and in
immunosupresive therapy in transplantology.
By biochemical approach, IMPDH was recently detected also in the nucleus. Although, it is
believed that IMPDH could be involved in gene transcription, its proper nuclear function is
enigmatic.
We were able to show the nuclear localization of IMPDH as detected by immunofluorescence
and immunoelectron microscopy. Whereas in the nuclei of non-treated cells the fluorescent
signal is low, in the cells treated by IMPDH inhibitors nuclear the rod-shaped structure becomes
clearly visible. Considering ultrastructure, the RR structures correspond to fibrous material
non-surrounded by biological membrane. Even though ultrastructural fibrous appearance of
nuclear Rods is similar to cytoplasmic one, the nuclear Rods are more straight, thinner and less
immunolabeled. By electron microscopy, we also confirmed the presence of the diffuse form of
TUESDAY-LATE POSTER PRESENTATIONS
IMPDH throughout the nucleus except the nucleolus in non-treated as well as treated cells.
All together, we identified a novel nuclear inclusion and described the ultrastructure of
cytoplasmic RR structures and nuclear Rods formed after treatment by IMPDH inhibitors.
2919
Role of Lamin B1 in nucleotide excision repair.
V. B. Butin-Israeli1, S. Adam1, R. Goldman1; 1Cell and Molecular Biology Feinberg School of
Medicine, Northwestern University, Chicago, IL
Alterations in the shape of the nucleus are major pathological hallmarks of the changes in cells
during cancer development and progression. We hypothesize that these alterations in nuclear
architecture are due to changes in the nuclear lamins, which may contribute to tumor
progression. For these studies we have used the MCF7 cell line derived from a mammary gland
carcinoma. Transient silencing of lamin B1 by ~85% using shRNA induced cell cycle arrest
without significant apoptosis. In addition, we detected that transient silencing of lamin B1 in
MCF7 cells leads to significant changes in DNA damage response, chromatin remodeling, gene
expression and cell cycle regulation. Cell viability/apoptosis analyses showed that silenced cells
became highly sensitive to UV irradiation. Further analysis indicated that LB1 silencing altered
expression of genes that participate in DNA damage signaling and repair mechanisms. We
detected transcription arrest of DDB1 and DDB2, factors involved in global nucleotide excision
repair (NER). Decreased expression of these genes affects the repair of UV induced DNA
damage and contributes to increased apoptosis. We concluded that silencing of lamin B1
triggers dramatic changes in gene expression through transcription regulation. Our preliminary
results suggest that LB1 may be take part in chromosomal domain organization and
transcription. Further research of LB1 and nuclear lamins may shed light on the regulation of
global transcription from chromosomal domains and how this may contribute to the tumor
development.
2920
Interaction of Fragile X Mental Retardation Protein (FMRP) with lamin A/C is impaired by
FPLD-causing point mutation R482W.
A. R. Oldenburg1, P. Collas1; 1Stem Cell Epigenetics Laboratory, Institute of Basic Medical
Sciences, University of Oslo, 0317, Norway
Nuclear lamins are essential for integrity and organization of various nuclear functions.
Mutations in the LMNA gene cause degenerative diseases collectively called laminopathies.
Many laminopathy-causing mutations are clustered in the immunoglobulin (Ig) fold of lamin A/C
(LMNA). Among these, the R482W mutation of LMNA causes familial partial lipodystrophy
(FPLD), manifested by loss and redistribution of subcutaneous adipose tissue. We set out to
identify proteins interacting with the Ig fold of LMNA by screening lysates of adipose stem cells
(ASCs) and HEK cells using LMNA peptide arrays covering the Ig fold. Mass spectrometry of
isolated protein complexes identified Fragile X mental retardation syndrome-related protein 1
(FXR1), which together with its homologues FXR2 and FMR forms a small gene family.
Interaction of full length LMNA with FXR1 and FMRP was confirmed by immunoprecipitation. A
fraction of FMRP colocalizes with LMNA at the nuclear periphery in a LMNA-dependent manner:
perinuclear recruitment of FMRP is enhanced by overexpression of LMNA and abrogated by
LMNA knock-down. Perinuclear FMRP recruitment is also abrogated by expression of
LMNA(R482W) or a LMNA mutant lacking the Ig fold. Pull-down of GFP-LMNA confirms weaker
binding of the LMNA mutants to FMRP. Whereas LMNA knock-down or expression of
LMNA(R482W) impairs adipogenic differentiation, FMRP knock-down has no effect. Thus
LMNA-dependent perinuclear targeting of FMRP is not essential for the adipogenic program.
TUESDAY-LATE POSTER PRESENTATIONS
This however may be due to partial functional redundancy between members of the FMRP
family. Lastly, skin fibroblasts from FPLD patients with a LMNA(R482W) mutation show
markedly reduced levels of FMRP, while levels of FXR1 are strongly enhanced. Increased
FXR1 is also detected in HEK cells expressing LMNA(R482W). Immunostaining reveals strongly
enhanced cytoplasmic labeling of FXR1 in FPLD patient cells compared to controls. This could
be recapitulated in HEK cells expressing LMNA(R482W) relative to wild-type LMNA. Our results
indicate a dysregulation of the adipogenic program caused by the LMNA(R482W) FPLD
mutation, and suggest an implication of impaired binding of LMNA to FMRP in preadipocytes.
2921
An Interaction between Telomeres and the Nucleoskeleton Affects Chromosome
Structure.
S. T. Kosak1, A. Wood1, C. Lucas1, E. Smith1, D. Scalzo2; 1Cell and Molecular Biology,
Northwestern University Feinberg School of Medicine, Chicago, IL, 2Fred Hutchinson Cancer
Research Center, Seattle, WA
A causal relationship between cellular aging, or senescence, and organismal aging has not
been established. Cellular senescence is the process by which cells permanently exit the cell
cycle after numerous mitotic divisions, while still maintaining metabolic activity. Several
molecular events can trigger cellular senescence, including the loss of telomeric DNA.
Telomeres are the nucleoprotein complexes that prevent the ends of linear eukaryotic
chromosomes from being degraded or recognized as damaged DNA. On the other hand, a
human model for organismal aging, the segmental progeroid Hutchinson Gilford Progeria
Syndrome (HGPS), is due to a mutation in LMNA, which encodes the A-type lamins. The
nuclear lamina is a proteinacious network underlying the inner nuclear membrane and
dispersed throughout the nucleoplasm. As an integral component of the nucleoskeleton, the
nuclear lamina functions in multiple nuclear activities, including DNA replication, transcription,
and chromatin organization. We present evidence that the basic paradigms of aging, cellular
and organismal, are functionally and physically intertwined in the interaction between the
nucleoskeleton (in the form of lamin A/C) and telomeres (in the form of telomere repeat binding
factor 2, TRF2). Moreover, telomeres end in protective t-loops, facilitated by TRF2 and formed
by the insertion of the 3’ overhang into telomeric DNA. We have two-color fluorescence in situ
hybridization and chromatin immunoprecipitation (ChIP) evidence indicating that t-loops can
form with interstitial telomere repeats (interstitial t-loops, ITL) megabase pairs from telomeres
proper. We demonstrate that lamin A/C is necessary for co-occupancy of interstitial telomere
repeats with TRF2, while loss of lamin A/C and the expression of progerin (the mutant form of
LMNA in HGPS) results in a significant reduction in ITL. We suggest that ITL may be important
in the structure of chromosomes during both mitosis and, with its abrogation, in disease states
as manifested in early-onset aging.
Cell Division II: Mitosis
2922
G1 arrest after prolonged prometaphase is due to partial activation of the apoptosis
pathway.
Y. Uetake1, G. Sluder1; 1University of Massachusetts Medical School, Worcester, MA
If prometaphase lasts 1.5 hr or more in untransformed human cells, all daughter cells
irreversibly arrest in G1 despite normal division of their mothers (Uetake and Sluder, 2010).
TUESDAY-LATE POSTER PRESENTATIONS
What aspect of prolonged prometaphase is sensed by the cell and what triggers the p53, p21
dependent arrest for the daughters has been a mystery. We find that exposing mother cells to
low (50µM) external concentrations of the reducing agent glutathione or their culture under low
oxygen (~3%) conditions allows many, but not all, daughter cells to proliferate after
prometaphase durations lasting >1.5 hr. Knockdown or chemical inhibition of the pro-apoptotic
factor Bax during prolonged (>1.5 hr) prometaphase for the mother cells allows some daughters
to progress through interphase to mitosis. When the anti-apoptotic protein MCL1 is knocked
down, daughter cells arrest in G1 after shorter (<1.5 hr) prolongations of prometaphase in the
mother cells. These observations suggest that mitotic cells are particularly sensitive to oxidative
stress and this stress leads to a partial but not lethal activation of the apoptotic pathway once
the mother cell has completed an otherwise normal mitosis.
2923
Novel Kinesin Motor Kif25 and its Role in Regulating Cell Division.
J. A. Decarreau1, M. Wagenbach1, L. Wordeman1; 1Physiology and Biophysics, University of
Washington, Seattle, WA
Correct orientation of the mitotic spindle during cell division is critically important in development
as well as in the regulation and maintenance of stem cell populations in tissues. Orienting the
spindle in the appropriate plane of division is an active process mediated by membraneanchored proteins and subject to regulation by mitotic kinases. Molecular motors are thought to
act as force generators that both orient and anchor the spindle in the proper plane of division.
We have found that depletion of a little studied kinesin family motor, Kif25, in human cells
causes misorientation of the mitotic spindle and defects in spindle assembly. We have
determined that Kif25 is a C-terminal kinesin motor although the structure and function of this
motor are poorly understood. Kif25 is located at the centrosome of dividing cells, much like
other C-terminal kinesin motors, probably owing to its minus-end directed microtubule motility.
We have shown that this novel motor binds to microtubules both in cells as well as in vitro and
have initial evidence that it is able to bind multiple microtubules simultaneously. This
microtubule cross-linking has been shown in other motors to promote antiparallel microtubule
sliding and pole focusing, although Kif25 does not appear to control microtubule focusing at the
poles of human cells. Live cell imaging of cells over expressing Kif25 shows defects in spindle
pole separation as well as a defect in mitotic timing. Overexpressing cells take significantly
longer to proceed through metaphase; we have initial evidence that this delay is caused by
activation of the mitotic spindle assembly checkpoint due to loss of tension between sister
kinetochores. In addition, cells that do proceed through metaphase show a higher incidence of
multinucleate cells indicating that regulation of Kif25 is important for maintaining the fidelity of
cell division. We propose that Kif25 is able to regulate mitotic spindle orientation by using its
microtubule sliding capability to produce force at the spindle pole. This force can act to balance
the forces produced within the spindle by other kinesin motors as well as forces imposed on the
spindle by cortically localized dynein. This force balance model can account for our initial data in
Kif25 knockdown as well as over expressing cells and presents a novel mechanism of control
for mitotic spindle orientation.
2924
TUESDAY-LATE POSTER PRESENTATIONS
Sub-cellular localization and functions of active AMPKalpha during the cell cycle in HeLa
cells.
I. Lee1, J-H. Lee1,2; 1Department of Biochemistry and Molecular Biology, Ajou University,
Suwon, Korea, 2Department of Molecular Science and Technology, Ajou University, Suwon,
Korea
AMP-activated protein kinase (AMPK) has critical roles in regulating growth and reprogramming
metabolism, and has also been connected to cellular processes such as autophagy, cell
polarity, and cell division. Furthermore, recent studies suggest that AMPK, seemingly
independent of cellular energy status, promotes mitotic progression. However, how these
events unfold at the molecular level is not well understood. Here, we show that AMPKalpha2
depletion causes the delay of G2/M transition in synchronized HeLa cells, evidenced by flow
cytometric and mitotic index analysis. Surprisingly, we also found that threonine172
phosphorylated AMPKalpha transiently appears to the perinuclear region of late G2 phase cells.
It is known that the Golgi apparatus is a network of polarized cisternae localized to the
perinuclear region and undergoes extensive vesiculation at the onset of mitosis. Therefore, we
investigated whether the pAMPKalpha-Thr172 co-localizes to Golgi apparatus. The
pAMPKalpha-Thr172 partially co-localizes with the Golgi matrix protein GM-130 in a transient
fashion during G2/M transition, when GM-130 plays an important role in Golgi fragmentation.
Interestingly, in HeLa cells depleted of AMPKalpha2, further fragmentation of the isolated Golgi
stacks is delayed during G2 phase and levels of pAMPKalpha-Thr172 is also decreased. These
data may shed new light on AMPKalpha2 function for Golgi-fragmentation that is initiated by cell
signaling cascades in the early phases of mitosis.
2925
Using the Oral Cancer Cell line UPCI:SCC078 to purify NuMA protein.
A. C. Rodrigues1, N. J. Quintyne1; 1Honors College, Florida Atlantic University, Jupiter, FL
Formation of multipolar spindles is closely linked to increased genomic instability and therefore
tumor progression. Hyperamplification of the centrosome, while a major contributor to
multipolarity, is insufficient to initiate the mitotic defect. A mechanism by which cells can cluster
extra centrosomes to form a bipolar spindle must also fail before multipolarity can occur.
Numerous proteins have been implicated as essential for this process with as many as 82
genes in mammals playing a role in centrosome clustering (Kwon et al., 2009). Our previous
studies have indicated that cytoplasmic dynein is a key factor in preventing multipolar spindle
formation, and that overexpression of the NuMA protein is sufficient to mislocalize dynein from
the spindle and abrogate the coalescence machinery. NuMA is an abundant component of
interphase nuclei and an essential player in mitotic spindle assembly and maintenance.
Therefore, it is essential that a specific concentration of NuMA be maintained in the cell as too
much or too little of the protein can prevent normal mitotic progression. Because the mechanism
by which NuMA can inhibit dynein is unclear, we are purifying NuMA to use in in vitro studies
with purified dynein, with the aim of better understanding when NuMA becomes harmful to the
actions of dynein on microtubules and in spindle formation and maintenance. Purifying NuMA
from recombinant sources have not been successful, so we have been purifying NuMA from a
native source by chromatography, modifying the method used in Kempf and coworkers (1994).
We are using the oral squamous cell carcinoma line UPCI:SCC078 as a source to begin with
more starting material: the UPCI:SCC078 cell line has nine copies of the NUMA1 gene.
Previously, we have encountered problems with purity and yield. After ion exchange and gel
filtration chromatography have been performed, several contaminants persist, but additional
rounds of chromatography reduce the yield to unusable amounts. Due to the high level of
membrane, we had previously concentrated on the supernatants from lysates, but with
TUESDAY-LATE POSTER PRESENTATIONS
modifications to our protocol are now including the NuMA-rich pellets, after having disrupted the
membranes. This increase in starting material is being used in conjunction to other modification
of the original protocol, yielding sufficient quantities of NuMA protein for biochemical analysis
with purified dynein.
2926
Tracking mitotic defects in oral cancer cells using siRNA-mediated knockdown and live
cell analysis.
R. N. Beltran1, J. D. Williams1, N. J. Quintyne1; 1Honors College, Florida Atlantic University,
Jupiter, FL
As tumors progress, there is a corresponding increase in genomic instability, derived from both
chromosomal rearrangement and chromosomal instability. Often, these manifest themselves as
defects in mitosis, frequently seen as lagging chromosomes, multipolar spindles and anaphase
bridges. Lagging chromosomes are the result of inaccurate chromosomal division in mitosis,
thus jeopardizing the genome of an organism’s offspring. Multipolarity causes aberrant spindles
with more than two poles to form in the cell, which can result in improper mitotic division, with
the same genetic consequences. Anaphase bridges are the result of improperly fused broken
chromosome ends and also contribute to genomic instability. We have been investigating the
causes and mechanisms of lagging chromosomes and multipolarity in two oral cancer cell lines,
UPCI:SCC070 and UPCI:SCC103. Previous work has shown that correct expression of a wide
array of proteins is necessary to prevent multipolar spindle formation. Two such proteins, NuMA
and HSET are mis-expressed in UPCI:SCC103 and UPCI:SCC070 respectively. Lagging
chromosomes derive from several errors, such as failure of a chromosome to attach to the
spindle, or the existence of an acentric chromosome. Our laboratory’s work shows that
treatment with certain carcinogens increase the rate of mitotic defect and antioxidants can
reduce that rate. To further our understanding of how these defects form and progress, we are
monitoring the progress of lagging chromosomes in UPCI:SCC103 cells with live cell analysis,
using GFP-tagged histone H2B to track their appearance and fate, so to distinguish between the
possible causes and resolutions of this mitotic defect. Additionally, we are using shRNAmediated knockdown of HSET in UPCI:SCC070 cells to examine if, like NuMA, having
excesses of the protein is equally detrimental to the cell as too little protein in terms of causing
mitotic defect.
2927
Golgi-derived Microtubule Assembly in Mitosis.
J-H. Wei1, J. Seemann1; 1Cell Biology, UT Southwestern Medical Center, Dallas, TX
Spindle assembly requires the coordinated action of multiple cellular structures to nucleate and
organize microtubules in a tightly controlled spatiotemporal manner. Among them the
contributions of centrosomes, chromosomes and microtubules have been extensively studied,
yet the involvement of membrane-bound organelles remains largely obscure. Here we provide
mechanistic evidence for a Golgi-based microtubule assembly pathway. Upon mitotic entry, the
Golgi is rapidly disassembled as a result of unstacking and vesiculation. Concomitant with the
morphological and biochemical changes, the Golgi matrix recruits specific mitotic regulators to
facilitate microtubule assembly around Golgi membranes. Our results reveal an active role for
the Golgi apparatus in regulating spindle formation to ensure faithful inheritance.
TUESDAY-LATE POSTER PRESENTATIONS
2928
Cell cycle regulation by deubiquitinating enzymes.
T. Das1, E. Kim2, E. Song1; 1Molecular Recognition Research Center, Korea Institute of Science
& Technology, Seoul, Korea, 2Center for Theragnosis, Korea Institute of Science & Technology,
Seoul, Korea
Ubiquitination is a post-translational modification that involves the covalent attachment of
ubiquitin (Ub) to cellular proteins. This process is carried out by three enzymes referred to as
E1, E2 and E3. Specific deubiquitinating enzymes (DUBs) can remove ubiquitin from
substrates. Ub-mediated modification has been shown to regulate the stability, function or
localization of their modified target proteins. In this study, we identified DUBs which cause
mitotic defect. We focused on DUB candidates whose depletion lead to significant spindle
checkpoint bypass in response to taxol treatment. Subsequently, we selected and analyzed the
role of Usp4, depletion of which showed mitotic defect such as chromosome missegregation
and multiple poles. We isolated Usp4 complex using mass spectrometry to identify substrates
and studied functional mechanism. We found that Usp4 regulate spliceosomal activity by
inhibiting the modification of spliceosomal proteins with ubiquitin chains. Loss of Usp4 interferes
with the accumulation of correctly spliced mRNAs, including those for a-tubulin and Bub1, and.
Therefore, these findings indicate that Usp4 are involved in cell cycle progression through
regulating the modification of spliceosomal proteins. [This work is supported by GRL Program
(20110021713) from the National Research Foundation of Korea and an institutional grant from
Korean Institute of Science and Technology]
2929
High-throughput siRNA screen for novel mitotic regulators.
E. Narvi1,2, M. Tambe1,2, P. Toivonen1, M. J. Kallio1,2; 1Medical Biotechnology, VTT Technical
Research Centre of Finland, Turku, Finland, 2Centre of Biotechnology, University of Turku,
Turku, Finland
Mitosis-targeting compounds are an evolving group of potential cancer drugs of which the most
promising ones are under clinical evaluation for their anti-cancer efficacy. Typically, the
compounds cause M-phase arrest followed by cell death or to a lesser extend premature forced
exit from mitosis. In order to identify novel targets for early phase drug discovery, we performed
a high-throughput siRNA screen covering the druggable genome with approximately 7000
genes. We focused on discovery of gene silencing conditions that perturbed normal mitosis.
Hela-cells expressing H2B-GFP were used enabling visual observation of the induced mitotic
defects. We identified several hit siRNAs that caused a transient mitotic arrest, inhibition of
cytokinesis, or escape from taxol imposed mitotic block.
Selected hits have been further validated using cell-based assays. The most potent mitosis
arresting hit is a gene that previously has not been implicated in regulation of M-phase. We
have termed this gene mitotic arrest factor 28, MAF28. The MAF28 silenced cells exhibit
pleiotropic M-phase defects that culminate to a transient M-phase delay and loss of cell viability.
Another hit gene is termed cytokinesis factor 20 (CF20) loss-of-function of which results in
polyploidy and multipolarity. The function of CF20 is previously connected to ERK1/2 signalling
cascade. A third hit gene is termed mitotic exit factor 18 (MEF18). Cells depleted of MEF18
rapidly overcome taxol block and exit mitosis prematurely without cytokinesis. The effect of
MEF18 is putatively mediated by interaction with the spindle checkpoint protein MAD2.
TUESDAY-LATE POSTER PRESENTATIONS
2930
A Substrate-Trapping Mutant of Ulp1 for the Identification of SUMO Protease Targets.
M. L. Guillotte1, J. D. Wells1, O. Kerscher1; 1Biology, The College of William and Mary,
Williamsburg, VA
Eukaryotic cells utilize the addition and removal of SUMO, a small ubiquitin-like modifier, to
modulate protein function. SUMO modification and demodification is facilitated by SUMO
ligases and SUMO proteases respectively. In the yeast Saccharomyces cerevisiae, the SUMO
protease Ulp1 is responsible for removing SUMO from target proteins and for processing
precursor SUMO into its conjugation-competent form. Yeast cells lacking Ulp1 are not viable
and arrest in the G2/M phase of the cell cycle. We hypothesize that this cell cycle arrest may be
due to the accumulation of specific Ulp1 substrates with a role in cell cycle progression. Using a
novel substrate-trapping mutant of Ulp1, Ulp1(3) (C580S), we have devised a strategy to affinity
purify sumoylated Ulp1 substrates. Here we describe the identification and functional
characterization of novel Ulp1 substrates including those with a potential role in cell cycle
progression.
2931
Spatial Control of the Kinesin-14 XCTK2 by the Ran-GTP Gradient.
L. N. Weaver1, S. Cai2, S. C. Ems-McClung3, C. E. Walczak3; 1Biology, Indiana University,
Bloomington, IN, 2Biochemistry, Indiana University, Bloomington, IN, 3Medical Sciences, Indiana
University, Bloomington, IN
The mitotic spindle is a highly dynamic structure that assembles to ensure equal segregation of
genetic material into two daughter cells. The ability of cells to nucleate microtubules (MTs) in the
vicinity of the chromosomes and to organize them into a bipolar MT array is a fundamental
mechanism of spindle assembly that appears to be conserved between cells with acentrosomal
spindles, as well as in centrosome-containing cells. Cues from chromatin, such as the Ran-GTP
gradient, initiate a cascade of downstream signaling that promotes the release spindle assembly
factors (SAFs) to form the spindle. We previously showed that the minus-end directed Kinesin14, XCTK2/HSET is a Ran-GTP regulated SAF that is important for spindle assembly. In
Xenopus extracts, XCTK2 is important for spindle pole organization and excess amounts of the
protein can increase the rate and extent of bipolar spindle formation. In HeLa cells,
overexpression of HSET results in long bipolar spindles, which was postulated to be due to the
protein’s ability to crosslink and slide MTs. In addition, knockdown of HSET allows for
chromosome congression in cells lacking K-fibers. Together our data support the idea that
XCTK2/HSET is a key Ran-regulated factor whose activity may be spatially controlled in the
spindle. We are currently testing the model that XCTK2/HSET mediates its function in the
spindle by directly regulating MT dynamics. We are also testing if this activity is dependent on
the spatial localization of the Ran-GTP gradient. Together these studies will elucidate how
spindles use kinesin motor proteins to locally control microtubule dynamics and organization.
2932
Mitotic RanGTP gradient is amplified by chromosomal gain and promotes mitotic spindle
assembly in aneuploid cells.
K. Hasegawa1, S. Ryu1, P. Kalab1; 1Laboratory of Molecular and Cellular Biology, National
Cancer Institute, National Institutes of Health, Bethesda, MD
In addition to its important role in regulating the transport between nucleus and the cytoplasm in
interphase cells, the small GTPase Ran was shown to have an important role in the activation of
many mitotic spindle assembly factors (SAFs). Owing to the chromatin-binding activity of RCC1,
TUESDAY-LATE POSTER PRESENTATIONS
the guanine nucleotide exchange factor for Ran, and the cytoplasmic localization of RanGAP1,
mitotic chromosomes are surrounded by a diffusional concentration gradient of RanGTP. As a
result, the RanGTP gradient-dependent activation of SAFs is thought to promote the nucleation
and organization of microtubules into mitotic spindles at the chromosomes. However, while the
mitotic RanGTP gradients were previously visualized with Foerster resonance energy transfer
(FRET) sensors in meiotic egg extracts, mouse oocytes and proliferative tissue culture cells, the
role of Ran in normal somatic cell mitosis is not known. Using FRET sensors optimized for
quantitative live cell imaging, here we show that while steep mitotic RanGTP gradients were
present in variety of rapidly growing normal or transformed cells, such as HeLa cells, slow
growing normal human primary cells, such as HFF-1 fibroblasts, displayed either strongly
reduced or non-detectable mitotic RanGTP gradients. We present correlative and experimental
evidence demonstrating that RCC1 expression levels and RCC1 binding to chromatin, which
depends on its N-terminal methylation via NRMT methyl transferase, are the key factors
supporting the rise of steep mitotic RanGTP gradients. We found that the chromosome
congression to metaphase plate was slower and more stochastic in HFF-1 cells (with no
detectable mitotic RanGTP gradients) than in HeLa cells. Also consistent with the proposed role
of the mitotic RanGTP gradients in mitotic spindle assembly and function, the disruption of
RanGTP gradients by three different methods (RanGAP1 RNAi and NRMT RNAi in HeLa cells;
disruption of RCC1 in temperature-sensitive tsBN2 cells) induced strong prometaphase delays.
Finally, we found that in vitro-induced cell-cell fusion of HFF-1 cells produced cells with steep
mitotic RanGTP gradients comparable to HeLa cells. These results indicate that chromosomal
gain can support mitosis progression in aneuploid cancer cells via RanGTP gradient-regulated
mechanisms.
2933
Regulation of the mitotic checkpoint by p31comet-BUBR1 and p31comet-TRIP13 interactions
K. Wang1, W. Ji1, A. Tipton1, H. Zhang1, B. Sturt1, S-T. Liu1; 1Department of Biological Sciences,
University of Toledo, Toledo, OH
Proper activation and silencing of the mitotic checkpoint is important for successful chromosome
segregation. p31comet is a protein dedicated to mitotic checkpoint silencing. Examining the
proteins that interact with p31comet is expected to provide novel insights into the mitotic
checkpoint mechanisms. Here we show that p31comet directly interacts with checkpoint protein
BUBR1 in vitro. The interaction is reminiscent of that between BUBR1 and C-MAD2, another
checkpoint protein that shares similar structural fold to p31comet. The p31comet:BUBR1 interaction
is subject to cell cycle regulation in vivo. During G2 phase BUBR1 interacts with CDC20 but not
p31comet, consistent with the observation that CDC20 binding to BUBR1 (1-486) reduces the
p31comet affinity towards BUBR1. As recently noticed by others, the p31comet level associated with
APC/C and BUBR1 was surprisingly high in prometaphase cells even though the mitotic
checkpoint is functional at this stage. Although the details remain to be clarified, some evidence
suggested that a C-MAD2:p31comet heterodimer could form and get recruited to BUBR1. The
heterodimer formation could result from increased intracellular concentration of C-MAD2 in
prometaphase cells. The working model is a BUBR1:C-MAD2:p31comet complex may bind and
position CDC20 for auto-ubiquitylation by the APC/C. Interestingly, BUBR1 (486-700) was also
observed to bind to CDC20, C-MAD2 and p31comet. C-MAD2 interacts with the N-terminus of
BUBR1 to form the mitotic checkpoint complex (MCC), the APC/C inhibitor. It will be
investigated whether BUBR1 (486-700) is selectively utilized to form the complex containing
p31comet in prometaphase cells to prevent premature APC/C activation. During anaphase when
the interactions of CDC20 with BUBR1 and MAD2 are weakened, p31comet may directly interact
with both BUBR1 and MAD2 to persistently inactivate the BUBR1:C-MAD2 interaction, leading
to MCC disassembly and checkpoint silencing. We have reported direct interaction between
TUESDAY-LATE POSTER PRESENTATIONS
p31comet and TRIP13 AAA ATPase. Whether TRIP13 is the missing link to coordinate ATP
hydrolysis and p31comet -mediated disruption of the MCC is being investigated.
2934
A lung cancer associated mutation in the coding sequence of survivin promotes cell
proliferation.
A. Aljaberi1, J. R. Webster1, S. P. Wheatley1; 1School of Biomedical Sciences, University of
Nottingham, Nottingham, United Kingdom
Cancer occurs when normal regulation of the cell division cycle is disrupted by genetic or
environmental factors. Thus, understanding the molecular mechanisms which regulate cell
division is vital for developing anti-cancer therapeutics. Survivin is a partner protein in the
Chromosomal Passenger Complex, which is a critical mitotic regulator of chromosomal
movements and cytokinesis. Survivin expression is normally restricted to G2 and M phases of
the cell cycle, however, in cancer, its expression is deregulated causing it to be high throughout
the cell cycle. Survivin is also a member of a family of proteins that inhibit apoptosis. Having
dual prosurvival roles, survivin is considered as an attractive target for novel oncotherapeutic
agents. The elevation of survivin expression in cancers is typically attributed to changes in
transcriptional regulation. To date the only cancer-associated polymorphism in the coding region
of the survivin gene that has been reported was identified in lung cancer in which lysine 129
was found to be substituted with glutamic acid, K129E. Being an exposed lysine, this site could
be regulated by post-translational modifications, particularly by ubiquitination and acetylation.
Here we recapitulate the K129E mutation in a controlled system and investigate whether this
change affects survivin function in mitosis or in the inhibition of apoptosis. We also investigate a
second version K129A that acts to mimic a constitutively acetylated state. Our preliminary data
suggest that although mutating K129 does not affect survivin localization during mitosis or
prevent survivin from inhibiting TRAIL-mediated apoptosis, substitution to a glutamic acid
(K129E) avidly supports proliferation, while mimicking acetylation (K129A) retards growth.
These data suggest that modification of survivin at K129 by acetylation regulates cell growth
rate, and that this regulation is lost in lung cancer when this specific lysine is mutated to a
glutamic acid.
2935
Cdc26 cooperates with Cdk1 phosphorylation to activate the APC-Cdc20.
L. Williams1, E. Ferguson2, A. D. Rudner2; 1Department of Biochemistry, Microbiology &
Immunology, University of Ottawa, Ottawa, ON, Canada, 2Biochemistry, Microbiology &
Immunology, University of Ottawa, Ottawa, ON, Canada
Cdk1 promotes the metaphase to anaphase transition by phosphorylating the anaphase
promoting complex (APC), a multisubunit ubiquitin ligase that targets specific mitotic regulators
for degradation by the proteasome. The degradation of these proteins is critical for accurate
sister chromatid segregation and mitotic exit.
We have shown previously that inhibition of Cdk1-dependent phosphorylation of the APC
stabilizes mitotic cyclins and Pds1 (securin), impairs sister chromatid segregation and arrests
cells in mitosis, suggesting that Cdk1-dependent phosphorylation of the APC may be essential
for mitotic APC activity. However, blocking phosphorylation of Cdk1 consensus sites on the
APC subunits Cdc16 and Cdc27 (the APC-A mutant), has only mild effects on mitotic
progression and APC activity in vitro.
The TPR repeats in Cdc16 and Cdc27 have been shown to form the binding site for the
APC activators Cdc20 and Cdh1, and an APC which lacks Cdk1 phosphorylation sites on
Cdc16 has reduced affinity for Cdc20. Cdc16 also binds to Cdc26, a small, non-essential
TUESDAY-LATE POSTER PRESENTATIONS
subunit of the APC that becomes essential for cell viability only at elevated temperatures.
Structural work has shown that the human Cdc26 binds to Cdc16 and forms an intra-molecular
TPR repeat. We have therefore tested if Cdc26 functions with APC phosphorylation to activate
the APC-Cdc20.
Genetic analysis indicates that deleting Cdc26 in the APC-A background lowers the
maximum permissive temperature of this strain. This interaction is not due to loss of APC
integrity, as has been suggested in some reports of cdc26Δ purifications. Furthermore, we have
created truncations of Cdc26 in an effort to isolate a Cdc20-dependent function of Cdc26. The
cdc261-52 mutant (residues 1-52) rescues the temperature sensitivity of a cdc26Δ mutant, but
still exhibits a genetic interaction in the APC-A background. We are currently using this mutant
to test the role of Cdc26 in Cdc20-dependent activation of the APC.
2936
Quantitative model of metaphase chromosome dynamics via viscoelastic bonds.
G. Civelekoglu-Scholey1, B. He2, M. Shen2, D. Cimini2; 1Department of Molecular and Cell
Biology, University of California, Davis, CA, 2Department of Biological Sciences, Virginia Tech,
Blacksburg, VA
Duplicated mitotic chromosomes aligned at the metaphase plate maintain dynamic attachments
to spindle microtubules via their kinetochores, and multiple motor and non-motor proteins
coordinate their behavior. Depending on the system, sister chromatids may display either of
two distinct behaviors, namely (i) the presence or (ii) the absence of oscillations about the
metaphase plate. Significantly, in PtK1 cells, both types of behavior are observed within the
same spindle, but how and why these distinct behaviors are manifested is unclear. In an effort
to address these questions in a combined experimental-quantitative framework, here, we
developed a new force-balance model to describe metaphase chromosome dynamics via
kinetochore-microtubule interactions mediated by non-motor viscoelastic linkages. Our model
reproduces all the key features of metaphase kinetochore dynamics in PtK1 cells, and suggests
that differences in the distribution of polar ejection forces at the periphery and in the middle of
PtK1 cell spindles underlie the observed dichotomy of chromosome behavior.
2937
A Zebrafish cardiac overload stress model for the investigation of cardiomyocyte
proliferation.
N. H. Lopez1, D. Mendoza1, B. Schoffstall1; 1Biology Department, Barry University, Miami
Shores, FL
Zebrafish (Danio rerio) hearts are able to fully regenerate after apex amputation or cryoinjury.
We have recently established a Zebrafish cardiac stress model without injury to examine a
similar proliferative response. Our studies have indicated that excessive cardiac overload
stress can be exerted on Zebrafish using a forced, fast-speed swimming exercise regimen over
a period of 10 weeks. This stress model results in significant overall enlargement of ventricles
via hyperplasia in the exercised fish. Enlargement appears to be efficient compensation;
physiology is similar to that of non-exercised controls. To study the process of hyperplasia
stimulation, we need to determine the time point at which active cell division is detectable during
several weeks of a forced exercise regimen. Because post-injury regeneration studies have
determined that full regeneration of the Zebrafish heart occurs by around day 31, our hypothesis
is that exercise stressed Zebrafish hearts are significantly remodeled via hyperplastic
enlargement by the end of 4 weeks of exercise. We collected samples at the end of 7 days, 14
days, 21 days, and 30 days of a forced excessive exercise regimen. Using quantitative realtime PCR to detect mRNA expression of proliferating cell nuclear antigen (PCNA), our
TUESDAY-LATE POSTER PRESENTATIONS
preliminary results indicate that most cell division appears to occur at around day 14 of
strenuous exercise. By the end of 30 days of cardiac overload, exercised hearts are 15% larger
than non-exercised controls. These preliminary data have helped us to target the second week
(~ day 14) of exercise for identification of genes involved in stimulation of hyperplasia. In
addition, we have found that the stress remodeling response using our Zebrafish cardiac stress
model occurs much in the same time frame (~30 days) as the regenerative response. We will
continue to use this model to identify molecules involved in both stimulation of hyperplasia and
cardiac stress response.
2938
Cdc14 phosphatase is not fully released and still keeps some in nucleolus till at the end
of exit.
S. Ramachandran1, J. Zhu1, R. Li1; 1Stowers Institute, Kansas, MO
Mitosis is the process by which a cell divides into two daughter cells that contain same number
of chromosomes as in the parent cell. For accurate chromosomes segregation, the kinetochores
of each pair of sister chromatids attach to microtubules originating from the opposite poles of
the cell. Cdc14 plays a major role in mitotic exit through two signaling networks, the Cdc14 early
anaphase release (FEAR) and the mitotic exit network (MEN), which ensures the proper
chromosome segregation between the mother and daughter cells. Most of the published
studies employed immunoflourescence staining to visualize Cdc14 in fixed cells, and this
method does not permit kinetic measurement with high temporal resolution at the single cell
level. Our live cell imaging analysis shows that Cdc14 phosphatase is not fully released and still
keeps some in nucleolus till at the end of exit.
Signal Transduction and Signaling Networks II
2939
Nuclear translocation of a G-protein coupled receptor mediates increased gene
expression in osteoblasts.
A. Di Benedetto1, C. Cuscito1, G. Colaianni1, R. Tamma1, C. D. Calvano2, C. G. Zambonin2, A.
Zallone1; 1Department of Basic Medical Sciences, University of Bari, Bari, Italy, 2Department of
Chemistry, University of Bari, Bari, Italy
The neuro-hypophiseal hormone oxytocin (OT) is a novel anabolic regulator of bone mass
(Tamma et al., PNAS 2009), upregulating expression of critical osteoblast transcription factors.
These effects are mediated by Oxytocin receptor, a GPCR expressed by osteoblasts. An
increasing number of reports indicates that GPCRs could be targeted to the nuclear membrane:
prostaglandin, endothelin and β-adrenergic receptors among others (Boivin et al., 2008).
Accordingly we found OTRs in osteoblast nuclear extracts after OT stimulation (15-30 min).
Confocal microscopy performed on intact cells either transfected with OTR-GFP or stained with
different monoclonal antibodies after fixation indicated a nuclear localization evident 20-40'
(min) after OT stimulation, data confirmed by immunogold staining at TEM. Exogenous OTRGFP, transfected in primary osteoblasts, colocalized with β-arrestin1/2 within 2-3’ after OT
treatment, thereafter was found in RAB5-positive endosomal vesicles followed, in a range of
time from 10' to 20' from OT treatment, by a colocalization with transportin-1. Eventually, at least
a part of the receptors was sorted to the nucleus, where OTR-GFP was evident by confocal
microscopy both in intact cells and in isolated nuclei. Indeed, to avoid artifact due to the extreme
flatness of cultured osteoblasts, nuclei were isolated with a technique that eliminates the
TUESDAY-LATE POSTER PRESENTATIONS
external nuclear membrane (Adebanjo 0.A. et al. Nature Cell Biol, 1999). MALDI-TOF analysis
of nuclear proteins immunoprecipitated with anti-OTR confirmed this finding; the spectra
analyzed with FindPept database revealed the presence of four peptides corresponding to OTR
intracellular loops. We hypothesized as possible role for OTR in nuclei the regulation of
transcription and/or transcription factors.In fact,in response to OT stimulus a physical interaction
of native OTRs with the osteoblast transcription factor Runx-2 and with the transcription coactivator Schnurri-2 was found by immunoprecipitation. The blockage of OTR endocytosis by βarrestins silencing, prevented OT induced mRNA up-regulation of genes as Osx, ATF-4, BSP
and Osteocalcin. Similarly by Transportin-1 silencing OTR nuclear localization as well as the
up-regulation of Osx, ATF-4, Osteocalcin and BSP mRNA in response to OT were impaired.
Taken together these data suggest that OT anabolic effect on bone could be dependent upon a
novel mechanism initiated by β-arrestin-mediated OTR internalization and followed by
Transportin1-dependent nuclear translocation.
2940
The H1HR agonist, HTMT dimaleate, increases small cholangiocyte proliferation during
liver regeneration after 70% partial hepatectomy.
F. Meng1,2, G. Alpini2,3, H. Francis2; 1Scott & White Healthcare, Temple, TX, 2Central Texas
Veterans Health Care System, Temple, TX, 3Texas A&M University, Temple, TX
Background: The liver is able to regenerate itself after injury or transplantation. Liver resection
or transplantation are the only treatment options for patients suffering from liver disease or liver
cancer. After 70% partial hepatectomy (PH), cholangiocytes proliferate to repopulate the liver,
peaking at days 3 and 5 and this effect is enhanced by treatment with the H1HR agonist.
Histamine exerts its actions via four G-protein coupled receptors, H1-H4. The H1HR agonist
increases small cholangiocyte growth via Ca2++- dependent mechanisms. Aim: To determine
the effect(s) of the H1HR agonist on small cholangiocyte proliferation during liver regeneration
after PH. In vivo studies: Normal rats were subjected to partial hepatectomy (PH) and treated
with vehicle (0.9% NaCl) or the HRH1 agonist (HTMT dimaleate [HTMT], 375 mg/kg/BW) for 3
and 5 days. Immunohistochemistry (IHC) was performed to evaluate the number of PCNApositive small cholangiocytes and intrahepatic bile duct mass (IBDM) by counting the number of
CK-19- positive small cholangiocytes in liver sections. IHC was performed to evaluate the
expression of VEGF-A and VEGFR-2 in liver sections. Apoptosis was evaluated in liver
sections by IHC for TUNEL positive cholangiocytes.
In vitro studies: Small murine
cholangiocytes (SMC) were utilized for in vitro studies and were cultured according to previous
methods. Wound healing studies were performed in SMC treated with 0.2% BSA (basal) or
HTMT (10 μM) for 0 – 18 hrs. Wounds were induced using a sterile needle and images
collected. SMC were treated with 0.2% BSA (basal) or HTMT (10 μM) for up to 72 hours and
protein lysate and mRNA was extracted. The gene and protein expression of H1HR and VEGFA was measured and proliferation was evaluated by MTT assays, BrDU staining and PCNA
western blotting. Results:
After 3 days of PH, HTMT treatment increases small cholangiocyte proliferation and IBDM as
seen by PCNA and CK-19 IHC. Similarly, HTMT treatment increased VEGF-A and VEGF-R2
expression when compared to animals treated with NaCl. TUNEL staining revealed little to no
cholangiocyte apoptosis in HTMT-treated animals.
In vitro, HTMT accelerated the wound healing time of SMCs compared to basal-treated SMCs.
The gene and protein expression of H1HR and VEGF-A were both increased in SMCs treated
with HTMT compared to basal-treatment. HTMT increased the proliferation of SMCs as shown
by MTT assays, BrDU staining and PCNA western blotting. Conclusions: The H1HR agonist,
HTMT accelerates the regrowth of the liver by increasing small cholangiocyte proliferation via
TUESDAY-LATE POSTER PRESENTATIONS
increased VEGF-A/R2 expression. Treatment with histamine receptor agonists may be a
therapeutic option for patients undergoing liver resection or transplantation.
2942
Alanine-87-Threonine polymorphism of the human P2Y11 receptor.
G. Reiser1, A. Shaaban1, M. Haas1; 1Otto-von-Guericke University, Magdeburg, Germany
A statistical link between the Alanine-87-Threonine (A87T) polymorphism of the human P2Y11
nucleotide receptor and risk for acute myocardial infarction as well as elevated levels of Creactive protein has been proposed (Amisten et. al 2007). In this study, the pharmacological and
functional characteristics of the P2Y11A87T receptor have been investigated in 1321N1
astrocytoma cells and HEK293 cells. For HEK293 cells stably expressing the P2Y11A87T
receptor, we show a reduced potency of ATP, BzATP and 2-MeS-ADP as well as a complete
loss of nucleotide-induced internalization. Furthermore, we found an increased ability for
receptor resensitization in HEK293 cells expressing the P2Y11A87T receptor after prolonged
receptor desensitization. In 1321N1 and HEK293 cells, the increase of nucleotide-induced
cAMP production was comparable between the P2Y11 receptor and the A87T variant. The ATPinduced elevation of phospho-ERK concentration was significantly lower in HEK293 cells
expressing the P2Y11A87T receptor. The data supports the hypothesis of a possible
physiological impact of this receptor polymorphism and therefore on the development of acute
myocardial infarction.
2943
Comparison of phenotypic change between A10 cell and primary rat aortic vascular
smooth muscle cell by cyclic stretch and stimulation of vasoactive substances.
E. Taira1, Y. Kondo1, Y. Irie1; 1Pharmacology, Iwate Medical School, Morioka, Japan
Vascular smooth muscle cells compose the tunica media of arteries and play a role to maintain
the strength of the vessel confronting with mechanical forces inherent in the cardiovascular
system. Vascular smooth muscle cells also change depending on blood pressure and blood
perfusion as part of cardiovascular system. Vascular smooth muscle cell phenotype is affected
by environmental influences, such as mechanical stress, vascular injury and vasoactive
substances. Vascular smooth muscle cell also makes a contribution on neointimal hyperplasia
of atherosclerosis. Differentiated vascular smooth muscle cell expresses some proteins as a
marker of contracting form. Undifferentiated vascular smooth muscle cell are able to proliferate,
migrate, and produce extracellular matrix components. Before now, many biological compounds
have been reported to change vascular smooth muscle cell phenotype. These compounds
include platelet-derived growth factor, angiotensin II, insulin-like growth factor-I, -II and
endthelin-1. But the modulation of expression of these receptors and the mechanism of the
modulation are still unclear. If they are to be elucidated, there is a possible of analyzing the
influences on the expression of the drug effect.
In this study, we examined the change of some vasoactive substance receptors expression in
A10 cells, cells derived from a rat aortic smooth muscle cell, and primary rat aortic vascular
smooth muscle cells under cyclic stretch. We also investigated the change of the two types
vascular smooth muscle cell phenotype under the cyclic stretch with or without these vasoactive
substances. We report that the change of several vasoactive substances receptors and related
proteins expression and phenotype of the two types vascular smooth muscle cell by cyclic
stretch and vasoactive substances exposure.
TUESDAY-LATE POSTER PRESENTATIONS
2944
MAST205 negatively regulates P2YR-mediated inflammatory responses via inhibition of
PLC-β2 activation.
J. K. Kim1, S. Lee1, S. Y. Choi1, H. Y. Moon1, Y-R. Yang1, J-M. Kim1, H. H. Lee1, O. Kwon2, I. S.
Kim1, S. H. Ryu2, H. M. Kwon1, P-G. Suh1; 1Ulsan National Institute of Science and Technology,
Ulsan, Korea, 2Pohang University of Science and Technology, Pohang, Korea
Phospholipase C-β (PLC-β) plays key roles in G-protein coupled receptor (GPCR)-mediated
signaling. Many studies have demonstrated that four PLC-β subtypes have different
physiological functions despite their similar structures. Because PLC-β subtypes possess
different PDZ-binding motifs at their C-terminus, they potentially interact with different PDZ
domain-containing protein (PDZ protein). In this study, we identified MAST205 (MicrotubuleAssociated Serine/Threonine protein kinase) as a novel PDZ protein which specifically interacts
with PLC-β2. Through this interaction, MAST205 phosphorylated and inhibited the enzymatic
activity of PLC-β2. As to the physiological meaning of this protein-protein interaction, we found
that knockdown of PLC-β2 significantly reduced, while that of MAST205 conversely enhanced,
the PLC activation upon P2 purinergic receptor (P2YR) stimulation in Raw264.7 cells, implying
the essential roles of MAST205 in negative regulation of P2YR-mediated PLC-β2 activation.
Moreover, knockdown of MAST205 potentiated PLC-β2-mediated MAPK activation in UDP
(P2Y6R specific endogenous agonist)-treated Raw264.7 cells and peritoneal macrophages,
which further affects expression and secretion of chemokines, such as CCL2 and CCL7.
Therefore, our results strongly suggest that MAST205 negatively regulates P2YR-mediated
inflammatory responses by primarily suppressing PLC-β2 / MAPK signaling in
monocyte/macrophage.
2945
Recruitment of ZAP-70 to subcellular compartments activates distinct TcR-specific
signaling pathways.
M. Ebner1, I. A. Yudushkin1, R. Vale2; 1MFPL, Vienna, Austria, 2UCSF, San Francisco, CA
T cell receptor complex (TcR) is constitutively internalized and recycled to the plasma
membrane, but the functional relevance of internalized TcR is unknown. Previously, we
demonstrated that phosphorylated TcR is present at the plasma membrane and the endosomal
compartment in T cells. Here, we addressed whether TcR can activate the immediate
downstream kinase ZAP-70 and sustain TcR-specific signaling in different subcellular
compartments.
Traditionally, the relevance of localized signaling is addressed either by global inhibition of
protein trafficking or protein mislocalization to an unrelated compartment. For most
transmembrane proteins, however, either strategy yields inconclusive or irrelevant results
because the plasma membrane and vesicles of the endosomal recycling system form a
continuous dynamic network. Instead, to discriminate between signaling elicited from the
plasma membrane or the endosomal pools, we examined the TcR-specific functional responses
upon recruitment of ZAP-70 to either compartment using the FKBP homodimerization approach.
We demonstrate that ZAP-70 phosphorylated on pY493 in the activation loop is trafficking
together with the internalized TcR upon T cell activation. Furthermore, independent of TcR
activation, ZAP-70 recruitment to early endosomes was sufficient to induce Erk1/2
phosphorylation, while recruitment to the plasma membrane induced PLCγ1 activation. ZAP-70
recruitment to mixed membrane compartments potentiated both responses and was sufficient to
induce expression of the early T cell activation marker CD69. Our data provide the first evidence
TUESDAY-LATE POSTER PRESENTATIONS
of compartment-specific TcR-proximal signaling and suggest that intracellular localization or
trafficking of the T cell receptor complex may contribute to TcR signaling specificity.
2946
Prediction of ligand binding to olfactory receptors.
L. Gelis1, S. Wolf2,3, E. M. Neuhaus4, H. Hatt1, K. Gerwert2,3; 1Cell Physiology, Ruhr-University
Bochum, Bochum, Germany, 2Biophysics, Ruhr-University Bochum, Bochum, Germany,
3
Biophysics, PICB Shanghai, Shanghai, China, 4Charite Berlin, Berlin, Germany
Transmembrane signal transduction by membrane proteins enables a cell to convert an
extracellular signal into one or more intracellular signals or responses. Many important cell
recognition and communication processes are mediated by a superfamily of transmembrane
proteins, the G protein-coupled receptors (GPCRs). GPCRs play a prominent role in sensing
extracellular signals, which cover a broad spectrum from physical stimuli (e.g. for vision),
transmitter and peptide molecules (e.g. hormones and cytokines) to chemosensory ligands (e.g.
for taste and smell). The largest gene family among the GPCRs comprise the olfactory
receptors (ORs) that belong to the class A (rhodopsin-like) GPCR family. Most characterized
ORs detect and discriminate multiple highly flexible odorant ligands and exhibit a rather broad
ligand spectrum. Interestingly, ORs show an ‘analog’ activation level, which is different for each
ligand. However, owing to difficulties in with functional OR expression in heterologous systems,
the molecular basis of ligand recognition in ORs is still poorly understood. A prerequisite for a
deeper understanding of odorant recognition and discrimination is the elucidation of the threedimensional structures of olfactory receptor's binding site. As neither X-ray nor NMR structural
models of ORs are available yet, we developed an alternative approach to gain insight into the
structural basis of ligand binding and the prerequisites for ligand-induced receptor activation.
Combining dynamic homology modeling and functional studies of site-directed receptor
mutants, we created a three-dimensional model of the human olfactory receptor hOR2AG1
based on a rhodopsin X-ray structure. By this interplay between theoretical prediction and
experimental verification, we provided a molecular model of the ligand binding niche of
hOR2AG1. The role of putative binding residues was experimentally analyzed by functional
characterization of site-directed receptor mutants by Ca2+-Imaging and experimental results
were compared to simulations. Thus, we could deduce a quantitative criterion for receptor
activation. Activating ligands exhibit a highly dynamic and fluctuating hydrogen bond pattern
with the receptor that offers a solution to cope with the high flexibility of the odorant ligands. We
could further show that the hydrogen bond formation frequency of the receptor/ligand pair is
directly coupled to receptor activation. Therefore, instead of a static, well-defined binding mode,
the dynamics of the protein/ligand complex is decisive for olfactory receptor activation. These
results helped us to identify a ligand selectivity filter for recognition of a minimal distinct local
molecular shape in hOR2AG1 that allows for correct prediction of the activation capability of
novel ligands. Moreover, the dynamic model correctly predicts alterations of receptor function
upon mutation for activation of ligands that do not activate the wild type receptor. Dynamic
homology modeling may be applied in the future for deorphanization of ORs and to provide a
valid basis for OR-based drug design, which we aim to demonstrate by dynamic modeling of
another, pharmacological relevant human OR.
TUESDAY-LATE POSTER PRESENTATIONS
2947
Single-molecule investigation of the intraflagellar transport of membrane signaling
proteins.
A. M. Kovacs1, S. Dutcher2, Y. M. Wang1; 1Physics, Washington University in St. Louis, St.
Louis, MO, 2Genetics, Washington University in St. Louis, St. Louis, MO
Cilia/flagella are ubiquitous hair-like cellular projections that play important roles in sensing and
responding to the environment. Their proper signal transduction functionalities are maintained
by the concerted action of flagellar membrane signaling proteins and their intraflagellar transport
(IFT) machinery - BBSomes and IFT particles. While the membrane signaling proteins collect
information regarding the cells’ environment, the IFT machinery has been implicated in
facilitating the cellular delivery of the information by translocating signaling proteins to and from
flagella and positioning them along the flagellar body. Unfortunately, the intraflagellar motion
characteristics of membrane proteins’ and IFT machinery can at best be described as being
fragmented and limited to rough measurements of average IFT speeds. Furthermore, which
components of their concerted motions contribute to signal transduction pathways have not
been studied. To address these questions, we use single-molecule fluorescence imaging
methods to study the intraflagellar motion of BBS4-GFP, IFT20-GFP, and a transmembrane
signaling protein, Pkd2-GFP. (i) In the flagellar entry region, Pkd2 mostly performs Brownian
diffusion, suggesting that flagellar signaling proteins enter the flagella mainly via IFT, rather than
lateral diffusion along membrane. (ii) In the flagellar body, each IFT train exhibits one speed
change of 400 and one of -400 nm/s per anterograde trip, which we interpret as a dropping off
and a picking up events of one membrane signaling protein, respectively. When not performing
IFT, the flagellar membrane signaling proteins diffuse with diffusion coefficient of 5×10^4 4×10^3 nm^2/s. This result indicates a 10-fold higher viscosity in flagellar membranes than
cellular membranes, contrary to current understanding. The average speed of an IFT train with
a BBSome attached is 300 nm/s slower than one without the BBSome, indicating that BBSome
carries at least one membrane signaling protein. (iii) At the flagellar tip, BBSomes diffuse with
their associated membrane signaling proteins for an average of 2 sec before the return trip,
rather than the alternatively model of dissociation from the microtubule and diffusing in the
flagellar lumen.
2948
The kinase domains of obscurin interact with intercellular adhesion proteins.
L-Y. R. Hu1, A. Kontrogianni-Konstantopoulos1; 1Biochemistry and Molecular Biology, University
of Maryland Baltimore, Baltimore, MD
Obscurins comprise a family of giant (~870-600kDa) and small (~250-55kDa) proteins that play
important roles in myofibrillogenesis, cytoskeletal organization and cell adhesion, and are
implicated in hypertrophic cardiomyopathy and tumorigenesis. Giant obscurins are composed
of tandem structural and signaling motifs, including two serine/threonine kinase domains, SK1
and SK2, present at the COOH-terminus of giant obscurin-B. Using biochemical and cellular
approaches, we show for the first time that both SK1 and SK2 possess enzymatic activities and
undergo auto-phosphorylation. SK2 can phosphorylate the cytoplasmic domain of N-cadherin,
a major component of adherens junctions, and SK1 can interact with the extracellular domain of
the β1-subunit of the Na+/K+-ATPase, that also resides in adherens junctions. Immunostaining
of non-permeabilized myofibers and cardiocytes revealed that some obscurin-kinase isoforms
localize extracellularly. Quantification of the exofacial expression of obscurin-kinase proteins
indicated that they occupy ~16% and ~5% of the sarcolemmal surface in myofibers and
cardiocytes, respectively. Treatment of heart lysates with Peptide:N-Glycosidase F revealed
that while giant obscurin-B localizes intracellularly possessing dual kinase activity, a small
TUESDAY-LATE POSTER PRESENTATIONS
obscurin-kinase isoform that contains SK1 localizes extracellularly where it undergoes Nglycosylation. Collectively, our studies demonstrate that the obscurin-kinase domains are
enzymatically active and may be involved in the regulation of cell adhesion.
2949
Identification of a Novel Phospho-Binding Motif in the Regulatory Subunit of Casein
Kinase 2 (CK2β) that Mediates an Interaction with the Epstein-Barr Virus EBNA1 Protein.
J. Y. Cao1, K. Shire1, L. Frappier1; 1Molecular Genetics, University of Toronto, Toronto, ON,
Canada
Epstein-Barr Virus (EBV) is a widespread human herpes virus whose latent infection is
associated with the development of variety of lymphoid and epithelial cancers. The EBV EBNA1
protein plays multiple roles in EBV latent infection including the promotion of cell survival and
transformation by inducing the degradation of the PML (promyelocytic leukemia) tumor
suppressor proteins that form the basis of PML nuclear bodies (PLoS Pathog. 4(10):e1000170),
which are known to suppress lytic infection of herpes viruses. We have previously shown that
EBNA1-mediated PML degradation involves interaction with the host protein kinase CK2
(Casein Kinase 2), resulting in increased CK2 phosphorylation of PML proteins (J Virol. 84,
11113). CK2 is a pleiotropic kinase comprised of two catalytic (CK2α and/or CK2α’) and two
regulatory (CK2β) subunits. In both GST pull-down assays and glycerol gradient sedimentation
analyses with purified proteins, EBNA1 bound CK2β but not CK2α, indicating that EBNA1 binds
directly to CK2 through the regulatory subunit. By co-immunoprecipitation of EBNA1 mutants
with CK2 from human cells, we mapped the CK2 binding region of EBNA1 and showed that
deletion of EBNA1 residues 387-394 selectively disrupted CK2 binding.
Here, we present evidence for a phospho-specific interaction between EBNA1 and CK2β.
Through point mutations of the EBNA1 387-394 region followed by co-IP analysis, we defined
the residues important for binding and showed that Ser393 is critical. Interestingly, Ser393 is
part of a Cdk consensus phosphorylation site and has been previously shown to be
phosphorylated in human cells (J Gen Virol. 90, 2251). We can detect phosphorylated EBNA1
by its shifted migration on SDS-PAGE and S393A mutation abrogates the shift. Moreover,
phosphatase-treatment of baculovirus-purified EBNA1 protein abrogated interaction with CK2β.
Taken together, the data suggest that CK2β interacts with phosphorylated EBNA1, and the
interaction is likely primed by phosphorylation of EBNA1 at Ser393. By examining the crystal
structure of human CK2 (PDB:1JWH), we noticed that CK2β contained a region that
coordinated phosphates from the crystallization buffer. Point-mutation of the four residues
involved (Lys147-Arg150) abrogated the interaction of CK2β to EBNA1 in vivo and in vitro, but
did not affect CK2β binding to CK2α. In conclusion, these findings suggest that we have
identified a novel phospho-binding motif in CK2β. This motif mediates interaction with
phosphorylated EBNA1, and may bind other proteins (cellular or viral) in a similar manner.
2950
The expression of diacylglycerol kinase theta during mouse embryonic development.
S. Ueda1, B. Tu-Sekine2, M. Yamanoue1, D. M. Raben2, Y. Shirai1; 1Department of
Agrobioscience, Kobe University, Kobe, Japan, 2Department of Biological Chemistry, The Johns
Hopkins University School of Medicine, Baltimore, MD
Diacylglycerol kinase (DGK) is a lipid kinase that phosphorylates diacylglycerol (DG) to produce
phosphatidic acid (PA), regulating lipid-mediated intracellular signal transduction. Of 10
isoforms, DGKθ is the sole member of type V subfamily. DGKθ was originally cloned from rat
brain, and its biological functions have been addressed in several studies. However, few
TUESDAY-LATE POSTER PRESENTATIONS
physiological roles of DGKθ are known, and the histological expression pattern has not yet been
analyzed except in the adult rat brain. Therefore, a detailed expression analysis has done using
mouse embryos to give a useful hint and to predict specific functions of DGKθ in biological
system.
To reveal the spatial and temporal expression of DGKθ, we performed immunohistochemical
staining on paraffin sections of E10.5 to E17.5 mouse embryos. We observed DGKθ protein
expression in the brain, liver, heart, stomach, kidney, intestinal tract, and epidermis in the E17.5.
Antibody specificity was confirmed by adding a blocking peptide for the DGKθ antibody. At early
developmental stages (E10.5 and E11.5), abundant expression of DGKθ was observed in the
neuroepithelium of brain and the ectodermal epithelium of branchial arch, and in the hindlimb
bud. It was also detected in the notochord, bronchus, hepatic primordium, midgut, bulbus cordis,
and atrial chamber. At E12.5, DGKθ was prominently expressed in the medulla oblongata, pons,
dorsal root ganglia and the roof of fore- and midbrain, as compared with the early stages. In
addition to its presence in the central nervous system (CNS), DGKθ was clearly detected in the
nasal cavity, primordia of the incisor tooth, lip, epidermis, and the follicle of vibrissa at E12.5 and
E14.5. At E17.5, the expression pattern was maintained in the CNS, as well as in the epithelia
of the respiratory apparatus and digestive apparatus.
These results suggest that DGKθ has important physiological roles not only in the brain, but
also in other tissues including the epidermis. Specifically, PKCη is predominantly expressed in
epithelial tissues and has been shown to interact with DGKθ in vitro. Most epithelia expressing
DGKθ also express PKCη, implying that DGKθ may be involved in epithelial differentiation via
regulation of PKCη.
2951
Characterization of dephosphorylation of the RNA-binding protein tristetraprolin by
PP2A.
I. O. Chase1, S. L. Clement2; 1Chemistry and Biochemistry, California Polytechnic State
University, San Luis Obispo, CA, 2Biological Sciences, California Polytechnic State University,
San Luis Obispo, CA
Post-transcriptional and post-translational processes play essential roles in regulating rapid
changes in gene expression. The RNA-binding protein tristetraprolin (TTP) mediates mRNA
decay by binding to adenylate-uridylate-rich elements (AREs) in their 3’ untranslated regions
and recruiting mRNA decay enzymes. The ability of TTP to recruit these decay factors is
sensitive to its reversible phosphorylation state induced by the p38 MAPK-activated kinase
MK2. Phosphorylated TTP recruits the adaptor protein 14-3-3, which impairs the ability of TTP
to activate mRNA decay. Consequently, the regulation of TTP phosphorylation allows cells to
quickly modify levels of ARE-containing transcripts encoding transcription factors, cytokines,
and proto-oncoproteins. While the phosphorylation of TTP has been well characterized, the
timing and mechanism of TTP dephosphorylation is poorly understood. TTP dephosphorylation
is presumed to occur by the action of the heterotrimeric protein phosphatase 2A (PP2A). This
enzyme is composed of three subunits: a scaffolding subunit (A), a catalytic subunit (C), and a
regulatory subunit (B, B’, or B’’) that dictates substrate specificity. The catalytic subunit of PP2A
has been shown to physically associate with TTP, while 14-3-3 is thought to prevent its
recruitment. In order to better understand the mechanism by which TTP is dephosphorylated,
we observed the phosphorylation status and relative levels of TTP, 14-3-3, and PP2A-C in
lipopolysaccharide (LPS) stimulated RAW 264.7 murine macrophages via Western blot. TTP
levels were found to be highest 12 hours after LPS exposure, and TTP dephosphorylation
commenced ~ 16 hours after LPS induction. PP2A-C levels increased approximately five-fold
during TTP dephosphorylation, while no clear trend was observed for 14-3-3. The correlation of
TTP dephosphorylation with increases in PP2A-C suggests this process may be regulated in
TUESDAY-LATE POSTER PRESENTATIONS
part by the modulation of catalytic subunit levels. In order to identify the PP2A regulatory
subunit responsible for targeting PP2A to TTP, experiments are currently underway to measure
relative levels of candidate PP2A regulatory subunits and investigate association between the
two proteins.
2952
Calcineurin dephosphorylates the yeast synaptojanin Inp53/Sjl3 to promote recovery
from osmotic stress.
E. Guiney1, J. Elias2, M. Cyert1; 1Biology Department, Stanford, Stanford, CA, 2Chemical and
Systems Biology, Stanford, Stanford, CA
Calcineurin (CN) is a highly conserved Ca2+ dependent serine/threonine protein phosphatase.
In Saccharomyces cerevisiae, CN is required for surviving a variety of stresses including ionic
toxicity (eg, Na+ and Li+), cell wall damage, and high pH. We are investigating the function and
localization of CN during the response to hyperosmotic stress, and sought to identify
physiological targets of CN-dependent signaling under these conditions. We found that in
response to hyperosmotic shock (>750mM KCl), the localization of CN-GFP changes from
diffuse localization in the cytoplasm to the bud tip, neck, and to cytoplasmic puncta. We took
two approaches to investigate the mechanism and functional relevance of this change in
localization. First, we examined CN localization after exposure to osmotic stress in a set of
deletion mutants that disrupt genes that function at the bud tip/neck or during osmotic stress.
This analysis showed that deletion of the bud neck localized actin cable nucleator Bnr1p
compromised the observed changes in CN localization. Depolymerization of the actin
cytoskeleton with Latrunculin-A for 10min before osmotic stress also prevented CN localization
to the bud tip/neck, suggesting that actin cables are required for CN trafficking during osmotic
stress.
Second, we used SILAC mass spectrometry to identify proteins that interact with CN
preferentially during osmotic stress, enabling molecular characterization of CN function in this
signaling condition. This approach identified the PIP2-phosphatase, Inp53 , homolog of the
mammalian CN substrate synaptojanin. We show that CN and Inp53 physically interact in vivo,
and that this interaction is substantially strengthened during hyperosmotic stress. This
interaction is also dependent on the presence of a PxIxIT docking motif in Inp53, and
phosphorylation of the Inp53 C-terminal domain is dependent on CN activity, consistent with
Inp53 being a yeast calcineurin substrate. We also show that cells partially compromised for
Inp53 function are sensitive to FK506. We are currently examining the effect of CN on
phosphoinositide abundance via whole cell labeling and chromatography. Finally, the actin
cytoskeleton depolarizes early in the osmotic stress response and its repolarization depends
partly on Inp53 activity; we found that inhibition of CN with FK506 caused a delay in the onset of
this repolarization. We propose a model of calcineurin activity during hyperosomotic stress:
immediately after osmotic stress, CN is specifically trafficked to the bud tip/neck via actin
cables; CN is then in position to promote actin repolarization and resumption of growth by
activation of Inp53. This work demonstrates a novel of a role for CN in the osmotic stress
response and in actin polarity, and is the first evidence of CN trafficking or scaffolding in yeast.
TUESDAY-LATE POSTER PRESENTATIONS
2953
Nanodiamond activates blood platelets and induces thromboembolism.
S. Kumari1, M. K. Singh2, S. K. Singh1, J. J. Grácio2, D. Dash1; 1Department of Biochemistry,
Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, 2Center for Mechanical
Technology & Automation, University of Aveiro, Aveiro, Portugal
Nanodiamond (ND) is considered to be one of the most attractive materials in nano dimension.
In view of its potential biomedical and industrial applications, thorough investigation of its
biocompatibility and biological interface becomes a research priority. Platelets are abundantly
circulating blood cells having major role in haemostasis and arterial thrombosis; hence, it is
crucial to determine the effect of ND on platelet patho-physiology and thrombogenecity. Here
we report for the first time that, carboxylated nanodiamond (cND) (4-10 nm) provoked significant
activation of human platelets by inducing rise in intracellular free calcium and content of tyrosine
phosphoproteome. Concomitantly, ND brought about prothrombotic changes in platelets as
revealed from increase in annexin V binding to platelet surface membrane, disruption in
mitochondrial transmembrane potential (ΔΨm) and generation of reactive oxygen species
(ROS). Administered intravenously in mouse, ND was found to evoke extensive pulmonary
thromboembolism, reflective of remarkable thrombogenic potential of this nanomaterial. Our
findings raise a concern on putative biomedical applications of ND pertaining to diagnostics and
therapeutics where its toxicity and prothrombotic properties should be critically evaluated.
2954
Mechanisms of inflammatory gene regulation by estrogen.
H. Tran1, C. Vallaster2, R. Karas2, G. Schnitzler2; 1Biology, Georgia State University, Atlanta,
GA, 2Molecular Cardiology Research Institute, Tufts University, Boston, MA
In premenopausal women, estrogen (E2) acts as a cardioprotectant, retarding atherosclerosis
and mitigating the inflammatory response in vascular tissue after damage. In earlier studies,
treatment of ovariectomized mice with E2 following vascular injury resulted in significantly
reduced injury compared to mice treated with a vehicle control. Our objective is to determine the
molecular mechanisms by which E2 attenuates the inflammatory response to vascular injury by
utilizing primary aortic smooth muscle cell (AoSMC) culture. Stimulation of AoSMCs with the
cytokine tumor necrosis factor-alpha (TNF-α) induces transcription of inflammatory marker
genes and treatment with E2 suppresses this effect. Pretreatment with E2 was sufficient in
suppressing this effect. Cells in which the non-genomic pathway is disrupted failed to suppress
inflammatory marker gene activation in response to TNF-α. Identification of the molecules that
mediate the rapid-signaling non-genomic effects of E2 will potentially aid in the development of
drugs that confer the beneficial anti-inflammatory and vasoprotective effects of estrogen.
2955
Localization analysis of MetE, a receptor for the Leaf-Opening Factor of the genus
Cassia.
S. Hamaguchi1, Y. Otsuka1, N. Kanzawa1; 1Dept of Materials and Life Sciences, Sophia
University, Tokyo, Japan
Leguminous plants open their leaves early in the morning and close them in the evening as if
they are sleeping. This periodic leaf movement is called nyctinasty and is controlled by circadian
clock and plant hormone. Furthermore, it is known that nyctinasty is regulated by changes in the
balance of concentrations between two endogenous bioactive substances: Leaf-Opening Factor
(LOF), which induces leaf opening, and Leaf-Closing Factor (LCF), which induces leaf closure.
These substances have been isolated from several leguminous plants and exhibit specificity for
TUESDAY-LATE POSTER PRESENTATIONS
plant genus. In addition, they have also been reported to localize in the motor cells, located in
the pulvini. Recently, methionine synthase (MetE) has been identified as a candidate for the
intracellular receptor of potassium isolespedezate which is a type of LOF for the genus Cassia.
However, the physiological meaning of the LOF binding to MetE is not yet revealed. Thus, we
have investigated the relevance of MetE and the nyctinastic movement towards the
understanding as to how leaves open. Our previous studies have shown that in Cassia
obtusifolia, the amount of MetE protein does not change with time and methionine synthase
activity of MetE also does not change regardless of the LOF-binding in vitro. Following these
results, we made a hypothesis that the binding of LOF and CoMetE induces changes in the
intracellular localization of MetE and eventually causes the leaves to open. To investigate this,
we examined localization of MetE in Cassia obtusifolia using a specific antibody. Western blot
analyses showed that MetE protein were present ubiquitously. Therefore, to observe the
subcellular localization of MetE in each tissue, indirect immunofluorescent observation was
performed on sections which were made in Steedman fs wax method using an anti-MetE
polyclonal antibody. As a result, MetE was mainly localized in the cytoplasm of the motor cells
of main and secondary pulvini involved in nyctinasty. In addition, apparent signals were also
observed in the nucleus of the cell. Careful inspection every six hours showed no change in the
localization of MetE in motor cells of the pulvini. From this, we concluded that the posttranslational modifications of MetE might be involved in the mechanism for opening the leaves.
In other tissues, MetE was localized in the cytoplasm and nucleus similarly. No nuclear
localization signal (NLS) was found on the sequence of MetE, suggesting that nuclear
localization of MetE is regulated by some unknown factors.
2956
Mapping the Period (PER) binding surface of the circadian clock protein Cryptochrome
(CRY).
K. E. Marz1, M. Jarvis1, A. Messelt1; 1Gustavus Adolphus College, Saint Peter, MN
In the mammalian circadian clock, central clock proteins PER and CRY inhibit transcriptional
activation by CLOCK and BMAL1, closing a negative feedback loop. They also interact with
other proteins in both the cytoplasm and the nucleus. In order to better understand the various,
and at times simultaneous, protein-protein interactions in which mCRY (mouse CRY) and
mPER (mouse PER) participate, we aimed to identify the surfaces of mCRY1 and mCRY2 that
bind mPER1 and mPER2. Previous research in other laboratories has identified three mCRY1
and mCRY2 mutations that reduce mCRY-mPER interactions. Based on homology models of
these proteins, the mutated residues are far apart, which suggests that they are near the edges
of a broad interacting surface. In order to compare PER binding by wild-type CRY and a panel
of site-directed CRY mutants, we expressed CRYs and PERs in HEK-293 cells, and used
immunocytochemistry to assess the extent to which the different CRYs increased PER nuclear
localization. We have identified a surface of mCRY1 that appears to be involved in binding both
mPER1 and mPER2.
2957
S-nitrosylated B23/nucleophosmin (NPM) regulates Siah1, preventing Siah1/GAPDHmediated neuronal cell death.
S. Lee1, K-H. Lee2, J-Y. Ahn1; 1Molecular Cell biology, Sungkyunkwan University School of
Medicine, Suwon, Korea, 2Anatomy, Sungkyunkwan University School of Medicine, Suwon,
Korea
The cell survival functions of the nucleolar protein nucleophosmin (NPM)/B23 have been
documented. Here we report a novel neuroprotective function of B23 through regulation of the
TUESDAY-LATE POSTER PRESENTATIONS
Siah1/GAPDH death cascade. B23 physiologically binds to both Siah1 and GAPDH, disrupting
the Siah1-GAPDH complex in the nucleus in response to NO stress. S-nitrosylation of B23 at
cysteine 275 by transnitrosylation from GAPDH diminishes S-nitrosylation of GAPDH and
reduces its interaction with Siah1. S-nitrosylation of B23 enhances B23-Siah1 binding and
mediates the neuroprotective actions of B23 by abrogating the E3 ligase activity of Siah1. In
mice, overexpression of B23 dramatically inhibits NMDA-mediated neurotoxicity whereas
expression of the C275S mutant, which is defective in binding to Siah1, and silencing of B23
does not prevent neurotoxicity. Thus, B23 regulates neuronal survival by preventing Siah1GAPDH death signaling under stress-induced conditions in the brain.
2958
Identification of Nuclear Localization Signal on O-GlcNAc transferase and its nuclear
import regulation.
H. Seo1, J. Ryum 1, J. Cho1; 1Yonsei University, Seoul, Korea
It has been reported that one of the downstream molecules generated from glucose is uridine
diphosphate-N-acetly glucosamine(UDP-GlcNAc) via the hexoamine biopsynthetic pathway
(HBP). The dynamic cycle of addition and removal of O-linked-N-acetlyglucosamine (O-GlcNAc)
to Ser/Thr residues is involved in regulating nuclear and cytoplasmic proteins.
Nucleocytoplasmic O-GlcNAc transferase (ncOGT) adds a single GlcNAc onto hydroxyl groups
of serine and threonine residues. Interestingly, O-GlcNAc glycosylation occurs in ncOGT itself
as well and several putative sites have been reported mainly within TPR domain. However, the
mechanism by which nuclear localization of O-GlcNAc transferase is not clear. Here, we
identified specific nuclear localization signals (NLS) in O-GlcNAc transferase that is required for
nuclear transport. The three amino acid domain in NLS inserts a non-diffusible protein to the
nucleus autonomously. Also, we show that ncOGT binds importin alpha proteins, and the
association between importin alpha proteins and ncOGT is interfered by O-GlcNAcylation on
TPR domain. This ongoing effort would give us clear understanding of the key enzyme of OGlcNAc metabolism.
2959
Regulation of phosphoglucomutase1 (PGM1) activity and elucidation of its function.
E. Bae1, D. Jung1, H. Kim2, E. Koh2, K. Kim2; 1Brain Korea 21 project for Medical Science,
Yonsei University, Seoul, Korea, 2Yonsei University, Seoul, Korea
Phosphoglucomutase1 (PGM1) catalyzes the interconversion between Glucose-1-phosphate
and Glucose-6-phosphate in carbohydrate metabolism. Glc-1-P and Glc-6-P act as key initiative
molecules for glycogenesis and glycolysis, respectively. So, PGM1 enzymatic activity is
important for both pathways. However, the specific regulation of its activity, other than its role in
the enzymatic process itself, is unknown. Here, we focused distinct four threonine residues at cterminus of PGM1. Using site-directed mutagenesis, we made several PGM1 mutants that each
threonine residues have replaced with glutamic acid or alanine residues. As a result, we found
the PGM1 mutant (T553E) has low protein expression through proteasomal degradation. In
addition, we showed that T562E mutant lost its activity. Furthermore, we investigated T562E
mutant-overexpressed HEK 293 cells showed reduced level of cellular O-GlcNAc modification
(O-GlcNAcylation). Using PGM1 mutants which have no enzyme activity, we realized OGlcNAcylation in several cells is dependent on PGM1 activity. The results imply that
posttranslational modification at C-terminus of PGM1 could regulate its activity and cellular
glycosylation.
TUESDAY-LATE POSTER PRESENTATIONS
2960
Regulation of enolase activity by acetylation.
D-Y. Jeong1, E. Bae1, H. Kim1, K-S. kim1; 1Brain Korea 21 Project for Medical Science, Yonsei
University, Seoul, Korea
Enolase is a metalloenzyme that catalyze the dehydration of 2-phospho-D-glycerate (2PG) to
phosphoenolpyruvate (PEP) in the second half of the glycolytic pathway. There are three
enolase isoforms, enolase1 (alpha-enolase), enolase2 (gamma-enolase) and enolase3 (betaenolase) with high sequence identity and tissue specific expression. Recently, it is reported that
many metabolic enzymes including enolase are acetylated. Although enolase1 in tumor cells is
known to be more acetylated than in normal tissues, roles of acetylation on enolase is not
known. We detected acetylated enolase1, enolase2 and enolase3 under the treatment of
trichostatin A (TSA), an inhibitor of histone deacetylases and also observed increased protein
levels. To eludicate the role of acetylation onenolases, enzymatic activity was measured utilizing
2PG and PEP as substrates and stability of enolase was tested. Furthermore, to identify the
acetylation sites, several putative lysine acetylation sites were mutated to arginine or glutamine
followed by enzymatic activity or protein stability was tested.
2961
A novel chemical glycoproteomics platform reveals O-GlcNAcylation of mitochondrial
voltage-dependent anion channel 2.
M. Boyce1, K. K. Palaniappan2, T. J. Smith1, M. J. Hangauer2, B. P. Smart2, A. A. Pitcher2, E. H.
Cheng3, C. R. Bertozzi2; 1Biochemistry, Duke University Medical Center, Durham, NC,
2
Chemistry, University of California, Berkeley, CA, 3Human Oncology and Pathogenesis
Program, Memorial Sloan-Kettering Cancer Center, New York, NY
Protein modification by O-linked β-N-acetylglucosamine (O-GlcNAc) is a critical cell signaling
modality, but identifying signal-specific O-GlcNAcylation events remains a significant
experimental challenge. We describe a new method for labeling and visualizing organelle- and
stimulus-specific O-GlcNAcylated proteins, and use it to identify the mitochondrial voltagedependent anion channel 2 (VDAC2) as an O-GlcNAc substrate. VDAC2-/- cells resist the
mitochondrial dysfunction and apoptosis caused by global O-GlcNAc perturbation,
demonstrating a functional connection between O-GlcNAc signaling and mitochondrial
physiology through VDAC2. More broadly, our method will enable the discovery of signalspecific O- GlcNAcylation events in a wide array of experimental contexts.
2962
Direct, single molecule observation of early lymphocyte signaling.
G. P. O'Donoghue1, R. Pielak1, A. Smoligovets1, J. Lin1, J. T. Groves2; 1Chemistry, UC Berkeley,
Berkeley, CA, 2HHMI, Chemistry, UC Berkeley, Berkeley, CA
T cells show a remarkable ability to quickly (in less than 10 seconds) respond to fewer than 10
agonist molecules even when structurally-similar self peptides outnumber agonist peptides by at
least 100:1. We recapitulate these phenomena using a highly controlled supported lipid bilayerlive cell junction. Using this approach we report direct, single molecule observations of T cell
receptor-peptide-MHC binding kinetics coupled with stochastic simulations in order to test serial
receptor triggering models within the context of our studies of membrane-proximal signal
amplification in living T cells.
TUESDAY-LATE POSTER PRESENTATIONS
2963
Unique localization of synaptic scaffolds in horizontal cell processes contacting
photoreceptors.
A. Vila1, C. M. Whitaker1, J. O'Brien1; 1Opthalmology and Visual Science, UTHSC, Houston, TX
Introducion: Synaptic processes and plasticity of synapses are mediated by large suites of
proteins. In most cases, many of these proteins are tethered together by synaptic scaffold
proteins. Scaffold proteins have a large number and typically a variety of protein interaction
domains that allow many different proteins to be assembled into functional complexes. As each
scaffold protein has a different set of protein interaction domains and a unique set of interacting
partners, the presence of synaptic scaffolds can provide insight into the molecular mechanisms
that regulate synaptic processes.
Methods: We examined the distribution of the MAGUK-containing synaptic scaffold proteins in
the rabbit outer plexiform layer (OPL) by multiple-label immunofluorescence and confocal
microscopy.
Known interacting partners were identified using NCBI-Gene and HPRD
databases to guide further immunofluorescence investigation. Horizontal cells (HC) were
labeled by microinjection of Neurobiotin.
Results: SAP102 immunoreactivity (IR) appeared as several round puncta in the lower part of
each cone pedicle and as a single weaker punctum in each rod spherule. The identity of the
cones was confirmed with GluR5, a marker that labels OFF bipolar cell dendrites at the base of
cone pedicles. SAP102-IR puncta were located slightly above GluR5, a finding suggesting that
SAP102 was located inside cone pedicles. In the rabbit, B-type horizontal cells send dendritic
processes into the invaginating synapses in cone pedicles while axon terminal processes enter
rod spherules. B-type HCs injected with Neurobiotin showed dendritic processes colocalized
with SAP102, confirming that B-type HCs contain the SAP102 clusters. Known SAP102 binding
partners, kainate receptor GluR6/7 and inward rectifier potassium channel KiR2.1, co-localized
with SAP102 in the tips of HC dendrites and axon terminals. In contrast, B-type HC connexin
Cx57 was present nearby in gap junctions but was not present in the synaptic complex.
Proteins in the synaptic complex may contribute both to feed forward signaling from
photoreceptors to HCs and in feedback signaling from HCs to photoreceptors. Ion channels
identified can contribute to ephaptic feedback signaling, but evidence for a gap junction
hemichannel for this purpose is still lacking in this species.
2964
Exosomal signaling analysis of cells activated by cold temperature perception.
S. Park1, S. Chun1; 1Department of Chemistry, Dongduk Women's University, Soeul, Korea
Exosomes are a discrete population of vesicles that are secreted from various cell types to the
extracellular media. Characterization of exosomes from different biological samples is useful in
understanding of cell-type-specific function and their communication. In order to find out protein
composition of exosomes that neuronal cell secretes in case of activation by low temperature
perception, we used human neuronal cell line. Exosomes were isolated from cells that were
incubated in 18 ¡É for 30 minutes, four times during 48 hours. The exosomal proteins were
analyzed by two-dimensional gel electrophoresis and MALDI/TOF mass spectrometry. In this
way, leucine-rich repeat-containing protein, immunoglobulin heavy chain variable region, solute
carrier family 25 member 31, phosphoinositide 3-kinase and zinc finger protein were identified
as target proteins transferred by exosome, when the cells were incubated at low temperature
condition
TUESDAY-LATE POSTER PRESENTATIONS
2965
Chemical biology defines a new role of cAMP microdomain during Gli trafficking.
C. H. Williams1, J. Hao2, A. Y. Frist2, C. Hong2; 1Cell and Developmental Biology, Vanderbilt
University, Nashville, TN, 2Division of Cardiovascular Medicine, Vanderbilt Medical Center,
Nashville, TN
Aberrant activation of hedgehog signals is involved in a wide range of pathologies including
various forms of cancer ranging from medulloblastoma to pancreatic adenocarcinoma.
Consequently, regulatory mechanisms of the hedgehog pathway have important therapeutic
implications. cAMP is known to modulate hedgehog signaling by regulating activation of the
hedgehog mediator Gli, but the mechanism remains largely undescribed. Here, we describe
eggmanone, a novel small molecule discovered in a zebrafish phenotypic screen as a
hedgehog inhibitor. Eggmanone is an unusual allosteric phosphodiesterase inhibitor with a
unique selectivity profile which disrupts the cilium-to-nucleus Gli trafficking by perturbing local
cAMP levels in the peri-cilium region around the basal body. Our chemical genetic study
utilizing a novel pharmaceutical class that specifically modulates local cAMP levels in the basal
body highlights the importance of this organelle in hedgehog regulation.
2966
New Near Infrared Cell Normalization Stains for Cell-based Assays.
K. Xing1, T. Urlacher1, X. Xu1, K. Schaepe1, N. Padhye1; 1LI-COR Biosciences, Lincoln, NE
Purpose: Normalization is important for quantitative cell-based immunoassays. Here we
present two near infrared cell stains and their use to normalize cell number in quantitative cellbased immunoassays. Methods: Two near infrared stains have been studied for their cell
normalization staining in different types of cells, including A431, RAW264.7, and NIH3T3 cells.
To visualize the staining, either live or fixed and permeabilized A431 cells were incubated with
the stains. After removing the excess stain, cells were detected with fluorescence microscope.
To investigate the linear relationship between fluorescence intensity and the cell number, serial
diluted cells were plated into 96-well plate and grown for 24 hours. Then the cells were fixed,
permeabilized and stained. Fluorescence was detected at 680nm or 780nm and quantified. To
demonstrate their compatibility with immunofluorescence assays, either EGF-stimulated A431
cells or PDGF-stimulated NIH3T3 cells were utilized to detect the appropriate targets together
with cell normalization staining by adding stains with secondary antibodies for the target
detection. Results: Fluorescence microscopy observed stains only in fixed and permeabilized
A431 cells and the fluorescence was more intense in the nuclei than in the cytoplasm. Both near
infrared stains displayed the linear relationship (with R2 more than 0.99) between the intensity of
fluorescence and the cell number in the range from 200 to 10240 cells/well. Cell staining with
either near infrared stains in the immunofluorescence assay were compatible with the
quantification of EGFR phosphorylation in EGF-stimulated A431 cells and Akt phosphorylation
in PDGF-stimulated NIH3T3 cells. Conclusion: Two new near infrared cell stains have been
demonstrated to be excellent for immunofluorescence cell-based assay normalization with
advantages of wide linear range, quantitative accuracy, and ease of use.
TUESDAY-LATE POSTER PRESENTATIONS
2967
The novel aberrant splicing A20 transcript contribute to rheumatoid arthritis (RA)
progression.
H. Byun1,2, H. Lee1,3, J. Jeon1, Y. Lee1, E-H. Jin2, J. Kim2, J. Hong1,2, J. Kim3, J. Seok1, S. Kang3,
W. Lee2, G. Hur1,2; 1Phamacology, College of Medicine, Chungnam National University,
Daejeon, Korea, 2Clinical Trial Center, Chungnam National University Hospital, Daejeon, Korea,
3
Division of Rheumatology, Chungnam National University Hospital, Daejeon, Korea
Aberrant splicing is one of the most significant components generating functional diversity in
many pathological conditions. The objective of this study was to anaylse the mutations or
aberrant splicing of A20 transcript the region encompassing ovarian tumour (OTU) domain
(which is functionally important as an inhibitor of NF-kB activation) in fibroblast-like synoviocytes
(FLSs) from RA patients. Alterations in A20 transcripts were determined through sequence
analysis of 10 clones of A20 cDNA in FLS from each of the five RA patients. The levels of
aberrant A20 transcript were measured by quantitative real-time RT-PCR with primers to
specifically recognize the inserted introns. The functional role of A20 and its aberrant variants
were examined by analysing NF-kB luciferase reporter activity and NF-kB-dependent target
gene expression. In RA FLS, we discovered four novel aberrant A20 transcripts, most of which
resulted from insertion of partial intron 2, intron 4 and/or deletion of exon 4. In each of these
FLSs, sequence analysis revealed that these aberrant insertional sequences were flanked by
consensus splice donor and acceptor sequences without nucleotide substitution, suggesting
alternative splicing as the likely mutational mechanism. These variants elicited a codon frame
shift by creating a premature translational stop codon, and eventually, disruption of OTU domain
(which is functionally important as an inhibitor of NF-kB activation) of A20. The expression level
of aberrant A20 transcript was correlated well with persisitently enhanced status of NF-kB
signalling, as evident by the phosphorylation of IkB-a and transcription of NF-kB target genes.
The results suggest that A20 inactivation by the novel aberrant splicing which may contribute to
RA progression by inducing persistent NF-kB activation. This work was supported by the
National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST)
(No. 2012-0005764), (KRF-2009-352-E00009), Chungnam National University Hospital
Research Fund 2010 and a grant from the National R&D Program for Cancer Control Ministry of
Health & Welfare, Repubic of Korea (No. 0720560). This study was also supported by a grant
(A070001) of the Korean Health Technology R&D Project, Ministry of Health & Welfare,
Republic of Korea.
Cell Polarity
2968
Apical vacuole formation by gastric parietal cells in primary culture — effect of low
extracellular Ca2+.
S. L. Nakada1, J. M. Crothers, Jr. 1, T. E. Machen1, J. G. Forte1; 1Molecular and Cell Biology, UC
Berkeley, Berkeley, CA
In primary culture, the gastric parietal cell’s deeply invaginated apical membrane, seen in
microscopy by phalloidin binding to F-actin (concentrated in microvilli and a subapical web), is
engulfed into the cell, separated from the basolateral membrane (which then becomes the
complete plasma membrane) and converted, from a lacy interconnected system of canaliculi,
into several separate vacuoles. In this study, vacuolar morphology was achieved by 71% of
parietal cells 8 hr after typical collagenase digestion of rabbit gastric mucosa, but the tight-
TUESDAY-LATE POSTER PRESENTATIONS
junctional protein ZO-1 was completely delocalized after ~2 hr, when cells were ready for
culturing. Use of low-Ca2+ medium (4 mM EGTA) to release cells quickly from gastric glands
yielded parietal cells in which ZO-1 was seen in a small spot or ring, a localization quickly lost if
these cells were then cultured in normal Ca2+, but remaining up to 20 hr if they were cultured in
low Ca2+. The cells in low Ca2+ mostly retained, at 20 hr, an intermediate morphology of many
bulbous canalicular expansions (“pre-vacuoles”), seemingly with narrow interconnections.
Histamine stimulation of 20-hr cells with intermediate morphology caused co-localization of
proton-pumping H,K-ATPase with canaliculi and pre-vacuoles, but little swelling of those
structures, consistent with a remaining apical pore through which secreted acid could escape.
Apparent canalicular interconnections, lack of stimulated swelling, and lingering ZO-1 staining
indicate inhibition of membrane fission processes that separate apical from basolateral
membrane and vacuoles from each other, suggesting an important role for extracellular Ca2+ in
these, and possibly other, endocytotic processes.
2969
The integrity of the PDK1 apical vesicular compartment is necessary to sustain atypical
PKC in polarized epithelial cells.
A. Mashukova1,2, R. Forteza2, F. Wald2, P. Salas2; 1College of Medical Sciences, Nova
Southeastern University, Davie, FL, 2Cell Biology, University of Miami, Miami, FL
Atypical PKC (aPKC) has a well-established role in the development of tight junctions and
epithelial polarity. We have shown that aPKC steady-state levels and active conformation are
dependent on intermediate filament (IF)-based rescue mechanism involving Hsp/Hsc70.
Furthermore, our previous experiments indicated that PDK1 might be involved not only in
phosphorylation of newly synthesized aPKC, but also in rephosphorylation of aPKC as a part of
the Hsp70 and IF-mediated refolding and rescue. Yet it is well established that active PDK1 is
associated to the plasma membrane via phosphatidylinositol (3,4,5)-trisphosphate (PIP3)–
dependent and –independent mechanisms, whereas the atypical PKC rescue mechanism that
we have identified occurs on IFs. Thus, to fully understand the aPKC rescue mechanism, it was
essential to determine the intracellular compartment in which aPKC rephosphorylation takes
place as well as subcellular localization of the kinase responsible for this process. To address
these questions we conducted confocal and transmission electron microscopy experiments on
differentiated Caco-2 cells and on the sections of mouse intestinal epithelium. We found that
PDK1 localized to the apical pole of the epithelial cells in the same region of the terminal web
IFs. Our data showed that PDK1 is associated with the apical plasma membrane and apical
endosomes, including apical recycling endosome (ARE). Moreover, PDK1 seems to distribute to
more than one vesicular compartment, as it also colocalized with apical vesicles carrying
transferrin (Tfn) as well as in the apical membrane. To independently characterize the apical
PDK1 membrane compartment, we conducted cell fractionation and separation of endosomal
compartments in sucrose gradients. PDK1 signal comigrated with Rab11—a marker of ARE and
with the Tfn-containing fractions, confirming the confocal microscopy results. In addition, we
found that dynamin knockdown inhibits PDK1 activity and results in steep decrease of aPKC
levels. Altogether, our data support two major conclusions: first, that PDK1 is necessary and
sufficient to aid the IF-based rescue of aPKC, and second, that PDK1 is exquisitely localized to
apical vesicles and apical plasma membrane in intestinal epithelial cells. These findings are
consistent with the model in which aPKC is refolded by IF-associated Hsp70 being immediately
phosphorylated by PDK1 in adjacent endosomes.
TUESDAY-LATE POSTER PRESENTATIONS
2970
Establishment and Characterization of Primary Epithelial Cells Displaying Acinar Markers
from Human Salivary Gland Tissue.
S-I. Jang1, H-L. Ong1, A. Gallo1, X. Liu1, W. Swaim1, M. Grisius1, I. Ambudkar1, G. Illei1, I.
Alevizos1; 1NIH, Bethesda, MD
Disorders of human salivary gland resulting from therapeutic radiation treatment for head and
neck cancers or from the autoimmune disease Sjögren's syndrome (SS) frequently resuls in the
reduction or complete loss of saliva secretion. Such irreversible dysfunction of the salivary
glands is attributed to the impairment of acinar cells, the major glandular cells of ion secreting
and fluid movement. Availability of primary epithelial cells from human salivary gland tissue
(pHSG) is critical for studying the underlying mechanisms of these irreversible disorders.
Previous attempts at isolating pHSG have involved a combination of enzymatic digestion
(collagenase, hyaluronidase, Liberase Blendzyme 3 and trypsin) and culture in a serum–free
medium supplemented with specific growth factors. Those cells however exhibit characteristics
and properties of striated and excretory duct cells, not acinar cells. We applied explant culture
techniques on minor salivary gland biopsies from health control patients and used for
optimization on growing primary acinar cultures. Explants were first maintained in Basal
Epithelial Medium containing low content of fetal bovine serum. pHSG cells were subsequently
grown in Keratinocyte Basal medium (KBM) supplemented with various growth factors.
Expression of acinar and ductal markers was monitored in pHSG cells using qPCR, Western
blotting, and immunofluorescence staining. Under serum-free and low calcium supplemented
KGM growth condition, the pHSG cells expressed the acinar markers AQP5, cystatin C, NKCC1
and α-amylase. These cells also expressed the progenitor cell marker keratin 5 and stained
positive for Nanog, a stem cells marker. Expression of duct cells markers, kallikrein, keratin 19,
was relatively low. Additionally, pHSG cells formed a monolayer on filter support and display
high level of “Transepithelial Electrical Resistance” suggesting tight junction formation for these
cells. Taken together, suitable growth conditions have been established to isolate and support
culture of acinar-like cells from human salivary gland. These primary epithelial cells can be
useful to study at the cellular and molecular levels the loss of salivary gland function in a variety
of pathologies.
2971
More than a mark: The role of Rsr1 in symmetry breaking in budding yeast.
S. E. Smith1,2, I. Mendes Pinto1, A. Das1, B. Slaughter1, R. Li1,2; 1Stowers Institute for Medical
Research, Kansas City, MO, 2Department of Molecular and Integrative Physiology, University of
Kansas Medical Center, Kansas City, KS
The question of how cells establish a polar axis is vital to a range of essential processes across
the spectrum of life, from cell migration in human development to polarized cell division in
budding yeast. The budding yeast Saccharomyces cerevisiae is an ideal model for the study of
symmetry breaking: Although yeast generally bud in a defined pattern guided by sites of
previous cell division (bud scars), they polarize with equal efficiency, albeit in random
orientations, when bud scar cues are eliminated or cells lose the ability to recognize them. The
conserved small GTPase Cdc42 is the central regulator of polarization in yeast. Activated by
the guanine nucleotide exchange factor (GEF) Cdc24, Cdc42GTP accumulates in a polar cap and
works through downstream effectors to direct polarized growth and cell division. Both Cdc24
and Cdc42GTP physically interact with the adaptor Bem1, which is thought to facilitate positive
feedback by recruiting GEF to a nascent cap of Cdc42GTP, resulting in amplification of the cap
through activation of nearby molecules of Cdc42. A frequently used method for abrogation of
bud scar signaling is the deletion of the Ras related GTPase Rsr1, which is thought to be an
TUESDAY-LATE POSTER PRESENTATIONS
intermediary between the bud scar and the polarization module. The requirement for Bem1 for
viability in the rsr1Δ background led to the proposal that Bem1 mediated positive feedback is
responsible for symmetry breaking polarization. In this work we examine a second symmetry
breaking background in which upstream bud scar components have been deleted, and find that
Rsr1 itself may have a role in symmetry breaking.
2972
A Novel Microtubule Binding Protein, MARKAP1, Regulates Stability of the GolgiNucleated Microtubules and Directional Cell Migration.
Y. Satoh1, K. Hayashi1,2, Y. Amano1, M. Takahashi3, I. Hayashi4, S. Ohno1, A. Suzuki1,2;
1
Department of Molecular Biology,Graduate School of Medical Science, Yokohama City
University, Yokohama-City, Japan, 2Division of Medical Bioscience.Graduate School of Medical
Science., Yokohama City University, Yokohama-City, Japan, 3Teikyo Heisei University, Ichihara,
Japan, 4Department of Supramolecular Biology, Graduate School of Nanobioscience,
Yokohama City University, Yokohama-City, Japan
In mammalian cells, the Golgi apparatus (GA) composed of stacks of flattened cisternae are
laterally connected to form a ribbon-like structure (the Golgi ribbon), and localized to the
perinuclear region and in close vicinity to the centrosomes. The critical importance of the
radially-emanating centromal microtubules (MTs) for the perinuclear positioning of the Golgi
ribbon have been recognized. Furthermore, recent studies established the indispensable role of
another subset of MTs, the Golgi-nucleated (GN-) MTs, for the assembly and maintenance of
the laterally-expanded structure of the Golgi ribbon. These studies further revealed that the
maintenance of the Golgi ribbon structures is essential for polarized vesicle transport and the
resultant directional cell migration. CLASPs and AKAP450 /CG-NAP were shown to play
indispensable roles for MT nucleation from the Golgi membranes. However, the detailed
molecular mechanisms regulating this novel MT subset remain to be clarified. Particularly, the
molecular basis of how GN-MTs are specifically stabilized is almost unknown.
We recently identified an uncharacterized coiled-coil protein named MARKAP1 (Par-1 /MARK
Associated Protein 1) as a novel MT-crosslinking protein. Here, we demonstrate that MARKAP1
is an important player involved in stabilization of the GN-MTs and formation of the Golgi ribbon
structure. Endogenous MARKAP1 in Hela-Kyoto and hTERT-RPE1 cells shows subcellular
localizations to the GA and the Golgi-associated MTs. MARKAP1 knockdown reduces
accumulation of acetylated (stabilized) MTs associated with the GA and transformed the Golgi
ribbon structure into a circular morphology around the centrosome. These defects of MARKAP1
knockdown cells are rescued by expression of RNAi-resistant MARKAP1, and this rescue
activity is dependent on the evolutionarily-conserved C-terminal MT-binding region of
MARKAP1, which is responsible for MT bundling and stabilization. As observed in CLASPs or
AKAP450 /CG-NAP -knockdown cells, MARKAP knockdown cells exhibit severe defects in
directional cell migration. Furthermore, MARKAP1 interacts with both CLASPs and AKAP450
/CG-NAP, and simultaneous depletion of CLASPs and AKAP450 /CG-NAP disrupt the GAlocalization of MARKAP1. Taken together, these results indicate that MARKAP1 is a novel MTbinding protein which cooperates with CLASPs and AKAP450 /CG-NAP to promote the
formation and the stabilization of GN-MTs and thus regulate polarized cell migration.
TUESDAY-LATE POSTER PRESENTATIONS
2973
Identification and characterization of novel epithelial cell polarity determinants in
Drosophila melanogaster.
G. Jeyanathan1, U. Tepass1; 1Cell and Systems Biology, University of Toronto, Toronto, ON,
Canada
The polarization of epithelial cells into distinct apical and basolateral domains is crucial for
proper animal development. Several findings over the last two decades have led me to the
understanding of the mechanisms that underlie epithelial cell polarity in flies. Recently,
intracellular trafficking of proteins has been implicated in the regulation of this polarity. However,
the exact mechanisms of how trafficking regulates polarity remains unclear. Moreover, not all of
the major regulators of epithelial cell polarity have been identified. To this extent, we performed
a genome wide deficiency screen looking for mutations that affect the normal distribution of the
apical protein, Crumbs. Of the 240 deficiency lines analysed three deficiency lines show
promising defects in the distribution of Crumbs. Two of these lines overlap with each other, and
show a similar phenotype where Crumbs accumulates in large vesicle-like structures that
resemble multi-vesicular bodies. Interestingly, only apical but not basolateral proteins
accumulate in these vesicular structures suggesting a polarity specific phenotype. We isolated a
gene in this region, shrub, to be the potential candidate due to its role in the formation of multivesicular bodies. Further characterisation of shrub suggests a role in the modulation of apical
polarity proteins in the membrane. The third deficiency, Df(2L)Exel6031, shows large holes in
the ventral epidermis of Drosophila embryos with fragmented distribution of Crumbs,
characteristic of mutations in many known epithelial polarity regulators. A cell cycle regulator,
fzy, was identified as a potential candidate responsible for the observed phenotype. In these
mutants, ventral epidermal cells are arrested in metaphase and are eventually lost from the
epidermis. It is possible that cell division poses an additional stress that sensitizes these cells
to polarity defects. This could be an explanation as to why the neuroectoderm that goes
through cell division for longer is more sensitive to defects in polarity. Further characterisation
of the defects in these three deficiency lines could potentially lead to the identification of novel
cell polarity determinants and to a better understanding of the link between epithelial cell polarity
and cellular machineries such as intracellular trafficking and cell division.
2974
A novel microtubule binding protein, MARKAP1, cooperates with PAR-1 for the
development of the lateral microtubule bundles in epithelial cells.
M. Akitsu1, Y. Satoh1, Y. Amano2, K. Yamashita1, A. Yamashita1, T. Maki3, I. Hayashi3, H.
Hirano3, S. Ohno1, A. Suzuki1,2; 1Molecular Biology, Yokohama City University, Graduate School
of Medical Science, Yokohama, Japan, 2Medical Bioscience, Yokohama City University,
Yokohama, Japan, 3Supramolecular Biology, Graduate School of Nanobioscience, Yokohama
City University, Yokohama, Japan
Spatial organization of microtubule (MT) cytoskeleton is critical for many cellular functions.
Particularly, it is clearly indicated in the developmental process of apicobasal polarity of
epithelial cells, which is intimately linked to the dramatic reorganization of MTs from a radial
array into a vertical alignment of non-centrosomal MT bundles along the lateral membrane and
a meshwork under the apical and basal membrane. However, little is known about the
underlying molecular mechanism of this MT remodeling upon polarization. The evolutionarilyconserved cell polarity-regulating kinase, PAR-1 is essential for maintenance of the dynamic
state of MTs, and plays indispensable roles to promote this process. Here, we demonstrate that
a novel PAR-1-binding protein, named MARKAP1 (PAR-1/MARK-associated protein 1)
cooperates with PAR-1 for the development of the lateral MT bundles of epithelial cells.
TUESDAY-LATE POSTER PRESENTATIONS
MARKAP1 specifically colocalizes with the lateral MT bundles in epithelial cells. The N-terminal
and C-terminal basic regions of MARKAP1 flanking the central coiled-coil region independently
interacts with MTs and induces MT bundling. MARKAP1 knockdown in MDCK cells impairs the
development of the lateral MT bundles and epithelia-specific columnar shape, and the Nterminal MT-binding region is required to restore these defects of knockdown cells. MT regrowth
assay after nocodazole treatment indicated that MARKAP1 is not required for the initial radial
growth of MTs from the apical centrosome, but is essential for the accumulation of noncentrosomal MTs at the sublateral regions. Taken together, these results suggest that
MARKAP1 promotes development of the lateral microtubule bundles by accumulating noncentrosomal MTs at the sublateral regions through its MT-crosslinking activity. MARKAP1 can
recruit PAR-1 to the lateral MT bundles, on which a minor subset of PAR-1 endogenously
colocalizes with MARKAP1. Furthermore, the PAR-1-binding is required for MARKAP1 to exert
complete activity to promote the lateral MT development. These results suggest a possibility
that PAR-1 cooperates with MARKAP1 by regulating MT dynamics in the lateral MT bundles
induced by MARKAP1.
2975
Actin stabilizer induces formation of motile thick actin bundles in fission yeast cells.
J. Kashiwazaki1, I. Mabuchi1; 1Department of Life Science, Faculty of Science, Gakushuin
University, Tokyo, Japan
Actin cytoskeleton has been known to be responsible for various cellular activities, such as cell
morphogenesis, polarized growth, and cytokinesis. In the fission yeast Schizosaccharomyces
pombe, actin filaments form three major structures; patches, cables, and the contractile ring.
During interphase, actin cables extend from the growing ends toward the cell equator. Although
it has been reported that actin cables are required for polarized growth of the cell, their detailed
behavior has not been clear. Recently, we found that anomalous thick actin bundles were
formed by treatment with an actin stabilizer and moved along long axis of the cell during
interphase. In this study, we tired to elucidate the mechanism of this movement and behavior of
actin cables during interphase. To identify factors which are involved in formation and/or
movement of the actin bundles, we treated various mutant cells with the actin stabilizer. We
found that tropomyosin is required for the thick actin bundle formation. Type V myosins and the
formin For3 are required for the rapid movement of the actin bundles. Next we investigated the
behavior of normal actin cables during interphase without actin stabilizer. There are two type V
myosins (Myo52/ Myo4 , Myo51/ Myo5) in fission yeast. Myo52 is known to transport the
membrane vesicles along actin cables to promote polarized growth during interphase. On the
other hand, both function and behavior of Myo51 during interphase have not been clear. We
examined localization of Myo51-GFP during interphase in detail. Myo51-GFP was observed as
filamentous structures which colocalized with actin cables. Next we investigated the behavior of
actin cables in the type V myosin-depleted cells. We found that in myo51Δmyo52Δ double
mutant cells, actin cables failed to elongate toward the cell equator and undulated at the cell
ends. Furthermore, the temperature sensitivity of the double mutant was severer than each
single mutant strain. These results suggest that type V myosins maintain proper orientation of
actin cables during interphase for polarized growth.
TUESDAY-LATE POSTER PRESENTATIONS
2976
Gravity-stress affects polar growth in plant cells.
Y. Chebli1, L. Pujol1, A. Shojaeifard1, J. J. van Loon2, A. Geitmann1; 1University of Montreal,
Montreal, QC, Canada, 2Dutch Experiment Support Center, European Space Agency, Nordwijk,
Netherlands
Plants are able to sense the magnitude and direction of gravity. Specially equipped cell types
within the plant body are known to accomplish this based on statolith sedimentation. However,
most plant cells do not possess statoliths, yet they respond to changes in gravity levels. To
understand the effect of gravity on the metabolism and cellular functioning of non-specialized
plant cells, we investigated the pollen tube, a cellular protuberance with highly polarized growth
pattern and extremely active metabolism. The effects of hyper-gravity and omnidirectional
gravity (micro-gravity) on intracellular trafficking and on cell wall assembly were assessed by
live cell imaging. Using an epi-fluorescence microscope mounted on the Large Diameter
Centrifuge at the European Space Agency, we were able to demonstrate that vesicular
trafficking is reduced under hyper-g conditions. Immuno-cytochemistry confirmed that both in
hyper and omnidirectional gravity conditions, the characteristic spatial profile of cellulose and
callose distribution in the pollen tube wall was altered, in accordance with a dose-dependent
effect of gravity stress on pollen tube diameter. Our findings suggest that the pollen tube
responds by modifying cell wall assembly to compensate for the altered mechanical load
caused by gravity stress.
2977
Mechanism of meiotic I spindle migration and symmetry breaking in mouse oocytes.
K. Yi1, B. Rubinstein1, J. R. Unruh1, F. Guo1, B. D. Slaughter1, R. Li1; 1Stowers Institute for
Medical Research, Kansas City, MO
Asymmetric spindle positioning and cortical polarity establishment ensure the successful
extrusion of small polar bodies during oocyte maturation. In meiosis I, the spindle is formed in
the cell center and then moves to a subcortical location. Pioneering work in the field
demonstrated that actin cytoskeleton and its nucleator Fmn2 are required for this process,
although the underlying mechanism is not well understood. Our previous observations that
Fmn2 localizes to the vicinity of the chromosomes and that an actin cloud is associated with
migrating spindle lead to a hypothesis that Fmn2 nucleated actin polymerization may push the
chromosomes/spindle toward the cortex. Here, we show that Fmn2 is recruited to the ER
structures surrounding the spindle during migration. We further explored the actin cytoskeletonmediated chromosomes/spindle migration and found that this process is driven sequentially by
Fmn2 and the Arp2/3 complex mediated actin-base forces. We will provide evidence that
supports a bi-phasic spindle migration model, in which consecutive actin based pushing forces
drive the chromosome/spindle migration.
TUESDAY-LATE POSTER PRESENTATIONS
Development and Morphogenesis II
2978
Regulation of stem cell niche organization and specification in the Drosophila ovary.
D. Godt1, T. Panchal1, E. Alchits1; 1Cell and Systems Biology, University of Toronto, Toronto,
ON, Canada
Cap cells are considered the main niche cells for the germline stem cells (GSCs) in the
Drosophila ovary. These oval-shaped cells are organized as a cap-like cluster at the tip of the
germarium where they are in direct contact to the GSCs. Above the cap cell cluster is a terminal
filament (TF), a stalk consisting of stacked-up disc-shaped cells. We present data indicating that
the ability of cap cells to organize into a cluster is a prerequisite for the formation of a large stem
cell niche that can sustain three GSCs. Cluster formation of cap cells depends on the somaspecific large Maf transcription factor Traffic jam (Tj). Even a moderate reduction of Tj function
results in a displacement of cap cells away from the germarium into the terminal filament. This
causes the formation of unusually long stalks. When cap cells form a stalk, they only can recruit
and maintain a single GSC. In the complete absence of Tj, cap cells do not only behave like TF
cells but also express markers in a TF cell-specific manner. The transformed cells are still able
to recruit a GSC at the prepupal stage but cannot maintain it. Furthermore, our findings suggest
that the primary role of the Notch pathway appears to be the regulation of the number of cap
cells whereas Tj regulates the specification and morphogenetic behavior of cap cells. Together
they regulate the size of the GSC niche.
2979
Role of Epididymis in the Recognition and Elimination of Defective Spermatozoa.
E. L. McLean1, G. E. Alvarez1, S. K. Nagdas1; 1Chemistry and Physics, Fayetteville State
University, Fayetteville, NC
Even though the epididymis produces an environment promoting sperm maturation and viability,
the presence of a nonviable sperm population in this region has been observed in many
species. Degenerating spermatozoa release enzymes or antigens that could have detrimental
effects on the viability of the neighboring cells and may be a source of autoantigens that could
induce an immune response if they escape from the blood-epididymis barrier. Anti-sperm
antibodies are not uncommon in men, they develop for reasons that are not understood and
they are a source of infertility that is difficult to correct. What are the protective strategies of the
epididymis that could prevent these negative impacts on male fertility? Previously, Nagdas et al
(Biol. Reprod., 63, 1428-36, 2000) identified a 64kDa glycoprotein (termed HEP64), secreted by
principal cells of the hamster proximal cauda epididymidis that specifically binds defective
spermatozoa. Both luminal fluid and sperm-associated HEP64 are assembled into disulfidelinked oligomers of ~260kDa and ~280kDa. Olson et al (JBC, 279, 51266-74, 2004) cloned
HEP64 and identified it as the fibrinogen-like protein-2 (fgl2). The objective of the present study
is to identify the subunit composition of the fgl2-containing oligomers.
Both coimmunoprecipitation analysis, using anti-fgl2, and sequential nonreduced followed by disulfidereduced, SDS-PAGE identified both fgl2 and a 31kDa polypeptide as subunits of each oligomer.
Likewise SDS-PAGE analysis of an epididymal fraction enriched for polymerized fgl2 also
identified a major polypeptide of 31kDa. Using proteomic analyses and a PCR-based cloning
strategy, the 31kDa polypeptide has been fully sequenced and identified as fibrinogen-like
protein-1 (fgl1). Northern blot analysis and in situ hybridization demonstrated low fgl1
expression in the caput and corpus regions, but high expression in principal cells of the proximal
cauda epididymidis, as previously found for fgl2. Immunostaining using a fgl1-specific
TUESDAY-LATE POSTER PRESENTATIONS
polyclonal antibody revealed that fgl1 also binds the defective sperm population in the cauda
epididymidis. This study is the first to show an association of fgl1 and fgl2 and demonstrates
that they are disulfide-linked subunits of the 260 and 280kDa oligomers secreted by the
epididymidis. Our results provide new insights into the mechanisms by which the potentially
unique epididymal protein functions in the recognition and elimination of defective spermatozoa.
Supported by NIH/NIGMS/ 1SC3GM096875-02 and FSU McNair Program.
2980
Mitochondrial Peroxiredoxin 3 protein expression during sperm development in
>Drosophila melanogaster.
T. M. Onorato1, J. E. Rollins2; 1Natural Sciences, LaGuardia Community College, Long Island
City, NY, 2Natural Sciences, College of Mount Saint Vincent, Riverdale, NY
Peroxiredoxin 3 (Prx3) is a mitochondrial thioredoxin-dependent peroxidase that neutralizes
excess peroxides, and helps protect against oxidative-stress-induced apoptosis. The gene DPx5037 in Drosophila melanogaster encodes for a homologue of mammalian Prx3. DPx-5037
mRNA is expressed in all stages of Drosophila development. However, Prx3 protein expression
in the Drosophila testes has not been documented. We utilized immunofluorescence
microscopy to examine the expression of Prx3 in the fly testis. Whole mounts and squashes
were prepared from testes dissected from 0-3 day old flies. No signal was detected in control
samples that were incubated without anti-Prx3 serum or heated anti-Prx3 serum. Signal was
detected when samples were incubated with anti-Prx3 serum. Prx3 appears to be expressed in
spermatogonia, spermatocytes, round haploid spermatids and elongating sperm. Prx3 signal is
detected in the nebenkern (fused mitochondria) as well as in the unfurling mitochondrial
derivative confirmed by concurrently staining the mitochondria with Mito-ID. In addition to
establishing Prx3 protein expression in the adult fly testis, we also examined whether Prx3 is
expressed in the larval testis. Whole mounts of testes dissected from wandering third instar
larvae were prepared. Prx3 protein was detected in the larval testis. In summary, our results
indicate that the Prx3 protein is expressed in both larval and adult fly testes. Prx3 localizes to
the nebenkern and the unfurling mitochondrial derivatives. Interestingly, the pattern of Prx3
protein expression in elongated sperm tails is similar to donjuan (a mitochondrial protein
expressed in post meiotic elongating spermatids). Further studies will be conducted to
determine the role of Prx3 in Drosophila sperm development and its role in testicular oxidative
stress.
2981
Stage-dependent analysis of mRNP proteins in oogenesis of Xenopus leavis.
B. Bahrani1, A. Keshvara1, J. Guenther 1, Z. Belak1, N. Ovsenek1; 1Anatomy and Cell Biology,
University of Saskatchewan, Saskatoon, SK, Canada
Early development relies on maternally transcribed mRNAs stored in complex with cytoplasmic
RNA-binding proteins in messenger ribonucleoprotein particles (mRNPs). The major mRNP
proteins are FRGY2A/B, RAP55, and Xp54. The significance of mRNPs in development as
regulators of maternal mRNA stability and selective translation during oogenesis and
embryogenesis is well established in the literature. The canonical view is that mRNP proteins
accumulate temporally in proportion to the quantity of maternal mRNA expressed from the
maternal genome in oocytes, and that they assemble into mRNP storage particles with long
term stability. However, a review of the literature shows that this model relies on a number of
assumptions that have never been fully tested experimentally. As a result, a number of
questions remain outstanding about the form and function of mRNPs throughout oogenesis and
TUESDAY-LATE POSTER PRESENTATIONS
early embryonic development. As a first step to addressing these gaps in our understanding of
mRNP biology, our objective was to examine in detail the stage-dependent expression patterns
of the major mRNP proteins during oogenesis, as well as their subcellular localization and
interactions with maternal mRNA. The resulting data contradicted the established model of
mRNP accumulation in several ways. The quantity of RAP55 increased progressively through
oogenesis, while FRGY2A/B and Xp54 peaked at stage III and declined through later stages. All
of the mRNP proteins were entirely localized to the cytoplasm throughout oogenesis, and we did
not observe partial nuclear localization of some mRNP proteins as described previously.
Analysis of oocyte lysates by size exclusion chromatography revealed that all four major mRNP
proteins were entirely associated with RNase-sensitive complexes in stage VI oocytes,
however, oligo-dT cellulose chromatography demonstrated that only a portion of mRNP proteins
were associated with poly-A+ mRNAs. For FRGY2A/B and XP54, the levels of mRNA-bound
proteins were highest in stages I-III then declined, reaching lowest levels at stage VI. In
contrast, RAP55 assembled into mRNPs from stage I to II, then remained relatively constant
through to stage VI. Taken together, our data show that mRNP proteins are most highly
expressed and associated with mRNA during stages of peak transcription (III/IV). This contrasts
with the previous model held that levels and mRNA association of mRNP proteins is highest in
stage VI, when oocytes contain maximal quantities of poly-A+ mRNA.
2982
Generation of Advanced Spermatogenic Cells from Non-human Primate Pluripotent Stem
Cells.
M. E. Merritt1, B. T. Phillips2, C. R. Simerly2, K. E. Orwig2, G. P. Schatten2, C. A. Easley1; 1Cell
Biology, Emory University School of Medicine Laboratory of Translational Cell Biology, Atlanta,
GA, 2OB-GYN and Reproductive Sciences, University of Pittsburgh Division of Developmental
and Regenerative Medicine, Pittsburgh, PA
We have recently shown that human embryonic (hESCs) and induced pluripotent stem cells
(hiPSCs) can be differentiated into advanced spermatogenic cells including round spermatids.
Here, we show that non-human primate, rhesus embryonic (nhpESCs), somatic cell nuclear
transfer-derived embryonic (nhpNT-ESCs), and induced pluripotent stem cells (nhpiPSCs) can
be differentiated into spermatogonial stem cell like cells that express PLZF, a marker for stem
and progenitor spermatogonia, and VASA, a known germ cell marker expressed by several
germ cell types. Differentiating nhp pluripotent stem cells also produce haploid cells that
express acrosin and protamine 1, proteins that are uniquely expressed on spermatids. We are
currently assessing whether these haploid spermatids are capable of fertilizing rhesus oocytes.
If successful, this model will provide the first evidence that functional gametes can be derived
from non-human primate pluripotent stem cells and may provide insight into potentially novel
methods for treating human male infertility.
2983
The Role of Smaug in Posttranscriptional Regulation.
L. E. Chen1, X. Li1, Q. Morris1, C. Smibert1,2, H. Lipshitz1; 1Molecular Genetics, University of
Toronto, Toronto, ON, Canada, 2Biochemistry, University of Toronto, Toronto, ON, Canada
Smaug is a sequence-specific RNA-binding protein (RBP) that regulates the translation and
stability of maternally loaded mRNAs in early Drosophila embryos. In this study, we have
performed RNP immunoprecipitation-microarrays (RIP-chip) to systematically identify mRNAs
that are associated with Smaug in vivo. 384 transcripts were found to be enriched in Smaug
immunoprecipitates. More than 65% of mRNAs associated with Smaug are also dependent on
Smaug for degradation. Smaug target transcripts are enriched for the stem-loop structures that
TUESDAY-LATE POSTER PRESENTATIONS
Smaug binds (Smaug recognition elements or SREs). They are also enriched in posterior and
pole cell localized transcripts in stage 4-5 embryos. Gene Ontology (GO) analysis revealed that
Smaug's targets are enriched for those encoding subunits of proteasome regulatory particles
and metabolic enzymes. Transcripts encoding these proteasome and metabolic proteins carry
SREs, consistent with direct regulation. Detailed analyses are being carried out on these
targets. Preliminary data suggest that Smaug regulates proteasome function and ubiquitinmediated protein degradation.
2984
Histone H2B ubiquitination in the mouse preimplantation embryos.
M. Ooga1, F. Aoki1; 1The University of Tokyo, Kashiwa, Japan
During preimplantation development, gene expression pattern is altered dynamically. It has
been known that ubiquitination of histone H2B (ubH2B) has crucial roles in the regulation of
transcriptional initiation and elongation in somatic cells. However, the roles of ubH2B in the
regulation of gene expression during preimplantation development have not been clarified.
Here, we investigated the dynamics of ubH2B during oogenesis and preimplantation
development. In the analysis by immunocytochemistry, ubH2B was not detected in fully-grown
and MII stage oocytes that are transcriptionally inert. On the other hand, ubH2B increased along
with the transcriptional activity after fertilization until 2-cell stage. ubH2B thus seemed to
correlate with transcriptional activity in these stages. The inhibition of transcription using αamanitin decreased the level of ubH2B in both oocytes and embryos, suggesting that ubH2B is
involved in the maintenance of transcriptionally active states of the chromatin in these cells. To
assess the significance of ubH2B in the regulation of preimplantation development,
Bre1A/RNF20, a H2B specific ubiquitin E3 ligase, was knocked down by using siRNA. In the
embryos which had been treated with the siRNA, the levels of Bre1A mRNA and ubH2B
decreased at 4-cell and morula stages. Intriguingly, these embryos normally developed until
morula stage but a half of them did not developed into the blastocyst stage. Thus, ubH2B is not
required for regulatory mechanisms of gene expression during early preimplantation
development but it becomes essential at the blastocyst stage.
2985
Characterization of the gene expression in the 1-cell stage embryos.
K. Abe1, R. Yamamoto1, G. Oka1, V. Franke2, K. Vlahoviček2, Y. Suzuki3, F. Aoki1; 1Department
of Integrated Biosciences, The University of Tokyo, Kashiwa, Japan, 2University of Zagreb,
Zagreb, Croatia, 3Department of Medical Genome Sciences, The University of Tokyo, Kashiwa,
Japan
It has been known that in the mouse embryos, the onset of gene expression occurs at the mid
to late 1-cell stage. However, the mechanism regulating this onset and the profile of expressed
genes remains to be clarified. We generated genome-wide gene expression profiles of oocytes
and pre-implantation embryos by next-generation sequencing. The analysis for the transcribed
regions revealed that transcription occurs across the genome including the intergenic regions at
1-cell stage and that the number of transcribed intergenic regions decreased at 2-cell stage.
Previous studies have shown that 1-cell stage embryos are in the transcriptionally permissive
state in which enhancers are not required for the gene expression, and that a transcriptionally
repressive state requiring the enhancers is established during the DNA replication at 2-cell
stage. We found that the widespread intergenic transcription is also repressed by DNA
replication at 2-cell stage, since the number of transcribed intergenic regions remained high in
the 2-cell stage embryos in which DNA replication had been inhibited with the treatment with
aphidicolin. Thus, it seems that dissipated transcription occurs in the permissive chromatin
TUESDAY-LATE POSTER PRESENTATIONS
without a stringent regulation at the 1-cell stage and it is restricted in the repressed chromatin in
the 2-cell stage embryos. The experiment of reporter gene assay also supported this
hypothesis. A luciferase reporter plasmid was transcribed without a gene promoter when it was
microinjected into the pronucleus of the 1-cell stage embryos, while it was not transcribed in the
2-cell stage embryos. We propose that transcription is regulated in a specific manner at the
onset of gene expression after fertilization.
2986
The profile of the genes transcribed at the onset of gene expression after fertilization.
R. Yamamoto1, K. Abe1, V. Franke2, K. Vlahovicek2, Y. Suzuki3, F. Aoki1; 1Department of
Integrated Biosciences, The University of Tokyo, Kashiwa, Japan, 2University of Zagreb,
Zagreb, Croatia, 3Department of Medical Genome Sciences, The University of Tokyo, Kashiwa,
Japan
In the oocytes, active transcription occurs during their growth phase. However, it ceases when
the oocytes grow into their full size. This transcriptionally silent state is kept during meiotic
maturation and even after fertilization, and then transcription is reinitiated at mid-1-cell stage in
mice. At this time, the gene expression pattern is changed from an oocyte-specific to embryospecific one. This process seems to be important to link the life to the next generation and
initiate the new program of gene expression in the embryos. However, little information has
been accumulated about the mechanism regulating the initiation of gene expression and even
about the genes expressed at the 1-cell stage, since their expression levels are so low that they
are masked by a large amount of maternal mRNAs which have been synthesized and
accumulated during oocyte growth. In this study, we obtained the transcriptome of oocytes and
1-cell stage embryos by next generation sequencing (RNA-Seq) to identify the genes expressed
at 1-cell stage, and to elucidate the mechanism regulating their expression. First, we identified
23 genes that are transcribed in the 1-cell stage embryos but almost not in the oocytes. When
the reads on RNA-Seq were mapped on the genome, we found in those 23 genes that the reads
were frequently mapped in their intron regions, suggesting that the transcripts are not efficiently
spliced at the 1-cell stage. We microinjected a pre-mRNA into the nucleus of the embryos and
confirmed that the splicing mechanism does not function in the 1-cell stage embryos: the intron
of pre-mRNA was spliced out in the oocytes and 2-cell stage embryos but not in the 1-cell stage
ones. Then, we took advantage of this feature to identify the genes transcribed at 1-cell stage
and obtained approximately 4000 genes. Our ongoing study is analyzing the regulatory regions
of these genes. Thus, we established the foundation to investigate the mechanism of gene
expression in the 1-cell stage embryos.
2987
Expression profile of receptor tyrosine kinases in human pluripotent stem cells.
M-Y. Son1, B. Seol1, Y. Cho1; 1Regenerative Medicine Research Center, KRIBB, Daejeon,
Korea
Human pluripotent stem cells (hPSCs) such as human embryonic stem cells and human
induced pluripotent stem cells have wide-range differentiation capacity and indefinite selfrenewal ability, and thereby contributing to understand human development and disease and
the development of personalized medicine. However, the factors that control the proliferation
and differentiation of hPSCs are still for the most part unknown. Receptor tyrosine kinases
(RTKs) and their downstream cellular pathways play crucial roles in various biological events
such as cell growth, differentiation and metabolism. In order to identify the key regulator
associated with the maintenance of hPSCs identity, we examined the expression profiles and
function of RTKs in hPSCs using reverse transcriptase-PCR with degenerate primers based on
TUESDAY-LATE POSTER PRESENTATIONS
the conserved sequences in the tyrosine kinase domain. We represent the first detailed
repertoire of RTK expressed in both undifferentiated and differentiated hPSCs. Furthermore, our
study showed that several RTKs were simultaneously activated under differentiation condition
using a human phospho-RTK to detect the relative levels of phosphorylation of 42 different
RTKs. We demonstrated that the inhibition of selected RTKs highly expressed in differentiated
hPSCs supported the maintenance of undifferentiated hPSCs as characterized by the typical
morphology and the expression of pluripotency markers. These findings suggest that there is
significant variability in the RTK expression pattern between stem cell self-renewal and
differentiation. This variability should be considered when selecting RTK inhibitors for the proper
maintenance and differentiation of hPSCs.
Acknowledgements
This work was supported by MHWFA (A084697), MEST/NRF (2010-0020272[3]), and
KRIBB/KRCF Research Initiative Program (NAP).
2988
Canonical Wnt/β-catenin signaling modulates chick retina regeneration.
J. Zhu1, J. Zavada1, A. Luz-Madrigal1, A. Burke1, K. Del Rio-Tsonis1; 1Zoology Department,
Miami University, Oxford, OH
The embryonic chick is able to regenerate its retina after retinectomy when exposed to fibroblast
growth factor (FGF). Regeneration occurs via the activation of stem/progenitor cells present in
the ciliary margin (CM) of the eye and by transdifferentiation of Retina Pigmented Epithelium
(RPE). Canonical Wnt/β-catenin signaling plays a critical role in the development of peripheral
eye structures which house retina stem/progenitor cells and in RPE specification during eye
development. Thus, it is important to study the role of canonical Wnt/β-catenin signaling during
retina regeneration from both cellular sources to better understand the molecular basis of retina
regeneration. Activation of canonical Wnt/β-catenin signaling assessed by nuclear β-catenin
immunostaining is detected in the CM as well as in the RPE in embryonic day 4 (E4) chick eyes.
At E5 and E6 when the RPE is unable to transdifferentiate and the CM has reduced
regenerative ability, nuclear β-catenin staining is reduced in the RPE and restricted in a
population of low proliferative retinal stem/progenitor cells in the CM region. After retina removal
at E4, β-catenin activation is reduced in the CM and increased in the RPE cells to protect their
phenotype. Intriguingly, Wnt signaling is completely absent in FGF-induced regeneration from
the CM and RPE transdifferentiation. When over-expressing a negative lef1 construct or
applying a selective Wnt pathway antagonist XAV939 to inhibit Wnt signaling in chick eyes after
retinectomy, regeneration is induced from both cellular sources in the absence of FGF. Robust
proliferation is observed in the regenerating retina and no cell death is detected by 7 days postretinectomy. The regenerated neuroepithelium differentiates into all major retina cell types with
proper lamination.
2989
A dual DNMT/HDAC inhibitor in pluripotency control.
J. Lee1, Y. Xia2, M-Y. Son1, G. Jin2, B. Seol1, M-J. Kim1, M. Son1, M. Lee3, D. Kim3, K. Lee2, Y.
Cho1; 1KRIBB, Daejeon, Korea, 2Dongguk University, Seoul, Korea, 3KAIST, Daejeon, Korea
Human somatic cells can be reprogrammed to embryonic stem cell (ESC) like induced
pluripotent stem cells (iPSCs) by defined factors. Several obstacles including low efficiency and
slow kinetics of reprogramming still have limited the potential application of iPSCs. Here, we
report that newly synthetic chemical compound, reprogramming stimulating compound (RSC)
133 enhances the efficiency of human iPSC generation identified using in-house chemical
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library of heterocyclic compounds screening. We show that RSC133 also improves the kinetics
of reprogramming by enhancing the rate of cell proliferation and the expression of ESC-enriched
genes, and blocking the cellular senescence pathways known to be a barrier to reprogramming.
By in silico reverse docking approach and experimental verification, we found that RSC133
modifies epigenetic chromatin remodeling regulated by the epigenetic modulators such as
DNMT and HDAC. Furthermore we demonstrate that RSC133 not only boosts the
reprogramming dynamics, but also facilitates the maintenance of pluripotent ESCs/iPSCs. Our
results identify RSC133 as a novel epigenetic regulator capable of accelerating cellular
reprogramming and supporting pluripotency of ESCs/iPSCs.
Acknowledgements
This work was supported by MEST/NRF grant (2010-0020272[3]), and KRIBB/KRCF Research
Initiative Program (NAP).
2990
The role of Stat3 signaling in cell fate decisions during repair of adult airway epithelial
cells.
T. Tadokoro1, L. Barak1, Y. Bai1, B. L. Hogan1; 1Department of Cell Biology, Duke University
Medical Center, Durham, NC
In the tracheal airway epithelium it is necessary to maintain the correct proportion of ciliated and
secretory cells. Although disruption of this organization is observed in pathologic phenotypes,
including goblet cell hyperplasia and fewer ciliated cells in asthma, cystic fibrosis and chronic
obstructive pulmonary disease, little is known about the signaling pathways that control the
differentiation of the epithelium. Thus, it is important to understand the precise function of the
different signaling pathways involved in the homeostasis of the mucociliary epithelium and its
repair after damage.
In previous studies, we showed that basal cells as a population are stem cells of the airway
epithelium that give rise to both ciliated cells and Clara secretory cells in vivo. Recently, we
found that isolated basal cells can self-renew and differentiate into ciliated cells and secretory
cells in vitro (sphere-forming assay). FDA-approved 1,514 compounds were screened with this
assay using basal cells from FoxJ1-GFP mice, and 85 compounds were found to inhibit
ciliogenesis. These included pyrimethamine, a drug used for protozoal infections, which is
known to inhibit STAT3 signaling. As phospho-STAT3 is upregulated during the repair of airway
epithelium following SO2 injury, we focused on the role of STAT3 signaling in this process.
In vitro studies showed that the cytokine, IL-6, which activates STAT3, promotes the
differentiation of ciliated cells in the sphere-forming assay: At day 13, flow cytometric analysis
showed that IL-6 treated spheres contained 21.0±0.4% ciliated cells compared with 15.6±0.8%
in controls (p<0.001). Gain-of-function experiment in vivo showed that STAT3 activation in basal
cells by conditional deletion of the feedback inhibitor, SOCS3, promotes differentiation of basal
cells to ciliated cells during regeneration after injury: The tracheal epithelium contained
30.8±0.7% ciliated cells in KRT5-CreER;SOCS3flx/flx;ROSAEYFP mice compared with 20.1±3.5%
ciliated cells in KRT5-CreER;ROSAEYFP control mice (p<0.01). Moreover, deletion of IL6ra, one
of the components of the IL-6 receptor, in Krt5+ basal cells results in a decrease of ciliated cells.
Taken together, our results support the hypothesis that STAT3 signaling plays a key role in
airway repair, especially in the differentiation of ciliated cells.
2991
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Development of ependymal cells in zebrafish embryos.
N-H. Choi1, M-K. Kim1, S-Y. Choi1; 1Chonnam National University Medical School, Gwangju,
Korea
Ependymal cells are glial cells in the CNS. They are multiciliated neuroepithelial cells that line
the ventricles of the brain and the central canal of the spinal cord, and feature microvilli and cilia
on the apical surface and zonula adherens on the lateral surface.
Cells in the subventricular zone have been reported to act as pluripotent progenitors in the
brain. Adult ependymal cells in the mouse spinal cord are derived from Nkx6.1 expressing
ventral neural progenitor cells. Ependymal cells are derived from radial glial cells in developing
mouse forebrain.
There is controversy over the stemness of ependymal cells. This controversy was due to, at
least in part, the absence of “absolutely specific markers” for ependymal cells.
Using zebrafish as a model organism, we will show
embryogenesis.
how ependymal cells develop during
2992
Hepatic biliary epithelial cells alter the differentiation potential during the development.
N. Tanimizu1, T. Mitaka1; 1Sapporo Medical University School of Medicine, Sapporo, Japan
During the development, epithelial cells originate from tissue stem cells and differentiate to
mature cells. In the liver, hepatocytes and cholangiocytes split from hepatoblasts, fetal liver
stem cells, at mid gestation. Cholangiocytes, biliary epithelial cells lining bile ducts, undergo
tubular morphogenesis in the late gestation and the perinatal period. Since duct structures are
evident by around 1 week after the birth, we thought that cholangiocytes might have been
matured by that time point. Recently, we isolated cholangiocytes as EpCAM+ cells from 1W
and 6W livers and compared their capability to differentiate into functional hepatocytes. We
found that cholangiocytes isolated from 1W liver showed hepatocyte morphology and expressed
hepatocyte markers including carbamoylphosphate synthetase I (CPSI) and tyrosine
aminotransferase (TAT) in the presence of oncostatin M (OSM) and Matrigel, whereas neither
morphological changes nor expression of hepatocyte markers were observed in culture of
cholangiocytes isolated from 6W liver. Therefore, cholangiocytes initially possess bidirectional
differentiation potential, though they lose such capability during the development. The loss of
differentiation potential is partly explained by that transcription factors important for hepatocytic
differentiation, such as HNF4α&#61472;and C/EBPα, are induced in 1W cholangiocytes but not
in 6W cells during the culture. Here, we further investigate molecular mechanisms determining
the differentiation potential of cholangiocytes during the development.
We examined gene expression profiles of 1W and 6W EpCAM+ cholangiocytes by microarray.
The data indicated that cholangiocyte markers were expressed less in 1W cholangiocytes than
in 6W ones. Among those genes, we focused on grainyhead like 2 (Grhl2), which we identified
as a transcription factor induces maturation of epithelial cells1). We introduced Grhl2 into
HPPL, a bipotential liver progenitor cell line, and have established HPPL clones expressing
Grhl2. We induced hepatic differentiation by adding OSM and Matrigel into the culture. The
results indicated that HPPL-Grhl2 expressed less hepatocyte markers as compared with the
control cells. This result suggests that Grhl2 not only induces epithelial maturation but also
determines the differentiation potential of liver epithelial cells.
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1) Senga, K., Mitaka, T., Mostov. KE,, Miyajima, A., and Tanimizu, N. Grhl2 regulates epithelial
morphogenesis by establishing functional tight junctions through upregulation of Cldn3, Cldn4,
and Rab25. Mol. Biol. Cell, 2012, 23, 2845-55.
2993
Effect of titanium nanotopography on mesenchymal stem cells cultured under
osteogenic and non-osteogenic conditions.
A. L. Rosa1, R. B. Kato1, F. S. de Oliveira1, L. S. Bellesini1, P. T. de Oliveira1, M. Q. Hassan2, M.
M. Beloti1; 1Cell Culture Laboratory, School of Dentistry of Ribeirao Preto, University of Sao
Paulo, Ribeirao Preto, Brazil, 2Department of General Dental Sciences, School of Dentistry,
University of Alabama at Birmingham, Birmingham, AL
Among different titanium (Ti) nanostructures, TiO2 nanotubes may induce osteoblast
differentiation of human mesenchymal stem cells (MSCs) in the absence of osteoinductive
factors. The aim of our study was to evaluate the osteoblast differentiation parameters of MSCs
grown on nanostructured Ti in both osteogenic and non-osteogenic conditions. Machined Ti
discs, 12 mm in diameter, were chemically treated with H2SO4/H2O2 for 2 hours. MSCs were
obtained from rat bone marrow, in conformity with the Committee of Ethics in Animal Research,
and expanded in growth media until subconfluence. First passage cells were cultured under
osteogenic, i.e.: growth media supplemented with ascorbic acid, beta-glycerophosphate and
dexamethasone, or non-osteogenic, i.e.: growth media, conditions (2×104 cells/disc) on
nanostructured and machined Ti discs for periods of up to 21 days. At 4, 10 and 17 days gene
expression of bone markers were analysed by real-time PCR and at 21 days calcium content
was evaluated using a colorimetric method based on mineralized extracellular matrix detection
by Alizarin red staining. Data were compared by Mann-Withney U-test, for independent samples
(n=4). In osteogenic milieu, nanostructured Ti increased (p≤0.05) gene expression of Runx2 and
bone sialoprotein (Bsp) at 10 days, and alkaline phosphatase (Alp), osteocalcin (Oc) and bone
morphogenetic protein 4 (Bmp4) at 10 and 17 days. In the absence of osteogenic factors, Ti
with nanotopography increased (p≤0.05) the gene expression of Runx2 at 4 and 17 days, and
Alp, Oc, Bsp and Bmp4 at 17 days. Calcium content was only detected in osteogenic conditions
and there were no statistically significant differences (p≥0.05) between nanostructured and
machined Ti. In conclusion, our results suggest that nanotopography may stimulate gene
expression of key bone markers during the time-course progression of differentiation from
MSCs to mature osteoblasts even in a non-osteogenic environment, making this surface
modification a promising approach to drive the fate of stem cells.
Acknowledgements: FAPESP (Grant # 2010/18395-3, 2010/19280-5)
2994
Regulation of COX2 by Cn/NFATc1 signaling pathway during early adipogenesis.
C. J. López-Victorio1, A. Beltrán-Langarica1, C. Velez-delValle1, W. Kuri-Harcuch1; 1Cell Biology,
Cinvestav IPN, Distrito Federal, Mexico
Obesity is a risk factor for several common diseases, including type II diabetes, metabolic
syndrome and cardiovascular diseases. Differentiation of preadipocytes into adipocytes is a
complex process in which adipogenic precursors undergo biochemical, morphological and
metabolic changes related to gene expression. Understanding the regulatory signals involved
during adipose conversion offers a useful insight for the recognition of potential therapeutic
targets. Adipogenesis conveys three major steps: commitment, clonal expansion of committed
cells, and phenotype expression. Our group has developed an in vitro model to study
commitment of 3T3-F442A cells. Cells are induced to differentiate in absence of adipogenic
TUESDAY-LATE POSTER PRESENTATIONS
serum by the addition of staurosporine and dexamethasone (StDex). StDex induces two welldefined stages of commitment: the first one consists in the induction of commitment and the
second in its stabilization, allowing the identification and analysis of gene regulation during this
process. In this study we analyzed the relation between Calcineurin signaling pathway
(Cn/NFATc1) and ptgs2 gene (COX2) during the early events of adipose commitment.
It has been reported that Cn acts as a molecular switch that negatively regulates adipose
differentiation by preventing the expression of critical pro-adipogenic transcription factors. Antiadipogenic Cn pathway includes NFATc1 participation as a nuclear effector, and Calmodulin A
(CaM) as a molecule needed for Cn activity.
We found during the induction of commitment that StDex transiently increases the expression of
pp3ca, nfatc1 and ptgs2 mRNAs and during the stabilization, it proceeds with the down
regulation in the expression of these, and the inhibition of Cn phosphatase activity in 3T3-F442A
cells promoting progression of adipogenesis. Also it has been reported that ptgs2 promoter
have sites of activation by NFATc1, that could explain the transitory increment of both mRNAs.
Moreover, the activation of Cn with ionomycin up regulates ptgs2 and nfatc1 during induction of
commitment. Furthermore, treatment with 0.125 mg/ml of celecoxib, a specific COX2 inhibitor,
does not modify adipose conversion, however triglycerides accumulation is decreased.
Combination of ionomicyn-celecoxib inhibits adipose conversion in about 30% more than
ionomicyn alone. Cn in 3T3-F442A cells activate multiple anti-adipogenic pathways, through
NFATc1 among which might include the expression and activation of ptgs2.
2995
Analyzing cell fate establishment in the pre-implantation mouse embryo using long-term
live imaging with two-photon microscopy.
K. McDole1, Y. Xiong2, P. Iglesias2, Y. Zheng1; 1Embryology, Carnegie Institution, Baltimore,
MD, 2Electrical and Computer Engineering, Johns Hopkins University, Baltimore, MD
The first lineages in the mouse embryo are specified after only five cell divisions, providing a
relatively simplistic model to study initial cell fate specification. By the 32-cell stage cells in the
pre-implantation embryo have differentiated into the inner cell mass (ICM), from which
embryonic tissues arise, and the trophectoderm (TE), which generates the placenta and other
extra-embryonic tissues. Segregation into these lineages is accompanied by a large amount of
cell movement, which combined with the sensitivity of the pre-implantation embryo to light
exposure has made visualizing and tracking cell behavior challenging. We demonstrate the use
of two-photon microscopy in providing superior spatiotemporal resolution as well as high viability
over days of imaging in order to track and quantify cellular behaviors. We have generated an
endogenous reporter of cell fate by fusing eGFP to the endogenous locus of Cdx2, an essential
transcription factor for trophectoderm specification. Using these tools we have identified a
unique cell population able to give rise to both ICM and TE and observed cell-fate changes
through migration late in the 32-cell stage. In addition to tracking individual cell position and cell
fate we demonstrate the ability to visualize and track cell-cell contacts and surface exposure by
the use of a membrane-bound GFP reporter. This reporter also allows us to visualize with highresolution membrane dynamics and membrane partitioning during division, as well as examine
in close detail the process of cavitation. These tools have allowed us to observe and quantify
early cell fate behaviors as well as track and describe the origins of the earliest cell lineages,
and will hopefully provide a framework for and encourage the use of high-resolution non-linear
microscopy in future developmental studies.
2996
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Glial migratory streams in the developing hindbrain: Regulation of glial precursor
differentiation by retinoic acid and notch signaling pathways.
M. S. Domowicz1, V. Palacios1, M. McGovern1, J. Henry1, N. B. Schwartz2; 1Department of
Pediatrics, University of Chicago, Chicago, IL, 2Department of Pediatrics and Department of
Biochemistry and Molecular Biology, University of Chicago, Chicago, IL
Compared to our knowledge of neurogenesis, relatively little is known about glial cell
specification and migration during central nervous system development. We have established a
novel chick hindbrain slice preparation which permits examination of gliogenesis in its native
environment, thus providing a means to study the signaling pathways involved in glial cell
specification and migration during development. Cells in the hindbrain slice preparations
mature in a manner which is similar to in vivo developmental timing and patterning. We
examined the effect of retinoic acid (RA) and NOTCH pathways, which are known to regulate
glial differentiation, on this preparation to determine how they influence glial precursor
commitment. Addition of exogenous trans-retinoic acid (t-RA) to slice cultures promotes
expression of glial fibrillary acidic protein (GFAP), an established marker for mature astrocytes.
This upregulation of GFAP by t-RA occurs in a concentration-dependent manner and maintains
the pattern of glial migratory streams observed in vivo. Inhibition of endogenous RA synthesis
with citral and DEAB reduces GFAP expression, supporting the notion that intracellularly
produced RA may modulate astrocytic differentiation in vivo. Levels and patterns of expression
of nestin, FGFR3 and NOTCH were unaltered in the presence of t-RA while the level of
aggrecan, a proteoglycan expressed by glial precursors, was downregulated by RA treatment.
We then examined the contribution of the NOTCH pathway to glial differentiation by treating the
cultures with DAPT, a γ-secretase inhibitor that blocks NOTCH activation. Inhibition of NOTCH
activation downregulates expression of aggrecan and GFAP, while upregulating expression of
proteolipid protein, a marker for developing oligodendrocytes. These results suggest that
astrocytic precursors have the potential to generate oligodendrocyte precursors when the
NOTCH pathway is inhibited. Treatment with both DAPT and t-RA inhibits expression of
astrocyte maturation markers, indicating that RA signaling act downstream of NOTCH signaling
during astrocyte differentiation. Taken together, our results suggest that NOTCH activation
regulates glial precursor commitment while RA influences the differentiation and matrix
deposition in the glial lineage, probably acting on partially committed precursors.
Cancer Cell Biology II
2997
The Role of CDK4/6 in Cancer Cells and the Potential Therapeutic Effects of CDK4/6
Inhibitors
Y. Arima1, H. Saya1;
1
Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine,
Keio University, Tokyo, Japan
The retinoblastoma protein (pRb) is an important tumor suppressor that regulates the
progression of the cell cycle from G1 to S phase. pRb is often mutated, expressed at very low
levels, or hyperphosphorylated by cyclin-dependent kinases (CDKs) in many cancers. We found
that the depletion of pRb induced the expression of ZEB, which is one of the transcriptional
repressors of the E-cadherin gene, and led to epithelial-mesenchymal transition (EMT) through
ZEB-dependent pathways in breast cancer cells. EMT is defined by the loss of epithelial
characteristics and the acquisition of a mesenchymal phenotype, and can be associated with an
TUESDAY-LATE POSTER PRESENTATIONS
invasive and metastatic potential in cancers. We also found that the depletion of ZEB in pRbinactive cells suppressed cell invasiveness and proliferation and induced epithelial marker
expressions. Then, we developed a screening program for inhibitors of ZEB1 expression and
identified several agents including CDK inhibitors. We confirmed that a CDK4/6 inhibitor
suppressed breast cancer cell proliferation and invasiveness in vitro, and that treatment with a
CDK4/6 inhibitor suppressed tumor growth in vivo using an orthotopic xenograft breast cancer
model in nude mice. In terms of the endogenous CDK inhibitor p16INK4a (p16), we found that
the depletion of p16 in estrogen receptor-negative breast cancer increased the CD44+/CD24Low/breast cancer stem cell population. Moreover, we determined that CDK4 inhibitors completely
blocked tumorsphere formations, which are a characteristic of stem-like cancer cells. Together,
our findings suggest that the CDK4/6 inhibitor affects tumor progression, not only through cell
cycle control, but also through suppression of EMT and stem-like properties of cancer cells.
2998
Cordycepin Shows Potent Anticancer Activity on Testicular Cancer by Regulating
Caspase Signaling Pathways.
B-S. Pan1, Y-F. Mu1, Y-K. Wang2, M-S. Lai1, E. So3, B-M. Huang1; 1Department of Cell Biology
and Anatomy, National Cheng Kung University, Tainan, Taiwan, 2Graduate Institute of
Biomedical Materials and Tissue Engineering, Taipei Medical University, Taipei, Taiwan,
3
Department of Anesthesia, An Nan Hospital, China Medical University, Tainan, Taiwan
It is the most common testicular cancer diagnosis in men between the ages of 15 and 35 years,
and radical orchidectomy combine with chemotherapy is the common protocol to treat testicular
and Leydig cell cancer. However, these drugs used for chemotherapy could cause drug toxicity,
side effects and serious impacts on the quality of life. The present study was designed to
determine the biological effects of 3'-deoxyadenosine, cordycepin, in testicular tumor cells for its
anti-tumor potential and the underlying mechanisms as a novel chemotherapeutic agent. The
detailed studies on the antitumor effects and possible underlying mechanisms of action of the
compounds were then performed. MTT test was used to investigate the effect of cordycepin on
cell viability in MA-10 mouse Leydig and TM4 Sertoli tumor cells. The results showed that
cordycepin-induced death effect on MA-10 and TM4 cells in a dose-dependent manner. We
next detect both early apoptotic and late apoptotic cells as determined by flow cytometry after
annexin V and PI staining were significantly increased when the cells were treated with the
cordycepin in MA-10 cells. Mechanistic studies revealed that cordycepin induced a marked
expression the cleavage of caspase-8, -9, -3, -6, -7 and PARP by different of concentration
treatments (control, 100 uM and 1mM) for 3, 6, 12 and 24 h, respectively. In conclusion,
cordycepin exhibits significant anticancer activity against testicular tumor cells, and will facilitate
the development of therapeutic drugs for the treatment of testicular cancer.
2999
Cynanchi atrati Radix Inhibits Inflammatory Cytokines through IKK-mediated NF-kB
Pathway and Apoptotic Cell Death.
J. Jeon1,2, K. Park1,2, H. Byun1,2, T. Zhang1, H. Lee1, G. Hur1,2; 1Department of Pharmacology,
Chungnam national university School of Medicine, Daejeon, Korea, 2Infection Signaling Network
Research Center, Chungnam national university School of Medicine, Daejeon, Korea
Nuclear factor-kappa B (NF-kB) is one of the most important transcription factors which regulate
the expression of genes critical for a variety of biological processes, including immune
responses, inflammatory reactions and apoptosis. As part of our search for NF-kB inhibitors
form natural products of herbaceous herb, the water extract of Cynanchi atrati Radix (WECR)
was identified as a novel, potent inhibitor of NF-kB activation induced by LPS and TNF. The
TUESDAY-LATE POSTER PRESENTATIONS
Cynanchi atrati Radix is a member of the Asclepiadaceae family, and has been used in
traditional medicine as an antifebrile and diuretic. To investigate the underlying molecular
mechanism involved in the WECR-mediated suppression of NF-kB signaling pathway, the effect
of WECR on the degradation of IkB-a and IKK activation in LPS-stimulated RAW 264.7 cells
was tested. WECR efficiently inhibited LPS-induced expression of proinflammatory mediators
including iNOS and COX-2. IKK kinase activity, IkB-a degradation, nuclear translocation of relA
(p65) and NF-kB transcriptional activity induced by LPS were suppressed by WECR.
Furthermore, WECR dramatically enhances the apoptotic response, as evident by the
combination with tumor necrosis factor (TNF) was able to induce the cytotoxic action through
caspase-dependent pathway. These results indicate that WECR has a potential to inhibit IKKmediated NF-kB activation, and is a valuable compound for modulating inflammatory or anticancerous conditions. This work was supported by the Infection Signaling Network Research
Center (No. 2012-0005764) at Chungnam National University, and a grant from the National
R&D Program for Cancer Control Ministry of Health & Welfare, Republic of Korea (No.
0720560).
3000
Antitumor efficacy of the salicylic acid on A549 cells.
M. Cengiz1, D. Vejselova1; 1Faculty of Science, Anadolu University, Eskişehir, Turkey
Acetylsalicylic acid is a well known non-steroidal anti-inflammatory drug that has long been used
as an anti-pyretic and analgesic. Recently, acetylsalicylic acid, a major component of
analgesics, and its metabolite salicylic acid have been shown to reduce the risk at some
cancers, but the mechanism of action is still unclear (1). Cancer is characterized by over
proliferation of cells or the inhibition of programmed cell death known as apoptosis (2). Here in,
we performed a systematic study on the cytotoxicity of salicylic acid at cell level.
Our aim to evaluate the cytotoxic effect of salicylic acid on A549, the human lung
adenocarcinoma type II, alveolar epithelial cell. The morphology, viability, mortality and
membrane integrity of A549 cells, were evaluated after exposure of 1500-9500µM salicylic acid
for 24 hours.
Stock solution of the compound was prepared in Roswell Park Memorial Institute medium
(RPMI, Gibco), and further dilutions were made with fresh culture medium. A549 cells were
plated at 105 cell/ml per well into 96 well plates. For detecting cytotoxic effect of salicylic acid,
A549 cells were treated with salicylic acid in the range of 1500-9500µM for 24 hours. The plates
were read with ELISA reader ( ELx808), using MTT test system in the 540 nm. MTT [ 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] colorimetric assay depends on the
activity of the mitochondrial succinate dehydrogenase enzyme that measure the reduction of
MTT and so assess the cell viability and proliferation. Each experiment and control group were
measured in triplicate for three times. IC50 value was determined as 6000µM. The percentage
of viability was calculated by the following formula:
Proliferation (%) = [(OD sample / OD control] x100
These results demonstrate that salicylic acid exibited potent antiproliferative effects inducing
apoptosis by concentration dependent manner in A549 cells. In the future our aim is to desing a
new nano-salicylic acid complex to use in cancer treatment.
References
1.
Charles giardianaa, hamid bouraresb, mehmet sait ýnana; nsaýd sandbutyrate sensitize
a human colorectal cancer cell line to tnf-α and fasiligation: the role of reactive oxygen species.
biochimica et biophysica acta (bba)- molecular cell research volume 1448, ýssue 3, 11 january
1999, pages 425-438
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2.
Amin, ruhul; kamitani, hideki; sultana, habiba; taniura, seijiro; ýslam, azrahul; sho,
atsuko; ýshibashi, minako; eling, thomas; watanabe, takashi aspirin an dindomethacin exibit
antiproliferative effects and induce apoptosis in t98g human glioblastoma cells, neurological
research, volume 25, number 4, june 2003, pp. 370-376 (7)
3001
Cytotoxic, antiproliferative and apoptotic effects of silver nitrate against H-ras
transformed 5RP7.
H. M. Kutlu1, A. Kaplan1, A. İşcan1; 1Faculty of Science, Anadolu University, Eskişehir, Turkey
Metal based drugs have successfully been used in the detection and treatment of a variety of
diseases (Thati et. al, 2009). Despite the advantages in early diagnostic and treatment of
cancer, there is essential to develop new alternatives drugs. In recent years, silver complexes
have showed anticancer activity in vitro studies (Boca et. al, 2011) .
Stock solution of Silver nitrate (SN) was prepared in dimethyl sulphoxide (DMSO; Sigma
Aldrich, Poole, UK). Cell viability was determined with MTT assay. H-Ras 5RP7 cells were
plated at 1x105 cell/ml per well into 96 well plates and was incubated in various concentrations
of silver nitrate during 24 hours at 37 °C in a humidified atmosphere of 5% CO2 in air. Plates
were read with ELISA reader (EL X 808), using test wavelength of 540 nm (n=3). For
ultrastructral changes TEM processed, 5RP7 cells growing in DMEM medium, fixed with 2,5%
glutaraldehyde in 0,1 M phosphate buffer (PBS) at pH 7.4 and and left in PBS overnight at +4
°C. After being embedded in agar and post fixation in %2 osmium tetroxide, cells were
dehydrated in graded ethanol: 70, 90, 96 and 100%. Then cells were embedded in EPON 812
epoxy and sectioned on ultramicrotome (LEICA UC6).
In the present study, the cytotoxic and apoptotic effects of (SN) was investigated in
5RP7 cell line using in vitro MTT test system. The 50% inhibition concentration (IC50) of 5RP7
cells was determined 6,75 µM for 24h exposure. Structural and ultrastructural changes of these
cells were showed with transmission electron microscopy (TEM). The viability, morphogical
changes and DNA fragmentation were assesed depending on IC50 values. These results
demonstrated that (SN) have high cytotoxicity and has induced apoptosis on H-ras 5RP7 cells.
References
1.
Boca, s.c., et al (2011), “chitosan-coated triangular silver nanoparticles as a novel class
of biocompatible, highly effective photothermal transducers for in vitro cancer cell therapy”,
cancer letters 311, 131–140.
2.
Thati, b., et al, (2009), “role of cell cycle events and apoptosis in mediating the anticancer activity of a silver(ý) complex of 4-hydroxy-3-nitro-coumarin-bis(phenanthroline) in
human malignant cancer cells”, european journal of pharmacology 602, 203–214.
3002
In vitro toxicity evaluation of salicylic acid on A549 cells
D. Vejselova1, M. cengiz1, M. kutlu1; 1Anadolu University, eskişehir, Turkey
Lung cancer is one of the most common types cases in the World. In spite of significant
advances in development of therapeutics, the incidence of cancer has been experiencing arise
in recent years. Anti-inflammatory drugs, such as acetylsalicylic acid and salicylic acid, present
anti-tumoral properties and induce apoptosis (1, 2).
We suggest that saliycylic acid may have effects on modulation of some intracellular enzyme
and promoting anti-tumoral activity.
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Stock solution of the compound was prepared in Roswell Park Memorial Institute medium
(RPMI, Gibco) and further dilutions were made with fresh culture medium. A549 cells were
plated at 105 cell/ml per well into 96 well plates. In order to detect the cytotoxicity of salicylic
acid, A549 cells were treated with salicylic acid in the range of 1500-9500µM for 24 hours. The
plates were read with ELISA reader ( ELx808), using MTT cytotoxicity test in the 540 nm. MTT [
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] colorimetric assay depends on
the activity of the mitochondrial succinate dehydrogenase enzyme that measure the reduction of
MTT and so assess the cell viability and proliferation. IC50 value was determined as 6000µM.
For confocal microscopy A549 cells which were given salicylic acid (IC50 concentration) were
incubated for 24 hours. After incubation, growth medium was removed and cells were washed
with PBS, fixed with HCI, washed again with PBS and finally stained with acridine orange. The
structural changes of A549 cells were shown with confocal microscopy. The viability,
morphological changes and DNA fragmentation of the cells were assesed depending on IC50
value. These results demonstrated that salicylic acid has exhibited potent antiproliferative
effects inducing apoptosis in A549 cells.
References
1.
Spitz ga, furtado cm, sola-penno m, zancan p (2009). acetylsalicylic acid and salicylic
acid decrease tumor cell viability and glucose and glucose metabolism modulating 6phosphofructo-1-kynase structure and activity, biochemical pharmacology;77,46-53
2.
Lee ý, kolata a, gewirtz a and stlogen k. antitumor efficancy of the cyotoxic rnase,
ranpirnase on a549 human lung cancer xenografts of nude (2007). anticancer research 27:299308.
3003
Protective effect of carvacrol in cyclophosphamide induced oxidative injury in rat testis.
A. Ayhanci1, Y. Tekin1, R. Uyar2, V. Sahintürk3, A. Musmul4, R. Ertekin5, S. Gunes1, S. Cetik1, E.
Eyiis1, M. Demirkaya1, M. Y. Piyale1, B. Demirbas1, Y. Bilgetekin1, İ. Can1; 1Biology, Faculty of
science and art, Eskisehir, Turkey, 2Physiology, faculty of medicine, Eskisehir, Turkey,
3
histology and embryology, faculty of medicine, Eskisehir, Turkey, 4statistics, faculty of
medicine, Eskisehir, Turkey, 5physical therapy and rehabilitation, faculty of medicine, Eskisehir,
Turkey
Cyclophosphamide (CP) is an anticancer drug with immunosuppressive properties. Its adverse
effects are partly connected to the induction of oxidative stres (1). The aim of the present study
was to evaluate the protective effect of Carvacrol (Car) on the biochemical changes, tissue
peroxidative damage and abnormal antioxidant levels in the rat testis during CP-induced injury.
Adult male Sprague Dawley rats were divided into four treatment groups: (I) control, (II) 150
mg/kg CP intraperitoneally (i.p), (III) 5 mg/kg Car once a day for 6 days by i.p, (IV) 10 mg/kg Car
once a day for 6 days by i.p, and (V or VI) CP plus Car (5 or 10, 72 h prior to CP administration).
Testicular toxicity, assessed by decreased enzymatic activities of lactate dehydrogenase (LDH)
and alkalyne phosphatase (ALP), was reversed with Car pretreatment. CP-exposed rats (group
II) showed abnormal levels of enzymes (superoxide dismutase, catalase) and antioxidants
(reduced glutathione) along with high malondialdehyde levels. In contrast, rats pretreated with
Car (group III or IV) showed normal lipid peroxidation and antioxidant defenses. These findings
indicate a cytoprotective role of Car in this experimental model of testicular toxicity.
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Keywords: Cyclophosphamide, Lipid peroxidation, Testicular toxicity, Oxidative stress,
Carvacrol, Antioxidants.
References
1.
Adnan Ayhanci1, Suzan Yaman1, Sila Appak2, and Sibel Gunes1 Hematoprotective
Effect of Seleno-L-Methionine on Cyclophosphamide Toxicity in Rats: Drug and Chemical
Toxicology, 2009; 32(4): 424–428.
2.
Elangovan Selvakumar, Chidambaram Prahalathan, Periyasamy Thandavan Sudharsan,
Palaninathan Varalakshmi. Protective effect of lipoic acid on cyclophosphamide-induced
testicular toxicity: Clinica Chimica Acta 367 (2006) 114 – 119.
3004
Regulation of Mitotic Cytoskeleton Dynamics and Cytokinesis by Integrin-Linked Kinase
in Retinoblastoma Cells.
A. Strikwerda 1, W. K. Sikkema 1, J. Mills1,2; 1Biology, Trinity Western University, Langley, BC,
Canada, 2Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada
During cell division integrin-linked kinase (ILK) has been shown to regulate microtubule
dynamics and centrosome clustering, processes involved in cell cycle progression, and
malignant transformation. In this study we examine the effects of downregulating ILK on mitotic
function in human retinoblastoma cell lines. These retinal cancer cells have elevated
expression of ILK and are caused by the loss of function of two gene alleles (Rb1) that encode
the retinoblastoma tumour suppressor, a principal controller of the cell cycle. We show that
inhibition of ILK activity results in a concentration-dependent increase in nuclear area and
multinucleated cells. Moreover, inhibition of ILK activity and expression increased the
accumulation of multinucleated cells over time. In these cells, aberrant cytokinesis and
karyokinesis correlate with altered mitotic spindle organization, decreased levels of cortical Factin and centrosome clustering. Downregulation of ILK also decreased BrdU labeling while
increasing the mitotic index. Live cell analysis indicate ILK downregulation causes an increase
in multipolar anaphases and failed cytokinesis (bipolar and multipolar). These studies extend
those indicating a critical function for ILK in mitotic cytoskeletal organization and describe a
novel role for ILK in cytokinesis of Rb deficient cells.
3005
Effect of acacetin on proliferation and apoptosis of squamous cell carcinoma HSC-3 via
mitochondrial stress and caspase activation mediated by MAPK-stimulated signaling
pathway.
J-D. Cha1, M-R. Jeong1, J-Y. Lim1, S-M. Hwang1, K-Y. Lee2; 1Institute of Jinan Red Ginseng,
Jinan-gun, Korea, 2Department of Oral Microbiology and Institute of Oral Bioscience, Chonbuk
National University, Jeonju, Korea
Acacetin (5,7-dihydroxy-40-methoxyflavone), present in safflower seeds, plants, flowers,
Cirisium rhinoceros Nakai, has been reported to be able to exert anti-peroxidative,
antiinflammatory, anti-plasmodial, and anti-proliferative activities by inducing apoptosis and
blocking the progression of cell cycles. The mechanism of acacetin-induced apoptosis of
squamous cell carcinoma HSC-3 was investigated. Acacetin caused 50% growth inhibition
(IC50) of HSC-3 cells at 25 ¥ìg/mL over 24 h in the MTT assay. Apoptosis was characterized by
DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-3 and
PARP (poly-ADP-ribose polymerase). Maximum caspase-3 activity was observed with 100
¥ìg/mL of acacetin for 24 h. Caspase-8 and -9 activation cascades mediated the activation of
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caspase-3. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the
Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release
of cytochrome c into the cytoplasm. Pretreatment with casapse-3 inhibitor (Z-DEVD-FMK),
caspase-8 inhibitor (Z-IETD-FMK) and Ac-LEHD-AMC (caspase-9) inhibited the acacetininduced loss of mitochondrial membrane potential and release of cytochrome c. The mitogenactivated protein kinases (MAPKs) were activated by acacetin. Moreover, pretreating the cells
with each of the caspase or MAPK specific inhibitors apparently inhibited acacetin-induced
cytotoxicity of HSC-3 cells. These results strongly suggest that acacetin might have cancer
chemopreventive and therapeutic potential, which is closely related to its ability to activate the
MAPK-mediated signaling pathways with the subsequent induction of a mitochondria- and
caspase-dependent mechanism.
3006
Anticancer Activity of a Combination of Cisplatin and Ecklonia cava extract in Ovarian
Cancer Cells and Xenograft Tumors.
Y. Yang1, J. Choi 1; 1Department of Life & Nanopharmaceutical Science, Kyung Hee University,
Seoul, Korea
Use of chemotherapeutic drug cisplatin is limited because of its nephrotoxicity. Therefore,
efforts continue for the discovery of novel combination therapies with cisplatin to reduce its side
effects. Ecklonia cava (EC) is an edible brown alga that contains high levels of phlorotannins,
which are unique marine polyphenolic compounds. In the present study, we investigated the
potential of EC extract, to increase cisplatin cytotoxicity in human ovarian cancer cells and
reduce kidney damage, along with the molecular mechanism of action.
The combination of cisplatin with EC extract was more effective than either agent alone in
ovarian cancer cells SKOV3 and A2780. The combination induced the activation of caspases-3,
-8, and -9 and pretreatment with caspase inhibitor significantly inhibited the combinationinduced apoptosis. Combination with EC extract inhibited the nuclear translocation of NFκB and
the expression of NFκB-dependent genes. We further demonstrated that EC extract induced a
significant increase in intracellular reactive oxygen species (ROS). Additionally, the antioxidant
N-acetyl-L-cysteine (NAC) significantly attenuated the EC extract-induced production of ROS,
activation of caspases, down-regulation of NFκB-dependent genes in ovarian cancer cells.
Moreover, combination of EC extract with cisplatin treatment also suppressed tumor growth in
SKOV3 bearing mouse model and reduced nephrotoxicity.
Taken together, these data suggest the therapeutic potential of EC extract and cisplatin for
ovarian cancer.
3007
Cryptotanshinone induces apoptosis in squamous cell carcinoma HSC-3.
M-R. Jeong1, J-D. Cha1, B-H. Park1, M-R. Choi1, K-M. Choi1; 1Institute of Jinan Red Ginseng,
Jinan-gun, Korea
Cryptotanshinone (CT) is a major active component of Salvia miltiorrhiza, which is recently
reported to have obvious anticancer activities against diverse cancer cells. The present study is
undertaken to elucidate the mechanisms of CT-induced cytotoxicity in HSC-3 cells. CT caused
cytotoxicity, which was reflected from apoptosis determined by MTT assay and flow cytometry.
CT stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogenactivated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK). Pre-incubation with
SP600125, a JNK inhibitor, SB203580, a p38 MAPK inhibitor, and PD98059, an ERK inhibitor
reduced CT-induced cytotoxicity. CT induced the apoptosis by up-regulation of downstream
target genes, and also cleaved caspase-3, whereas SP600125 and SB203580 caused
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cytochrome c release from mitochondria, and cleaved caspase-3 after a co-treatment with CT.
Taken together, these results suggest that CT can induce apoptosis by activation of p38 and
JNK MAP kinase pathways associated with caspase-3. Moreover, these findings indicate that
JNK- and p38 kinase-mediated mitochondrial pathways might be involved in CT-induced
apoptosis and enhance our understanding of the anticancer function of CT in herbal medicine.
3008
A systems investigation of the recuing slippage network under antimitotic drugs.
H-C. Huang1; 1Institute of Molecular and Cellular Biology, National Taiwan University, Taipei,
Taiwan
Antimitotic drugs (e.g. taxanes) are a mainstay of current chemotherapy. Understanding of why
some cancer cells are able to resist the drugs, or in other words the origins of cell-to-cell
variability in death decisions, is important not only from a basic science perspective, but also
from a cancer therapeutic perspective, as it will provide mechanistic insights into new spectrum
of drug targets. With an integrative experimental and computational (¡¥systems¡¦) approach, we
found that the heterogeneous phenotypic response to antimitotic drugs results from a stochastic
competition between two independent networks, one (¡¥death¡¦) leading to the activation of
death, the other (¡¥slippage¡¦) slowly degrading the master mitotic cyclin (cyclin B) to rescue
cells from the permanent mitotic arrest. In this study, we investigated the molecular mechanism
of the rescuing slippage network. With A549 and HeLa as the model cell lines for fast and slow
slippage kinetics, respectively, and immunoblot to experimentally probe endogenous protein
states, we found that gradual degradation is not specific to cyclin B, but a general phenomenon
of APC/C substrates (e.g. Securin). We also found that during prolonged mitosis, Cdk1mediated phosphorylation of Cdc27 (i.e. effective Cdk1 activity) goes down with cyclin B
degradation. In addition, with mass-action based computational models, we found that a leaky
APC/C-mediated proteolysis signal linked to Cdk1-mediated phosphorylation could explain the
observed single-cell degradation kinetics using cyclin B fluorescent reporter. These data
suggest that slippage network may be a simple slow-downed version of the unperturbed mitotic
exit and shed lights on the normal network, especially how protein degradation and
phosphorylation work together to give rise to the overall cellular kinetics during mitosis.
3009
A comparison of Ku0063794, a dual mTORC1 and mTORC2 inhibitor, and temsirolimus in
preclinical renal cell carcinoma models.
H. Zhang1, D. Berel1, Y. Wang1, P. Li1, N. A. Bhowmick1, R. A. Figlin1, H. L. Kim1; 1Cedars-Sinai
Medical Center, Los Angeles, CA
Rapamycin analogs, temsirolimus and everolimus, are approved for the treatment of advance
renal cell carcinoma (RCC). Currently approved agents inhibit mechanistic target of rapamycin
(mTOR) complex 1 (mTORC1). However, the mTOR kinase exists in two distinct multiprotein
complexes, mTORC1 and mTORC2, and both complexes may be critical regulators of cell
metabolism, growth and proliferation. Furthermore, it has been proposed that drug resistance
develops due to compensatory activation of mTORC2 signaling during treatment with
temsirolimus or everolimus. We evaluated Ku0063794, which is a small molecule that inhibits
both mTOR complexes. Ku0063794 was compared to temsirolimus in preclinical models for
renal cell carcinoma. Ku0063794 was effective in inhibiting the phosphorylation of signaling
proteins downstream of both mTORC1 and mTORC2, including p70 S6K, 4E-BP1 and Akt.
Ku0063794 was more effective than temsirolimus in decreasing the viability and growth of RCC
cell lines, Caki-1 and 786-O, in vitro by inducing cell cycle arrest and autophagy, but not
apoptosis. However, in a xenograft model there was no difference in the inhibition of tumor
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growth by Ku0063794 or temsirolimus. A potential explanation is that temsirolimus has
additional effects on the tumor microenvironment. Consistent with this possibility, temsirolimus,
but not Ku0063794, decreased tumor angiogenesis in vivo, and decreased the viability of
HUVEC (Human Umbilical Vein Endothelial Cells) cells in vitro at pharmacologically relevant
concentrations. Furthermore, expression levels of VEGF and PDGF were lower in Caki-1 and
786-O cells treated with temsirolimus than cells treated with Ku0063794.
3010
KSK05104, a Novel IKK Inhibitor, Induced Apoptosis via Mitochondria dysfunction and
ER-stress in Human Colon Adenocarcinoma HT-29 Cells.
H. Jung1, K. chung2, K. Lee2; 1Pharmaceutical Biochemistry, Kyung-Hee University, Seoul,
Korea, 2, Life & Nanopharmaceutical Science, Kyung-Hee University, seoul, Korea
In the present study, we investigated the effect of KSK05104, on the apoptotic activity and the
molecular mechanism of the action in Human Colon Adenocarcinoma HT-29 Cells. Treatment
with KSK05104 significantly increased externalization of phosphatidylserine residues as shown
by Annexin V staining. In addition, KSK05104 induced the activations of caspase-8, -9, -3, and
cleavage of poly(ADP ribose) polymerase (PARP-1) in HT29 cells. Pretreatment wit z-VAD-fmk
(a broad-caspase inhibitor) partly suppressed KSK05104-induced apoptosis. Furthermore,
KSK05104 was found to induce the release of both cytochrome c and apoptosis-inducing factor
(AIF) by damaging mitochondria, which resulted in caspase-dependent and caspaseindependent apoptotic cell death, respectively. Moreover, we demonstrated that KSK05104
triggered endoplasmic reticulum (ER) stress, as shown by changes in cytosol-calcium level,
activation of mu-calpain and caspase-12, and up-regulation of glucose-regulated protein 78
(GRP78). KSK05104 induced apoptosis was substantially reduced in the presence of an
intracellular calcium chelator BAPTA/AM. Taken together, these results suggest that both
mitochondrial dysfunction and ER stress contribute to KSK05104-induced apoptosis of Human
Colon Adenocarcinoma HT-29 Cells.
3011
CBD inhibits breast cancer growth through the generation of reactive oxygen species.
P. Sarma1, C. Casem1, S. McAllister1; 1Cancer Research, California Pacific Medican Center,
San Francisco, CA
Cancer cells possess heightened rates of cell proliferation due to aberrant cell-cycle
checkpoints. Cannabidiol (CBD) was shown to down-regulate expression of Id-1, an inhibitor of
basic helix-loop-helix transcription factor, reducing angiogenesis and metastatic progression.
CBD is a human cannabinoid receptor, CB2, antagonist. We studied that CBD was able to
efficiently stimulate reactive oxygen species (ROS) production in breast cancer cells linking
previously obtained data on cell viability inhibition. The CB1/CB2 receptor partial agonist delta9tetrahydrocannabinol could not produce significant amounts of ROS until a very high
concentration. Using human breast cancer cell lines, mainly MDA-MB 231, we determined the
effects of CBD on ROS production in a concentration-dependent manner. These effects were
observed to occur across most breast cancer cell lines (human and mouse) in our study. We
also analyzed CB2 receptor interactions with CBD, CB2 receptor selective antagonist
SR144528 and alpha-tocopherol(TOC or vitamin E)for possible reversals. The CB2 receptor
antagonism could only partially reverse cell viability inhibition as well as ROS production with
CBD-treated cells. However, TOC could fully reverse cell viability inhibition and stimulation of
ROS production. To determine CBD stimulation of ROS as a feature of breast cancer cells, we
studied these effects in non-transformed human fibroblast cells. The non-transformed cells
produced negligible amount of ROS, suggesting that CBD must be acting through mechanisms
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unique to transformed cells. Here, we report that CBD inhibits metastatic progression through
efficient ROS stimulation in breast cancer cells which is mediated through possible constitutive
interactions in the human cannabinoid system, other than the mechanism of the CB1/CB2
receptors.
3012
Oxytocin transiently upregulates ERK, p38 and Protein Kinase C activity in Hs578T
breast cancer cells.
E. L. Schenhals1, T. Laakko Train1; 1Biology, Elon University, Elon, NC
Oxytocin (OT) is a peptide hormone best known for its roles in lactation and parturition. It has
been hypothesized that OT production in the breast may be protective against developing
breast cancer. OT and OT receptors (OTR) have been demonstrated in a number of human
breast tumor cell lines, including MCF7 and Hs578T. However, the effect of OT on cellular
signaling in breast tumor cells is not yet fully understood. Present studies were conducted to
determine the role of the OT system in Hs578T breast tumor cells. Hs578T cells were treated
with 50 nM OT for 5, 10, 30, or 60 minutes. Western Blot analysis for the active,
phosphorylated, mitogen activated protein kinases ERK and p38 showed that phosphorylation
was maximal after 5 minutes and returned to background levels after 30 minutes of exposure to
OT. In addition, protein kinase C (PKC) activity was determined by using an antibody against
phosphorylated PKC substrates. PKC substrate phosphorylation also indicated the greatest
activity after 5 minutes exposure, with a return to background levels after 30 minutes. To
determine the concentration of oxytocin that induces optimal signaling, Hs578T cells were
treated with increasing concentrations of OT for 5 minutes (50 nM, 100 nM, 1 μM, and 10 μM).
Maximal phosphorylation of PKC substrates was observed with exposure to 100 nM OT, while
10 μM treatment did not demonstrate significant PKC activity. These results show that OT
causes a rapid and transient increase in PKC, p38 and ERK activation. Further studies will
investigate whether ERK activation is mediated by PKC and whether this transient increase in
signaling modulates cellular responses such as growth, death or migration.
3013
Investigating the Effects of BIBR1532 and Related Analogs on Telomerase Activity in
Human Prostate Cancer Cells.
A. Chowdhary1, A. Menon2, A. Szarecka1, R. Smart3, W. Schroeder3, S. Sridhar1; 1Cell and
Molecular Biology, Grand Valley State University, Allendale, MI, 2Biomedical Engineering,
University of Michigan, Ann Arbor, MI, 3Chemistry, Grand Valley State University, Allendale, MI
Telomeres are double stranded repeats of TTAGGG sequence that cap the ends of
chromosomes and provide genetic stability and immortality to cancer cells. Uncontrolled cellular
proliferation of cancer cells is associated with the maintenance of telomeres in DNA. In normal
cells the length of telomeres decrease with each successive cell division. When the length
becomes too short the cells cannot divide and hence become senescent or die. An enzyme
telomerase prevents the degradation of telomeres by adding bases to the ends of the telomere.
Cancer cells employ this enzyme telomerase to maintain immortality. Assessing telomerase
activity and its inhibition has become an attractive target for new cancer therapeutics. Synthetic
telomerase inhibitor, BIBR1532, has shown growth arrest in tumor cells. In our study BIBR1532,
a mixed-type competitive inhibitor, and its synthetic analogues (WS6-48, WS4-43A, WS5-29,
WS8-3, WS11-41, WS7-6, WS8-9, WS12-16, WS12-43, WS12-44, WS12-45, and WS12-48)
were tested for anti-proliferative and migratory activity on metastatic prostate cancer cells. In our
preliminary studies we have identfied 6 of these compounds that are highly active against
proliferation. We also carried out docking studies using Swissdock for three our synthetic
TUESDAY-LATE POSTER PRESENTATIONS
analogues with TERT (Telomerase reverse transcriptase) domain of the telomerase in T.
thermophila. The binding energies of ligands 8-9 and 7-6 are seen to be in the order of -10 J
and -9 J, while the binding energies of ligand 8-3 are only on the order of -8 J. This is consistent
with the observation from our drug study that WS8-9 and WS7-6 showed high anti-proliferative
potential whereas WS8-3 showed comparatively lower anti-proliferative potential. Our next step
would be to carry out the TRAP (telomerase repeat amplification protocol) assay to quantify the
change in telomerase at different concentrations of the selected 6 compounds in prostate
cancer parental cells. If these studies show promising results, we will further research the effect
that BIBR1532 and its synthetic analogues have on other metastatic cell lines.
3014
A novel antimitotic compound, ACEA100246, displays potent anti-tumoral and antiangiogenesis activities both in vitro and in vivo.
L. Zhao1, K. Wang1, L. Mao1, J. liu1, X. Zhang2, S. Ji2, B. Walsh3, E. Murphy3, W. Xu2, D.
Cheresh3, Y. Abassi1, X. Wang1, X. Xu1; 1ACEA Biosciences Inc., San Diego, CA, 2ACEA
Biosciences (Hangzhou) Inc., Hangzhou, China, 3University of California San Diego, La Jolla,
CA
ACEA100246 is a synthetic molecule derived from a phenotypic screen (using impedancebased time-dependent cell response profiling) for anti-mitotic compounds. ACEA100246
displays potent anti-proliferative activity in vitro against cancer cell lines from various
organs/tissues and triggers cell cycle arrest in G2/M phase leading to apoptosis. ACEA100246
inhibits tubulin polymerization in a concentration-dependent manner in vitro and disrupts the
microtubule network in cultured cancer cells. In competitive binding assays, ACEA100246
competes with [3H]colchicine but not [3H]vinblastine for binding to tubulin. ACEA100246 also
displays anti-proliferative activity against multidrug-resistance cancer cells. In addition,
ACEA100246 demonstrates anti-angiogenesis properties in 3D endothelial tube formation, as
well as developmental angiogenesis in the zebrafish embryo. ACEA100246 at an oral dose of
80 mg/kg (qd) inhibited tumor weight at 63.6%, 40.0%, and 45.5% in A375 skin malignant
melanoma, H460 lung cancer and HCT116 colorectal carcinoma xenograft models, respectively.
Because of its synthetic nature, its oral applicability, its potent in vitro and in vivo anti-tumoral
activity, its efficacy against multidrug resistant tumors, and its anti-angiogenesis properties,
ACEA100246 may have significant potential for clinical development.
3015
Antitumor agent DMXAA stimulates mouse macrophage to secrete TNF-alpha by
intracellular calcium signaling.
S. Kim1, L. Peshkin1, T. Mitchison1; 1Systems Biology, Harvard Medical School, Boston, MA
DMXAA, a new chemotherapeutic drug has been shown promising anti-cancer activity in tumor
mouse model. Especially it stimulates immune cells to secrete cytokines, including TNF-α. This
cytokine secretion is important for the anti-tumor potential of DMXAA, as tumors in TNF-α or
TNF-αreceptor-knockout mice do not respond to DMXAA treatment. However, how it stimulates
immune cells remains unclear. To address this question, we used microarrays to profile
transcription in Raw264.7 cells, a mouse macrophage cell line, in the presence and absence of
DMXAA. Since transcription inhibitors such as actinomycin D and DRB (5,6-Dichloro-1-β-Dribofuranosylbenzimidazole ) completely blocked DMXAA induced TNF-a secretion, we asked
which transcription factors are activated by DMXAA. We found 427 differentially expressed
genes when comparing DMXAA treated and untreated cells. We subsequently analyzed the
promoter regions of the genes to find the common transcription factors which can bind in those
regions using the Pscan (cite) Ver. 1.1 tool. The 950 base pair region upstream of each gene
TUESDAY-LATE POSTER PRESENTATIONS
was scanned up to 50 bp into the gene. From the analysis, we found NF-κB and IRF3 which
are known to be activated by DMXAA showing our promoter analysis was reliable. Also, we
found multiple calcium related transcription factors. To confirm whether calcium related
transcription factors are required for DMXAA induced TNF-α secretion, we treated RAW264.7
cells with an intracellular calcium chelator, BAPTA-AM, or an extracellular calcium chelator,
EGTA, prior to DMXAA treatment. Interestingly, BAPTA-AM but not EGTA blocked DMXAA
induced TNF-α secretion indicating that intracellular calcium is important for TNF-α transcription.
Furthermore, we found an increase in intracellular calcium levels upon DMXAA treatment using
two different calcium sensors, gCaMP3 and fluo-4 AM. Taken together, these results indicate
that DMXAA activates calcium regulated transcription factors by increasing intracellular calcium
levels, which is critical for TNF-α secretion.
3016
Evaluation of Anti-tumor Activity Isolated From Selected Herbs And Spices.
D. N. Pearson1, J. M. Tuscano2, T. Z. Woldemariam1; 1Pharmaceutical and Biomedical
Sciences, California Northstate University, College of Pharmacy, Rancho Cordova, CA,
2
Hematology and Oncology, UC Davis Cancer Center, Sacramento, CA
Spices and flavoring plants rich in health-promoting phytochemicals are currently gaining much
attention as a possible source of cancer chemopreventive compounds. A library of compounds
derived from commonly employed herbs and spices was generated and assessed for possible
in vitro cytotoxic activity against a panel of lymphoma cell lines and the non-small cell lung
cancer cell line A549. Bioactivity-guided fractionation of the methanol extracts yielded several
fractions composed of compounds which displayed significant reductions in cell viability were
observed. The mechanism by which the compounds are inducing cell death in the cancer cells
is currently under investigation. We present the isolation of biologically active compounds
derived from pumpkin and clove along with structural determination and identification of the
compounds.
3017
JS-K, an NO-releasing prodrug, impairs endocytosis by blocking autophagosomelysosome fusion.
W. Yoo1, J-B. Yoon1; 1Translational Research Center for Protein Function Control, Department
of Biochemistry, College of Life Science and Biotechnology, Yonsei university, Seoul, Korea
Protein turnover is precisely regulated to influence cell growth and survival. The ubiquitinproteasome system (UPS) and autophagy are two major intracellular protein degradation
pathways. Since cancerous cells often display elevated protein synthesis and by-product
disposal, inhibition of the protein degradation pathways is an emerging approach for cancer
therapy. At present the proteasome inhibitor Velcade, the drug for multiple myeloma, is the only
FDA-approved drug related to intracellular protein degradation pathways.
To screen small-molecule inhibitors that selectively block ubiquitin-like protein (UBL)
deconjugation steps, we developed cell-based assays for measuring activities of deconjugating
proteases. The assay employs reporter substrates composed of UBL fused to GFP that
becomes unstable upon cleavage by UBL deconjugating proteases. The assay designed for
LC3-deconjugating protease ATG4 was validated by performing a pilot screen with a small
collection of compounds enriched in bioactive molecules (n = 1280 for Lopac¢â library). We
identified JS-K, a nitric oxide prodrug, as a novel autophagy inhibitor by using the cell-based
assay. JS-K has been shown to have anti-tumor activity in both in vitro and in vivo models of
various cancers. However, molecular mechanisms of anti-tumor activity of JS-K and its target
molecules are not clearly demonstrated. Here we report that JS-K impairs autophagy-lysosome
TUESDAY-LATE POSTER PRESENTATIONS
system, but not UPS. JS-K blocks fusion between autophagosomes and lysosomes.
Furthermore, JS-K inhibits endocytosis-mediated degradation of epidermal growth factor
receptors. Many of the effects of nitric oxide are mediated by the direct modification of cysteine
residues of target proteins. To identify and characterize JS-K mediated S-nitrosylated target
proteins involved in impairment of autophagy-lysosome system, we are currently performing
experiments using the biotin-switch technique. Preliminary results and their implications will be
presented.
3018
Mesenchymal Stem Cells Promote Growth and Angiogenesis of Tumors in Mice.
S-C. Hung1,2; 1Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan,
2
Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei,
Taiwan
Though the early integration of mesenchymal stem cells (MSCs) into tumor-associated stroma
of cancer has been demonstrated, the functional contributions and underlying mechanisms of
these cells to tumor growth and angiogenesis remain to be clarified. Using a xenograft model,
human colorectal cancer cells, MSCs, and their cell mixture were introduced to a subcutaneous
site of immunodeficient mice. The tumor growth rate and angiogenesis of each transplantation
was then compared. We demonstrate that a variety of colorectal cancer cells, when mixed with
otherwise non-tumorigenic MSCs, increase the tumor growth rate and angiogenesis more than
that when mixed with carcinoma-associated fibroblasts or normal colonic fibroblasts. The
secretion of IL-6 from MSCs increases the secretion of endothelin-1 in cancer cells, which
induces the activation of Akt and ERK in endothelial cells, thereby enhancing their capacities for
recruitment and angiogenesis to tumor. The IL-6/endothelin-1/ Akt or ERK pathway of tumorstroma interaction can be targeted by an antibody against IL-6 or Lentiviral-mediated RNAi
against IL-6 in MSCs, by inhibition or knockdown of endothelin-1 in cancer cells, or by inhibition
of ERK and Akt in host endothelial cells. These demonstrate that attempts to interrupt the
interaction of MSCs and cancer cells help to abrogate angiogenesis and inhibit tumor growth in
tumors form by cancer cells admixed with MSCs. These data demonstrate that the tumor
microenvironment, namely, MSCs-secreted IL-6, may enrich the proangiognic factors secreted
by cancer cells to increase angiogenesis and tumor growth and that targeting this interaction
may lead to novel therapeutic and preventive strategies.
3019
The involvement of topoisomerase II in the carcinogenic cadmium-induced DNA
breakage and mutagenesis.
Y-C. Yang1, S-M. Chou1, T-K. Li1; 1Microbiology, Taipei, Taiwan
Cadmium (Cd2+), a toxic metal, has many industrial and daily applications. Like other
carcinogenic metals, it has been not only reported to be cytotoxic, but also classified as a
carcinogen. However, Cd2+ is not directly genotoxic and mutagenic in the bacterial system and
is only weakly mutagenic in mammalian cells. Thus, indirect mechanisms have often been
implicated in the Cd2+ carcinogenicity. In our previous study, we have found that topoisomerase
II (TOP2) might be involved in the Cd2+-induced DNA breakage. Interestingly, TOP2-mediated
DNA breakage has also been reported to be mutagenic and carcinogenic. In my proposal, we
sought to determine the potential role of TOP2 in the Cd2+-induced DNA breakage,
mutagenesis and tumor formation. We have found that Cd2+ could induce formation of TOP2
cleavable complex (TOP2cc). Both DNA breaks and cytotoxicity are reduced in shTOP2b cells,
indicating that Cd2+ might target TOP2b preferentially. Cd2+-induced plasmid integration is also
reduced in shTOP2b cells, suggesting an important role of TOP2b in Cd2+-induced DNA
TUESDAY-LATE POSTER PRESENTATIONS
sequence rearrangements. To examine the role of TOP2b in Cd2+-induced carcinogenesis, a
two-stage mouse skin carcinogenesis model was used. In this model, we found that the
incidence of Cd2+-induced melanoma on the skin of drug-treated mice is significantly higher in
wild-type than in skin-specific top2b-knockout mice. This data thus suggest that Cd2+
carcinogenesis is, at least in part, TOP2b dependent. Overall, these results indicate that Cd2+
induces TOP2b-mediated DNA damage and DNA sequence rearrangements, resulting in Cd2+
carcinogenesis.
3020
Calcium influx-induced proteolytic cleavage of topoisomerase 2β.
S-M. Chou1, Y-C. Yang1, Y-H. Liang1, T-K. Li1; 1Graduate Institute of Microbiology, Taipei,
Taiwan
In cells, Ca2+ homeostasis affects many kinases and proteases as well as subsequent cellular
responses upon different stimuli. Here, we used a Ca2+ ionophore, ionomycin, to induce Ca2+
influx and mimic the microenvironment of elevated intracellular [Ca2+]. We had reported hTOP1
is a novel nuclear substrate of calpains with the possible involvement of calapin 2. Interestingly,
hTOP2β but not hTOP2α was rapidly cleaved upon cellular exposure to ionomycin treatment.
The cellular location of hTOP2β was also affected after ionomycin treatment. This proteolytic
cleavage of hTOP2β requires the activation of calpains in the presence of Ca2+-influx
suggesting hTOP2β is a novel substrate to calpains. In addition, either overexpression of natural
calpain inhibitor calpastatin or calpain 2-targeting siRNA could dramatically abolish the Ca2+
influx¡Vinduced cleavage of hTOP2β. Corresponding to the above observations, cells with
calpain 2 knockdown had more protein level of hTOP2β than si-vector control cells implying that
calpains might regulate the stability of hTOP2β protein. In addition, this calpain 2-mediated
cleavage of hTOP2β might have a potential function to improve cell resistance to VP-16 (TOP2
poison) treatment. Consistently, we found calpain 2 knockdown cells are more sensitive to VP16 treatment. Together, our results suggest that Ca2+ regulates hTOP2βstability and could serve
as a sensitivity determent for TOP2 drugs.
3021
A flow cytometry investigation into the effect of Sema3a on the angiogenic signaling
cascade of VEGF-A.
A. Lim1; 1Pepperdine University, Malibu, CA
Vascular development during angiogenesis may be affected by the local composition of growth
factors. Semaphorin 3a (Sema3a), a signaling molecule known to stop vessel sprouting and
cause leakiness, shares the co-receptor neuropilin-1 (NP-1) with vascular endothelial growth
factor A (VEGF-A), a prominent inducer of vessel sprouting and growth. While these signals do
not compete directly for a binding site on NP-1, they induce antagonistic endothelial cell
behavior: growth (VEGF-A) or regression (Sema3a). In this study, the effect of Sema3a on the
VEGF-A signaling cascade in human umbilical vascular endothelial cells (HUVECs) was
investigated. We hypothesized that Sema3a would depress the response of the VEGF-A signal
cascade. We used flow cytometry and fluorescent probes to measure delta-like ligand 4, VEGF
receptor 2, and Notch1 response changes between Sema3a and VEGF-A. One or both signals
were added to cultured HUVECs at equal concentrations. In general, Sema3a depressed
expression of the signal cascade versus VEGF-A, which agrees with the established
understanding of their competing effects. The combined Sema3a/VEGF-A treatment results
were equivocal where some results appeared intermediate between the individual treatments
while others appeared closer to the VEGF-A-only treatment. Similar results were found when we
analyzed the median fluorescence values, which are a relative estimate of surface marker
TUESDAY-LATE POSTER PRESENTATIONS
concentration. Given the protocol used, the dual signal results might be explained by an
increased HUVEC sensitivity to VEGF-A versus Sema3a. Continued study of the interaction
between semaphorin and VEGF signaling could potentially be used to optimize angiogenic
therapy-based treatments of various cancers.
3022
A new rodent model to study MNU-induced prostate cancer: Promotional effects of
Testosterone and High-fat diet.
B. F. Goncalves1, S. P. Campos2, L. R. Falleiros-Jr2, S. R. Taboga2; 1Unicamp, Campinas,
Brazil, 2Unesp, São José do Rio Preto, Brazil
Induction of prostate tumors by the association of N-methyl-N-nitrosurea (MNU) and
promotional agents has been considered a good model to study prostate cancer in different
rodent strains. Thus, in this work it was investigated the role of testosterone and high-fat diet on
MNU-induced prostate carcinogenesis in gerbil (Meriones unguiculatus), a new rodent model
proposed to study this pathology. Adult gerbils (90 days) were divided in four groups: M (MNU
only), MT (MNU+testosterone) and MD (MNU+high-fat diet), which received a single dose of
carcinogen (50mg/Kg) and I, formed by intact animals. Additionally, MT and MD groups
received weekly doses of testosterone cypionate (2mg/Kg) and high-fat diet respectively. After 6
months of treatment, ventral (VP) and dorsolateral (DLP) prostate lobes were removed and
processed for light microscopy. Methodologies involved immunohistochemical (PCNA, α-actin,
vimentin and MMP-2), quantitative and statistical analysis of prostatic lesions incidence and
multiplicity. The induction methodology based on carcinogenic effects of MNU promoted at least
a twofold increase in neoplastic lesions in VP and DLP enabling investigation of epithelial and
stromal markers of prostate cancer. Regardless of the treatment administered VP was more
susceptible to development of neoplasias in comparison to DLP. However, testosterone and
high-fat diet increased the incidence and the number of premalignant and malignant MNUinduced lesions, which were strongly proliferative. Prostate lesions of M, MD and MT showed
high vimentin expression in both lobes indicating an increase and recruitment of cells with
fibroblast phenotype to tumor sites. In peritumoral stroma of MT and MD were commonly
observed clusters of carcinoma associated fibroblasts (CAFs) which were probably involved in
stromal reaction that took place at neoplastic sites. Administration of testosterone or high-fat
diet also stimulated the development of malignant lesions confirmed by disruption of smooth
muscle layer and local stromal invasion. Additionally, in such groups some CAFs and tumor
cells expressed high levels of matrix metalloproteinase-2 (MMP-2), a protease that mediates
extracellular matrix degradation. MNU-induced lesions expressed low levels of this protease.
Together these results suggest the greater invasive potential of tumors stimulated by MNU plus
testosterone or high-fat diet. Thus we conclude that; 1)Testosterone and diet act as tumor
promoters that contribute to more aggressive prostate lesions after MNU inoculation; 2)
Induced-neoplasias expressed markers related to reactive stroma which favor tumor invasion;
3) The gerbil provides a good model for prostate cancer study since it allows the investigation of
advanced steps of carcinogenesis in shorter latency periods and in different prostatic
lobes.Support: CAPES, CNPq and FAPESP.
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3023
Reversion of the mesenchymal cell phenotype in mouse tumorigenic keratinocytes via
HoxA5.
H. Layman1, S. Afghani1, S. Afghani1, I. Cuevas1, L. Coussens2, N. Boudreau1; 1Surgery,
University of California, San Francisco, San Francisco, CA, 2Cell and Developmental Biology,
Oregon Health and Science University, Portland, OR
Hox genes, a conserved subgroup of the homeobox superfamily, have critical roles in
development, embryogenesis, organogenesis, and angiogenesis; not surprisingly, alterations in
Hox gene expression have been reported in tumorigenesis and malignancy. We have previously
demonstrated that Homeobox gene A5 (HoxA5) is critical in homeostasis in adult vasculature
and is lost in proliferating, cancerous vasculature. In vitro, we showed that HoxA5 stabilizes
adherens junctions via increased Akt1, and sustained expression of HoxA5 upregulates antiangiogenic thrombospondin-2 in active angiogenic endothelial cells (HMEC-1). Additionally, we
found, in a mouse skin squamous cell carcinoma model, that HoxA5 reduces capillary density
by 33%, reduces vascular leakage, and delays tumor progression. In addition, in situ
hybridization revealed that HoxA5 was also expressed in superficial, differentiated
keratinocytes, but not in proliferative basal cells. Therefore we investigated whether HoxA5
could also act to limit proliferation of tumorigenic kerartinocytes. We utilized non-transformed
and tumorigenic keratinocytes, CN5 and B9 respectively, and assessed how sustained
expression of HoxA5 affects migration, EMT status, and invasion. CN5 and B9 cells were stably
nucleofected with pBabe-HoxA5 and we determined that the relative rate of migration was
significantly reduced (29.6%) after 12 hours in malignant B9 cells. Additionally, we found that
HoxA5 decreased markers of EMT in tumorigenic B9 cells, by restoring epithelial markers Ecadherin and ZO-1 with a concomitant reduction of mesenchymal markers N-cadherin and
vimentin. Moreover, HoxA5 expression in B9 cells was also linked to reduced expression of
mediators of EMT including TGFβ, Snail, and ZEB2. Lastly, we performed a Matrigel invasion
assay and showed that HoxA5 significantly reduced invasion of B9 cells over 36 hours. Taken
together our results suggest that HoxA5, a potent vascular stabilizing factor, can directly impair
the tumorigenic phenotype of adjacent keratinocytes and coordinately stabilize the tumor
microenvironment.
3024
A possibility of suppressing fibrogenesis-related gene in hsp60-inhibited A549 by
transfection of siRNA.
P. Young Hoon1, L. Yong Hyun1, K. Sua1, L. Seul Ki Na1, K. Dae Young1, H. You Jin1;
1
Department of Biological Science, College of BioNano Technology, Gachon University,
Incheon, Korea
Heat shock protein 60 (Hsp60) is one of stress proteins induced when cells are exposed to
certain stress factors like heat, physical pressure, electricity and etc. Widely known as
chaperonine for its specific feature in protein folding, hsp60 controls transportation of protein
within mitochondria. They are shown to be expressed excessively in various cancers like breast
cancer, liver cancer, cervix cancer and so forth. Fibrosis which refers to the condition when
extracellular matrix is accumulated with tenascin, fibronectin and other cytoskeleton molecules
is important in that it can develop further to the cancer.
We wanted to find out whether there was a relationship between hsp60 and fibrosis. We,
therefore, inhibited hsp60 protein expression by inserting small-interference RNA (siRNA) into
A549, lung carcinoma cell line and 293T, normal kidney epithelial cell line. After transfection, we
incubated cells in normal media and media with TGF-β for 2-3days to induce fibrosis within
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cells. Consequently, We contrasted fibrosis progression of both group by fluorescence
microscope and confirmed an expression of HSP60 and other fibrosis related proteins through
Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR) and Western Blot.
Our result showed that there was a positive relationship between the expression of hsp60 and
fibrosis. Therefore, We believe this fact implies us of a possibility of suppressing fibrosis through
mediating hsp60.
3025
HPSE-1 role in tumor rate development and morphology, and cancer cell migration in
nude mice.
T. M. Augusto1, A. H. Torres2, G. O. Barbosa1, H. F. Carvalho1; 1Unicamp, Campinas, Brazil,
2
Universidad CES, Medellin, Colombia
Prostate cancer is responsible for a great number of non- accidental deaths among men,
especially with aging. During prostate tumor growth and metastasis, the extracellular matrix
(ECM) is remodeled by cancer cells offering a better microenvironment for their survival,
migration and colonization of other tissues. Cancer cells degrade ECM components using a
variety of enzymes such as heparanase-1 (HPSE1) and matrix metalloproteinases. HPSE1
overexpression has been linked to tumor growth, metastasis potential and reduced post-surgical
survival. In the present study the role of HPSE1 expression in tumor development, organ
targeting by three cancer cells lines (PC3, LNCaP and DU145) in contrast with the non-tumoral
epithelial cell line RWPE-1 were assessed. HPSE1 expression in the four cell lines was
evaluated at the time that BalbC/nude mice were subcutaneously injected in the dorsum with
1.5x106 cells in culture medium; and in the tumor mass by immunostaining and molecular
analysis. Findings were also correlated with morphological analysis of tumor tissue. 2x10 6 PC3
and DU145 cells where labeled with CellTracker™ Red CMTPX and injected in the mice caudal
vein. After 7 days, different organs were frozen sectioned and observed for stained cells in
fluorescence microscope. Although HPSE1 was expressed in all cell lines, it was significantly
higher in the PC3 cell line. PC3 formed compact tumors with the greatest velocity rate of growth,
DU145 formed cystic tumors and LNCaP formed a highly vascularized tumor masses. HPSE1
expression was highly expressed in all tumors. RWPE1, the non-tumoral cell line did not formed
tumor in nude mice. On the other hand, RWPE1 transfected with HPSE1-constructions
produced a small tumor growth. HPSE1 mRNA was measured in the tumors and a greater
contribution by the host cells was observed. Analysis of the frozen sections showed that PC3
cells were preferentially located in liver and spleen while DU145 cells were found in the lung,
liver and spleen. These results showed that HPSE1 is an important enzyme that contributes to
tumor formation as observed in overexpressed RWPE1 cell line and cancer cell migration during
the metastasis processes. Finantial Support: FAPESP (2011/08559-1).
3026
Crosstalk of Wnt and HGF signaling increases growth of Colon Cancer Initiating Cells.
G. T. Chen1, S. Sprowl1, A. Konstrum2, A. Newman3, L. Alonzo4, M. Moya4, S. George4, E. Hui4,
C. C. Hughes3, J. Lowengrub2, R. A. Edwards5, M. L. Waterman1; 1Microbiology and Molecular
Genetics, UC Irvine, Irvine, CA, 2Mathematics, UC Irvine, Irvine, CA, 3Molecular Biology and
Biochemistry, UC Irvine, Irvine, CA, 4Bioengineering, UC Irvine, Irvine, CA, 5Pathology, UC
Irvine, Irvine, CA
Wnt signaling directs cell differentiation, embryonic development, and cell polarity. Signals are
commonly defined as β-catenin-dependent (“canonical”) and β-catenin-independent (“non-
TUESDAY-LATE POSTER PRESENTATIONS
canonical”), though both defy traditional categorization, with individual Wnt ligands capable of
activating multiple pathways.
Over eighty percent of colorectal carcinomas have genetic mutations that stabilize β-catenin,
leading to sustained canonical signaling and Wnt target gene expression. Despite being
constitutively active, aberrant Wnt signaling is sensitive to modulation by crosstalk from other
signals such as Hepatocyte Growth Factor (HGF) and Wnt ligands. These signals originate from
multiple sources in the tumor microenvironment, including myofibroblasts and the cancer cells
themselves.
HGF regulates cell growth and motility through the c-Met receptor tyrosine kinase, making it a
key factor in the development and metastasis of many cancers. The effect of HGF crosstalk on
the growth and progression of Wnt-driven colon tumors is currently unknown, as are the
molecular mechanisms that mediate influences on β-catenin. We utilized colon cancer initiating
cells (CCICs) embedded in fibrin gels to address these unknowns. CCICs are patient-derived
cells with multipotent properties that recapitulate the histology of the parental tumor in xenograft.
Single CCICs were seeded into fibrin and treated with a range of HGF concentrations. A
quantitative assessment of colony area was used as a measure of growth rate. We observed a
non-linear dose-response of HGF on CCIC colony growth and cell scattering; low doses of HGF
stimulated proliferation while high doses of HGF showed no change over control. Co-treatment
of the CCICs with HGF and the Wnt secretion inhibitor IWP2 led to a synergistic increase in cell
scattering, suggesting that autocrine secretion of Wnt(s) opposes HGF-directed cell scatter.
Colon myofibroblast-conditioned media exerted a greater effect on CCIC colony growth and
scattering compared to HGF alone, suggesting that CMFs either secrete an optimal amount of
HGF or other factors that synergize with HGF.
To assess how CMFs influence CCIC colony growth and Wnt signaling in a physiologically
relevant environment, we used new 3D tumor microchamber devices seeded with endothelial
progenitor cells, fibroblasts, and CCICs transduced with a lentiviral Wnt reporter driving
destabilized GFP (TOPdGFP). Within the chamber, endothelial cells form a microvessel network
which anastamoses with microfluidic channels to create an actively perfused vascular network.
We observe that CCICs require CMFs for growth and survival and that GFP expression is
heterogeneous - suggesting that microenvironmental signals drive complex patterns of Wnt
activity. Currently, we are identifying the Wnt ligands that mediate the HGF/Wnt effects and
defining the influence of HGF-secreting fibroblasts on the heterogenous levels of Wnt activity.
3027
Aberrant regulation of checkpoint response in mouse embryonic stem cells under iron
deprived conditions.
L. Raskova Kafkova1, L. Calabkova1, V. Divoky1; 1Department of Biology, Faculty of Medicine
and Dentistry, Palacky University, Olomouc, Czech Republic
Iron chelation therapy is used in patients with myelodysplastic syndrome and also with primary
myelofibrosis to prevent complications of iron overload. The impact of chelation therapy on
cancer cell and cancer stem cell iron metabolism is apparent but not well understood.
As a model of cancer stem cells we used mouse embryonic stem cells (mESCs), naturally
immortalized pluripotent cells with high proliferation rate and rapid cell cycle progression. To
remove iron ions from the cultivation medium, we used deferoxamine mesylate (DFO), a large
lipophilic molecule that does not enter cells through the cell membrane. In somatic cells, iron
depletion in cultivation medium typically results in a G1/S arrest, rarely in G2/M arrest. These
cell cycle arrests are governed by checkpoint pathways, where ATR, ATM, Chk1 and Chk2
TUESDAY-LATE POSTER PRESENTATIONS
kinases make the key core. In mESCs, checkpoint kinases were not activated under iron
deprived conditions; instead, we observed direct activation of apoptosis without any cell cycle
abrogation. Next we studied how apoptotic response is activated and further mediated in
mESCs cultivated in iron deprived medium. We found activation of caspase-3 that, however,
was not associated with changes in mitochondrial membrane potential in these cells. We
suggest that apoptosis is mediated through the significant increase of p21 protein level.
Ribonucleotide reductase (RR) is an enzyme which supplies cells with deoxyribonucleotides
(dNTPs) for DNA synthesis. In aforementioned culture conditions the activity of RR significantly
decreased. We hypothesize and experimentally support that lack of dNTPs results in replication
stress, which leads to increase of double strand breaks (DSB), and DSB evoke apoptosis
through activation of gammaH2AX-EYA1-JNK pathway in these cells.
In conclusion, we propose that iron chelation in mESCs results in direct apoptosis instead of cell
cycle arrest described in somatic cells.
Grant support: Projects No. NT12218, P305-11-1745, and institutional project LF_2012_016.
3028
Role of Sirt1 in human neural stem cells by modulating p53 dependent apoptosis under
oncogenic challenges.
J. Lee1, H. Cha1; 1Life Science, Sogang Univ., Seoul, Korea
Given that Sirt1 functions as a p53 deacetylase modulating p53 transcriptional activity, the
tumorigenic effect of Sirt1 may be associated to deregulation of p53. Previously, we had
reported that oncogenic challenge with H-Ras and v-myc is sufficient to induce tumorigenic
transformation of human neural stem cells (hNSCs) but not the differentiated oligodendrocytes,
suggesting that neural stem cells are more susceptible to oncogenic insults. The attenuated p53
response by oncogenic stimulation was responsible for the vulnerable oncogenic
transformation. Herein, we showed the evidence that Sirt1 is induced during oncogenic
stimulation and critical for oncogenic transformation of hNSCs. Knock-down of Sirt1 in stable vmyc-expressing neural stem cells promotes cellular senescence and endowed clear resistance
to oncogenic transformation. Moreover, depletion of Sirt1 in transformed neural stem cells but
not U87 glioma cell line, results in spontaneous mitochondrial apoptosis and loss of tumorigenic
potential in vitro and in vivo in p53 dependent manner. Increased transcription of pro-apoptotic
factors was concurrent in the depletion of Sirt1 in transformed neural stem cells, suggesting that
retrieval of p53 activity by loss of Sirt1 would be strongly associated to the mitochondrial
apoptosis, implying that higher Sirt1 expression in hNSCs during transformation is responsible
for both oncogenic susceptibility and tumorigenic potential of transformed neural stem cells.
3029
Relationship between the expression of F-Actin and Nanog, an embryonic stem cell gene,
in prostate cancer cells.
M. Karabork1, U. Kavgaci1, O. E. Tok2, N. Lack1, R. G. Aktas1; 1Medicine, Koc University,
Istanbul, Turkey, 2Marmara University, Istanbul, Turkey
Tumor development resembles abnormal embryogenesis. It has been shown that the embryonic
stem cell (ESC) self-renewal gene NANOG is expressed by certain types of cancer cells, in
other words cancer stem cells. However; role of these cells in tumor development has remained
unclear. . The recent studies demonstrates NANOG-positive cells between PC3 cells, a cell line
which was derived from a patient who had prostate adenocarcinoma. There are reports
emphasizing the importance of stem cells for tumoral growth and invasion of prostate cancer.
F-actin, another protein which is critical for cell adhesion, migration and division; has been
TUESDAY-LATE POSTER PRESENTATIONS
reported as primary sensor for epidermal stem cell differentiation. These findings led us to
examine the relationship between the expression of F-actin and NANOG in cancer cells.
PC3 cells were seeded on coverslips. They were grown in Minimum Essential Medium (ATCC)
which contain 10% fetal serum bovine. Cells, which were 60%, 80%, 100% confluent as well as
the cells on third day after confluency, were fixed with acetone. After fixation, they were first
labeled with fluorescent phallotoxins (Invitrogen). Same cells were also labeled with NANOG
(D73G4, Cell Signaling) as primary antibody and then anti-rabbit Dylight-550 (Abcam).
Axioscope fluorescence microscope and Eclipse 90i Nikon confocal microscope were used for
examination of the expression of F-actin and NANOG.
Results shows that the number of the cells which are labeled with either actin or NANOG are
limited during 60% and 80% confluency. FITC-phalloidin labeled cells increase during
cultivation. Distribution of actin in the cell also differs. At the beginning of the experiment,
filaments labeled with F-actin is seen throughout the cytoplasm. When their confluency
increase, peripheric location of actin in the cell becomes dominant. Cytoplasmic staining for
NANOG is evident for the cells in different days during cultivation. Colocalization experiments
demonstrate that most labeled cells contain both actin and NANOG.
References
1.
Jeter CR et al. Functional Evidence that the Self-Renewal Gene NANOG Regulates
Human Tumor Development, Stem Cells. 27(5): 993–1005, 2009.
2.
Jeter CR et al. NANOG promotes cancer stem cell characteristics and prostate cancer
resistance to androgen deprivation. Oncogene. 8; 30(36): 3833–3845, 2011.
3.
Stricker C et al. Mechanics of the F-actin cytoskeleton. J Biomechanics. 43 : 9–14, 2010.
4.
Connelly, J. T.et al. Actin and serum response factor transduce physical cues from the
microenvironment to regulate epidermal stem cell fate decisions. Nature Cell Biol. 12, 711–718,
2010.
3030
Standardization for culture conditions in the processing, characterization and
differentiation of mesenchymal stem cells to osteoblasts from patients with
osteosarcoma.
L. F. Acevedo1, G. Gutierrez1, J. M. Araujo1, G. Benitez 1, I. Palma1, O. Rodriguez1, H. Diaz1, A.
Parra1; 1Research, Instituto Politecnico Nacional, Mexico City, Mexico
Mesenchymal stem cells (MSCs or CTM) are able to self replicate and differentiate into a variety
of cell lineages, including osteoblast, chondrocytes, adipocytes, endothelial cells, muscle, and
recently described into neuronal cells. Due to its characteristics of viability, multipotency,
plasticity and the capacity for self-renewal, theoretically makes the first choice for the treatment
of many diseases, as well as repair and regeneration of bone tissue. Moreover, various growth
factors potentiate the differentiation and bone regeneration in vitro, among which include: the
BMP-7, IGF-1, EGF, bFGF and PE-1. The aim of this study was to conduct a comparative
analysis of three separate culture conditions for proliferation and differentiation of mesenchymal
stem cells from bone marrow of patients with osteosarcoma (CTMMO) to osteoblasts with: 1)
osteogenic medium, composed by α-MEM, dexamethasone, β-glycerophosphate and 15% FBS,
2) differentiator medium, which is a commercial medium (NH OsteoDiff Medium, Miltenyi
Biotec), and 3) supplemented osteogenic medium, separately with each of the growth factors:
bFGF, EGF, IGF-I, BMP-7 and EP-1. Experiments showed a greater proliferative effect and
differentiating with osteogenic medium with added growth factors, such is the case of the
supplemented medium osteogenic with EGF (10ng/mL) and PE-1 (35ng/mL) which showed a
proliferative effect higher than the other conditions, and in the presence of bFGF (5ng/mL)
showed an effect on osteoblast differentiation, with a significant increase in alkaline
TUESDAY-LATE POSTER PRESENTATIONS
phosphatase activity. Therefore, the best culture conditions to differentiate CTMMO into
osteoblasts is osteogenic medium supplemented with bFGF.
3031
Galectin-3 enhanced in vitro tumorigenecity of colon cancer stem cells.
C-C. Wei1,2, Y-W. Yen1, P-H. Huang3, E-Y. Huang4, K-L. Wu5, H-Y. Chen1, K. D. Yang1;
1
Department of Medical Research and Development, Show Chwan Health Care System,
Changhua County, Taiwan, 2Institute of Biotechnology, Central Taiwan University of Science
and Technology, Taichung, Taiwan, 3Department of Medical Research, Kaohsiung Chang Gung
Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan,
4
Department of Radiation Oncology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung
University College of Medicine, Kaohsiung, Taiwan, 5Department of Hepatogastroenterology,
Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine,
Kaohsiung, Taiwan
There is increasing evidence that cancer stem cells (CSC) play critical roles in the development,
progression, and recurrence of cancer. CSC is proposed to persist in tumors as a distinct
population and cause relapse and metastasis by giving rise to new tumors. Elevated levels of
galectin (Gal)-1 and Gal-3 were observed in blood or tumorous tissue of patients with several
tumors, including colorectal cancers. Their intracellular and extracellular effects have been
strongly implicated in cancer malignancy. The involvement of Gal-1 and Gal-3 in CSC- drived
tumor progression is still unclear; therefore, we aimed to clarify the effects of Gal-1 and Gal-3 on
CSC. To investigate how Gal-1 and Gal-3 contribute to CSC-involved tumor malignancy, we
selected CSC from colon cancer cell lines, colo-205 and DLD-1, by positive selection of cells
with three colon CSC markers expression, CD44, CD133 and CD326. The purity of DLD-1 and
colo-205 CSC determined by flowcytometric assay is more than 99% after repeated selection of
CD44+CD133+CD326+ colo-205 and DLD-1 cells. In contrast, the non-CSC (NCSC) fraction
contains less than 1% cells with expression of all these three markers. We then determined the
expression profiles of Gal-1 and Gal-3 followed by analyzing their tumorigenic effects on DLD-1
NCSC and CSC. The CSC expressed higher surface and extracellular Gal-3 compared with
NCSC. However, there is no significant difference of surface, intra- and extra-cellular Gal-1
expression and intracellular Gal-3 expression between these two cell populations. There was
significantly different in the expression of stemness-related genes and WNT signaling between
NCSC and CSC. The CSC also showed higher colony formation, sphere formation and
migration abilities, which can be further enhanced after Gal-1 and Gal-3 treatment. Knockdown
of Gal-3 in CSC abolished these abilities. These results suggest that increased Gal-3 produced
by rare CSC in colon cancer contribute to tumorigenecity by promoting tumor initiation,
anchorage-independent growth abilities and migration in an autocrine or paracrine manner.
Cell Biology of the Neuron
3032
A calcium regulated protein interaction at the microtubule plus end plays an important
role in retrograde axonal transport.
M. Kapur1, W. Wang1, M. T. Maloney1, Y. Yang1; 1Department of Neurology, Stanford University,
Stanford, CA
Neurons are particularly reliant on transport as a consequence of their extremely polarized
morphology. Highlighting this point, disruption of transport has been attributed to many
TUESDAY-LATE POSTER PRESENTATIONS
neurodegenerative diseases. In recent years, tremendous progress has been made towards a
better understanding of the fundamental elements of axonal transport. However, the regulatory
mechanisms that control its dynamics remain largely unknown. BPAG1n4 has previously been
shown to play an essential role in retrograde axonal transport by virtue of its interactions with
the dynactin subunit p150Glued, and with endosomes via retrolinkin (Liu et al., 2003, 2007). Its
ability to interact with the main components of the transport system make it ideally placed to
coordinate retrograde transport. The microtubule plus end, the putative site for loading and
unloading of cargo, is an ideal location at which signaling, such as calcium transients, can
spatially and temporally modulate the transport of specific cargoes. Here, we identify an
extremely rapid, calcium regulated switch between microtubule plus end interaction and lattice
binding in the carboxyl terminus of BPAG1n4. This switch is EF-hand dependent, and mutations
of the EF-hands abolish this dynamic behavior. Using single molecule labeling coupled with live
imaging and tracking, we show that the disruption of cargo docking onto the plus end through
the expression of the isolated plus end binding domain results in a substantial impairment in the
processivity of retrograde transport in DRG neurons. This study represents the first example of
a calcium-regulated transition between the microtubule plus end and lattice which plays a critical
role in maintaining the processive motility of long-range retrograde axonal transport. Our
discovery of a rapid calcium sensitive switch controlling microtubule association provides
exciting new insight into the molecular mechanisms that regulate essential microtubule based
cellular processes.
3033
Refinement of axonal projections in the developing visual system.
J. D. Luu1, T-W. Cheng1, H-J. Cheng1,2; 1Center for Neuroscience, UC Davis, Davis, CA,
2
Graduate Institute of Brain and Mind Sciences, National Taiwan University, Taipei, Taiwan
Functional sensory systems comprise precise neuronal connections across multiple regions of
the brain. In the visual system, information is relayed from retina, through dorsal lateral
geniculate nucleus (dLGN), to primary visual cortex (V1) and then to various cortical and
subcortical regions. During development, the initial axonal projections from retina to dLGN
(retina-dLGN projections) are refined to form precise connections at first postnatal week. The
dLGN-V1 projections and the V1-spinal cord (visual cortical spinal tract (CST)) projections are
refined during the third postnatal week. The emergence of precise retina-dLGN connections
requires spontaneous retinal activity before eye-opening in mice. Since the refinement (or
pruning) of visual CST parallels the occurrence of visually evoked activity and spontaneous
retinal activity, it is unclear whether neural activities are necessary for the pruning. Here we
report that disruptions of the intrinsic retinal activities perturb visual CST pruning. However, this
pruning is independent of evoked visual stimulation after eye-opening. Our finding suggests that
the refinement of the entire visual pathway requires patterned spontaneous retinal activity.
3034
Effects of ixabepilone, vincristine, paclitaxel, and eribulin on fast axonal transport and
kinesin-1 driven microtubule gliding: Implications for chemotherapy induced peripheral
neuropathy.
N. LaPointe1,2, G. Morfini3, S. Brady3, S. C. Feinstein1,2, L. Wilson1,2, M. Jordan1,2;1Neuroscience
Research Institute, University of California Santa Barbara, Santa Barbara, CA, 2Molecular
Cellular and Developmental Biology, University of California Santa Barbara, Santa Barbara, CA,
3
The Department of Anatomy and Cell Biology, University of Illinois Chicago, Chicago, IL
A common and severe side effect of cancer chemotherapy with microtubule-targeted drugs is
peripheral neuropathy. The underlying mechanism is unknown, but may involve drug-induced
TUESDAY-LATE POSTER PRESENTATIONS
inhibition of microtubule-based fast axonal transport. Here, we directly assessed the effects of
four chemically diverse microtubule-targeted chemotherapeutic drugs, eribulin, vincristine,
paclitaxel, and ixabepilone, on fast axonal transport of vesicles in isolated demembranated
squid axoplasm. We found that all four drugs inhibited anterograde (conventional kinesin
dependent) fast axonal transport. The magnitude of the inhibitory effect varied by drug and with
drug concentration: eribulin (-10% at 1 µM, -13% at 10 µM); vincristine (-27% at 1 µM);
paclitaxel (-12% at 1 µM, -17% at 10 µM), and ixabepilone (-27% at 1 µM, -37% at 10 µM).
Retrograde (cytoplasmic dynein dependent) fast axonal transport was inhibited by vincristine (19% at 1 µM), paclitaxel (-10% at 10 µM), and ixabepilone (-22% at 1 µM, -38% at 10 µM), but
not by eribulin. When treated axoplasms were probed with an antibody to tubulin and processed
for immunofluorescence microscopy, the periphery of the axoplasm showed drug effects
including tubulin aggregation (ixabepilone and paclitaxel) and microtubule depolymerization
(eribulin and vincristine), demonstrating that these drugs act on endogenous squid tubulin. In
contrast to the axoplasm periphery, the axoplasm interior was similar in treated and control
axoplasm. Next, to determine whether the context of axoplasm was necessary to observe
inhibitory effects of these drugs on transport, we tested the same four drugs using a microtubule
gliding assay, a simplified in vitro system consisting of recombinant kinesin-1 and microtubules
assembled from purified tubulin. We found that eribulin and paclitaxel had no significant effect
on kinesin-driven microtubule gliding in this assay, whereas gliding was slowed by ixabepilone (17% at 10 µM) and vincristine (-13% at 10 µM). The observation that some inhibitory effects on
kinesin-driven motility were recapitulated in this in vitro system indicates that at least some
microtubule-targeted drugs directly interfere with the interaction between motor proteins and
microtubules. However, our results also indicate that the in vitro system does not fully capture
all the effects of the drugs, leaving open the possibility that these drugs have additional effects
within the context of the axoplasm. Our results support the hypothesis that inhibition of fast
axonal transport is a major cause of the neurotoxicity induced by microtubule-targeted drugs.
Understanding the basis of these inhibitory effects may lead to the prevention or cure of
chemotherapy induced peripheral neuropathy.
3035
Role of Tetraspanin proteins in organizing a multi-subunit receptor complex for neurite
growth inhibitory Nogo-A-Delta20.
N. K. Thiede-Stan1, B. Tews1, M. Schwab1; 1Brain Research Institute UZH and ETH Zurich,
Zurich, Switzerland
The myelin-associated neurite growth inhibitory membrane protein Nogo-A restricts neurite
outgrowth and axonal regeneration with two different domains of the molecule. The Nogo-66domain inhibits outgrowth through binding to the receptor complex of NgR1, LINGO1 and p75 or
TROY and leads to RhoA activation. Interaction partners of the inhibitory Nogo-A-specific
fragment Delta20 have not been determined yet. A yeast two-hybrid screen in our laboratory
revealed a G-protein-coupled receptor family and a 4-transmembrane-spanning protein
(Tetraspanin-3, TSPAN-3) as novel binding partners of Delta20. As tetraspanins are described
in particular as organizer of functional microdomains, we investigate the role of Tetraspanin-3 as
a potential organizer of the Nogo-A-Delta20 multi-subunit receptor complex, with regard to its
role in the formation of a scaffold/ signaling platform, the internalization of the complex partner
and activation of downstream signaling. Binding studies and co-localization experiments show
interaction of Tetraspanin-3, Nogo-A-Delta20 and the signal-transducing GPCR. The complex
assembly is spatiotemporally regulated by Nogo-A-Delta20 at the cell surface. Interestingly,
Tetraspanin-3 remains on the cell surface while the GCPR is internalized together with Nogo-A
subsequent to binding. The underlying functional aspects of Tetraspanin-3 in a multi-subunit
receptor complex for Nogo-A-Delta20 are currently under investigation.
TUESDAY-LATE POSTER PRESENTATIONS
3036
Snapin mediates retrograde axonal transport of BDNF-TrkB signaling endosomes
essential for dendrite growth of cortical neurons.
B. Zhou1, Q. Cai1,2, Y. Xie1, Z-H. Sheng1;
1
Synaptic Functions Section, NINDS, NIH, Bethesda, MD, 2Cell Biology and Neuroscience,
Rutgers, The State University of New Jersey, Piscataway, NJ
Neurotrophin signaling is crucial for neuron growth. While “signaling endosomes” hypothesis is
one of accepted models, the molecular machinery that drives retrograde axonal transport of
signaling endosomes is largely unknown. BDNF is one of the well-studied neurotrophic factors
regulating dendrite outgrowth and branching. However, it remains unclear whether retrograde
axonal transport of TrkB signaling endosomes has a direct impact on dendritic growth in
cultured CNS neurons. In particular, mechanisms recruiting dynein to TrkB signaling
endosomes have not been elucidated. Our previous study reveals that Snapin acts as a dynein
adaptor by binding to dynein intermediate chain (DIC) and mediates retrograde axonal transport
of late endosomes, a process essential for the maturation of the autophagy-lysosomal system in
neurons (Cai et al., Neuron 2010). Here, using snapin deficient mice and gene rescue
experiments combined with compartmentalized cultures of live cortical neurons, we demonstrate
that Snapin mediates retrograde axonal transport of TrkB signaling endosomes. Such a role is
essential for dendritic growth of cortical neurons. Deleting snapin or disrupting Snapin-dynein
interaction abolishes TrkB retrograde transport, impairs BDNF-induced retrograde signaling
from axonal terminals to the nucleus, and decreases dendritic growth. Such defects were
rescued by reintroducing snapin gene. Our study indicates that Snapin-dynein coupling is one of
the primary mechanisms driving BDNF-TrkB retrograde transport, thus providing new
mechanistic insights into the regulation of neuronal growth and survival (Zhou et al., Cell
Reports 2012) (Supported by Intramural Research Program of NINDS, NIH)
3037
SATB1 in the basolateral amygdala is required for regulation of endosome trafficking
genes and normal anxiety behavior.
M. A. Balamotis1, B. Davy1, N. Tamburg1, M. S. Golub2, T. Kohwi-Shigematsu1, Y. Kohwi1;
1
Life Sciences, Lawrence Berkeley National Labs, Berkeley, CA, 2Internal Medicine, University
of California, Davis, Davis, CA
The amygdala is a key forebrain structure necessary for processing and retaining emotionally
important information from fear and anxiety-related experiences. Gene expression and
behavioral response are linked events with many factors likely contributing to this relationship.
We found that the genome organizer protein, SATB1, is expressed in the amygdala and the
cortex in the postnatal mouse brain. SATB1 regulates temporal expression of immediate early
genes (IEGs) during early postnatal cortical development. We examined whether SATB1
mediates neuronal response to environmental stimuli in adult mice at the level of chromatin
organization and gene expression. In neurons, SATB1 adopts a network-like protein distribution
and binds to specialized genomic sites near target genes to coordinate chromatin folding, which
allows multiple genomic loci to become tethered into a regulatory core to which chromatinmodifying and transcription factors can be recruited. To address if gene expression regulated by
SATB1 plays an important role in the function of the adult amygdala, we generated a conditional
knockout (CKO) mouse line to remove Satb1 postnatally from the anterior basolateral amygdala
(BLA). We found that CKO mice exhibited greatly reduced anxiety response. When SATB1 was
lost, multiple groups of functionally related genes became incorrectly regulated, with endosome
trafficking genes the most affected. In response to acute water stress stimulation, many of these
same trafficking genes in BLA of CKO mice showed improper induction potential. Additionally
TUESDAY-LATE POSTER PRESENTATIONS
under stimulating conditions, other gene groups responsible for controlling cell membrane
projections and cytoskeleton structure were aberrantly expressed in the BLA of CKO mice,
suggesting a possible defect in stimulation-induced neuronal plasticity due to the absence of
SATB1. This work identifies SATB1 as a new, indispensible component of amygdala function by
assigning it a here-to unknown role as a regulator of endosome trafficking genes in the brain
and a molecular element in anxiety response.
3038
Pharmacological inhibition of γ-Glutamylcysteine Synthetase Induces Amyloidogenic
Processing of Amyloid Precursor Protein.
R. D. Quiroz-Baez1,2, K. Hernández-Ortega2, C. Arias2; 1Departamento de Investigación Básica,
Instituto Nacional de Geriatría, México, Mexico, 2Departamento de Medicina Genómica y
Toxicología Ambiental, Instituto de Investigaciones Biomédicas, UNAM, México, Mexico
The Amyloid-β peptides (Aβ) has been considered a key element in the progression of
Alzheimer’s disease (AD) in both familiar and sporadic cases. Although amyloid plaques are not
directly related with severity of disease, growing evidence suggests that soluble o