Abstracts
ORAL PRESENTATIONS
Jennifer Lippincott-Schwartz President / Wallace Marshall Program Chair
Michael Marks Local Organizer
SUNDAY-ORAL PRESENTATIONS
ORAL PRESENTATIONS- Sunday, December 07
Symposium 1: Self Organization and the Origin of Life
S1
Self-Organization: From Cell Biology to Marine Ecosystems.
E. Karsenti1;
1
Director research, EMBL, Heidelberg, Germany
Just at the end of world war II in 1944, Erwin Schrodinger published a visionary small book named “What
is Life?” At this time (not long ago!) the molecular understanding of life was still rudimentary and
Schrödinger started by addressing the possibility of understanding life in the realm of chemical and
physical laws. He surely believed that it was. To him the key question of life was obviously that of the
origin of order as in physics and he stated, “There are two ways to produce orderliness”: - Statistical:
processes producing the emergence of order from disorder - Clockwork: processes producing order
from order. Today, 70 years later, we have learned that life can indeed be explained in chemical and
physical terms. Moreover, we can actually identify what embodies the two principles described by
Schrödinger: Order from order comes from the duplication of the “aperiodic crystal” of Schrödinger
(DNA) or from the transmission of structures and order from disorder emerges from the collective
behavior of information rich molecules like proteins that lead to complex shapes and functions through
self-organization processes. But at a different scale, that of ecology and evolution, a similar situation
exists. Organisms transmit their order, but the collective effects of environment and organism
interactions shape ecosystems patterns at any instant and evolution on very long time scales.
The theory of self organization indicates that order can indeed emerge from complex dynamic
interactions at virtually all space and time scales even if the forces involved are entirely different.
Therefore, at any scale if one wants to understand self organization processes, the key is 1) to identify
the “agents” and the dynamic parameters associated with them within a coherent referent and 2) how
these agents interact. In a sense, working on self-organization processes in living matter requires the
understanding of complex dynamic networks. To illustrate this, I will recapitulate briefly some of my
work on the mitotic spindles and how various self-organization mechanisms contribute to the assembly
of microtubules in complex functional patterns within cells. Then I will switch scale entirely and describe
how we have started to address the issue of plankton ecosystems self-organization in the world oceans.
In both cases, collective behaviors and reaction diffusion processes are at work. Yet the scales are
hugely different as well as the agents!
SUNDAY-ORAL PRESENTATIONS
S2
A Solid State Conceptualization of Information Transfer from Gene to Message to
Protein.
S. McKnight1;
1
Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX
The vast majority of DNA and RNA regulatory proteins consist of two parts. One such part enables direct
recognition of DNA or RNA, is well folded, and is represented by canonical domains including zinc
fingers, homeoboxes, leucine zippers, RNA recognition motifs, KH domains and pumilio domains. The
other part is typified by poorly folded, low complexity sequences whose mechanistic basis of function
has long been enigmatic. When incubated at high concentration, certain of these low complexity
domains can polymerize into amyloid-like fibers. My presentation will outline studies indicating that low
complexity sequence polymers may represent the organizational basis for the formation of nuclear and
cytoplasmic puncta including nuclear speckles, P granules, stress granules and neuronal granules. It is
our speculation that from the birth of a transcript in the nucleus to its ultimate translation in the
cytoplasm, the entire pathway of information flow is guided by movement of the message through a
solid state pathway of polymeric fibers. This “informational cytoskeleton” is regulated in a dynamic
manner by post-translational modification including phosphorylation, and can be impinged in disease
states via mutational events that either enhance fiber stability or clog the dynamic behavior of puncta.
S3
In vitro reconstitution of protein gradients and oscillations as spatial cues for cell
division.
P. Schwille1;
1
Max Planck Institute for Biochemistry, Martinsried, Germany
In order to elucidate the most fundamental features and functional modules for cell division, we have
been concerned with the spatial self-organization of the E.coli divisome. In particular, the MinCDE
proteins represent an archetypical gradient-forming system by reaction-diffusion, which displays the
fascinating feature of temporally oscillating between the poles of the cell on a minutes time scale, as a
cellular pacemaker. Reconstituted in vitro in the presence of a membrane, the Min proteins show
remarkable dynamic pattern formation in the form of travelling waves, which can spatially orient
themselves by sensing the geometrical features of the membrane. Transferring this system into
membrane-clad microcompartments mimicking cellular shape, we demonstrate that distinct timeaveraged protein concentration gradients are established, which act as spatial cues for the positioning of
downstream processes in bacterial cell division, such as the defined assembly of Z protorings in the
middle of the compartments. In particular, we show that compartment geometry plays a major role in
Min gradient establishment, and provide evidence for a geometry-mediated mechanism to partition Min
proteins during the actual process of septum closure.
SUNDAY-ORAL PRESENTATIONS
Symposium 2: Cells in Motion
S4
A physicist's view of some filopodial features.
P. Bassereau1;
1
PhysicoChimie Curie, Institut Curie, Paris, France
Filopodia are thin and dynamic tubular plasma membrane protrusions that cells use for sensing their
surroundings and for their motion. They can also exert retraction force upon adhesion to their tip, a
process utilized by pathogens to invade cells [1]. These cell structures result from the subtitle balance
between the forces generated by the actin cytoskeleton, the plasma membrane and different proteins
that connect both. A comprehensive physical model of the functioning of these structures does not exist
yet. However, I will present some physical clues explaining how the retraction force is produced [2, 3]
and how I-BAR domain proteins such as IRSp53 can contribute to the stabilization of the filopodia by a
preferential binding to highly curved membrane where it forms a scaffold [4]. For this, I will show in vivo
data based on force measurements on filopodia tip with optical tweezers and in vitro experiments with
reconstituted IRSp53 on membrane nanotubes.
References:
1.
Romero, S., Grompone, G., Carayol, N., Mounier, J., Guadagnini, S., Prevost, M.-C., Sansonetti,
P.J., and Tran Van Nhieu, G. (2011). ATP-Mediated Erk1/2 Activation Stimulates Bacterial Capture by
Filopodia, which Precedes Shigella Invasion of Epithelial Cells. Cell Host & Microbe 9, 508-519.
2.
Romero, S., Quatela, A., Bornschlögl, T., Guadagnini, S., Bassereau, P., and Tran Van Nhieu, G.
(2012). Filopodium retraction is controlled by adhesion to its tip. J. Cell Sci. 125 4999-5004.
3.
Bornschlögl, T., Romero, S., Vestergaard, C., Joanny, J.F., Tran Van Nhieu, G., and Bassereau, P.
(2013). Filopodia retraction force is generated by cortical actin dynamics and controlled by reversible
tethering at the tip. Proc. Natl Acad. Sci. USA 110, 18928-18933
4.
Prevost, C., Manzi, J., Zhao, H., Lappalainen, P., Callan-Jones, A., and Bassereau, P. (in
preparation).
S5
Functional specificity of integrin-based adhesions in cell function is defined by
actin nucleators.
C. Waterman1, L. Case1, V.S. Swaminathan1, C.T. Skau1;
1
National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD
Integrin-based adhesive structures perform a diverse array of cell functions that involve the regulated
interaction of the cell with its extracelluar environment. Integrin-based organelles performing these
functions include focal adhesions that mediate mechano-sensing and cell migration, nascent adhesions
that mediate haptotaxis, fibrillar adhesions that are critical to extracellular matrix remodeling, and
ventral lamellipodia that mediate maintenance of endothelial barrier function during the innate immune
response. The structure and function of each of these adhesive organelles in intimately linked to the
SUNDAY-ORAL PRESENTATIONS
cortical actin cytoskeleton. Although it is well- accepted that different members of the integrin receptor
superfamily contribute to the functional specificity of integrins, it is not known whether the actin
cytoskeleton contributes to the ability of adhesive organelles to specify distinct cell functions. We
hypothesized that distinct actin filament nucleators contribute to the structural and functional
specificity of integrin-based adhesive organelles, and sought to identify the molecular mechanism of
actin assembly that mediates their formation. We tested this hypothesis by utilizing high resolution light
microscopy of living cells, molecular perturbations of distinct Arp2/3 and formin family actin nucleators,
and assays for specific adhesive cell functions. We and others find that the nucleator of flat branched
dendritic actin networks, Arp2/3, is required for stabilizing lamellipodia during haptotactic migration,
and is signaled by vascular permeability factors to heal holes in endothelial cells during leukocyte
transmigration. We further show that two distinct formin family nucleators of bundled actin are
responsible for forming distinct adhesive structures in fibroblasts. The inverted formin INF2 is required
for forming dorsal strsss fibers connected to focal adhesion and mediating their transition to fibrillar
adhesions that produce organized extracellular matrices. In contrast, the formin FMN2 is critical to
formation of a novel class of subnuclear adhesions that mediate nuclear positioning during wound
healing and 3D cell migration. Thus, integrin superfamily members and distinct actin nucleators
cooperate to define the functional specificity of adhesive organelles.
ePoster Talks Session 1: Cell Motion and Mechanobiology 1
E1
Passive cell membrane regulation in response to mechanical stimuli.
A. Kosmalska1,2, L. Casares Garcia1,2, A. Elosegui-Artola1, J.J. Thottacherry3, S. Mayor3, M. Arroyo4, D.
Navajas1,2,5, X. Trepat1,2,6, N. Gauthier7, P. Roca-Cusachs1,2; 1Institute for Bioengineering of Catalonia
(IBEC), Barcelona, Spain, 2University of Barcelona, Barcelona, Spain, 3National Centre for Biological
Sciences (NCBS), TIFR, Bangalore, India, 4LaCàN, Universitat Politècnica de Catalunya-BarcelonaTech,
Barcelona, Spain, 5Ciber Enfermedades Respiratorias, Bunyola, Spain, 6Institució Catalana de Recerca i
Estudis Avançats (ICREA),, Barcelona, Spain, 7Mechanobiology Institute, Singapore, Singapore
Cell processes in any physiological environment ubiquitously involve changes in cell shape, which must
be accommodated by their physical envelope – the bilayer membrane. However, the fundamental
biophysical laws by which the cell membrane allows for and responds to shape changes remain unclear.
Here we show that in response to mechanical stimuli cells rearrange their membrane through a fast
passive physical process prior to any active remodeling. Upon application of biaxial stretch, cells recruit
membrane area by flattening existing ruffles in lamellipodia. Upon subsequent stretch release,
membrane area is stored in the form of tubular invaginations at the cell-substrate interface. In contrast,
membrane adaptation in response to changes in medium osmolarity is driven not by area requirements,
but rather by the storage of liquid flows expelled by cells. This leads to the formation of the large
membrane invaginations in the shape of spherical caps usually described as vacuole-like dilations (VLDs).
In both cases, we demonstrate that the shape of the invaginations can be explained by passive
minimization of membrane elastic and adhesive energies, constrained by the requirement to store
SUNDAY-ORAL PRESENTATIONS
membrane area (in the case of stretch) or volume (in the case of osmotic shocks). Further, we show that
by reducing the requirement for adhesion and elastic energy, existing invaginations can act as seeds for
additional deformation. By being passive and not depending on specific molecular components, the
principles unveiled provide a general mechanism for the adaptation and rearrangement of a significant
portion of cell membrane area. Additionally, the local curvature induced by membrane invaginations
could potentially trigger downstream mechanotransduction events by affecting curvature-sensitive
proteins.
E2
Cell migration in mechanically resistive environment.
N. Srivastava1, A. Kabla1, R. Kay2; 1Department of Engineering, University of Cambridge, Cambridge,
United Kingdom, 2Laboratory of Molecular Biology, MRC, Cambridge, United Kingdom
1.
2.
3.
4.
5.
The mechanical environment of a cell influences its migration. It has been observed in particular that
certain cell types such as Dictyostelium discoideum and cancer cells tend to migrate by forming blebs
instead of pseudopodia/lamellipodia in mechanically resistive environments1–3. However, very little is
known about the mechanisms governing this transition between different modes of migration4,5⁠. In
this work, we aim to disentangle the contributions of two key mechanical factors that might trigger this
switch, the environment's stiffness (the extent of local deformation of extra-cellular matrix by the cell)
and its state of stress (amount of pre-existing tension or compression). We use Dictyostelium
discoideum as a model to study these questions. A custom built device, the "cell squasher", is used to
compress an agarose gel of known stiffness against a glass cover slip under a dynamically controlled and
uniform loading condition. A chemotactic gradient (using cAMP) is used to direct the cells under the
agarose while monitoring cell's migration and recording its morphology and trajectory. Our preliminary
results indicate that switching to bleb mode of migration can be triggered by an increase of either the
external loading or the gel stiffness. This transition is also associated with a decrease of cell speed.
Increasing the load even further triggers the rounding up of the cells and dramatically slows down the
retraction of blebs; migration ceased in most of the cases. The setup is currently applied to study the
dynamics of the lamelipodia/bleb transition. This work may be of significant importance in
understanding the initiation of cancer metastasis where cells often migrate using blebs in environments
often highly deformed by the growth of the tumour5⁠.
References:
Zatulovskiy, E., Tyson, R., Bretschneider, T. & Kay, R. R. Bleb-driven chemotaxis of Dictyostelium cells. J.
Cell Biol. 204, 1027–44 (2014).
Charras, G. & Paluch, E. Blebs lead the way: how to migrate without lamellipodia. Nat. Rev. Mol. Cell
Biol. 9, 730–6 (2008).
Tyson, R. A., Zatulovskiy, E., Kay, R. R. & Bretschneider, T. How blebs and pseudopods cooperate during
chemotaxis. Proc. Natl. Acad. Sci. 1322291111– (2014). doi:10.1073/pnas.1322291111
Bergert, M., Chandradoss, S. D., Desai, R. A. & Paluch, E. Cell mechanics control rapid transitions
between blebs and lamellipodia during migration. Proc. Natl. Acad. Sci. U. S. A. 109, 14434–9 (2012).
Tozluoğlu, M. et al. Matrix geometry determines optimal cancer cell migration strategy and modulates
response to interventions. Nat. Cell Biol. 15, 1–14 (2013).
SUNDAY-ORAL PRESENTATIONS
E3
Topography and stiffness of endothelial layers direct intraluminal crawling of T
cells.
J. Doh1, K. Song1; 1Department of Mechanical Engineering, POSTECH, Pohang, Korea
Immune cells circulating in inflamed blood vessels undergo series of adhesion cascades to leave out of
blood vessels to infiltrate into inflamed tissues. It has been shown that leukocytes crawl substantial
amount of distances before they undergo transendothelial migration (TEM), but biochemical/biophysical
cues directing the crawling of leukocytes have not been clear. While both endothelial cell (EC) junctions
and flow can affect crawling direction of leukocytes, experimental settings currently widely used do not
allow systematic examination on which factor has predominant roles on determining intraluminal
crawling direction. To directly address this problem, we fabricated well-aligned EC layers by culturing
ECs on nanogrooved surfaces and applied shear flow either parallel or perpendicular to the direction of
EC orientation. Regardless of flow direction, T cells crawled along the EC orientation. Then, we further
identified guiding cues on ECs and found out that T cells tend to crawl along the valleys of topographical
landscapes of EC layers while avoiding nuclei of ECs. To directly test whether T cell crawling is guided by
the topography of EC layers, EC layers were fixed, dried, and replicated with UV-curable resin
poly(urethane acrylate) (PUA). T cells on PUA replicas coated with key adhesion molecules for crawling
also crawled along the EC orientation, suggesting topography of EC layers is one of the major factors
guiding T cell crawling. However, T cells on PUA replicas frequently crossed over nuclei of ECs, meaning
that the tendency of avoiding nuclei of ECs by crawling T cells cannot be explained by topography effect.
When lamin A/C of ECs were knocked down by siRNA to reduce nucleus stiffness of ECs, crawling T cells
no longer avoided nuclei of ECs, meaning T cells sense stiff nuclei of underlying ECs during crawling. By
using pharmacological inhibitors for arp 2/3 and cdc42, we found out that T cells utilize lamellipodia to
sense topography of EC layers and invasive filopodia to sense stiff nuclei of ECs. Importantly, inhibitortreated T cells crawled much longer distances than untreated T cells before they underwent TEM,
suggesting that lamellipodia- and filopodia-mediated biophysical environments sensing of T cells are
critical for optimal intraluminal path finding.
E4
Rigidity sensing by tropomyosin-regulated nanometer steps in local contractions.
H. Wolfenson1, G. Meacci1, S. Liu1, M.R. Stachowiak2, T. Iskratsch1, S. Ghassemi1, P. Roca-Cusachs3,4, B.
O'Shaughnessy2, J. Hone1, M.P. Sheetz5,6; 1Columbia University, New York, NY, 2Department of Chemical
Engineering, Columbia University, New York, NY, 3Institute for Bioengineering of Catalonia (IBEC),
Barcelona, Spain, 4University of Barcelona, Barcelona, Spain, 5Mechanobiology Institute, National
University of Singapore, Singapore, NY, 6Biological Sciences, Columbia University, New York, NY
The rigidity of the extracellular matrix (ECM) affects processes such as cell migration, apoptosis,
proliferation, and differentiation. To sense the rigidity of the ECM, the leading edge of the cell protrudes
forward, encounters a new matrix, and pulls on it via nascent integrin adhesions. At its most basic sense,
rigidity sensing is taken as the decision to reinforce the adhesions during the initial period of force
SUNDAY-ORAL PRESENTATIONS
application. Thus, with increased rigidity, cells build stronger adhesions that can withstand higher forces.
This indicates the existence of a tight regulation mechanism between rigidity, force production, and
adhesion reinforcement, all occurring at the sub-micrometer level within a short time window of up to
few tens of seconds. However, it is unknown how this rigidity/force feedback loop is regulated.
Addressing this question requires ultra-high resolution analysis of cellular forces, both in time and space.
Thus, in the current work, we measured myosin force production within cells at the nanometer and
millisecond level during rigidity sensing. Using arrays of Polydimethylsiloxane (PDMS) micropillars as
substrates for spreading of mouse embryonic fibroblasts, we found that local contractile units that bear
resemblance to sarcomeres pulled opposing pillars in nano-level myosin-II-generated step-wise
contractions. Surprisingly, cells produced the same step size of 2.1-2.4 nm, regardless of rigidity. What
correlated with rigidity was the number of steps taken before reaching a ~20 pN force level, which
activated the recruitment of α-actinin to the pillars. The constant step size suggested a structural
restriction for the myosin motors; indeed, when we knocked-down tropomyosin-1 (a known a
regulatory protein that wraps around actin filaments), the step-wise movements were dramatically
altered and larger steps were observed. Further, after tropomyosin-1 knock-down, the cells could not
sense the rigidity properly, indicating that tropomyosin-1 is directly involved in rigidity sensing.
Importantly, tropomyosin-1 was previously implicated as a tumor suppressor whose extremely low
expression levels in malignant cells allows them to grow on soft agar (i.e., anchorage-independent
growth). When we tested a non-malignant vs. malignant cell line, we verified that the absence of
tropomyosin correlated with higher forces, suggesting that anchorage-independent growth depends
upon the production of high forces that activate the signaling cascades required for proliferation even
on soft matrices.
E5
Zyxin mediated regulation of airway smooth muscle function may be involved in
asthmatic airway hypercontractility. S.R. Rosner1, C.D. Pascoe2, E. Blankman3, C.C. Jensen3, R.
Panganiban4, R. Krishnan5, C.Y. Seow2, J. Park4, Q. Lu1, M.C. Beckerle3, P.D. Pare2, J.J. Fredberg4, M.A.
Smith3;
1
Department of Environmental Health, Harvard School of Public Health, Boston, MA, 2St Paul's Hospital
Center for Lung Innovation, University of British Columbia, Vancouver, BC, 3Huntsman Cancer Institute,
University of Utah, Salt Lake City, UT, 4Harvard School of Public Health, Boston, MA, 5Center for Vascular
Biology Research, Beth Israel Deaconess Medical Center, Boston, MA
Morbidity and mortality due to asthma arise mainly from loss of airway patency as a result of airway
hypercontractility. Airway hypercontractility is the result of excessive contraction of airway smooth
muscle. Airway hypercontractility in the non-asthmatic lung is readily reversed by the taking of a deep
breath, which results in bronchodilation. A deep breath stretches the contracted airway smooth muscle
causing it to fluidize rapidly and then resolidify slowly. On the other hand, airway hypercontractility in
the asthmatic lung is generally refractory to the dilative effect of a deep breath. In the asthmatic case,
the airway smooth muscle enters what is called a latch state, wherein the parenchymal tissue lacks
sufficient tethering force to stretch the airway smooth muscle. The molecular mechanisms of stretch
SUNDAY-ORAL PRESENTATIONS
induced airway smooth muscle fluidization and resolidification are unknown, as is the mechanism of its
failure in asthmatics.
The LIM-domain scaffolding protein zyxin has been shown to be involved in repairing, remodeling and
reinforcing actin stress fibers in response to mechanical strain. Thus, zyxin is essential for generation of
peak traction forces. Zyxin is rapidly recruited to sites of stress fiber strain, where it enlists actin
regulatory proteins to effect repair. Due to its role in regulation of the actin cytoskeleton’s response to
force, we hypothesized zyxin might be involved in the regulation of the airway smooth muscle response
to stretch. In this work we show that zyxin plays a critical role in reinforcing the actin cytoskeleton of
airway smooth muscle in response to stretch. We demonstrate that zyxin responds to physiologic levels
of isotropic stretch through rapid recruitment to sites of acute strain on stress fibers, and in murine
airway smooth muscle, loss of zyxin results in abrogated resolidification following single or multiple
stretches. In precision cut lung slices from wild type or zyxin null mice, the airways from zyxin null mice
dilate more rapidly in response to stretch. Furthermore, zyxin expression is increased in the lungs of
humans who experienced fatal asthma attacks.
Together, these data show a role for zyxin in the regulation of airway smooth muscle response to
stretch, and suggest a connection to airway hypercontractility in human asthmatics.
E6
Malignant melanoma cells dynamically construct a tumor biofilm that promotes
survival in response to drug treatment and hypoxia.
K. Tanner1, A. Afasizheva1;
1
NCI/NIH, Bethesda, MD
Cutaneous melanoma cells secrete and assemble a myriad of extracellular matrix (ECM) molecules into
dense fibrillar and globular networks. This process is dynamic, mimicking biofilm formation by bacteria
in response to nutrient deficiencies, antibiotic treatment and oxidative stress. We hypothesize that the
melanoma ‘tumor biofilm’ is similarly adaptive and protective in response to external insults, including
pharmaceutical intervention and hypoxic stress. Using quantitative proteomics, we identified the
components and determined the temporal evolution of the tumor biofilm. Isogenic melanoma lines
were cultured in 3D to recapitulate tumor morphology and interrogated for the establishment of a de
novo tumor microenvironment as a function of dimension, oxygen tension and drug treatment. We
recreated an established tumor and drug regimen using a modified 3D soft agarose assay to evaluate
anchorage independent proliferation. Modulation of the observed tumor biofilm in culture was
monitored using confocal microscopy, protein expression analysis and immunofluorescence, and its
physiological impact on primary tumor establishment was examined in a mouse subcutaneous injection
model. Fibronectin was found to be one of the key architectural components, regulating drug efficacy
for a broad spectrum of drug therapies. Stable cell lines engineered to secrete minimal levels of
fibronectin became sensitive to cisplatin, BRAF, and ERK inhibition as clonally derived 3D tumor
aggregates, emphasizing the importance of 3D architecture in tumor proliferation. Reduced levels of
tumor-generated fibronectin also impeded successful tumor formation in mice. We reason that this
architectural complexity provides the degree of plasticity and flexibility that allows for better colonial
SUNDAY-ORAL PRESENTATIONS
adaptability and survival in response to external perturbations, such as hypoxic stress and drug
treatments
E7
Coordination of actin-based activities in the front and back of migrating cells.
N. Ramalingam1, C. Franke2, E. Jaschinski3, M. Winterhoff2, Y. Lu4, S. Bruehmann2, A. Junemann2, H.
Meier2, A. Noegel5,6,7,8, A. Csiszar3, I. Weber9, H. Zhao4, M. Schleicher10, R. Merkel3, J. Faix2;
1
Columbia University, New York, NY, 2Hannover Med School, Hannover, Germany, 3Institute of Complex
Systems 7, Forschungszentrum Juelich, Juelich, Germany, 4Institute of Biotechnology, University of
Helsinki, Helsinki, Finland, 5Institute for Biochemistry, Medical Faculty, University of Cologne, Cologne,
Germany, 6Cologne Center for Genomics (CCG), Cologne, Germany, 7Center for Molecular Medicine
Cologne (CMMC), Cologne, Germany, 8Cluster of Excellence, Cellular Stress Responses in AgingAssociated Disease, Cologne, Germany, 9Dept. Molecular Biology, Ruder Boškovic Institute, Zagreb,
Croatia, 10Ludwig-Maximilians Univ, Munchen, Germany
Eukaryotic cells move by highly polarized protrusion of membrane sheets filled with a dense meshwork
of actin filaments termed lamellipodia. It is widely appreciated that SCAR/WAVE-complex-activated and
ARP2/3-complex-mediated actin nucleation beneath the plasma membrane is crucial for the assembly of
actin in protruding lamellipodia. However, it has remained elusive how cells prevent the formation of
actin-based protrusions at the lateral sides and their back. Here, we used the model system
Dictyostelium discoideum, displaying similar motile properties as neutrophils, to address this
fundamental question. In a systematic search for factors accumulating in the trailing edge of migrating
cells we identified an as yet uncharacterized Diaphanous-related formin. Notably, the formin-null
mutants displayed a markedly faster random migration as compared to wild type. This phenotype is
highly reminiscent of mutants lacking the IQGAP-related protein DGAP1. Interestingly, DGAP1 is tightly
associated with the heterodimeric actin-bundling protein cortexillin, and both, DGAP1 and cortexillin
also accumulate in the back of migrating cells. Microcapillary aspiration assays with single or double
mutants suggest that a moderate reduction of cortical tension, such as in formin- or DGAP1-null cells, is
the reason for enhanced motility, whereas further decrease of cortical tension is deleterious for
coordinated cell movement. Together, these findings strongly indicate that formin-mediated actin
filament nucleation/elongation as well as the actin-bundling activities act in concert to locally increase
the mechanical rigidity of the cortical actin meshwork to inhibit cell protrusions. Moreover, IQGAP
appears to prevent SCAR/WAVE-induced ARP2/3-complex activation in the trailing cells by sequestering
active Rac1. Finally, we demonstrate how proper localization of these components is accomplished.
SUNDAY-ORAL PRESENTATIONS
ePoster Talks Session 2: Microtubules and Microtubule-Related
Motors
E8
Structural insights into the motility generation and regulation of cilia and flagella
by cryo-electron tomography.
J. Lin1, D. Nicastro1;
1
Department of Biology, Rosenstiel Basic Medical Sciences Research Center, Brandeis University,
Waltham, MA
Cilia and flagella play important roles in normal development and health of many eukaryotes, including
humans [1, 2]. Their motility is driven by thousands of dynein motors [3, 4] that are coordinated by
regulatory complexes, such as the Nexin-dynein regulatory complex and I1 dynein [5-8]. Our recent cryoelectron tomography study of intact sea urchin sperm flagella that were rapidly frozen while actively
beating, revealed the 3D structures of dynein in three distinct post- and pre-powerstroke conformations
[9]. These results provided important new insights into the structural basis of dynein’s force generation
[9], but how ciliary dyneins are regulated in detail and their different roles in generating ciliary motility
remain unclear. Here, we analyzed cryo-tomograms of actively beating sea urchin sperm flagella to
determine the 3D structures and conformational changes of all major regulatory complexes and dynein
isoforms in relation to different regions of the sinusoidal wave of beating flagella (i.e., curved regions of
principal or reverse bend, or straight regions between bends). For some of the dynein isoforms and
predicted regulatory complexes we resolved distinct structures and distribution patterns of different
conformations that correlate well with specific regions along the flagellar waves. Our comprehensive
high-resolution analysis provides a new understanding of the molecular mechanisms underlying ciliary
and flagellar motility and suggests a model that shifts previous hypotheses.
References: [1] Fliegauf et al. (2007) Nat Rev Mol Cell Biol. 8:880-93; [2] Mitchell (2007) Adv Exp Med
Biol. 607:130-40; [3] Summers and Gibbons. (1971) Proc. Nat. Acad. Sci. USA. 68:3092-6; [4] Sale and
Satir (1977) Proc. Nat. Acad. Sci. USA. 74:2045-9; [5] Huang et al. (1982) Cell. 28:115-24; [6] Piperno et
al. (1994) J Cell Biol. 125:1109-17; [7] Bower et al. (2009) Mol Biol Cell. 20:3055-3063; [8] Toba et al.
(2010) Mol Biol Cell. 22:342-53; [9] Lin et al. (2014) Nat Cell Biol. 16:479-85.
E9
TACC3 is a microtubule plus-end tracking protein that promotes axon elongation
and also regulates microtubule plus-end dynamics in multiple embryonic cell
types.
B.U. Nwagbara1, B. Erdogan1, E. Bearce1, P. Ebbert1, M. Evans1, E. Rutherford1, L.A. Lowery1;
1
Biology, Boston College, Chestnut Hill, MA
Proper neural connections, essential to nervous system function, depend upon precise navigation by the
neuronal growth cone. A fundamental problem in growth cone cell biology is how guidance pathways
are integrated to coordinate cytoskeletal dynamics, thus driving accurate steering. To address this
SUNDAY-ORAL PRESENTATIONS
question, we focus on the plus-ends of microtubules, which explore the growth cone periphery and play
a key role in growth cone steering. Microtubule plus-end dynamics are regulated by a conserved family
of proteins called ‘plus-end-tracking proteins’ (+TIPs). Yet, it is still unclear how +TIPs interact with each
other and with plus-ends to control microtubule behavior, especially in the developing nervous system.
The centrosome-associated protein TACC3, a member of the transforming acidic coiled coil (TACC)
domain family, has been previously implicated in regulating several aspects of microtubule dynamics.
However, TACC3 has not been shown to function as a +TIP in vertebrates. Here, we show that TACC3
promotes axon outgrowth and also regulates microtubule dynamics by increasing microtubule plus-end
velocities in vivo. We also demonstrate that TACC3 acts as a +TIP in multiple embryonic cell types, and
that this requires the conserved C-terminal TACC domain. Using high-resolution live-imaging data of
tagged +TIPs, we reveal that TACC3 localizes to the extreme microtubule plus-end, where it lies distal to
the microtubule polymerization marker, EB1, and directly overlaps with the microtubule polymerase,
XMAP215. TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to
microtubule plus-ends. Together, our results implicate TACC3 as a +TIP that functions with XMAP215 to
regulate microtubule plus-end dynamics.
E10
NuSAP stabilizes kinetochore microtubules by attenuating MCAK
depolymerization activity.
C. Li1, Y. Liou1;
1
Department of Biological Science, National University of Singapore, Singapore, Singapore
NuSAP (Nuclear and Spindle Associated Protein), a novel Microtubule Associated Protein (MAP),
functions as a microtubule stabilizer. Depletion of NuSAP leads to severe mitotic defects. However, it
remains unclear how NuSAP stabilizes microtubules in cells during mitosis. In this paper, we dissect the
function of NuSAP in stabilizing kinetochore microtubules during metaphase and present the first
evidence of the direct interaction of NuSAP and a MT depolymerizer, MCAK. NuSAP tightly regulates the
localization, dynamics and depolymerization activity of MCAK. Furthermore, we demonstrate that
Aurora B kinase regulates the functional complex of NuSAP and MCAK at kinetochore region. Aurora B
significantly enhances the interaction of NuSAP with MCAK and further modulates the effects of NuSAP
on the localization and depolymerization activity of MCAK. Thus, we provide new insights into the
complex regulation of kinetochore microtubules dynamics during metaphase to ensure proper tension
at kinetochore and establish the attachment of kinetochore microtubules to promote precise cell
division.
SUNDAY-ORAL PRESENTATIONS
E11
Measurement of the force that centers the mitotic spindle in the early C. elegans
embryo using magnetic tweezers.
C. Garzon-Coral1, H. Fantana1, J. Howard2;
1
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, 2Molecular Biophysics
and Biochemistry, Yale University, New Haven, CT
Because the plane of cell division bisects the mitotic spindle, the positioning of the spindle specifies the
size and location of the two daughter cells. Little is known, however, about the mechanical processes
underlying spindle positioning. To study this mechanism, we applied calibrated magnetic forces to the
spindle via super-paramagnetic beads inserted into the cytoplasm of one- and two-cell C. elegans
embryos.
At metaphase in the one-cell embryo, a 20 pN force displaced the mitotic spindle pole of one-cell
embryos approximately 1 μm away from the anterior-posterior axis over 10-20 seconds. By tracking the
bead displacement, we found that the spindle behaved roughly as a damped spring with a spring
constant of 18 ± 12 pN/μm and a drag coefficient of 127 ± 65 pN∙s/μm (mean ± SD).
The centering stiffness was two-fold higher in the two-cell embryo, consistent with a centering
mechanism that scales inversely with cell size. The stiffness increased when the number of microtubules
reaching the cortex was increased two fold using RNAi against the depolymerizing kinesin klp-7, but
remained roughly the same when the interaction time of microtubules with the cortex was increased
using RNAi against efa-6, a cortical catastrophe factor and when cortical pulling forces were reduced by
RNAi against gpr-1/2, an activator of the cortical force generators. Furthermore, the stiffness was fivefold higher during anaphase in both the one- and two-cell stage embryos, indicating that the centering
forces change during the cell cycle.
Taken together, our results constrain molecular models of centering. The gpr-1/2 RNAi knockdown
results rule out a role for cortical forces pulling on the spindle via astral microtubules and the scaling
with cell argues against pulling by cytoplasmic factors. On the other hand, the results are consistent with
centering being mediated by astral microtubules pushing against the cortex as the centering forces
scales with the number of microtubules, the cell size and the pushing time of microtubules against the
cortex.
Moreover, these results help us to understand how change in mechanical properties is linked to the
function of the spindle in metaphase and anaphase.
SUNDAY-ORAL PRESENTATIONS
E12
A molecular mechanism for the anterograde transport of dynein in the axon.
A.E. Twelvetrees1,2, G. Schiavo2, E.L. Holzbaur3;
1
Physiology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, 2Sobell
Department of Motor Neuroscience and Movement Disorders, University College London Institute of
Neurology, London, United Kingdom, 3Department of Physiology, University of Pennsylvania, Perelman
School of Medicine, Philadelphia, PA
Microtubules in the axon are uniformly orientated with their plus ends directed away from the soma
and towards the axon terminal. Thus, the minus end directed microtubule motor, cytoplasmic dynein,
has no ability to reach the axon terminal under its own ATP hydrolysis power and must be actively
transported. This process is critical to neuronal function as without the presence of dynein in axon
terminals there is no facility for essential retrograde trafficking events that are key to neuronal survival.
In neurones cultured from DIC1-GFP knock in mice (which express a GFP tagged form of the neuron
specific dynein intermediate chain subunit, DIC1, at endogenous levels) dynein accumulates in axon
terminals. By live cell TIRF-M, imaging in axons of known polarity, we observe many dynein-GFP motile
events in both anterograde and retrograde directions. Previous work in the lab had identified direct
interactions between kinesin-1 and the DIC subunit. Building on this observation we find multiple points
of protein-protein interaction between DICs and both the light chain and heavy chain subunits of
kinesin-1. These direct interactions are consistent with activation of kinesin by dynein as well as being
specific to neuronal spliceforms of the DIC subunits. Consistently, we find dynein and kinesin form an
endogenous complex in vesicle free brain fractions. Using FRAP analysis we find that net rates of
anterograde transport for dynein populations in the axon are the same as those that typically describe
slow axonal transport. We propose that neuron specific isoforms of dynein subunits support the direct
activation of kinesin by dynein in order to power the anterograde transport of dynein in the axon.
E13
14-3-3 proteins tune non-muscle myosin II assembly, providing a possible bridge
between cell mechanics and cancer metastasis.
H. West-Foyle1, J. Osborne1, D. Robinson1,2;
1
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, 2Department
of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD
The 14-3-3 family comprises a group of small acidic regulatory proteins which are essential, ubiquitous,
and highly conserved across eukaryotic species. These proteins have roles in numerous core cellular
processes such as DNA damage repair, cell cycle control, apoptosis prevention, and certain signaling
pathways. Generally 14-3-3s act as tumor suppressors and consequently, are down-regulated in many
cancers. By contrast, overexpression of 14-3-3s sigma, epsilon, zeta, and eta correlate with a higher
metastatic potential and poorer clinical outcomes in breast, liver, and pancreatic cancers. The literature
has no unified theory on how 14-3-3 proteins that are normally tumor suppressors become oncogenes.
However, we have uncovered a role for 14-3-3s in regulating the assembly of non-muscle myosin II. This
SUNDAY-ORAL PRESENTATIONS
demonstrates that 14-3-3s could tune cell mechanics directly, and therefore, contribute to the
progression of metastatic cancers. Here, we examine the entire landscape of myosin II assembly
regulation by 14-3-3 in Dictyostelium (one 14-3-3, one non-muscle myosin II) and humans (seven 14-3-3s
and three non-muscle myosin IIs). In Dictyostelium, 14-3-3 mediates a pathway between microtubules
and racE small GTPase to regulate myosin II assembly, and 14-3-3’s expression levels negatively
correlate with BTF accumulation. In vitro assembly assays using purified myosin II tail fragments and 143-3 demonstrate that this interaction is direct and phosphorylation-independent. We then extended our
analysis to the seven human 14-3-3 paralogs and the three non-muscle myosin IIs. We found that the
seven human paralogs of 14-3-3 affect the assembly of myosin II filaments in different ways, broadly
clustering into those that cause myosin II overassembly and those that cause its solubilization.
Assemblers and solubilizers can directly compete to govern the overall level of myosin II assembly.
Examining assembled myosin II filaments by EM confirms that the average filament size correlates with
the overall assembly level. Furthermore, we mapped three critical residues which differ between the
two classes and discovered that mutating any of these residues converts an assembler to a solubilizer.
Our findings demonstrate a novel phosphorylation-independent method for regulating myosin II
assembly that is mechanistically conserved from amoebas to humans. These findings imply that altered
14-3-3 expression profiles could directly modulate cell mechanics in metastatic cancers, which would be
of great interest for basic and clinical sciences alike.
E14
Kinesin regulation dynamics through cargo delivery, a single molecule
investigation.
A.P. Kovacs1, J.M. Kessler1, H. Lin2, S.K. Dutcher2, Y.M. Wang1;
1
Washington University School of Medicine, Saint Louis, MO, 2Genetics, Washington University School of
Medicine, Saint Louis, MO
Kinesins are microtubule-based motors that deliver cargo to their destinies in a highly regulated manner.
Although in recent years numerous regulators of cargo delivery have been identified, the regulation
mechanism of kinesin through the cargo delivery and recycling process is not known. By performing
single molecule fluorescence imaging measurements in Chlamydomonas flagella of 200 nm in diameter
and microns in length that contains 9 sets of microtubule doublets, we tracked the intraflagellar
transport (IFT) train and BBSome cargo and kinesin-2 motors through the cargo delivery process and
obtained the aforementioned dynamics. Upon arrival at the microtubule plus end at the flagellar tip, (1)
intact IFT train and BBSome cargo together dissociate from kinesins and microtubules and diffuse and
reorganize along flagellar membrane for a mean of 2.3 sec before commensurating retrograde travel.
(2) Kinesin motors remain bound to and diffuse along microtubules for 1.3 sec before dissociating into
flagellar lumen for recycling.
SUNDAY-ORAL PRESENTATIONS
ePoster Talks Session 3: Stem Cells, Tissues, Organs, and Pathogens
E15
A Chlamydia effector manipulates host microtubule dynamics to promote
bacterial survival.
M. Dumoux1, A. Menny1, D. Delacour2, R. Hayward1;
1
Biological sciences, Institute of structural and molecular biology (Birkbeck college-UCL), London, United
Kingdom, 2Institut Jacques Monod - CNRS, Paris, France
Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes sexually transmitted
disease worldwide and is the leading cause of acquired blindness (trachoma) in developing nations.
Chlamydiae employ a type III secretion system to deliver virulence effector proteins into host cells.
These effectors subvert fundamental cellular processes, allowing bacterial intracellular survival and
replication within a specialized membrane-bound compartment termed an inclusion. We discovered
that C.trachomatis dramatically reorganizes the microtubule cytoskeleton within infected cells to form a
filamentous superstructure. This comprises a microtubule scaffold surrounding the inclusion and an
associated nest of microtubules that originate at the inclusion and extend towards the plasma
membrane. Using a bioinformatics approach, we identified a chlamydial effector (inclusion protein
acting on microtubules; IPAM) that shares limited sequence similarity with eukaryotic centrosomal and
microtubule-binding proteins. Using pull-down and co-immunoprecipitation assays we demonstrate that
IPAM, a hydrophobic effector known to associate with the inclusion membrane, interacts with host
centrosomal protein 170kDa (CEP170), a centrosome and kinesin-interacting protein, via a domain
exposed to the cytosol. Ectopic expression of this IPAM domain impairs microtubule dynamics. This
defect was restored by simultaneous CEP170 knock-down, demonstrating functional interplay between
IPAM and CEP170 in cells. CEP170 is essential for chlamydial control of host microtubule dynamics and
organelle repositioning during infection, and is necessary for inclusion morphogenesis and bacterial
infectivity. Moreover, as IPAM stimulates CEP170 functions in Chlamydia-infected cells, this reveals
potential physiological roles of CEP170 repressed in non-dividing uninfected cells. Together our data
show how a chlamydial type III secretion effector stimulates a host target to promote the reorganization
of the microtubule cytoskeleton in infected cells, an event required for inclusion biogenesis and
bacterial intracellular survival. In turn this provides broader insight into the control of cellular
microtubule dynamics.
E16
Deficient protein palmitoylation in the deadly fungus Cryptococcus neoformans
affects pathogenesis by altering fungal interactions with host cells.
F.H. Santiago-Tirado1, M. Yang1, T.L. Doering1;
1
Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO
Cryptococcus neoformans is the most common fungal cause of meningitis, responsible for over 625,000
deaths every year. Most of these deaths are in immunocompromised patients, historically in the setting
SUNDAY-ORAL PRESENTATIONS
of AIDS, but currently also affecting patients with other underlying conditions such as cancer or organ
transplant. C. neoformans is a facultative intracellular pathogen, and adherence to and uptake by host
phagocytes during infection are key events that are central in its pathogenesis. Fungal engulfment by
host cells and subsequent intracellular proliferation has been implicated in latency, dissemination, and
virulence, but the full complement of C. neoformans gene products that participate in these processes
has not been defined. To address this question, we used an automated high content imaging method to
quantify the interactions between a human macrophage-like cell line and mutant fungi from a partial
deletion collection. We identified multiple genes whose deletion led to alterations in the adherence
and/or phagocytic indexes. One of the genes identified encode a protein S-acyl transferase, one of a
family of DHHC domain-containing proteins that catalyzes lipid modification of proteins. Deletion of this
gene, termed PFA4, results in enhanced adherence to and phagocytosis by human macrophages.
Mutant cells lacking PFA4 exhibit morphological defects that are exacerbated under host-like conditions,
and are sensitive to a variety of cell wall stress conditions in vitro. Consistent with these observations,
they have a profound defect in intracellular growth and are avirulent in a mouse model of cryptococcal
infection. These results suggest a surface defect but, interestingly, the mutant cells show no obvious
defect in the polysaccharide capsule that surrounds their cell walls, which is a major virulence factor.
They do, however, display altered cell wall structure and increased exposure of surface glycans
compared to wild type. Defects in lipid modification may cause mislocalization or degradation of the
substrate proteins, leading to their dysfunction. We hypothesize that C. neoformans uses palmitoylation
to regulate its surface composition and thereby modulate its interactions with the host. Our current
efforts are directed at identifying the specific protein substrate(s) responsible for the changes we
observe in the pfa4 mutant. Little is known about the role palmitoylation plays in virulence of this or
other pathogenic fungi, hence these findings open a new area of investigation and promises novel
avenues for therapeutics based on the role of palmitoylation in fungal pathogenesis.
E17
Distinct Actin Nucleation Mechanisms are Required for the Differentiation
Programs of EMT and Embryonic Stem Cells.
M.K. Rana1, B.K. Grillo-Hill1, C. Choi2, D.L. Barber1;
1
Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, 2Department of
Radiation Oncology, Samsung Medical Center, Seoul, Korea
Marked advancements have been made toward identifying transcriptional events controlling the
programs of epithelial to mesenchymal differentiation (EMT) and embryonic stem cell (ESC)
differentiation. However, both programs require dramatic remodeling of actin filaments and we know
little about how this remodeling is regulated, particularly the role of actin nucleators that generate
distinct actin architectures. We found that formins are necessary for EMT but the Arp2/3 complex is
necessary for ESC differentiation. The broad spectrum formin inhibitor SMIFH2 but not the Arp2/3
complex inhibitor CK666 blocked EMT of human lung A549 cells and mouse mammary NMuMG cells
induced by TGF-β. SMIFH2 blocked the change in cell shape to a mesenchymal morphology, remodeling
of actin filaments to actin stress fibers, loss of cell-cell contacts, decreased expression of E-cadherin,
increased expression of fibronectin and nuclear translocation of the transcription co-factor MRTF. We
SUNDAY-ORAL PRESENTATIONS
also found the surprising result that SMIFH2 inhibited proximal signaling by TGF-β, as indicated by
decreased phosphorylation of Smad2 (pSmad2), a TGF-β receptor substrate. We tested shRNA for 9 of
the 15 human formin family members and found that silencing expression of only two, FHOD1 and
formin-1, inhibited EMT, but through distinct mechanisms. FHOD1 shRNA blocked the assembly of actin
stress fibers and increased phosphorylation of myosin light chain that were rescued by expression of
wild type FHOD1; however, pSmad2 was not blocked. Formin-1 shRNA blocked loss of cell-cell contacts,
internalization of E-cadherin and nuclear translocation of MRTF but not the assembly of actin stress
fibers. We are testing whether formin-1 is necessary for increased pSmad2. In contrast to EMT, CK666
but not the inactive analog CK689 or SMIFH2 inhibited mouse ESC differentiation. Using a dual reporter
line with self-renewing, undifferentiated cells expressing mCherry and differentiated cells expressing
GFP, we used FACS analysis to show that at 72 h of differentiation there were no differences between
controls, CK689 or SMIFH2 but with CK666 there were 50% more self-renewing cells and 35% fewer
differentiated cells. Differentiated cells had substantially more and larger membrane protrusions and
distinct asymmetric clustered actin filaments at protrusions. These changes were completely blocked by
CK666 but not SMIFH2. To our knowledge, neither Arp2/3 complex regulation of stem cell
differentiation nor formin-dependent Smad signaling has been reported. We predict that requirements
for formin-dependent EMT compared with Arp2/3-dependent ESC differentiation reflect distinct needs
of the differentiation program, specifically the need for increased cell contractility with EMT but not ESC
differentiation.
E18
Lack of arginylation causes stem cells’ inability to maintain pluripotency, leading
to abnormalities during embryogenesis.
S. Kurosaka1, N.A. Leu2, A.S. Kashina3;
1
University of Hiroshima, Hiroshima, Japan, 2Animal Biology, University of Pennsylvania, Philadelphia,
PA, 3Department of Animal Biology School of Veterinary Medicine, University of Pennsylvania,
Philadelphia, PA
Mice lacking arginyltransferase Ate1 die during embryogenesis with severe malformations in the heart,
including thin myocardium, septation and outflow tract defects. The mechanisms of these
malformations have been unknown and cannot be recapitulated in any conditional model with Ate1
knockout in heart-forming cell lineages, suggesting that these defects may originate prior to lineage
specification. To investigate this possibility we have first attempted to generate Ate1 knockout stem
cells, but failed to do so on multiple attempts, suggesting that these cells may have viability issues in
culture or other reasons that preclude them from being generated. We next used primary embryonic
fibroblasts derived from E12.5 wild type and Ate1 knockout embryos to generate induced pluripotent
stem (iPS) cells by transfecting them with an expression cassette containing combination of
reprogramming factors known to induce pluripotency (Oct4, Sox2, and Klf4). This approach was
successful in yielding multiple Ate1 knockout iPS lines. These lines could maintain pluripotency in
culture, however they showed a decrease in the intracellular level of several other pluripotent markers
compared to the control. Moreover, removal of the expression cassette resulted in their inability to
maintain pluripotency and induced their spontaneous differentiation into cardiomyocytes over the
SUNDAY-ORAL PRESENTATIONS
course of several days. Chimeras generated from these iPS cells died in development with prominent
malformations and morphogenic defects. These data suggest that Ate1 is required to maintain normal
intracellular levels of pluripotency factors in embryonic stem cells during early embryogenesis. We
propose that cardiovascular malformations seen in Ate1 knockout embryos result from premature
differentiation of cardiac progenitors into non-proliferating cardiomyocytes, which can occur before the
progenitors can expand sufficiently to ensure normal cell numbers in the developing myocardium and
septae. This effect is likely universal and may affect other tissues and organs that do not have a chance
to develop due to the timing of Ate1 knockout embryonic lethality.
E19
Super-resolution microscopy reveals a role for the tetraspanin CD82 in regulating
integrin molecular clustering.
C.M. Termini1, M.L. Cotter1, K.D. Marjon1, K.A. Lidke2, J.M. Gillette1;
1
Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM,
2
Department of Physics and Astronomy, University of New Mexico, Albuquerque, NM
Hematopoietic stem and progenitor cell (HSPC) adhesion to the surrounding microenvironment is critical
for maintaining HSPC proliferation, differentiation, and survival. However, our present understanding of
the mechanisms that regulate this adhesive interaction remains incomplete. Previously, our lab
identified the tetraspanin, CD82, as a critical regulator of HSPC adhesion and homing to the bone
marrow microenvironment. More specifically, we found that that CD34+ cells treated with monoclonal
CD82 blocking antibodies display reduced homing and engraftment in vivo. Tetraspanins, like CD82, are
membrane scaffold proteins that promote the organization of membrane proteins, such as integrins,
which contribute to cellular adhesion and migration. Therefore, we hypothesize that CD82 regulates
HSPC adhesion by modulating integrin organization and density at the nanoscale. In order to test this
hypothesis, we generated stable KG1a cells that overexpress wild-type CD82 (CD82OE). We found that
the CD82OE cells display increased adhesion to fibronectin when compared to control cells. Pre-treating
CD82OE cells with LDV, an α4β1 specific blocking peptide, inhibited the CD82-mediated increase in
adhesion, suggesting the involvement of the α4β1 integrin in HSPC adhesion. In order to determine how
molecular clustering contributes to HSPC adhesion, we used the super-resolution microscopy technique,
dSTORM. We quantified CD82 clustering by pair-autocorrelation analysis and determined that there is
an increase in CD82 cluster size in CD82OE cells when compared to the control cells. The palmitoylation
of tetraspanins has been shown to regulate lateral membrane protein packing, which is critical for cell
adhesion and migration. In order to test the role of CD82 palmitoylation in modulating membrane
complex organization and cell adhesion, we also generated KG1a cells that overexpress a mutant form
of CD82 that cannot be palmitoylated (Palm-CD82OE). As predicted, we found that the Palm-CD82OE
cells display reduced cell adhesion and significantly smaller CD82 clusters than CD82OE cells.
Additionally, we used the DBSCAN clustering algorithm to quantify the α4 cluster area from our superresolution data, finding that CD82OE cells display decreased α4 cluster area when compared to control
and Palm-CD82OE cells. Interestingly, we found an increase in the number of α4 molecular locations per
0.01 um2 in CD82OE cells when compared to control and Palm-CD82OE cells. Taken together, these data
suggest that CD82 regulates α4 molecular density and palmitoylation of CD82 is critical for its regulation
SUNDAY-ORAL PRESENTATIONS
of tightly packed α4 clusters within the plasma membrane. Additionally, we will discuss how CD82
expression and mutation control β1 integrin activation and signaling.
E20
Mechanism Regulating Synchronous Collagen Expression and Trafficking during
Stem Cell to Chondrocyte Differentiation.
G. Unlu1, E.W. Knapik2;
1
Vanderbilt University, Nashville, TN, 2Medicine, Vanderbilt University Medical Center, Nashville, TN
Transition from the stem cell phase to a differentiated cell type is characterized by retooling cellular
machinery, including regulatory molecules, structural proteins, and their transport machinery. To
understand how the differentiation process provisions for synchronized changes in cellular functions
during development, we used a robust in vivo model of zebrafish neural crest stem cells, which
differentiate into chondrocytes and then produce, transport, and secrete procollagen II.
Although procollagen synthesis and trafficking are well studied, little is known about how collagenspecific transport machinery and collagen cargo are made synchronously available during vertebrate
development and what mechanisms restrain this process from occurring in stem cells.
We have previously shown that procollagen transport requires COPII-inner coat components sec23a and
Sec24D to promote collagen secretion (1,2), and a transcription factor, Creb3L2 to activate the
expression of these COPII adaptors (3). We hypothesize that a stem cell factor temporarily restricts both
collagen expression and trafficking machinery in the undifferentiated stem cell state; and its
downregulation during chondrogenic differentiation allows for synchronous upregulation of collagen
expression and secretion programs. Our in silico analyses suggested that a neural crest stem cell factor
may bind to the promoter region of Creb3L2 to suppress collagen secretion as well as the promoter of a
major chondrogenic factor Sox9 to inhibit collagen expression during cartilage development.
Using in vivo mosiac analysis, we find that overexpression of the stem cell factor in differentiated
zebrafish chondrocytes and human fibroblasts downregulates both collagen expression and secretion.
We demonstrate that overexpression of the stem cell factor leads to reduction in CREB3L2 promoter
activity as detected by luciferase-based assays as well as decreased transcript levels of CREB3L2 and
components of ER-to-Golgi (COPII) trafficking machinery, especially SEC23A and SEC24D. The changes at
the transcript levels are matched by intracellular accumulation of type-I and type-II collagens. Our data
support a model in which a neural crest stem cell factor acts as a master regulator of collagen
expression and intracellular trafficking machinery; and its developmentally driven downregulation leads
to synchronous upregulation of collagen cargo and its transport machinery during differentiation
References: (1) Lang, et.al., Nature Genetics 2006. (2) Sarmah, et.al., PLoS One, 2010. (3) Melville, et.al.,
Disease Models and Mechanisms, 2011.
E21
Anti-chaperone activity of human α-defensin HNP1 against bacterial toxins.
E. Kudryashova1, R. Quintyn1, S.M. Seveau2, W. Lu3, V. Wysocki1, D.S. Kudryashov1,4;
1
The Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH,
SUNDAY-ORAL PRESENTATIONS
2
Microbiology, The Ohio State University, Columbus, OH, 3THe Department of Chemistry and
Biochemistry, University of Maryland, Baltimore, MD, 4Molecular and Cellular Developmental Biology
Program, The Ohio State University, Columbus, OH
Defensins are short cationic, amphiphilic, cysteine-rich peptides that constitute the front line immune
defense against various pathogens. In addition to exerting direct antibacterial activities, defensins
inactivate several classes of unrelated bacterial exotoxins. To date, no coherent mechanism has been
proposed that would explain defensins’ enigmatic efficiency towards various toxins. We show that
binding of α-defensin HNP1 to affected bacterial toxins causes their local unfolding, potentiates their
thermal melting and precipitation, exposes new regions for proteolysis, and increases susceptibility to
collisional quenchers, without causing similar affects on tested mammalian structural and enzymatic
proteins. We propose that protein susceptibility to inactivation by defensins is contingent to their
thermolability and conformational plasticity and that the defensin-induced unfolding is a key element in
the general mechanism of toxin inactivation by human α-defensins.
ePoster Talks Session 4: Cell Signaling and Decision-Making
E22
Transmitotic persistence of Wnt pathway activity diversifies gene expression in
C. elegans embryos.
A. Zacharias1, T. Walton1, E. Preston1, J. Murray1,2;
1
Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, 2Penn Genome
Frontiers Insitute, University of Pennsylvania, Philadelphia, PA
Quantitative differences in signaling pathway activity are a powerful potential mechanism for
diversifying cell fates during development. However, few examples of this mechanism have been
identified in vivo, in part due to the challenges of performing quantitative assays. The objective of this
study was to evaluate whether C. elegans embryos use quantitative differences Wnt pathway activity to
regulate gene expression. The Wnt signaling pathway plays a conserved role during animal
development, transcriptionally regulating distinct targets in different stages and cell types (i.e. contexts).
This dependence of targets on context could reflect not only interactions with differentially expressed
transcription factors, but also context-specific differences in the activity of the Wnt pathway itself. We
investigated the role of Wnt pathway activity in target expression by using time-lapse microscopy and
automated lineage tracing of Caenorhabditis elegans embryos to quantify expression of Wnt ligands,
target genes, and nuclear localization of transcriptional effectors in vivo at single cell resolution
throughout development. We measured the Wnt pathway-dependence of candidate targets and
identified over a dozen important developmental regulators as new Wnt targets. We found that most
targets require the Wnt-effector transcription factor POP-1/TCF for either activation or repression but
not both. Contrary to existing models, we observed that Wnt-mediated transcriptional activation is
strongest in cells that received a Wnt signal in two or more consecutive divisions. We found that these
repeatedly signaled cells have higher nuclear β-catenin concentrations and are more likely to express
SUNDAY-ORAL PRESENTATIONS
targets that require POP-1 for transcriptional activation. Taken together, these results suggest that the
persistence of Wnt signaling across mitosis can integrate lineage history and allow Wnt to activate
distinct targets in different developmental contexts.
E23
NADPH oxidase activity is required for axonal development in zebrafish.
C.J. Weaver1, Y.F. Leung1, D.M. Suter1;
1
Department of Biological Sciences, Purdue University, West Lafayette, IN
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases produce bursts of reactive oxygen
species (ROS). Long thought to be involved exclusively in host defense, NADPH oxidase-derived ROS are
now appreciated as critical signaling intermediates in a growing number of cell signaling pathways.
However, their role in axonal growth and guidance has not been extensively studied. The zebrafish
(Danio rerio), because of excellent live-imaging capability and well-developed molecular genetics, is an
extremely powerful model system for studying axonal development in vivo. Our current study is the first
to detail the expression of the known zebrafish NADPH oxidase (Nox) isoforms, Nox1, Nox2, Nox5 and
Duox during development. Using quantitative PCR, we show that all Nox isoforms are expressed during
the first two days of development. Our results indicate that expression levels differ greatly among Nox
isoforms and over time. We used in situ hybridization to investigate Nox isoform expression in the brain,
retina, and spinal cord. In support of the qPCR results, we detected expression of all four Nox isoforms in
all brain tissues. Surprisingly, individual Nox isoforms are not tissue specific, but expressed broadly
throughout the brain and spinal cord. We next investigated the functional significance of NADPH
oxidases in axonal growth and guidance by applying two broad-spectrum Nox inhibitors, VAS2870 and
Celastrol, during critical periods of axonal development. Both the anterior commissure and the optic
nerve showed diminished axonal growth and the formation of mistargeted axons when NADPH oxidases
were inhibited. This demonstrates that despite broad expression, inhibition of NADPH oxidases only
interferes with the development of specific axons. In order to explore the function of individual Nox
isoforms during axonal development, we have used a CRISPR/Cas9-based method to generate the first
specific loss-of-function mutants for Nox1, Nox2, Nox5 and Duox in zebrafish. Upon the completion of
genetic and phenotypic analysis, we will assess axonal growth and guidance in each mutant line. This
study is the first to demonstrate an in vivo role for NADPH oxidase-derived ROS in axonal development.
E24
Desmoplastic 3D-adhesion structures orchestrate a tumor-associated ECM
induced myofibroblastic phenotype.
J. Franco-Barraza1, T. Luong1, N. Shah1, R. Madhani1, G. Cukierman2, K. Alpaugh1, K. Devarajan1, J.
Hoffman1, R. Uzzo1, E. Dulaimi1, E. Cukierman1;
1
Fox Chase Cancer Center, Philadelphia, PA, 2University of North Carolina, Chapel Hill, NC
The desmoplastic microenvironment plays a pivotal role in tumor development/progression. This
fibrotic niche is produced by myofibroblastic cells and is commonly observed in tumors such as
SUNDAY-ORAL PRESENTATIONS
pancreatic adenocarcinoma (PDAC) and renal cell carcinoma (RCC). We have shown that quiescent
fibroblasts become activated, undergoing a desmoplastic (i.e., myofibroblastic) phenotypic switch
triggered by tumor-associated (TA) but not by normal fibroblast-derived extracellular matrix (ECM).
However, the molecular mechanisms responsible for this process remain unclear.
Using syngeneic human fibroblasts harvested from patient-matched normal and tumor samples, we
produced a human mimetic pancreatic and renal 3D stromal system to study the role of myofibroblastic
activator TGFβ and ECM integrin receptors αvβ5 and α5β1 in the TA-ECM-induced phenotypic switch. We
assessed the role of known integrin downstream effectors, non-receptor tyrosine kinases FAK and Src,
and tested their roles in the observed TA-ECM-induced phenotypic switch. Approaches included the use
of specific inhibitor and activator drugs as well as genetic manipulations. Our data suggest that while
TGFβ is indeed needed for the production of the particular TA-ECM, it is no longer required during the
matrix induction of the phenotypic switch. Results suggest a cross-talk between integrins αvβ5 and α5β1
necessary for maintaining the phenotypic switch.
We uncovered a mechanism whereby αvβ5-integrin activity, in a Src/FAK-dependent manner, triggers the
redistribution of FAK-independent α5β1-integrin activity away from desmoplastic 3D-adhesion
structures. This in turn prevents this integrin from inhibiting the TA-ECM-dependent phenotypic switch.
To validate the in vitro results, we used a multi-fluorescent labeling approach to simultaneously detect
tumoral vs. stromal compartments and question the localization and activity levels of α5β1-integrin in
the original pathological tissue samples. Finally to establish clinical relevance, the same approach was
combined with a novel computational code applied, as a batch analysis, on a large human renal and
pancreatic tissue cohort.
We conclude that outcome-predictive and TGFβ-dependent matrix-induced stroma activation is
maintained by Src/FAK-dependent αvβ5-integrin activity. This activity triggers the exclusion of
myofibroblastic-inhibitory and FAK-independent α5β1-integrin activity from altered 3D-adhesions. We
propose that this desmoplastic mechanism may not only comprise a clinically important occurrence but
also a new bio-marker to assess stromal index in cancer patients.
E25
Haptotaxis requires a Rac1/WAVE/Arp2/3-based pathway that regulates
differential lamellipodial dynamics.
S.J. King1, S.B. Asokan1, C. Wu1,2, J.D. Rotty1, K.T. Chan1,3, I.P. Lebedeva1,2, J.E. Bear1;
1
UNC Lineberger Comprehensive Cancer Center, Department of Cell Biology and Physiology, University
of North Carolina, Chapel Hill, NC, 2Howard Hughes Medical Institute, Chevy Chase, MD, 3Howard
Hughes Medical Institute, Chapel Hill, NC
Cells in vivo migrate in response to directional cues such as differing concentrations of extracellular
matrix (haptotaxis); however, a mechanistic understanding of how cells sense and respond to these
haptotactic cues is lacking. In order to generate the forces required to drive cell migration, actin
filaments must be nucleated and organized in a precise manner. One primary mechanism by which new
actin filaments are nucleated is the Arp2/3 complex. Using a small molecule inhibitor of the Arp2/3
complex, CK666, and Arp2/3-depleted cells we have previously shown that the Arp2/3 complex is
essential for haptotaxis in fibroblasts on a variety of extracellular matrices (Wu, C., S. B. Asokan et al,
SUNDAY-ORAL PRESENTATIONS
Cell, 2012). In order to dissect the mechanism of haptotaxis on fibronectin, an agnostic approach using
microfluidic chambers that allow direct imaging of fibroblast haptotaxis on gradients of fibronectin has
been utilized. The requirement for the Arp2/3 complex for haptotaxis on fibronectin was confirmed
using CK666 on vascular smooth muscle cells, indicating that this result is not cell type specific. It has
been proposed that the alignment and maturation of focal adhesions could regulate haptotaxis.
However, our experiments suggest that fibronectin based haptotaxis of fibroblasts does not appear to
be reliant upon a specific integrin pairing or upon the presence of ‘mature’ focal adhesions. One
important nucleation promoting factor (NPF) for Arp2/3 is the WAVE complex. Our data suggests that
the WAVE complex is the primary NPF required for fibronectin based haptotaxis, with N-WASP and
WASH being dispensable. Rac1 is required for WAVE activation, lamellipodial formation and is a
component of focal complexes. Inhibition or depletion of Rac1 abrogates fibronectin haptotaxis.
Additionally, inhibition of other focal complex components, Src or FAK, also blocks fibroblast haptotaxis
on fibronectin, implicating focal complexes in the regulation of fibronectin haptotaxis. Kymography has
revealed differential protrusion dynamics across haptotaxing cells, which have the potential to provide
the force necessary for differential migration. Furthermore, FRAP studies investigating actin dynamics
during haptotaxis have revealed that there are differential actin dynamics across a cell on a gradient on
fibronectin that is not present in a cell on uniform fibronectin or upon Arp2/3 inhibition. Our current
hypothesis is that differential engagement of integrins triggers a Src/Rac/WAVE pathway that leads to
Arp2/3 activation. Resulting in differential actin dynamics and protrusion dynamics, ultimately leading to
directed migration towards higher concentrations of extracellular matrices.
E26
Dynamics and Shaping of the BMP Signaling Gradient by the BMP Antagonists
during DV axial Patterning.
J.M. Zinski1, W. Dou2, Y. Huang2, D. Umulis2, M.C. Mullins1;
1
Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA,
2
Agricultural and Biological Engineering, Purdue University, West Lafayette, IN
A gradient of Bone Morphogenetic Protein (BMP) signaling patterns the Dorsal-Ventral (DV) axis of the
vertebrate embryo. However, its shape and dynamics during zebrafish DV patterning have not been
quantified. Furthermore, how the known extracellular BMP regulators shape the gradient in time and
space is not known. To measure the BMP signaling gradient, we quantified the nuclear intensities of
fluorescently stained Phosphorylated-Smad5 (P-Smad) in every cell of the embryo at 30-minute intervals
from blastula through early gastrula stages. We used automated algorithms to identify the thousands of
individual nuclei present at each embryonic time-point, and to measure their corresponding P-Smad
intensities. In WT embryos, we show that the P-Smad gradient intensifies and steepens in the late
blastula before stabilizing in the early gastrula. We compared the BMP signaling gradients in WT and
BMP antagonist loss-of-function embryos of Chordin (Chd), Noggin (Nog), and Follistatin (Flst) to
determine their spatiotemporal functions. Though Chd primarily inhibits BMP signaling, it’s homologue
Sog has been shown to enhance BMP signaling by transporting BMP ligand in Drosophila DV patterning
and crossvein formation. However, we find that Chd only inhibits BMP signaling laterally in DV
patterning in the gastrula. This region-specific inhibition allows Chd to steepen the BMP gradient. In
SUNDAY-ORAL PRESENTATIONS
contrast, loss of Nog and Flst have no effect on the gradient. However, the loss of all three antagonists
causes a massive embryo-wide increase in BMP signal, far exceeding the lateral-only increase seen when
Chd alone is absent. To elucidate mechanisms explaining these results, we generated a mathematical
model of the system and screened thousands networks consisting of different rates of production,
diffusion, degradation, and binding between the BMP ligand, Chd, Nog, Flst, and Tolloid/Bmp1a. Our
results suggest that: 1) in a WT system, Chd functions as the primary sink for BMP ligand. 2) When Chd is
absent, Nog and Flst take over as the primary sink for the BMP ligand. 3) When all three antagonists are
absent, no extracellular sink exists, and BMP signaling increases embryo-wide.
E27
FRET biosensors reveal Ca2+/PKA crosstalk through AKAP targeting dynamics.
M.B. Schott1, F.N. Thompson Gonowolo1, B.D. Grove1;
1
Basic Sciences, University of North Dakota, Grand Forks, ND
A-Kinase Anchoring Proteins (AKAPs) play a critical role in cellular homeostasis by scaffolding cAMPdependent Protein Kinase A (PKA) and other signaling enzymes in proximity to downstream effectors.
Although they are named “anchoring proteins”, some AKAPs do not assemble static enzyme complexes
but form dynamic signalosomes that traffic to different subcellular compartments in response to stimuli.
Gravin (AKAP12), a multivalent scaffold linked to a variety of cellular functions, anchors PKA and other
enzymes to the plasma membrane but is known to undergo redistribution to the cytosol upon
intracellular Ca2+ elevation. We postulate that gravin redistribution represents a novel crosstalk
mechanism between Ca2+-dependent and cAMP-dependent signaling pathways. To assess this, we
measured the impact of gravin-V5/His expression on compartmentalized PKA activity using the PKA FRET
biosensor AKAR3. In cultured AN3 CA cells which lack endogenous gravin, expression of gravin-V5/His
caused an elevation in forskolin-stimulated PKA activity in AKAR3 constructs targeted to the plasma
membrane compared to control cells lacking gravin or expressing a ΔPKA-gravin construct missing the
PKA-binding domain. In contrast, gravin-V5/His expression caused a decrease in forskolin-stimulated
PKA activity in cytosolic AKAR3 constructs compared to control cells lacking gravin or expressing ΔPKAgravin. Interestingly, gravin localization at the cell periphery was dramatically impaired by the mutation
of a calmodulin-binding consensus sequence within gravin’s CB4 domain. Similar to control cells, this
mutant gravin construct had no effect on membrane and cytosolic PKA activity. Finally, pretreatment
with the calcium-elevating agent thapsigargin caused the redistribution of gravin away from the plasma
membrane and prevented gravin-mediated elevation in plasma membrane PKA activity in response to
forskolin. These results reveal that gravin shapes the subcellular profile of PKA activity and support the
hypothesis that gravin mediates crosstalk between Ca2+ and cAMP-dependent signaling pathways. Based
on these results, AKAP localization dynamics may represent an important paradigm for the regulation of
cellular signaling networks.
SUNDAY-ORAL PRESENTATIONS
E28
A cytoskeleton-based memory in chemotactic leukocytes.
H. Prentice-Mott1, Y. Meroz2, A. Carlson2, M. Levine3, G. Charras4, L. Mahadevan2, J. Shah1;
1
Harvard Medical School, Boston, MA, 2Harvard School of Engineering and Applied Sciences, Cambridge,
MA, 3Biophysics, Harvard University, Cambridge, United States, 4London Centre for Nanotechnology,
University College London, London, United Kingdom
The directional response of cells to asymmetric chemical environments involves a complex sequence of
steps including: gradient sensing, internal polarization and migration. To understand how these modules
act to direct cell motion requires quantitative measurements of the cellular response and quantitative
control of the external chemical environment. We used microfluidics to generate confining
microchannels in which the front and back of the cell can be independently exposed to separate
chemoattractant concentrations, which are stable over time. Cells exposed to high, static differences
with low background (C0=0 nM, ∆C=100 nM) exhibited persistent polarization, as assessed by PH-Akt
asymmetry. Smaller differences (C0=0 nM, ∆C=3 nM) or increased background (C0=50 nM, ∆C=50 nM)
resulted in fluctuations between polarized and unpolarized states. The direction of polarization was
biased toward the higher concentration. When exposed to uniform concentrations of chemokine (C0=0,
3, 10, 100 nM, ∆C=0 nM), cells exhibited similar polarization fluctuations. Unexpectedly, the direction of
polarization for all concentrations except C0=0 was biased towards the initial direction of polarization,
indicating a directional “memory”. To further test this, cells were exposed to a controlled dynamic
environment, in which a uniform concentration (C0=10 nM, ∆C=0 nM) was first introduced, then
changed to no chemoattractant (C0=0 nM, ∆C=0 nM) which caused most cells to depolarize. Subsequent
reintroduction of the chemoattractant (C0=10 nM, ∆C=0 nM) after 2 minutes of no chemoattractant
caused ~90% of the cells to re-polarize in the same direction as initially polarized. If the chemokine was
removed for 10 minutes, this number decreased to ~70%. We measured the decay of internal cellular
asymmetries during the no chemoattractant phase and observed dramatic differences in timescales for
molecules such as PIP3 and Myosin light chain which were fast (10 seconds), versus the ERM protein
moesin (5 minutes). Chemical disruption of the long-lived moesin (NSC668394) or microtubule
(colcemid) structures during the no chemoattractant phase, resulted in decreased memory of
polarization after reintroduction of the chemoattractant and washout of the drug (60% or 55%,
respectively). We developed a phenomenological model incorporating two layers of polarization, a
short-lived membrane layer (pm) and a long-lived cytoskeletal layer (pc). The cytoskeletal polarization is
driven by the time-integrated pm, while the direction of the membrane polarization is driven by both pc
and external concentration differences. This simple model reproduced the observed directional
memory. Cells may use this directional memory to navigate the complex chemical environment found in
tissues.
SUNDAY-ORAL PRESENTATIONS
ePoster Talks Session 5: Cell Organization and Polarity
E29
Conformationally promiscuous prion-like domains coordinate RNP granule
condensation/dissolution dynamics during stress.
S. Kroschwald1, S. Alberti1;
1
MPI-CBG, Dresden, Germany
The formation of large supramolecular assemblies, such as stress-inducible RNP granules, is a poorly
understood process. Here, we perform an extensive analysis of P bodies and stress granules in budding
yeast and find that they have very distinct material properties: whereas P bodies behave as liquid
droplets, stress granules adopt a solid material state, which originates from co-aggregation with
misfolded proteins. Maintenance of the liquid P body state depends on the continuous action of protein
disaggregases, and interference with this process results in the entrapment of P body components in
stress granules. We further demonstrate that RNP assembly can be nucleated through multiple
pathways and requires scaffolding factors such as misfolded proteins or RNAs. Low complexity domains
can also drive RNP granule assembly, and we find that these domains do not undergo prion-like
conformational conversions but function as versatile molecular adaptors that promiscuously interact
with other low complexity domains or misfolded proteins. In summary, our findings reveal a high degree
of adaptability and redundancy in RNP granule biogenesis, which is regulated and maintained by ATPdriven disaggregating machines.
E30
Cellular chirality arising from the self-organization of the actin cytoskeleton.
Y. Tee1, T. Shemesh2, V. Thiagarajan1, R.F. Hariadi3, K.L. Anderson4, C. Page4, N. Volkmann4, D. Hanein4, S.
Sivaramakrishnan3, M. Kozlov5, A.D. Bershadsky1,2;
1
Mechanobiology Institute, National University of Singapore, Singapore, Singapore, 2Department of
Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel, 3Cell and Developmental Biology,
University of Michigan, Ann Arbor, MI, 4Bioinformatics and Systems Biology Program, Sanford Burnham
Medical Research Institute, La Jolla, CA, 5Physiology and Pharmacology, Sackler Faculty of Medicine, Tel
Aviv Univ, Tel Aviv, Israel
The development of a chiral pattern of actomyosin was revealed by studying the self-organization of the
actin cytoskeleton in human fibroblasts confined to cell-sized circular adhesive islands. We identified
several distinct stages of actin cytoskeleton evolution, starting with the formation of a radially
symmetrical system of actin bundles. Myosin-II-deficient alpha-actinin-enriched radial fibers grew
centripetally from peripheral focal adhesions, while myosin-IIA-enriched transverse fibers moved
centripetally along radial fibers. Formin inhibitor blocked the formation of radial fibers while myosin II
inhibitor suppressed formation of both radial and transverse fibers. This radial pattern evolved
spontaneously into the chiral pattern as a result of the synchronous and unidirectional tilting of all radial
fibers. Subsequently transverse fiber movement acquires a tangential component. We propose a novel
SUNDAY-ORAL PRESENTATIONS
mechanism of myosin IIA-driven retrograde flow where contractile stresses within transverse fibers
drive their movement along radial fibers. Computational modeling demonstrated that this mechanism
can explain the pattern transition from radial to chiral. Remarkably, the chiral pattern is characterized by
a defined handedness. This directionality could be reversed by overexpressing full-length alpha-actinin1. Expression of a truncated alpha-actinin-1 construct lacking actin binding domain abolished formation
of radial fibers, but resulted in the formation of a new type of long actin fibers containing myosin IIA and
anchored at focal adhesions. These actin fibers also tilt unidirectionally, preserving the handedness of
the direction of tilt similarly to the radial fibers in control cells. Expression of an alpha-actinin fragment
containing the integrin-binding site also perturbed the development of radial fibers. Depletion of alphaactinin-1 via siRNAs resulted in phenotypes analogous to those observed in cells expressing these
dominant-negative alpha-actinin-1 constructs. Taken together, these results suggest that while the
crosslinking function of alpha-actinin-1 is not essential for the initiation of the chiral asymmetry of the
actin cytoskeleton, it is essential for the alteration of the chirality seen in alpha-actinin-1 overexpressing
cells. We hypothesize that the handedness of the chiral pattern depends on the intrinsic helical
symmetry of actin filaments, which is transformed (with the aid of formin and alpha-actinin-1) into a
unidirectional torque that triggers the transition of unstable radial system to chiral rotation. Thus, selforganization of the actin cytoskeleton can provide built-in machinery that potentially allows cells to
distinguish between left and right.
E31
RhoGTPase Regulatory Signatures Define Distinct Stages of Synaptic
Development.
K.A. Newell-Litwa1, S. Martin-Vilchez2, H. Asmussen1, L. Whitmore1, A.R. Horwitz1;
1
Cell Biology, University of Virginia, Charlottesville, VA, 2Cell Biology, University of Virgina,
Charlottesville, VA
Small RhoGTPases organize the actomyosin cytoskeleton to drive changes in post-synaptic spine shape
that support learning and memory. RhoGTPase signaling pathways are major targets of synaptic
disorders, such as Autism. During normal synaptic development, the small RhoGTPase, Rac, promotes
the formation of filopodia-like spine precursors, which subsequently mature through RhoA-dependent
myosin II activation into polarized mushroom-shape spines. Further excitatory stimulation associated
with long-term potentiation leads to Rac-driven spine head expansion. We sought to identify novel
regulators of synaptic RhoGTPase activity, and to determine whether different upstream RhoGTPase
regulatory proteins, including GEF activators, GAP inactivators, and inhibitory GDIs, regulate specific
stages of synaptic development. We performed an in-silico screen of genes identified from published
synaptic proteomes and the SFARI database for association of these genes with Autism copy number
variants (CNVs) to identify known and putative RhoGTPase regulatory proteins. This approach identified
several candidate actin regulatory proteins, the majority of which localized to known Autism CNVs. We
used shRNA to down-regulate expression of specific RhoGTPase regulatory proteins in rat primary
hippocampal neurons during either early spine formation or subsequent maturation. Our data
demonstrate that unique RhoGTPase regulators mediate distinct stages of synaptic development.
Specifically, the Rac-GEF beta-Pix, ArhGAP23 and Frabin, affected early spine precursor formation, but
SUNDAY-ORAL PRESENTATIONS
not subsequent maturation. However, GDI-mediated attenuation of RhoGTPase activity stabilized
mature spines; knockdown of either GDI-alpha or gamma resulted in significantly longer spines. The
novel RhoGAP, ArhGAP39, regulated spine length and density specifically during spine maturation.
Notably, RNA expression of these GDIs and ArhGAP39 increased significantly during neuronal
development. We further characterized the function of the putative RhoGAP, ArhGAP39, in migratory
CHO.K1 fibroblasts, which also express ArhGAP39. GFP-ArhGAP39 resulted in robust stress fiber
formation and focal adhesion maturation, consistent with GAP activity towards Rac, resulting in a
corresponding increase in RhoA-mediated myosin activation. Alternately, knockdown of ArhGAP39
resulted in round cells with an extensive lamellipodia associated with increased small nascent
adhesions, indicative of increased Rac activity, which we confirmed with a Rac FRET biosensor. Our study
demonstrates that specific combinations of RhoGTPase regulatory proteins temporally balance Rac and
RhoA activity to regulate post-synaptic spine development, and further shows that ArhGAP39 functions
as a novel Rac GAP in post-synaptic spine maturation.
E32
Septin filaments recognize micron-scale positive plasma membrane curvature.
A.A. Bridges1, P. Occhipinti2, A.S. Gladfelter2;
1
Dartmouth College, Hanover, NH, 2Biological Sciences, Dartmouth College, Hanover, NH
Septins are conserved filament forming GTPases that maintain cell polarity by restricting diffusion of
proteins in the plasma membrane and endoplasmic reticulum while acting as a molecular scaffold for
cytosolic proteins. Septin higher-order structures form on and associate with the plasma membrane and
cells carrying mutated septin genes display irregular cell polarity, abnormal cell shape, are defective at
cytokinesis, and are generally inviable. Though it has been shown that Cdc42 drives the accumulation of
septins at incipient sites of cell polarity, properties of the plasma membrane that influence and maintain
their localization have not been established. Here, we show that septins preferentially bind the plasma
membrane in numerous cell types at sites of micron-scale positive curvature, a common topology of
polarized cells. In regions of the cell devoid of positive curvature, septin filaments preferentially
minimize interacting with negative plasma membrane curvature. Using phospholipid bilayer coated
glass microspheres we show that curvature recognition is an intrinsic property of septin filaments. This
work demonstrates that septins are a filamentous system capable of sensing plasma membrane shape
on the micron-scale. Utilizing this property, septins respond to large scale changes in cell shape and
communicate these changes to the cytoplasm.
SUNDAY-ORAL PRESENTATIONS
E33
Fusion to fluorescent proteins causes previously unrecognized effects on gap
junction mobility and Nexus arrangement.
R.F. Stout Jr.1, D.C. Spray1;
1
Dominic P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY
Gap junctions are comprised of connexins that form channels connecting adjacent cells. They also act as
the core of a signaling complex called the Nexus. Much insight into the structure and dynamic
morphology of the Nexus has been gained from the use of electron and optical microscopy. Fluorescent
protein fusions to connexins are important tools for studying the unusually rapid, responsive, and
regulated cycle of connexin production, channel formation, channel regulation and degradation.
We used connexin-fluorescent protein fusions with live confocal microscopy to perform fluorescence
recovery after photobleach (FRAP) experiments. We used new-generation fluorescent proteins to reveal
two previously unrecognized qualitative characteristics of the Nexus: A) Plaque formation does not
require plaque stability; B) Mixing of different connexin types within the Nexus is affected by the type
and site-of-attachment of fluorescent proteins. Specifically, we find that the degree of mixing of
different gap junction types varies not only with connexin type as previously described, but also varies
according to the fusion-site of the fluorescent protein tag. We found that two immobile connexins can
remain separated into distinct parts of the same gap junction plaque, as previously described. However,
when we co-transfected stable connexin 43 (Cx43) with more mobile connexins (superfolderGFP-Cx30 or
Cx26-mVenus, and others) the gap junctions mixed when included in the same gap junction Nexus
structure.
We found that Cx26 and Cx30 gap junctions are significantly more mobile than Cx43. We also found that
Cx26 is significantly more mobile when tagged by the monomerized superfolderGFP (msfGFP) variant
(V207K) than the non-monmerized superfolder (GFP 43.5+/-10% and 6+/-2% normalized FRAP at 30
seconds post-bleach for Cx26-msfGFP and Cx26-sfGFP, respectively). This effect was attenuated but still
significant when the fluorescent protein tag is fused to the N-terminus of Cx26 instead of the Cterminus. The effects of the fluorescent protein tag were similar for Cx30. However, Cx43 displays very
little or no mobility regardless of fluorescent protein type or site of fusion to a fluorescent protein.
We used several methods to quantitatively compare the mobility of gap junctions using data generated
in FRAP experiments. We find that the unique morphology of the Nexus requires that results obtained
through standard single focal plane FRAP experimentation be verified by 3D-timelapse imaging. These
results have major implications for our understanding of the gap junction Nexus structure. Supported by
the following grants 5T32NS007439-14, NS041282.
SUNDAY-ORAL PRESENTATIONS
E34
Farnesylated LKB1 regulates cell polarization and invasion through a 3D collagen
microenvironment in a kinase-independent manner.
S. Wilkinson1, A. Marcus2;
1
Graduate Program in Cancer Biology, Emory University, Atlanta, GA, 2Hematology and Medical
Oncology, Emory University, Atlanta, GA
The tumor suppressor LKB1 is a serine/threonine kinase that serves as a master regulator of cell polarity
in epithelial tissue, and is mutated in 30% of non-small cell lung cancer tumors. LKB1 contains a Cterminal farnesylation motif, which allows for post-translational addition of a hydrophobic farnesyl
group and insertion into the plasma membrane. Although LKB1 contains a farnesylation motif, the
functional significance of this motif is largely unknown. We generated lung cancer cell lines stably
expressing GFP-tagged: wildtype LKB1, a farnesylation motif mutant LKB1, and the various domains of
LKB1, including the C-terminal domain (CTD, lacking kinase activity) and the CTD containing the
farnesylation motif mutant, and then embedded spheroids of these cells in a 3D collagen matrix. Using
confocal microscopy and live-cell imaging, we show that lung cancer cells re-expressing wildtype LKB1
acquire a unidirectional polarity, while cells expressing the farnesylation-mutant LKB1 are unable to
polarize. Importantly, we show that 3D polarization is regulated through the LKB1 CTD alone, also in a
farnesylation-dependent manner. These data indicate that farnesylated LKB1 promotes polarization
during 3D motility through its C-terminal domain in a kinase-independent manner. We next sought to
identify the role of LKB1 farnesylation in regulating invasion through the 3D collagen microenvironment.
After acquiring a unidirectional polarity, cells re-expressing either wildtype LKB1 or the LKB1 CTD invade
into the collagen microenvironment. Strikingly, we show that cells re-expressing LKB1 with the
farnesylation motif mutation exhibit significantly reduced invasion into the collagen microenvironment,
with the few cells invading exhibiting no polarization. Taken together, these data implicate LKB1
farnesylation as critical for promoting cell polarization and potentially providing a mechanism of cell
invasion into the collagen microenvironment. This information has the potential to lead to novel
information regarding the mechanisms of LKB1 farnesylation in regulating polarization and invasion,
ultimately providing new insight into the tumor suppressive function of LKB1.
E35
Transbilayer coupling via long acyl chains and immobilized phosphatidylserine
mediate actin-based nanoclusters of outer leaflet lipids.
A.A. Anilkumar1, R. Raghupathy1, A. Polley2, R. Vishwakarma3, Z. Guo4, M. Rao2,5, S. Mayor5;
1
Cellular organisation and signalling, National Centre for Biological Sciences, Bangalore, India, 2Raman
Research Institute, Bangalore, India, 3Indian Institute of Integrative Medicine, bangalore, India,
4
Department of Chemistry, Wayne State Univ, Detroit, MI, 5National Centre for Biological Sciences
(NCBS), TIFR, Bangalore, India
A significant percentage (10%) of eukaryotic membrane proteins are attached to GPI anchors which help
them attach to the outer leaflet of the plasma membrane. The significance of the GPI anchor lies in the
SUNDAY-ORAL PRESENTATIONS
fact that it helps to localize these molecules to cholesterol and sphingolipid enriched domains. Work
over several years from our laboratory has shown that a dynamic actin cytoskeleton plays a major role in
the organization of GPI-APs into cholesterol-sensitive nanoclusters( Gowrishankar et al., 2012). A key
question is how GPI-APs at the outer leaflet of the membrane bilayer couples to the actin cytoskeleton
at the inner leaflet. A likely possibility is that dynamic actin filaments couple to the inner leaflet lipids
either directly or indirectly, which in turn are connected to the outer leaflet via a transbilayer coupling
mechanism involving long acyl chain lipids both in the GPI-anchors and the coupling lipid species at the
inner leaflet. Phosphatidylserine (PS) which is known to interact with a number of actin binding proteins
is the prime candidate for such a lipid species. We also hypothesize that these microdomains could lead
to the local ordering of the membrane which could act as sorting platforms for a number of signaling
molecules.
We study nanocluster organization of GPI-APs at the cell surface by FRET (specifically homo-FRET)
between fluorescently labeled GPI-APs. We then make perturbations in lipid structure of the GPI-anchor
by manipulating their acyl chains of GPI-AP by utilizing mutations in enzymes that have a role in fatty
acid remodeling. We have also explored the inner leaflet linker that facilitates the transbilayer coupling
by exogeneously adding different lipid species of varying chain lengths in cells deficient for PS
biosynthesis. Atomistic molecular dynamic simulations of asymmetric, multicomponent membrane
bilayers have also been used to explore the role of long acyl chain in the coupling of the inner and outer
leaflet lipids. Our results suggest that long-saturated acyl chains in the GPI-anchor, and in PS at the inner
leaflet lipid with adequate amount of cholesterol are necessary ingredients for nanocluster formation.
These results provide evidence for a transbilayer link between the outer leaflet and the inner-leaflet
required for GPI-AP nanoclustering.
References
Gowrishankar, K., Ghosh, S., Saha, S., C, R., Mayor, S., and Rao, M. (2012). Active remodeling of cortical
actin regulates spatiotemporal organization of cell surface molecules. Cell 149, 1353–1367.
ePoster Talks Session 6: Membrane Traffic: Dynamics and Regulation
1
E36
Trafficking of the Kir2.1 potassium transporter is regulated by the ubiquitin ligase
Rsp5 and a select subset of alpha-arrestins in yeast.
A.F. O'Donnell1,2, T. Mackie3, A. Kolb3, A. Dempsey4, C. Szent-Gyorgyi4, M.P. Bruchez4, J.L. Brodsky5;
1
Cell Biology, Univ Pittsburgh, Pittsburgh, PA, 2Dept. of Molecular and Cell Biology, University of
California at Berkeley, Berkeley, CA, 3Dept. of Biological Sciences, University of Pittsburgh, Pittsburgh,
PA, 4Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, PA, 5Department
of Biological Sciences, University of Pittsburgh, Pittsburgh, PA
To ensure optimal cell growth and survival, protein composition at the plasma membrane is tightly
regulated, with both controlled protein internalization and selective protein targeting to the cell surface
in response to environmental changes. To identify factors that regulate this selective protein trafficking,
SUNDAY-ORAL PRESENTATIONS
we developed a yeast expression system for the Kir2.1 potassium channel, which maintains potassium
homeostasis in heart cells and is thereby critical for cardiac function. By monitoring the growth of Kir2.1
yeast on low potassium medium, Kir2.1 activity at the cell surface can be assessed. Using a highthroughput screening methodology we identified the ESCRTs and retromer complex as novel regulators
of Kir2.1 trafficking. We now show that three specific alpha-arrestins – Ldb19/Art1, Aly1/Art6, and
Aly2/Art3 – control Kir2.1 trafficking. In marked contrast to the well-described role for alpha-arrestins as
endocytic adaptors, these alpha-arrestins promote Kir2.1 trafficking to the cell surface, increasing Kir2.1
activity at the plasma membrane and raising intracellular potassium levels. Consistent with the alphaarrestin requirement, we also demonstrate that the ubiquitin ligase, Rsp5, and the protein phosphatase
calcineurin regulate Kir2.1 plasma membrane targeting. Furthermore, by fusing a single chain antibody
to Kir2.1 and employing live cell fluorescence microscopy, we can selectively monitor Kir2.1 cell surface
localization or its intracellular distribution, thus providing a complementary tool to delineate the
intracellular sorting pathway for this channel. Current work is focused on using the yeast model to
define additional factors and regulatory elements required for alpha-arrestin-mediated trafficking of
Kir2.1 to the cell surface. Together, these findings identify Kir2.1 as new alpha-arrestin substrate and lay
the foundation to further define the molecular mechanisms governing alpha-arrestin-mediated
intracellular sorting.
E37
Differential regulation of organelle dynamics by tubulin acetylation in polarized
epithelia.
K.A. Toops1,2, L. Tan1,3, A. Lakkaraju1,2,3;
1
Ophthalmology and Visual Sciences, University Wisconsin-Madison, Madison, WI, 2McPherson Eye
Research Institute, Madison, WI, 3Pharmaceutical Sciences, University Wisconsin-Madison, Madison, WI
Intracellular trafficking is coordinated by the actin and microtubule cytoskeletons and associated motor
proteins. Organelle-specific recruitment of motor proteins is accomplished in part by post-translational
modifications of α-tubulin such as acetylation and detyrosination. Here, we used high-speed live imaging
to monitor organelle dynamics in the prototypical polarized MDCK cell line and in adult primary retinal
pigment epithelial (RPE) monolayers. A key function of the RPE is the daily phagocytosis and degradation
of shed photoreceptor outer segments, necessary for photoreceptor health and for vision. Over a
lifetime, this high metabolic activity leads to the accumulation of visual cycle by-products called
lipofuscin bisretinoids within the RPE endo-lysosomal system. Lipofuscin bisretinoids have been
implicated in the pathogenesis of numerous retinal diseases including age-related macular
degeneration, the most common cause of vision loss among older adults today. We have shown that
bisretinoids trap cholesterol and bis(monoacylglycero)phosphate, an acid sphingomyelinase cofactor in
RPE lysosomes. Acid sphingomyelinase activation increases cellular ceramide, which promotes tubulin
acetylation on stabilized microtubules. Live imaging using spinning disc confocal microscopy revealed
that long-range displacement of LC3-labeled autophagosomes and LAMP2-labeled late
endosomes/lysosomes (LE/Lys) is significantly impaired in cells with abnormally acetylated
microtubules. This results in incomplete autophagic flux and accumulation of canonical autophagic
substrates. Calcium-induced fusion of LE/Lys with the plasma membrane is critical for membrane repair
SUNDAY-ORAL PRESENTATIONS
and removal of cellular debris. In RPE with lipofuscin bisretinoids or in MDCK with excess cholesterol, we
observed decreased LE/Lys exocytosis after exposure to calcium ionophores or pore-forming toxins.
Therefore, constrained LE/Lys trafficking due to tubulin acetylation interferes with both autophagy and
membrane repair. Inhibition of acid sphingomyelinase decreased ceramide and acetylated tubulin
levels, restored autophagy and LE/Lys exocytosis in RPE and MDCK cells. In contrast to its effect on
LE/Lys and autophagosome motility, tubulin acetylation accelerated the trafficking of rab11-labeled
apical recycling endosomes. This was associated with increased apical secretion of exosomes containing
flotillin-1. Our data show that tubulin acetylation decreases transport of organelles in the degradative
route and increases trafficking of organelles in the recycling route. Studies are currently underway to
elucidate how selective recruitment of motors and scaffolding proteins drives organelle-specific motility
in polarized epithelia and how this might contribute to disease processes.
E38
Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm.
S. Lakdawala1, Y. Wu2, P. Wawrzusin2, A. Broadbent1, J. Kabat3, E. Lamirande1, E. Fodor4, N. AltanBonnet5, H. Shroff2, K. Subbarao1;
1
Laboratory of Infectious Diseases, NIH/NIAID, Bethesda, MD, 2Section on High Resolution Optical
Imaging, NIH/NIBIB, Bethesda, MD, 3Research Technologies Branch, NIH/NIAID, Bethesda, MD, 4Sir
William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom, 5NIH/NHLBI, Bethesda,
MD
Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of
viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells,
while progeny virions bud from the plasma membrane. Rab11a-containing recycling endosomes have
been implicated in the transport of vRNA from the nucleus to the plasma membrane. However, the
intracellular dynamics of vRNA assembly into progeny virions is not well understood. Here we used
recent advances in microscopy to explore vRNA assembly and transport during a productive infection.
We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization
(FISH) and observed that foci containing more than one vRNA segment were found at the external
nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually.
Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in
every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the
nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the
viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live
cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza
vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by
inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and
traveling together en-route to the plasma membrane. Our data strongly support a model in which vRNA
segments are exported from the nucleus as complexes that assemble en-route to the plasma membrane
through dynamic colocalization events in the cytoplasm.
SUNDAY-ORAL PRESENTATIONS
E39
A Novel Rab10 Regulated Complex Essential for the Autophagic Engulfment of
Lipid Droplets.
Z. Li1, R. Schulze2, H. Cao2, M.A. McNiven2;
1
Mayo Graduate School, Rochester, MN, 2Mayo Clinic, Rochester, MN
Autophagic digestion of lipid droplets (LDs) is a central mechanism by which cells catabolize lipids as an
energy source. Currently, the mechanisms by which autophagosomes (APs) physically bind and
envelope LDs for subsequent digestion are poorly defined. In this study we report that the small
GTPase, Rab10, implicated previously in the Golgi to plasma membrane transport of nascent secretory
cargo, plays an essential role in the autophagy of LDs in hepatocytes. We find that Rab10 activity is
amplified significantly in cells stimulated to undergo autophagy, concomitant with an increased
association of this GTPase to the LD-AP interface or “synapse”. Importantly, disruption of Rab10
function by siRNA knock down or expression of a GTPase defective protein leads to LD accumulation.
Finally, activation of Rab10 during an autophagic stimulus increases its association with EHD2 and
EHBP1, two effector proteins known to support membrane remodeling within the endocytic pathway.
These proteins are recruited to the LD-AP synapse in a Rab10- dependent manner and are essential for
autophagic-based LD breakdown. These findings identify a novel protein complex essential for the
engulfment of LDs during the autophagic process.
E40
PI3K/Akt signaling regulates ciliogenesis initiation via Rab11-effector vesicular
trafficking switch.
V. Walia1, C. Insinna (Kettenhofen)1, Q. Lu1, S. Specht1, C.J. Westlake2;
1
LCDS, National Cancer Institute-Frederick, Frederick, MD, 2National Cancer Institute-Frederick,
Frederick, MD
Defects in primary cilium formation and signaling are associated with a growing list of genetic diseases
and have been linked to certain cancers. A critical step in the initiation of ciliogenesis requires pre-ciliary
vesicle trafficking to the mother centriole. Previously, we showed that serum starvation induced rapid
association of Rabin8, a Rab8 GEF, with Rab11 pre-ciliary vesicles, a step needed to initiate ciliogenesis.
We hypothesized that pre-ciliary trafficking depends on growth factor signaling. To test this hypothesis,
we screened growth factors in RPE cells cultured in serum-free condition and identified lysophosphatidic
acid (LPA) and TGF-b as novel inhibitors of ciliogenesis. Interestingly, only LPA blocked Rabin8 pre-ciliary
trafficking suggesting that LPA functions specifically at the ciliogenesis initiation step. We show that the
LPAR1 receptor, but not LPAR2-5, functions in the inhibition of pre-ciliary trafficking and ciliogenesis.
Using chemical inhibitors against LPAR1 downstream pathways, we determined that inhibition of
PI3K/Akt signaling induced Rabin8 pre-ciliary trafficking and promoted ciliogenesis, whereas other
LPAR1 signaling pathways (Ras, Rac, or PLC) had no effect. Inhibition of PI3K/Akt signaling also promotes
ciliogenesis in other cell lines suggesting that this pathway is a global regulator of cilia formation. To
understand how Akt regulates pre-ciliary trafficking, we examined Akt-phosphorylation on Rab11 and
SUNDAY-ORAL PRESENTATIONS
Rabin8. While Rabin8 was found to be an Akt substrate, there was no observed effect of this site on
Rab11 binding or ciliogenesis. We next theorized that Akt-phosphorylation regulates Rab11-Rabin8
interaction indirectly via a Rab-effector switch. RNAi studies for Rab11-effectors demonstrated that
WDR44/Rabphillin-11 ablation promotes Rabin8 centrosomal trafficking in the presence of serum.
WDR44 is the first reported Rab11 effector but its function is not well understood. We mapped Akt sites
on WDR44 within the Rab11 binding domain and showed that phosphorylation status of WDR44
coincided with Rab11-Rabin8 interaction upon serum starvation. Importantly, a phospho-mimetic
WDR44 mutant showed stronger binding to Rab11 and inhibited ciliation upon serum starvation.
Together these findings uncover a novel role for a growth factor signaling network mediated by Akt
kinase and its downstream substrate, WDR44, in the regulation of pre-ciliary vesicle trafficking and
ciliogenesis initiation.
E41
TRIM72/MG53 directly modulates phosphatidylinositol-4,5-bisphosphate 3-kinase
signaling to mediate vesicular trafficking during endocytosis and exocytosis.
S. Bhattacharya1, J. Alloush1, E. Beck1, L. Gushchina1, N.L. Weisleder1;
1
Davis Heart and Lung Research Institute, Ohio State University Medical Center, Columbus, OH
Tripartite motif (TRIM) proteins constitute a family of proteins that all contain a canonical RING finger,
B-box and coiled-coil domains which participate in several cellular processes. TRIM72, also known as
mitsugumin 53 (MG53), is an essential component of the membrane repair machinery in striated
muscles. The TRIM72/MG53 knockout mouse displays compromised muscle membrane repair and
trim72-/- mice develop myopathy. Due to its essential role in the membrane repair process in striated
muscle, TRIM72/MG53 has recently been considered as a therapeutic candidate for the treatment of
muscular dystrophy. Following membrane disruption, TRIM72/MG53 translocates to the site of injury to
allow resealing of the damaged area. The precise molecular mechanism by which TRIM72/MG53
translocate to the injury site and reseal the damage membrane is clearly not known. Previous studies
established that membrane damage increases vesicular trafficking to the site of membrane injury, which
is followed by vesicular fusion to patch the damaged portion of the membrane. Previous studies indicate
that both endocytosis and exocytosis are important in the membrane repair process, and also that
TRIM72/MG53 expression can appear in vesicles that are released from myocytes. In this study we
examined the role of TRIM72/MG53 in vesicular endocytosis and exocytosis in C2C12 myoblasts
overexpressing TRIM72/MG53. The extent of vesicular endocytosis and exocytosis was measured as
internalization of fluorescently tagged dextran and internalization/recycling of fluorescent transferrin
using laser scanning confocal microscopy and fluorescence-activated cell sorting (FACS), respectively.
Overexpression of TRIM72/MG53 in C2C12 myoblasts increased the total amount of endocytosis of
labeled dextran into the myoblasts. Exocytosis as assayed by transferrin internalization and recycling
was also increased by expression of TRIM72/MG53. Our recent findings show that TRIM72/MG53
overexpression is sufficient to activate the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)
signaling pathway. Therefore, we hypothesized that this TRIM72/MG53 mediated vesicular translocation
may be regulated by a PI3K-dependent pathway. To test this hypothesis, dextran uptake and transferrin
internalization/recycling were measured in the presence of wortmannin. Wortmannin significantly
SUNDAY-ORAL PRESENTATIONS
attenuated dextran uptake and transferrin internalization/recycling in C2C12 myoblasts. To control for
any off-target effects of chemical inhibitors, molecular approaches were used to introduce constitutively
active PI3K and protein kinase B (AKT) constructs which increased dextran and transferrin trafficking in
C2C12 myoblasts similar to TRIM72/MG53. Dominant negative PI3K and AKT constructs prevented
TRIM72/MG53 mediated increases in endocytosis and exocytosis. We conclude that TRIM72/MG53 is an
important regulator of the PI3K/AKT signaling pathway and positively regulates translocation of vesicles
to the site of membrane injury.
E42
Dynamic modulation of cortical actin at the immunological synapse controls
cytotoxic granule secretion.
A.T. Ritter1,2, E. Betzig3, G.M. Griffiths4, J. Lippincott-Schwartz5,6;
1
Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and
Human Development, NIH, Bethesda, MD, 2Cambridge Institute for Medical Research, University of
Cambridge, Cambridge, United Kingdom, 3HHMI/Janelia Farm Research Campus, Ashburn, VA, 4Univ
Cambridge, Cambridge, England, 5Physiology Course at Marine Biological Laboratory, Woods Hole, MA,
6
Cell Biology and Metabolism Program, NICHD, NIH, Bethesda, MD
Cytotoxic T lymphocytes (CTLs) are an integral part of the immune system as they are responsible for
destroying virally infected and tumorigenic cells throughout the body. CTL undergo a dramatic
reorganization of the actin and microtubule cytoskeletons upon target recognition, facilitating the
polarized secretion of a potent cocktail of lytic proteins that induce target apoptosis. These cytolytic
proteins are housed in specialized lysosome-related organelles called “lytic granules.” It is known that
CTL direct lytic granule trafficking specifically in the direction of the target by polarizing the centrosome
to the CTL/target interface. However, how CTL regulate secretion of lytic granules is still unclear.
The actin cortical cytoskeleton plays a role in regulating exocytosis in a number of professional secretory
cells. It can act as a barrier-- preventing secretory granules from reaching the plasma membrane. We
previously showed that actin clears away from the plasma membrane at the interface between the T cell
and the target. However, controversy remains as to the role of actin in secretion at the synapse. Using
newly developed Lattice Light Sheet Microscopy, we show that actin cortical density is reduced at the
synapse within 1 minute after target recognition. We also find that lytic granules cluster at the
centrosome prior to its delivery to the synapse. We hypothesize that the rapid formation and
maintenance of an area of reduced cortical actin density at the synapse after target recognition could
provide a platform to mediate immediate and facile secretion of lytic granules which are delivered to
the synapse in conjunction with the centrosome. Supporting this hypothesis, we also note that CTL
manipulate the actin cortical cytoskeleton to prevent secretion. Using TIRF microscopy, we show that
following secretion of a few granules, actin cortical density dramatically increases at the synapse.
Surprisingly, actin cortical density remains reduced at the synapse lacking proteins that are required to
secrete lytic granules, indicating that this actin “wall” forms only after lytic granule secretion.
Depolymerization of the actin “wall” with low doses of Latrunculin A results in immediate lytic granule
secretion. This suggests that this actin wall could be serving as a barrier to prevent sustained lytic
granule secretion toward an individual target cell. CTL are serial killers. In order for this to be true, CTL
SUNDAY-ORAL PRESENTATIONS
must regulate the number of granules that are secreted in each target encounter. This way, they
maintain a complement of granules to be used against subsequent target cells.
E.E. Just Award Lecture
G1
Tuning signals that regulate mast cell function.
A. August1;
1
Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University,
Ithaca, NY
Signals emanating from the Fc epsilon receptor on mast cells are positively regulated by the Tec family
kinases Itk and Btk. In this lecture, I will discuss how these same kinases act to tune the mast cell
response to signals from bacterially derived LPS acting via the TLR4 receptor. I will show that depending
on the receptor being used, these kinases can send either positive or negative signals.
Bruce Alberts Award for Excellence in Science Education
G2
Four decades of bringing diversity into science.
E. Fowlks1;
1
Department of Biological Sciences, Hampton University, Hampton, VA
Observation of the absence of African Americans at scientific meetings forms the basis for using
research to interest students in pursuing careers in science. At UC Berkeley in 1970, in the midst of
political and social unrest, and against the backdrop of the dearth of diversity in science, I developed a
plan for returning to an HBCU with the notion of providing students with access to biology and
medicine. I wanted students to learn science by doing science. Confronting me, however, was the
shortage of the resources for implementation: bold ideas and an abundance of enthusiasm could not
substitute for the lack of cutting-edge facilities required to make biology come to life in the classroom
and the laboratory. Advances in teaching and learning provided the rationale for developing a student
research and training facility. Through persistent effort, funds were secured, and empty space soon gave
way to an ultra-modern molecular biology research and training infrastructure. Drawing upon the
Socratic approach to instruction, my teaching style began to evolve without the use of conventional
lectures. I found that engaging students seemingly captures their interest more, as they learn that
research is the origin of the concepts in their textbooks. Classical as well as molecular biology
discoveries are discussed and analyzed as the framework of course content. Exploring the cell theory,
the laws of genetics, evolution through natural selection, the DNA double helical structure, the universal
genetic code, and the semi-conservative mechanism of DNA replication are all examples of how biology
can be exciting and meaningful. Learning biology through researchis a major guiding principle that draws
students’ attention. As biology is explored, important connections begin to emerge for students; they
SUNDAY-ORAL PRESENTATIONS
realize that all organisms are related due to common ancestry and that the nearly universal genetic code
provides support for evolution. Another concept studied is how the DNA double-helix illustrates the
interplay of biology, chemistry, computer science, physics, and mathematics. Moreover, students are
guided to explore living systems at the intersection of these core sciences to learn about resultant
discoveries. As students study genomics and bioinformatics, it becomes clear that biology is an
information science. In the classroom and in the laboratory, students are mentored and encouraged to
attend scientific conferences, participate in summer internship programs, and apply to graduate and
professional schools. For over four decades, the use of research in the classroom as a teaching strategy,
the incorporation of recent advances in teaching and learning, embracing Bio2010, and Vision and
Change have all played a role in increasing diversity in science.
Minisymposium 1: Actin Cytoskeleton Structure and Dynamics
M1
GMFβ controls branched actin content and lamellipodial retraction in fibroblasts.
E.M. Haynes1, S.B. Asokan1, S.J. King1, C. Wu1,2, I.P. Lebedeva1,2, J.E. Bear1;
1
UNC Lineberger Comprehensive Cancer Center, Department of Cell Biology and Physiology, University
of North Carolina, Chapel Hill, NC, 2Howard Hughes Medical Institute, Chevy Chase, MD
The process of cell migration is integral to both organism development and disease. In order to migrate,
cells rely on the lamellipodium, a fan-like structure comprised of Arp2/3 nucleated branched actin which
generates the force necessary for leading edge protrusion. Polymerization of actin at the leading edge is
relatively well understood; however, much less is known about branched actin disassembly/remodeling
and how it influences migration. In order to respond to extracellular cues, a cell must be able to quickly
remodel its actin cytoskeleton, a process which relies on actin severing and pruning of branches. The
cofilin homolog GMF has been implicated in disassembly of branched actin mainly by in vitro work. We
sought to understand the role of the ubiquitous mammalian GMF isoform, GMFβ, in lamellipodial
dynamics and cell migration. We found that GMFβ (but not GMFγ) is expressed in fibroblasts and
localizes to the leading edge of the cell. Modulation of GMFβ by depletion or overexpression resulted in
significant phenotypes in lamellipodial dynamics, branched actin content and migration. Specifically,
depletion of GMFβ resulted in larger lamellipodia with reduced ability to retract, and a higher fraction of
the cell periphery positive for Arp2/3. GMFβ overexpressing cells were smaller and had a reduced
fraction of Arp2/3 positive cell periphery. These data are consistent with the notion of GMFβ as an
antagonist of branched actin, but does not differentiate between GMFβ’s two proposed mechanisms of
action: debranching and preventing nucleation promoting factors from binding to Arp2/3. In order to
address whether branch lifetime itself was affected by GMFβ, we performed a wash-in using the Arp2/3
inhibitor CK666. Based on previous studies (Hetrick et al 2013) CK666 binds free Arp2/3 and prevents
formation of new branches, but branches that are already formed will not disassemble in the presence
of CK666. GMFβ depletion increased the time required for Arp2/3 signal to dissipate from the cell edge
after CK666 wash-in, suggesting that GMFβ is important for debranching. In addition, we created a
mutation in GMFβ analogous to a mutation in budding yeast GMF (Ydenberg et al 2013) which severely
SUNDAY-ORAL PRESENTATIONS
reduces debranching activity. This mutant cannot rescue the observed phenotypes of depletion of wild
type GMFβ. Finally, we tested if the changes we observed in the branched actin content in these cells
affected directional migration. While both GMFβ depleted and GMFβ overexpressing cells could detect a
gradient of soluble cue (chemotaxis), we found that both cell lines were unable to detect a gradient of
bound extracellular matrix (haptotaxis). This corroborates previous results from our lab that the
branched actin generated by Arp2/3 is necessary for haptotaxis.
M2
A temporally ordered mechanism for actin filament disassembly involving
mammalian Coronin, Cofilin, AIP and Srv2/CAP.
S. Jansen1, A. Collins1, D. Breitsprecher1, B.L. Goode2;
1
Brandeis University, Waltham, MA, 2Department of Biology, Rosenstiel Basic Medical Sciences Research
Center, Brandeis University, Waltham, MA
The actin cytoskeleton is a self-organizing, dynamic polymer system that drives important biological
processes such as endocytosis, cell migration and morphogenesis, and relies on balanced actin filament
assembly and disassembly. How actin disassembly is regulated is still not well understood. One of the
most well characterized components of the disassembly machinery is Cofilin/ADF, which binds
cooperatively to filaments and induces severing. However, Cofilin binds F-actin with surprisingly slow
kinetics, has relatively inefficient severing activity, and produces free (uncapped) barbed ends, which
leads to assembly instead of disassembly. These enigmas have long suggested that additional cellular
factors may be required in vivo for rapid and efficient F-actin disassembly. Here, we used three-color
TIRF microscopy single molecule analysis to investigate the mechanism by which mammalian Coronin,
AIP1 and Srv2/CAP (cyclase-associated protein) work with Cofilin to promote actin disassembly. We
show that these four proteins work in a specific order on ADP-F-actin and together produce ultra-fast
disassembly. Coronin binds first to filaments and rapidly recruits Cofilin. Then, AIP1 arrives and induces
highly efficient severing. After severing, AIP1 remains associated with the newly generated barbed ends,
blocking growth and promoting disassembly even under assembly conditions. Srv2/CAP makes a distinct
mechanistic contribution to disassembly by binding autonomously to filament sides and altering F-actin
structure to enhance Cofilin-dependent severing. Finally, in a reconstituted assembly-disassembly
system, we could directly visualize formins rapidly polymerizing filaments, counterbalanced by equally
rapid severing and depolymerization by the disassembly factors working in concert.
M3
Establishing Mechanical Polarization of the Cell Cortex.
P.W. Oakes1, M.L. Gardel1;
1
James Franck Institute, Institute for Biophysical Dynamics and Physics Department, University of
Chicago, Chicago, IL
Polarization plays an important role in a diverse array of physiological processes, including adhesion,
migration and cell spreading. Polarized cells spatially regulate protrusive activity, focal adhesion
SUNDAY-ORAL PRESENTATIONS
assembly, and traction forces to guide cell migration and tissue morphogenesis. This polarization
originates within the actomyosin cortex but the physical and molecular mechanisms are largely
unknown. Using micropatterning to control cell shape in conjunction with genetic perturbations to alter
the actin cytoskeleton, we identified mechanisms of symmetry breaking within the actin cortex of
fibroblasts that establish polarization. We first demonstrated that changes in cell shape promoted a
redistribution of traction stresses, but did not alter the total contractile work of the cell. We then
identified geometric factors that induced polarization in adherent cells and found that polarization was
coincident with the formation of peripheral bundles within the lamella. Hypothesizing that peripheral
bundle formation played an important role in mechanical polarization, we sought to identify factors
regulating their assembly. Reduced expression of activity of these factors impaired peripheral bundle
assembly and, consequently, the onset of cytoskeletal polarization. Our work demonstrates an
important mechanical role for peripheral bundle assembly in establishing polarization of adherent cells
and creating spatial heterogeneity in the cortex. These processes thus act as self-reinforcing
mechanisms through which cells work to actively maintain their shape. In all, these works suggest that
the actin cytoskeleton organization could serve as a mechanical mechanism to scale molecular
interactions into cellular scale phenomena and polarize cellular activity.
M4
Actin network crosslinking ensures spatial integration during cell polarization.
A. Pitaval1, F. Senger1, H. Ennomani*1, L. Blanchoin1, L. Kurzawa1, M. Théry2;
1
iRTSV, CEA, Grenoble, France, 2iRTSV, CEA, Paris, France
Cells adapt their shape and cytoskeleton architecture to the adhesive cues from their extra-cellular
microenvironment. They also displayed the remarkable capacity to modulate the position of their
organelles and orient their polarity with respect to these spatial cues. Focal adhesions assembly and
actin network growth are known to depend on local adhesive cues.
How these local responses are spatially integrated is still unknown. How this integration process defines
cell’s symmetry axes and body plan with respect to microenvironment geometry also remains to be
uncovered.
Here we plated cells on spatially controlled extra-cellular environments by using surface micropatterning
and showed that actin filaments crosslinking by alpha-actin ensures the physical connection between
the distinct cell parts. This connectivity supports the transmission of mechanical forces from one cell
side to the other. Cell adhesions, actin retrograde flow and actin network architecture conform to the
overall spatial distribution of extracellular matrix thanks to this mechanical connection. In the absence
of alpha-actinin, the symmetries of intracellular organization no longer correspond to extracellular
symmetries; the nucleus becomes mispositioned and cell polarity misoriented. The basic and elementary
role of crosslinking in spatial integration was further confirmed by monitoring the contraction of
reconstituted actin rings in vitro.
SUNDAY-ORAL PRESENTATIONS
M5
Septins are essential for tissue integrity during epithelial morphogenesis.
M. Mavrakis1, Y. Azou-Gros1, A. Kress2, S. Brasselet2, T. Lecuit1;
1
Institut de Biologie du Développement de Marseille, CNRS UMR 7288, Marseille, France, 2Institut
Fresnel, CNRS UMR 7249, Marseille, France
Septins are highly conserved cytoskeletal proteins that have been mostly studied in dividing cells.
Besides their role in cytokinesis, recent studies suggest that septins might regulate the organization of
the actin cortex in nondividing cells. However, the precise contribution of septins to the architecture of
the cortex in nondividing cells is still unknown, and the molecular functions of septins altogether are
poorly understood. To test the role of septins in actin-driven cell shape changes in vivo, we studied
epithelial tissue morphogenesis using the gastrulating Drosophila embryo as a model system. To study
the architecture of the actin cortex and the overal organization of the epithelial tissue during
morphogenetic movements, we used a combination of live two-photon imaging, transmission and
scanning electron microscopy and polarization-resolved fluorescence microscopy in wild-type and
septin-deficient whole embryos. Importantly, septins appear to have a major role in maintaining the
integrity of the remodeling epithelial tissue. In the absence of septins, epithelial polarity is lost, the
bases of epithelial cells open up, cell membranes undergo excessive blebbing and are eventually
fragmented leading to the complete loss of cell integrity and the collapse of the epithelial tissue within
an hour after the onset of gastrulation. We will discuss these findings in the context of our recent report
on the capacity of septins to cross-link and organize actin filaments in contractile rings (Mavrakis et al,
2014).
M6
Structured illumination microscopy reveals that focal adhesions are composed of
linear subunits.
S. Hu1, Y. Tee1, A. Kabla2, R. Zaidel-Bar1, A.D. Bershadsky3,4, P. Hersen1,5;
1
Mechanobiology Institute, Singapore, Singapore, 2Department of Engineering, University of Cambridge,
Cambridge, United Kingdom, 3Department of Molecular Cell Biology, Weizmann Institute of Science,
Rehovot, Israel, 4Mechanobiology Institute, National University of Singapore, Singapore, Singapore,
5
Université Paris Diderot, Laboratoire Matière et Systèmes Complexes, UMR 7057 CNRS, Paris, France
The cells ability to mechanically interact with the extracellular matrix (ECM) is a fundamental feature of
adherent eukaryotic cells, and is required for cell migration, proliferation, and differentiation. Adhesion
is mediated by protein complexes, called focal adhesions. Recent progress in super resolution
microscopy revealed that they have an internal organization, yet such methods do not allow observing
the formation and dynamics of focal adhesions’ internal structure. Here, we combine structured
illumination microscopy (SIM) with total internal reflection fluorescence microscopy (TIRF) to be able to
observe focal adhesions at high spatial resolution in live cells. We show that focal adhesion proteins
inside focal patches are distributed along elongated subunits, typically 300 nm wide, separated by 300
nm and individually connected to actin cables. These focal adhesions subunits growth speed is in
SUNDAY-ORAL PRESENTATIONS
average of 0.3 µm/min. We further show that these structures are intimately linked to actin radial fiber
formation. Taken together, our study reveals that, when observed with an appropriate resolution in
both space and time, the minimal units of adhesion appear to be elongated, dynamic structures. We
anticipate this ultrastructure to be relevant when studying the mechano-biological attributes of focal
adhesions and their behavior in response to mechanical stress.
M7
Nanoscale organization of cadherin-mediated adhesion complexes formed on
planar cadherin-coated surfaces.
C. Bertocchi1, T. Sailov1, M. Baird2, R. Mege3, B. Ladoux3,4, M.W. Davidson5, P. Kanchanawong1,6;
1
MBI, National University of Singapore, Singapore, Singapore, 2Florida State University, Tallahassee, FL,
3
Institut Jacques Monod, Université Paris Diderot CNRS, Paris, France, 4Mechanobiology Institute,
National University of Singapore, Singapore, Singapore, 5National High Magnetic Field Laboratory and
Department of Biological Science, Florida State University, Tallahassee, FL, 6Department of Biomedical
Engineering, National University of Singapore, Singapore, Singapore
Cadherin-mediated junctions are of fundamental importance for multicellularity and tissue homeostasis.
Although cadherins are part of a supramolecular complex that performs diverse functions such as
mechanotransduction, signaling and dynamic regulation and linkages to the actin cytoskeleton, still
much remains unknown about the ultrastructural basis of these functions. Although superresolution
microscopy offers a potential avenue for dissecting the nanoscale organization within these complexes,
the highly variable 3-D geometry of the native cell-cell junctions is sub-optimal for high resolution optics.
Therefore, we employed a planarized substrate which presents uniformly oriented cadherin
extracellular domains that mimic many attributes of true cell-cell junctions such as cadherins clustering,
recruitment of canonical cadherin associated proteins, and importantly the connection to the actin
cytoskeleton. To elucidate the nanoscale architecture underlying distinct cadherin-mediated complexes,
we applied superresolution microscopy techniques to determine proteins position and orientation in
C2C12 myoblast (N-cadherin) and MDCK cells (E-cadherin) cultured on respective cadherin biomimetic
surfaces. We observed that distinct proteins within the cadherin-mediated complexes appear to be
differentially partitioned along the vertical (z) axis at the nanoscale. Proteins that reside in close
proximity to the cadherin cytoplasmic tails (N-cadherin: z = 47.4 ±5.9nm; E-cadherin: z= 46.9 ±4.7nm,
relative to the substrate) include p120-catenin (z = 47.9 ±8.3nm in C2C12; 45.0 ±4.3nm in MDCK) and acatenin (n-terminal probe) (z = 47.0 ±10.3nm in C2C12; 41.5 ±7.5nm in MDCK). The actin cytoskeleton is
distinctly separated from the cadherin-associated partition by ~30 nm, and is closely associated with
actin-binding proteins such as a-actinin, eplin, VASP, and zyxin. Interestingly, vinculin was observed to
span between these two vertical partitions, suggesting that it is oriented in a polarized manner, and
likely in an open and active conformation. Similar hierarchies of nanoscale protein organization between
the two different cell types suggest a commonality between the nature of the physical links between
actin and E-/N-cadherin. However, we observed a greater extent of vertical stratification in N-cadherinbased complexes. In particular, vinculin appears to be highly extended, with at least ~30 nm contour
length, consistent with the large and elongated morphology of the cadherin clusters in the more
contractile myoblast cells. Our results suggest how cadherins and associated proteins are assembled at
SUNDAY-ORAL PRESENTATIONS
the nanoscale level on a planar format, which likely recapitulates how they are organized in native cellcell contacts to interface with the actin cytoskeleton.
M8
A novel actin-adhesion structure involved in nuclear positioning requires the
formin FMN2.
C.T. Skau1, C. Waterman1;
1
National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD
Active asymmetric positioning of the nucleus in interphase cells is critical to cell functions including cell
migration, particularly in complex 3D environments. Previous studies have highlighted the importance of
integrin and Rho signaling in controlling nuclear orientation in migrating fibroblasts. Although these
studies establish that integrins, actin, and actin-binding proteins at the nuclear envelope are critical for
nuclear positioning in adherent cells, it is not known if this is mediated by linking the nucleus to the
previously-characterized adhesion/actin system used for migration, or if nucleus-ECM coupling is
accomplished via a specialized cytoskeletal system. We tested the hypothesis that a dedicated adhesionactin system is responsible for maintenance of nuclear position in adherent, migrating cells. We
examined the interplay between actin, adhesions, and the nucleus in fibroblasts using fluorescence
microscopy. We identified novel adhesive structures located underneath the nucleus termed subnuclear
adhesions that are compositionally and dynamically distinct from the canonical focal adhesions at the
leading edge. First, subnuclear adhesions have reduced levels of signaling and actin-binding proteins
found in leading edge adhesions but have high levels of fibrillar adhesion proteins, although they are not
required for fibrillogenesis. Additionally, unlike focal adhesions at the protruding edge, assembly of the
long-lived subnuclear adhesions is coordinated with movement of the nucleus and independent of the
leading edge. Furthermore, evidence from nuclear displacement experiments indicates that the number
and placement of these adhesions is controlled by the nucleus. We also show that a specific set of actin
fibers connecting two subnuclear adhesions can control nuclear shape by physically impinging on the
nucleus. These subnuclear fibers have elevated levels of the IIB isoform of myosin as compared with
dorsal stress fibers, and are less dependent on the contractile activity of myosin than dorsal stress
fibers. We previously identified the actin nucleation factor formin FMN2 in a screen of adhesion
components. We now find that FMN2 localizes underneath the nucleus, and that the rapid dynamics of
FMN2 partially depend on myosin activity. Critically, FMN2 is essential for both subnuclear actin and
adhesions. Cells lacking FMN2 and subnuclear adhesions migrate poorly in 3D environments and exhibit
defects in nuclear positioning, including failure to reorient during wound healing. Together, our data
reveal the critical role of an actin nucleator in a previously unidentified mechanism for control of nuclear
position via a novel adhesive structure linking the actin cytoskeleton, which connects to the nucleus, to
the extracellular matrix.
SUNDAY-ORAL PRESENTATIONS
M9
Formin FH2 domains accelerate ATP hydrolysis prior to polymerization: insights
into formin-specific effects on actin structure.
P. Gurel1, H. Higgs1;
1
Department of Biochemistry, Geisel School of Medicine at Dartmouth College, Hanover, NH
Formins are a class of actin assembly factors, and the 15 mammalian formins have wide-spread roles in
many actin-based processes. Central to their effects on actin is the dimeric FH2 domain, which can
accelerate nucleation of new actin filaments, then modulate elongation by remaining bound to the
filament barbed end. There is considerable variation between FH2 domains in both nucleation and
elongation efficiencies, and one formin, INF2, can actually enhance depolymerization. While nearatomic resolution structures of actin-bound FH2 domains are available, we do not understand how FH2
domains change actin to enhance nucleation, elongation and, in the case of INF2, depolymerization.
Here, we show that formin FH2 domains cause an acceleration of actin’s ATPase activity even when
actin is in the monomeric state (bound to latrunculin). The magnitude of this acceleration scales roughly
with the nucleation efficiency of the FH2 domain, with FH2 domains that potently nucleate being the
most acceleratory to ATP hydrolysis from actin monomers. This effect had been predicted for the
barbed end-capping molecule, cytochalasin D (Goddette & Frieden (1986) J. Biol. Chem.), and we recapitulate these results to show that FH2 domains act in a quantitatively similar manner. For some
formins, the region C-terminal to the FH2 provides further acceleration of ATP hydrolysis. We use
analytical ultracentrifugation and electron microscopy to show that actin is not polymerized under these
conditions. In the case of INF2, increased ATP hydrolysis rate appears to play a role in its
depolymerization activity. When added to pre-polymerized filaments, INF2 causes rapid
depolymerization to a state in which filaments are no longer present, yet the system continues to
hydrolyze ATP in the non-filamentous state. In summary, we reveal a fundamental effect of FH2
domains, to enhance the ATPase activity of actin monomers, suggesting that formins convert the actin
monomer to the “flattened” ATPase-competent conformation (Mizuno et al (2010) Science) prior to its
addition to the barbed end. Individual formins such as INF2 can tailor this activity for their specific
purposes.
M10
Potent inhibition of Diaphanous formins by actin oligomers produced by the
Actin Crosslinking Domain toxin of V.cholerae.
D. Heisler1, E. Kudryashova1, D. Grinevich1, S. Kotha2, J. Winkelman3, C. Suarez4, P. Narasimham2, D.R.
Kovar4,5, D.S. Kudryashov1,6;
1
The Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH,
2
Department of Internal Medicine, Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, The
Ohio State University, Columbus, OH, 3Molecular Genetics and Cell Biology, Univeristy of Chicago,
Chicago, IL, 4Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL, 5Biochemistry and
Molecular Biology, University of Chicago, Chicago, IL, 6Molecular and Cellular Developmental Biology
SUNDAY-ORAL PRESENTATIONS
Program, The Ohio State University, Columbus, OH
Actin Crosslinking Domain (ACD) is produced by pathogenic strains of Vibrio cholerae, Vibrio vulnificus,
and Aeromonas hydrophila. Upon delivery to the cytoplasm of host cells via Type I or Type VI Secretion
systems, ACD catalyzes the formation of an amide bond between K50 and E270 of actin monomers,
resulting in the formation of actin oligomers of varying sizes. According to the current view on the ACD
pathogenesis, the ACD activity leads to a slow failure of the cytoskeleton due to the gradual
accumulation of non-functional oligomers leading to depletion of both monomeric and filamentous
actin pools. Instead, we propose that ACD employs a hitherto unprecedented toxicity mechanism by
converting cytoplasmic actin into highly toxic oligomers that specifically target key steps of actin
dynamics. Particularly, we demonstrate that the oligomers inhibit with sub-nanomolar affinity actin
polymerization controlled by Diaphanous formins. TIRFM experiments reveal that the formin controlled
elongation is blocked in a concentration dependent manner in the presence of nanomolar
concentrations of the oligomers. Profilin plays a key role in the observed inhibition and mDia1 formin
constructs with fewer proline-rich regions in their FH1 domains are inhibited by the oligomers less
efficiently. Accordingly, we demonstrate that at least some vital functions of the cytoskeleton (e.g.
integrity of epithelial barriers) are deeply impaired when only a small fraction of cytoplasmic actin is
crosslinked into oligomers. The effects are reproduced by formin-, but not ARP2/3 complex-specific
small molecule inhibitors. Intriguingly, the pool-down analysis of HeLa cell lysates using double-tagged
actin demonstrates that the oligomers exhibit selectivity towards certain human formins, suggesting the
possibility of creating specific ACD-based and/or ACD mechanism-inspired formin inhibitors. To
conclude, we propose that delivery of only a few copies of ACD into the cytoplasm of the host cell
enables this enzyme to produce actin oligomers that possess the unique combination of properties that
is neither observed in G- nor F-actin. Specifically, a tandem organization of the ACD-crosslinked actin
oligomers and their ability to interact with G-actin binding proteins (e.g. profilin) confers them an
abnormally high affinity to tandem G-actin binding proteins (e.g. tandem proline-rich and WH2 domains
of formins, VASP, and NPFs) and enables them to actively interfere with the specific nucleation and/or
elongation activities of these proteins. Thereby, ACD initiates a toxicity cascade that employs actin
oligomers as second messengers, whose novel, “gain-of-function” inhibitory properties potently
“poison” cellular functions orchestrated by formin-mediated actin polymerization.
M11
Actin is required for IFT regulation in Chlamydomonas reinhardtii.
P. Avasthi1, M. Onishi2, J. Karpiak3, R. Yamamoto4, L. Mackinder5, M.C. Jonikas5, W.S. Sale4, B. Shoichet3,
J. Pringle2, W. Marshall6;
1
Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA, 2Genetics,
Stanford Univ Med Ctr, Stanford, CA, 3Pharmaceutical Chemistry, University of California, San Francisco,
San Francisco, CA, 4Department of Cell Biology, Emory University School of Medicine, Atlanta, GA, 5Plant
Biology, Carnegie Institution for Science, Stanford, CA, 6Biochemistry and Biophysics Dept., University of
California, San Francisco, San Francisco, CA
SUNDAY-ORAL PRESENTATIONS
Assembly of cilia/flagella require the bidirectional transport of motors and cargo, termed intraflagellar
transport (IFT). Recruitment of IFT machinery to the base of flagella is increased in shorter, rapidly
assembling flagella and is reduced at the base of longer, slowly assembling flagella that are approaching
their final steady state length. Disruption of the actin network has been shown to affect ciliary
formation, length and number but the mechanisms are unknown. We utilized the green alga
Chlamydomonas reinhardtii to investigate potential mechanisms by which actin might regulate
cilia/flagella formation. In Chlamydomonas, actin is found in the inner dynein complexes within flagella
as well as in the cell body. While, previous attempts to localize filamentous actin in vegetative cells have
failed, we have localized filaments to both perinuclear and anterior regions using expression of a tagged
filament binding peptide, Lifeact-Venus. We also show that actin disruption using small molecule
inhibitors and a null actin mutant results in impaired flagellar regeneration, IFT injection and
recruitment of IFT material to the flagellar base, with recruitment lacking the normal dependence on
flagellar length. Each phenotype is recapitulated when using a myosin II inhibitor and we have modeled
the potential binding of this inhibitor to a Chlamydomonas myosin. Mid-cell localization of a tagged
version of this myosin is disrupted when flagellar regeneration is induced. Our data suggest actin and
myosin are involved in establishing the length dependence of flagellar assembly via IFT regulation.
Minisymposium 2: Cell Dysfunction in Cancer
M12
Wnt5A promotes an adaptive, senescent-like stress response, while continuing to
drive invasion in melanoma cells.
M.R. Webster1, M. Xu2, K.A. Kinzler2, A. Kaur1, J. Appleton1, M.P. O'Connell1, K. Marchbank1, A. Valiga1,
V.M. Dang1, M. Perego1, G. Zang1, A. Slipicevic1, F. Keeney3, E. Lehrmann2, W. Wood III2, K. Becker2, A.
Kossenkov4, D. Frederick5, K. Flaherty5, X. Xu6, M. Herlyn1, M.E. Murphy7, A.T. Weeraratna1;
1
Tumor Microenvironment and Metastasis, The Wistar Institute, Philadelphia, PA, 2The National Institute
on Aging, Baltimore, MD, 3Microscopy, The Wistar Institute, Philadelphia, PA, 4Bioinformatics, The
Wistar Institute, Philadelphia, PA, 5Massachusetts General Hospital Cancer Center, Boston, MA,
6
Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, 7Molecular and Cellular
Oncogenesis, The Wistar Institute, Philadelphia, PA
We describe a previously unexplored adaptive stress response driven by Wnt5A in highly invasive
melanoma cells. We show that highly invasive melanoma cells express functional p53 and p21, and
consequently undergo a G2/M growth arrest when exposed to stress such as irradiation and BRAFV600E
targeted therapy. This arrest heralds the onset of an increase of senescence markers including
senescence-associated beta-galactosidase activity (SA-beta-gal), senescence-associated heterochromatic
foci (SAHF), promyelocytic bodies (PML) and modified chromatin, as defined by the presence of the
Histone H3 trimethyl Lys9 (H3K9Me) marks. These cells, which are positive for senescence markers
retain invasive capacity, and are capable of forming new colonies, both in vivo and in vitro. Silencing
Wnt5A reduces expression of these senescence markers and decreases invasiveness. Mechanistically,
we show that this is governed by the non-canonical Wnt molecule, Wnt5A via p21. It is these Wnt5A
SUNDAY-ORAL PRESENTATIONS
high, highly metastatic cells that undergo a senescent-like arrest upon induction of stress, yet retain all
the hallmarks and capabilities of very aggressive cells. We propose that this adaptive stress response
may be a way for tumors to evade genotoxic damage and targeted therapy, and may select for a
subpopulation of cells, which are highly invasive. Importantly, Wnt5A is emerging as a biomarker of not
only metastasis in melanoma, but also of therapy resistance.
M13
VAMP3-regulated delivery of MT1-MMP to tumor-derived microvesicles.
J. Clancy1, A.E. Sedgwick1, C. Rosse2, M. Method3, P. Chavrier2, C. D'Souza-Schorey1;
1
Biological Sciences, University of Notre Dame, Notre Dame, IN, 2Cell Biology, Institut Curie, Paris,
France, 3Michiana Hematology Oncology, Northern Indiana Cancer Consortium, Mishawaka, IN
Cells of multiple origins have been known for some time to shed distinct types of extracellular vesicles.
Among these, are structures known as microvesicles, which are formed by the outward budding and
pinching of the plasma membrane. Shed vesicles, through their ability to carry and transfer bioactive
cargo throughout the body, may act to alter the extracellular environment or mediate paracrine
signaling. Nucleic acids, functional proteins and lipid components can all be transferred to recipient cells
or deposited in the extracellular space in this manner. Furthermore, the identification of microvesicles in
body fluids has heightened interest in their potential to serve as disease biomarkers. We have previously
reported on the cargo content of tumor-derived microvesicles (TMVs) that are released in an ARF6dependent manner. Despite the growing understanding of extracellular vesicle biogenesis, function, and
cargo content, little is known about mechanisms regulating cargo delivery and selective enrichment of
vesicular contents. Here, we demonstrate for the first time an association between vesicle-associated
membrane protein 3 (VAMP3) and membrane type 1 matrix metalloprotease (MT1-MMP) that in
rounded, amoeboid-like, invasive cells regulates the delivery of microvesicle cargo. Delivery of MT1MMP is dependent on the association of VAMP3 with the tetraspannin CD9, and the transport of
functional MT1-MMP into nascent TMVs is critical for the maintenance of invasive capabilities in
amoeboid-like cells. Knockdown of endogenous VAMP3 depletes shed vesicles of MT1-MMP and
drastically reduces the ability of otherwise invasive LOX melanoma cells to invade cross-linked collagen
matrices highlighting the importance of functional TMVs in cell invasion. Finally, we describe TMVs
showing structural and functional similarity to those above that are isolated from the peripheral bodily
fluids of ovarian cancer patients. Together these studies demonstrate the importance of TMV cargo
sorting in the dynamic course of cell invasion and disease progression.
M14
Autophagy regulates focal adhesion turnover in transformed cells.
C.M. Kenific1, S. Stehbens2, K. Woo1, J. Ye1, T. Wittmann3, J. Debnath1;
1
Department of Pathology, University of California, San Francisco, San Francisco, CA, 2Queensland
University of Technology, Queensland, Australia, 3Department of Cell and Tissue Biology, University of
California, San Francisco, San Francisco, CA
SUNDAY-ORAL PRESENTATIONS
Autophagy is a process of lysosomal-mediated degradation that has traditionally been viewed as a
stress-adaptation pathway that promotes tumor cell survival. However, recent evidence suggests that
autophagy serves broader functions during cancer progression. Here, we demonstrate a new role for
autophagy in regulating focal adhesion (FA) turnover in transformed cells.
Autophagy inhibition via RNAi-mediated depletion of essential autophagy regulators (ATGs) decreases
single-cell migratory rate of cells in a wound healing assay, and wound edge cells have significantly
enlarged FAs upon autophagy inhibition. Because FA assembly and disassembly, or turnover, regulates
migratory rate we hypothesized that increased FA size was due to impaired FA turnover. Using spinning
disk confocal (SDC) microscopy to image FA dynamics in live cells expressing Paxillin-mCherry, to mark
FAs, we observed that ATG depletion decreased FA assembly and disassembly rates, while increasing FA
lifetime, suggesting that stabilization of FAs upon autophagy inhibition impairs motility. To further
analyze the relationship between autophagy and FAs, we determined if autophagosomes localize near
FAs. Total internal reflection fluorescence microscopy of live cells revealed that autophagosomes, as
marked by GFP-LC3, are found in proximity to FAs. Furthermore, SDC imaging showed that
autophagosomes associate with dynamic FAs primarily during the disassembly phase of the turnover
cycle.
Because autophagy is pathway of cellular degradation, these findings led us to hypothesize that
autophagy facilitates FA disassembly by locally degrading FA components. To test this prediction, we
focused on establishing if autophagy cargo receptors, which mediate selective autophagic degradation
of cellular substrates by linking cargo to autophagosomes, modulate FA turnover. We found that similar
to ATG depletion, knockdown of the cargo receptor NBR1 impairs FA turnover. Conversely,
overexpression of NBR1 decreases FA lifetime, and GFP-NBR1 associates with dynamic FAs. These data
suggest that NBR1-mediated selective autophagy controls FAs in transformed cells. We are now testing
this model more rigorously using autophagy-defective mutants of NBR1.
In addition to regulating motility, cell-matrix adhesions also promote growth signals that drive
metastatic colonization. Thus, enhanced cell-matrix adhesion due to increased FAs upon autophagy
inhibition may support metastasis. Accordingly, ATG depletion in a polyoma middle T (PyMT) breast
carcinoma cell line leads to enlarged FAs in vitro and enhanced pulmonary metastasis in vivo. We are
now determining if increased cell-matrix adhesion is a mechanism by which autophagy inhibition
potentiates metastasis and if NBR1 regulates metastasis of PyMT cells.
M15
Entosis is induced by nutrient deprivation of tumor cells.
J. Hamann1, J. Balin1, M.H. Overholtzer1;
1
Cell Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY
Cell engulfment mechanisms maintain metazoan tissue homeostasis by removing dying cells and
pathogenic organisms. However recent evidence suggests that engulfment mechanisms also target
viable cells and could thereby regulate rates of physiologic cell turnover in some contexts. One
mechanism that targets live cells for engulfment in human tumors is called entosis. By entosis, human
tumor cells use the machinery of cell-cell adhesion to ingest their live neighbors, in a manner dependent
on Rho-GTPase signaling and actomyosin. Live cells that are ingested by this mechanism undergo a non-
SUNDAY-ORAL PRESENTATIONS
cell-autonomous form of cell death, resulting from the autophagy protein-dependent maturation of
entotic vacuoles within engulfing cells that leads to lysosome fusion.
We have shown that entotic cell death can limit transformed growth, suggesting a potential mechanism
of tumor suppression linked to the engulfment and killing of live cells. Alternatively, we have also shown
that entosis promotes the development of aneuploidy, suggesting that this process could promote
tumor progression in the long term. Consistent with this, cell-in-cell structures resembling those formed
by entosis are observed most frequently in high-grade breast tumors that exhibit high rates of
aneuploidy and aggressive clinical characteristics. We have also recently found that entosis can support
the survival of cells cultured under starvation conditions by supplying engulfing cells with nutrients,
indicating an additional potential pro-tumorigenic role of entosis linked to the support of cell survival in
poorly vascularized tumors. While entosis can supply cells with nutrients, whether nutrient deprivation
could promote this process in tumors is unknown.
Here we examined if nutrient deprivation by glucose withdrawal, as well as by manipulation of the
cellular pathways that regulate glucose signaling and uptake, can influence entosis. We find that glucose
withdrawal, as well as phosphoinositide 3-kinase (PI3K) inhibition, induce entosis in MCF-7 and MDAMB-231 breast tumor cell populations, in a manner requiring activity of the cellular energy sensor
AMPK. We further find that AMPK activation, even in the presence of high ATP levels, is sufficient to
induce this form of cell engulfment, and that nutrient deprivation-mediated induction of entosis leads to
the appearance of polyploid tumor cells. These data demonstrate that entosis is controlled by nutrient
signaling pathways, and suggest that nutrient deprivation could be one mechanism that induces this
process in human tumors.
M16
ARF6/JIP3-JIP4/dynactin-dynein/MT1-MMP axis controls a matrix invasion
program during breast cancer progression.
P. Chavrier1, V. Marchesin1, A. Castro-Castro1, C. Lodillinsky1, J. Cyrta1, L. Furhmann1, A. VincentSalomon2, M. Irondelle1, A. Guichard1;
1
Cell Biology, Institut Curie, Paris, France, 2Pathology, Institut Curie, Paris, France
Initial steps in breast cancer dissemination require tumor epithelial cells to cross tissue barriers through
a matrix invasion program involving the trans-membrane type 1 matrix metalloproteinase (MT1-MMP).
Proteolytic remodeling of the extracellular matrix environment by tumor cells requires formation of
actin-based protrusions of the plasma membrane called invadopodia, where MT1-MMP accumulates
through exocytosis from a late endosome storage compartment. The small GTP-binding protein ARF6
controls endosomal membrane traffic and has been implicated in the matrix invasion program of breast
tumor cells by promoting epithelial-to-mesenchymal transition and invadopodia formation. JIP3 and JIP4
are large coiled-coil proteins controlling microtubule-based motility by binding to kinesin-1 and
dynactin/dynein in an ARF6-controlled manner. We addressed the contribution of ARF6 and its
downstream effectors JIP3 and JIP4 to the regulation of MT1-MMP endosomal trafficking and
consequences for the invasive potential of breast tumor cells. Depletion of ARF6 or JIP3/JIP4 attenuated
matrix remodeling and invasive potential of breast tumor cell lines in a 3D type collagen environment in
vitro, reduced exocytosis of MT1-MMP from late endosomes and correlated with MT1-MMP-positive
SUNDAY-ORAL PRESENTATIONS
endosome mispositioning through a mechanism involving microtubule minus-end directed
dynactin/dynein motor activity. ARF6 was overexpressed in clinical samples of invasive ductal breast
cancers, accumulated to the plasma membrane of breast tumor cells and correlated with elevated levels
of plasma membrane MT1-MMP in a subset of high-grade triple-negative breast cancers. Our results
identify ARF6 as a regulator of dynamic microtubule-based trafficking of MT1-MMP-containing
endosomes to support exocytosis and surface expression of MT1-MMP essential for pericellular matrix
remodeling during breast cancer invasion.
M17
Endothelial podosome rosettes regulate vascular branching in tumor
angiogenesis.
G. Seano1,2,3, G. Chiaverina1,2, P. Gagliardi1,2, L. di Blasio1,2, A. Puliafito1,2, C. Bouvard4, R. Sessa1,2, G.
Tarone5, L. Sorokin6, D. Helley7, R. Jain3, G. Serini1,2, F. Bussolino1,2, L. Primo1,2;
1
Department of Oncology, University of Torino, Torino, Italy, 2Candiolo Cancer Institute-FPO, Candiolo,
Italy, 3Radiation Oncology, Harvard Medical School - MGH, Boston, MA, 4UMR-S 765, Université Paris
Descartes, Paris, France, 5Department of Molecular Biotechnology and Health Sciences, University of
Torino, Torino, Italy, 6Institute of Physiological Chemistry and Pathobiochemistry, Muenster University,
Muenster, Germany, 7UMR-S 970, Université Paris Descartes, Paris, France
The mechanism, by which angiogenic endothelial cells break the physical barrier of vascular basement
membrane and consequently sprout, forming new vessels in mature tissues, is unclear. Here, we show
that angiogenic endothelium is characterized by the presence of functional podosome rosettes. These
extracellular matrix-degrading and adhesive structures are precursor of de novo branching points and
represent a key event in the formation of new blood vessels. VEGF-A stimulation induces the formation
of endothelial podosome rosettes by up-regulating integrin α6β1; in contrast, the binding of α6β1
integrin to vascular basement membrane laminin impairs the formation of podosome rosettes by
restricting α6β1 integrin to focal adhesions and hampering its translocation to podosomes. Using ex vivo
sprouting angiogenesis assay, transgenic and knock-out mouse models and human tumor samples
analysis, we provide the first evidence that endothelial podosome rosettes control blood vessels
branching and are critical regulators of pathological angiogenesis.
M18
Multiclonal Seeding is a Frequent Route to Metastatic Spread.
K. Cheung1, V. Padmanaban1, K. Schipper1, V. Silvestri1, A. Ewald2;
1
Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, 2Department of Cell Biology,
Johns Hopkins University School of Medicine, Baltimore, MD
Introduction: A foundational concept in metastasis research is that primary tumors first dissociate into
single cancer cells in order to seed distant sites. The requirement for a single-cell seed forms an
important tenet for common molecular models of metastasis including the epithelial-mesenchymal
transition. However, the experimental evidence for the single-cell seed model of metastasis remains
SUNDAY-ORAL PRESENTATIONS
limited. Furthermore, circulating tumor cell clusters are detectable in the bloodstream and therefore
multiclonal seeding events are also possible. In this study, we experimentally isolated the relative
contributions of single and multiclonal seeds and uncovered that multiclonal seeding is a frequent route
to metastatic spread.
Methods: We devised a lineage tracing strategy using Rainbow transgenic mice to reveal the clonal
origin of lung metastases in MMTV-PyMT mice, a commonly used mouse model of metastatic breast
cancer. Primary PyMT tumor organoids that express a genetically encoded Rainbow construct were
treated with Cre in order to induce random assortment of color labeling, and then injected
orthotopically into the mammary fat pad of host mice. Lung metastases were then scored for the
number of unique colors.
Results: If lung metastases arise exclusively from single seeds then each lung metastases should express
only one color. Surprisingly, we observed frequent multicolor lung metastases (mean 32%, maximum
61%, N=998 metastases). By implanting tumor organoids labeled with distinct colors into separate flanks
of host mice, we observed that these multiclonal seeding events occur predominantly in a single step
rather than by serial colonization of single cells. To address why multiclonal seeding might be
advantageous, we flow sorted trypsinized tumor cells, gating by size. This enabled us to obtain fractions
of single cells and cell clusters of increasing size distributions. Interestingly, we observed that cell
clusters had 10-fold higher colony formation and superior survival relative to isolated single cells. Work
is underway to test the lung colonization potential of single cells versus cell clusters using tail-vein
metastasis assays.
Conclusions: Our results challenge the requirement for single cell seeding in metastatic colonization and
establish the importance of a parallel route for metastatic spread involving collective multiclonal
dissemination. Furthermore, our data suggest a fundamental mechanism for cell growth and survival
mediated by cell-cell contacts. We suggest that disrupting cell contacts may limit cancer cell survival and
metastatic spread.
M19
Intravital microscopy suggests that induction of mitotic arrest by antimitotic
cancer drugs does not predict their efficacy in fibrosarcoma mouse xenograft
tumors.
S. Florian1, D.R. Chittajallu2, R.H. Kohler3, J.D. Orth4, R. Weissleder1,3, G. Danuser5, T.J. Mitchison1;
1
Department of Systems Biology, Harvard Medical School, Boston, MA, 2Department of Cell Biology,
Harvard Medical School, Boston, MA, 3Center for Systems Biology, Massachusetts General Hospital,
Boston, MA, 4MCD Biology, University of Colorado Boulder, Boulder, CO, 5Department of Cell Biology,
University of Texas Southwestern Medical Center, Dallas, TX
Despite immense progress in targeted cancer drug design, to date, the most important drugs in
chemotherapy remain traditional cytotoxic drugs which are thought to kill proliferating cells, no matter
if malignant of not. This "antiproliferative" hypothesis is based on results from the 1970s coming from
mouse leukemia models or cell culture experiments.
SUNDAY-ORAL PRESENTATIONS
Microtubule binding drugs are clinically important antiproliferative drugs which arrest cultured cancer
cells in mitosis through activation of the spindle assembly checkpoint (SAC). As opposed to empirically
found microtubule binding drugs, newer drugs designed to more specifically induce mitotic arrest have
repeatedly failed in clinical trials, suggesting that the antitumor effect of microtubule binding drugs is
still not well understood. Alas, tissue culture assays are not predictive of their respective clinical success,
suggesting that more realistic models are needed to understand why there are such huge differences in
antitumor efficacy.
Therefore, we developed an integrated in vivo microscopy and image analysis pipeline of mouse
xenografts to compare the cell cycle effects of microtubule drugs to those of newer, targeted antimitotic
drugs in the form Ispinesib, an inhibitor of the mitotic kinesin Eg5. Image analysis with a custom
developed software for automated three dimensional segmentation and cell cycle classification revealed
that the microtubule interacting drugs Paclitaxel and Eribulin which are in wide clinical use induce
similar mitotic arrest as the Eg5 inhibitor Ispinesib which failed clinical trials. Nevertheless, similar to the
clinical situation, they seem to be more effective in reducing tumor cell density than Ispinesib in our
system. This suggests that, rather than mitotic arrest, a different mechanism, intrinsically coupled to
microtubule binding, may be responsible for the antitumor effect of microtubule drugs in vivo.
M20
Cancer-wide survey of centrosomes reveals centriole fragmentation as a novel
origin of supernumerary centrosomes.
G. Marteil1, A. Guerrero2, S. Godinho3, P. Machado1,4, J. Loncarek5, S. Mendonça1, I. Fonseca1, D.
Pellman6, M. Bettencourt-Dias1;
1
Instituto Gulbenkian de Ciência, Oeiras, Portugal, 2Instituto de Biotecnología Universidad Nacional
Autónoma de México, Mexico, Mexico, 3Barts Cancer institute, London, United Kingdom, 4EMBL,
Heidelberg, Germany, 5Laboratory of Protein Dynamics and Signaling, NIH/NCI-Frederick, Frederick, MD,
6
Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA
A century ago, Theodor Boveri suggested that abnormalities in the major microtubule organising center
of animal cells, the centrosome, lead to abnormal cell division and tumorigenesis. Those abnormalities
have since been observed in cancer in vivo, but their incidence and causes remain poorly characterised.
To address those important questions, we implemented a three-step screen and developed a state-ofthe-art algorithm to score centriole number and length in 3D in the NCI-60 panel of cancer cell lines that
represents cancer diversity. Importantly, we observed that the majority of these cell lines shows
increase in both centriole number and length, and uncovered an important link between centrosome
amplification and invasive capacity. Our work also suggests a novel origin of centriole amplification in
cancer, which we subsequently validated with complementary approaches: as centriole length control is
deregulated in cancer cells, centrioles can grow more than 3 fold and form an abnormal structure that
fragments and generates smaller, functional centrioles. Centriole fragments then behave as microtubule
organising centers leading to abnormal mitosis. The fact that centriole abnormalities are widespread in
cancer, but not in normal cells, and the identification of novel causes of those abnormalities solidifies
and suggests new avenues to the use of centrosomes in the clinic as diagnostic, prognostic and
therapeutic tools.
SUNDAY-ORAL PRESENTATIONS
Minisymposium 3: Cell Organization and Polarity
M21
Role of sterol-rich membrane domains in fission yeast cell polarity establishment
and maintenance.
T. Makushok1, P. Alves2, S.M. Huisman3, D. Brunner4;
1
Biochemistry and Biophysics Dept., University of California, San Francisco, San Francisco, CA, 2IGBMC,
Illkirch, France, 3UZH, Zurich, Switzerland, 4Institute of Molecular Life Sciences, University of Zürich,
Zurich, Switzerland
Fission yeast (Schizosaccharomyces pombe) is a model organism that is widely used for studying cell
polarization. Polarization in fission yeast involves cytoskeleton-mediated positioning of growth sites. A
complex consisting of the polarity factors Tea1 and Tea4 is transported on microtubules to the cell end
regions. Tea4 interacts with the actin polymerization promoter For3. Selective activation of For3 in the
cell end regions makes them rich in F-actin that contributes to localized cell growth. In this study, sterolrich membrane (SRM) domains present at the growth sites are introduced as a new element in the
picture, and their role in cell polarity establishment is analyzed. Since SRMs are absent from the plasma
membrane in starved cells, imaging of cells recovering from starvation using the novel SRM marker GFPTna1 was performed to follow SRM domain formation de novo. Automated image analysis software was
developed to analyze and correlate cell growth and SRM dynamics with an unprecedented level of
precision. The results show that properly formed SRM domains are essential for fission yeast growth.
SRMs, and with them the growth machinery, have to polarize before cell growth initiation. F-actin is
required for selective removal of SRM domains in the cell middle region, and thus for polarizing SRMs.
Fast removal of SRM domains in the cell middle region is not due to increased endocytic activity
(mediated by F-actin). Tea1 controls the localization of polarized growth via Tea4 by affecting the
positioning of SRM domains. Tea1 and Tea4 are essential for the stability of the SRM domains not
associated with active growth sites. The importance of the microtubule cytoskeleton for the stability of
SRM positioning stems from its role in the transport of the Tea1-Tea4 complex to the cell end regions.
Tea1∆ and tea4∆ cells, known to grow monopolarly, grow faster at individual cell ends than wild type
cells. Tea1 and Tea4 are required for proper timing of growth initiation. The proteins associated with the
actin cytoskeleton, For3 and its activator Bud6, are important for the stability of SRM domains at both
cell ends. For3 is also important for growth speed stabilization. Thus, a complex feedback loop links
SRMs and cell growth. SRMs are essential for the polarization of the growth machinery, probably serving
as platforms for its recruitment. The growth machinery, in turn, seems to stabilize SRM domains at sites
that have initiated growth. The results of this study show that SRMs are a critical factor in de novo cell
polarization, and not merely a player in its maintenance.
SUNDAY-ORAL PRESENTATIONS
M22
A Cell Life under Confinement: how immune cells manage to move, squeeze and
survive.
H. Thiam1, M. Raab1, M. Le Berre1, Y. Liu1, M. Piel1;
1
UMR 144, Institut Curie, Paris, France
The quest to understand how the mechanical and geometrical environment of cells impacts their
behavior and fate has been a major force driving the recent development of new technologies in cell
biology research. Despite rapid advances in this field, many challenges remain in order to bridge the gap
between the classical and simple cell culture plate and the biological reality of actual tissues. In tissues,
cells have their physical space constrained by neighboring cells and extracellular matrix. In the recent
years, we have developed simple and versatile devices to precisely and dynamically control this
confinement parameter in cultured cells. I will present results we obtained on the effect of confinement
on cell migration focusing on two questions: how modulating confinement and adhesion of slow
mesenchymal migrating cells can make them switch to a fast amoeboid like migration behavior and how
cells can squeeze their nucleus when migrating through small gaps. I will conclude presenting intruiguing
results showing that large deformations of migrating cells induce strong damages to their nucleus raising
the question of how immune cells such as dendritic cells can combine high motility and long term
survival.
M23
Spontaneous and electric field-induced polarization and motility initiation utilize
different mechanochemical pathways.
Y. Sun1, Y. Sun1, K. Zhu1, B. Draper2, M. Zhao1, A. Mogilner3;
1
School of Medicine, University of California, Davis, CA, 2Mol Cell Biology, University of California, Davis,
CA, 3Courant Inst and Dept of Biology, New York University, New York, NY
Cell polarization and motility initiation are key processes in development, immune response and wound
healing. Many questions about cell polarization remain open: is polarization governed by chemical or
mechanical pathways? Are polarization, directional cues’ sensing and motility interdependent? What are
detailed molecular and mechanical mechanisms of motility initiation? Stationary symmetric fish
keratocytes break symmetry spontaneously and start moving within tens of minutes. It is known that
the spontaneous symmetry break starts from the prospective rear when elevated contractility increases
centripetal actin flow. The motile cells respond to an electric field (EF) and move to cathode. In the
process of polarization, cells exhibit waves of protrusion-contraction. We observed that EF drastically
accelerates keratocytes’ polarization and motility initiation. While biochemically perturbing myosin or
PI3K-signaling disables the spontaneous polarization, EF induced polarization of the perturbed cells.
Surprisingly, while spontaneously polarized cells move persistently for hours, the EF-induced motile cells
loose polarity and become stationary as soon as the EF is switched off. Microscopy demonstrated that
while actin and myosin in stationary and motile cells are distributed similarly without and in the
presence of EF, adhesion (vinculin) distributions are different: in spontaneously motile cells, vinculin is
SUNDAY-ORAL PRESENTATIONS
swept to the rear, while in EF-induced cells it is biased to the front. Pharmacological perturbations of the
EF-induced motile cells showed that (i) there is an alternative, myosin-independent, mechanical
polarization pathway based on pushing the front by actin growth and retracting the rear by membrane
tension; (ii) signaling through PI3 kinase complements two mechanical pathways in cell polarization; (iii)
EF bypasses the default rear-contraction polarization pathway by rapidly orienting protrusion to the
front and weakening adhesions at the rear. In addition to untangling complex mechanochemistry of
polarization and directional sensing of epithelial cells, physiological implications of our findings are that
EF overrides the spontaneous motility and controls motility and directionality of healthy and perturbed
cells in wounds and developmental processes very tightly through biasing adhesion machinery.
M24
The mechanism of maternal centriole elimination in starfish oocytes.
J. Pinto1, J. König2, T. Müller-Reichert3, P. Lenart4;
1
Cell Biology and Biophysics, EMBL, Heidelberg, Germany, 2TU Dresden, Dresden, Germany, 3Medical
Faculty "Carl Gustav Carus", TU Dresden, Dresden, Germany, 4EMBL, Heidelberg, Germany
Centriole duplication and segregation is tightly coupled to the cell cycle in somatic cells to preserve
centriole number over cell generations. However, at fertilization, upon gamete fusion, mechanisms have
to exist to prevent the surplus of centrioles in the embryo that would lead to multipolar spindles and
aneuploidy during early development. Therefore, centrioles are eliminated from the oocyte and only the
sperm provides active centrioles to the zygote. Although this process is essential and common to animal
species, the mechanisms underlying centriole elimination in female gametes are still poorly understood.
In most species, centrioles are eliminated during the long prophase of female meiosis that is difficult to
access experimentally. In contrast, in starfish (P. miniata) oocytes, centriole elimination occurs during
the rapid and synchronous meiotic divisions that can be easily followed by live-imaging. Therefore, to
visualize centrioles in vivo, we established GFP markers and found that two pairs of centrioles are
present at meiosis onset and contribute to spindle organization. Out of these two pairs, one is extruded
into the first polar body at the end of the first meiotic division (MI). As no S-phase exists between MI
and the second meiosis (MII), single centrioles form the poles of the MII spindle. Remarkably, by using
the mother centriole specific marker Odf2-mEGFP, we found that the older, mother centriole always
localizes to the spindle pole facing the plasma membrane. Consequently, the mother centriole is
extruded into the second polar body, whereas a single daughter centriole remains in the mature egg.
We additionally show, by tracking centrioles in live cells and by correlative electron microscopy, that the
mother centriole is specifically transported to the plasma membrane at the end of MI, where it remains
stably anchored. This likely occurs via the mother centriole specific appendages, in a mechanism that
might be related to basal body formation. Additionally, the specific extrusion of mother centrioles is
functionally important, because if the centrioles are artificially retained in the egg (by inhibition of the
polar body extrusion), only the mother centrioles remain active, recruit microtubules and cause
multipolar spindles in the zygote.
Taken together, we propose the first comprehensive model for centriole elimination in animal oocytes
that relies on the specific extrusion of the mother centrioles. We show that known and conserved
features of the mother centrioles are employed in the oocyte for spindle positioning, membrane
SUNDAY-ORAL PRESENTATIONS
attachment and finally their own extrusion. Our observations suggest that the single daughter centriole
remaining in the mature egg is not able to duplicate, leading to its elimination.
M25
Deconstruction of a bio-mechanical ratchet during tissue morphogenesis.
A. Munjal1, T. Lecuit1, J. Philippe1;
1
Institut de Biologie du Développement de Marseille, CNRS UMR 7288, Marseille, France
Myosin II motors generate forces that power cell shape changes during tissue morphogenesis. Such
actomyosin networks function like a mechanical ratchet where phases of pulsed contraction and cell
deformation alternate with phases of stabilization. Drosophila ectoderm extension exemplifies this
differential behavior whereby pulses of acto-myosin flow towards junctions aligned in dorso-ventral axis
leading to their shrinkage in order to facilitate cell intercalation(1). A planar polarized pool of Myosin II
enriched at these junctions stabilizes the deformation resulting in an irreversible process(2). The
mechanisms for regulation of these networks remain unknown. We investigated the role of dynamic
phosphorylation of Myosin II regulatory light chain (RLC) by the Rho1-ROCK pathway. We find that
spatial control over RLC phospho-cycles by Rok and Myosin II phosphatase mediates planar-polarized
accumulation of Myosin II through regulation of its recruitment and dissociation. Investigating Myosin II
temporal dynamics, we report that Myosin II pulses involve de novo recruitment of mini-filaments
concomitant with their contraction, followed by disassembly. Interestingly, Myosin II pulses with its
regulators Rho1, ROCK and Myosin II phosphatase. We refute a model where Myosin II pulsatility is
enslaved by the upstream biochemical pacemaker Rho1. Instead, we propose a bio-mechanical feedback
model postulating emergence of pulses through self-organization of its components. We find that
pulsatility is set by the balance of recruitment of Myosin II by advection- mediated concentration of
upstream regulators, and a delayed phosphatase-dependent depletion of Myosin II.
1. Rauzi, M., Lenne, P.F. & Lecuit, T. Planar polarized actomyosin contractile flows control epithelial
junction remodelling. Nature 468, 1110-1114 (2010).
2. Bertet, C., Sulak, L. & Lecuit, T. Myosin-dependent junction remodelling controls planar cell
intercalation and axis elongation. Nature 429, 667-671 (2004).
M26
A new model for the origin and topological organisation of the eukaryotic cell.
B. Baum1, D. Baum2;
1
Laboratory for Molecular Cell Biology, University College London, London, United Kingdom,
2
Department of Botany and Wisconsin Institute for Discovery, University of Wisconsin, Madison, WI
The emergence of the eukaryotic cell with its nucleus, endomembrane system and organelles
represented a leap in complexity beyond anything seen in prokaryotes. Although this has long been
recognized as the single most profound change in cellular organization during the history of life on
earth, the origins of the eukaryotic cell remain poorly understood. Models have always assumed that
the nucleus and endomembrane system evolved within the cytoplasm of a prokaryotic cell. Here,
SUNDAY-ORAL PRESENTATIONS
drawing on diverse aspects of cell biology and phylogenetic data, we invert this idea. We propose that
an ancestral prokaryotic cell (an eocyte), homologous to the modern-day nucleus, extruded membranebound blebs through pores in its cell wall, which functioned to facilitate material exchange with protomitochondria. The cytoplasm was then formed through the expansion of blebs around protomitochondria, with continuous space between the blebs giving rise to the endoplasmic reticulum, which
later evolved into the eukaryotic secretory system. Importantly, this theory offers cell biologists a
coherent framework by which to understand eukaryotic cell organisation, helps to explain a number of
previously enigmatic features of eukaryotic cell biology, including the relative autonomy of nuclei in
syncytia, makes a number of predictions, including a novel mechanism of interphase nuclear pore
insertion, and has implications for the origins of cell polarity.
M27
Physical mechanisms of protein segregation at membrane interfaces.
E.M. Schmid1, M.H. Bakalar1, K. Choudhuri2, J. Weichsel3, H. Ann4, P.L. Geissler3, M.L. Dustin5, D.A.
Fletcher6,7,8;
1
Bioengineering, University of California, Berkeley, Berkeley, CA, 2Department of Pathology, New York
University Medical Center, New York, NY, 3Chemistry, University of California, Berkeley, Berkeley, CA,
4
Bionengineering, University of California, Berkeley, Berkeley, CA, 5NDORMS, University Oxford/Kennedy
Inst Rheumatol, Headington, England, 6Department of Bioengineering, University of California, Berkeley,
Berkeley, CA, 7Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA, 8Physical
Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA
Membrane interfaces formed at junctions between cells are often associated with characteristic
patterns of membrane protein organization, such as E-cadherin localization to the edge of tight
junctions and CD45 exclusion from signaling foci in immunological synapses. Ligand induced receptor
clustering and the cortical cytoskeleton dynamics can both participate in spatial organization of
membrane proteins in cells, leaving the effect of the membrane interface itself unclear. To address this
question, we reconstituted membrane interfaces in vitro using giant unilamellar vesicles decorated with
synthetic proteins. We find that a fluid, deformable membrane interface linked by protein-protein
binding can drive segregation of non-binding proteins in a size-dependent manner, without
contributions from other cellular components. Interestingly, the membrane interfaces do not fully
exclude larger non-binding proteins nor fully permit smaller non-binding proteins. Combining this
protein segregation data with measurements of the interface distance and Monte Carlo simulations, we
show that protein segregation at membrane interfaces is dependent on both thermally-driven height
fluctuations of the flexible membrane and lateral crowding of the binding proteins. This simple,
sensitive, and highly effective mechanism of spatial organization at membrane interfaces may have
implications for protein organization in a diverse range of cellular contexts ranging from contact points
between membrane-bound organelles to focal adhesions between adherent cells and the extracellular
matrix.
SUNDAY-ORAL PRESENTATIONS
M28
Nuclear size control depends on spatial information within the cell in Xenopus
laevis egg extracts.
Y. Hara1, C.A. Merten1;
1
EMBL, Heidelberg, Germany
Nuclear size changes dynamically during development and has long been observed to correlate with cell
size. However, the underlying molecular mechanisms controlling nuclear scaling have remained largely
unknown. Here, we combine an in vitro cell-free system of Xenopus laevis egg extract with microfluidic
devices to systematically analyze the effect of spatial constraints. The speed of nuclear expansion
depended on the available space surrounding the nucleus up to a threshold volume in the nanoliter
range, herein referred to as the nuclear domain. Under spatial constraints smaller than the size of
nuclear domain, the accumulation of membranes around the nucleus was limiting nuclear expansion.
Detailed biochemical studies revealed that mechanisms sensing the space are not mainly regulated by
simple diffusion of nuclear components, but rather by microtubules accumulating membranes via the
motor protein dynein. This mechanism helps understanding how spatial information surrounding the
nucleus, like positioning of the nucleus inside the cell, can control the nuclear expansion in vivo.
M29
A mechanism of polarized inheritance of proteome damage during yeast
asymmetric cell division.
C. Zhou1, R. Li2, B.D. Slaughter2, J. Unruh2;
1
Rong Li lab, Stowers Institute for Medical Research, Kansas City, MO, 2Stowers Institute for Medical
Research, Kansas City, MO
Formation and propagating of protein aggregates cause many aging-related and amyloid diseases. In
yeast, the process of replicative aging is naturally associated with accumulation of damaged proteins
aggregates that are distributed asymmetrically during cell division. In a crowed cytosolic environment
with many organelles, aggregate formation can be a consequence of non-native protein structures being
introduced into these well-organized intracellular compartments. How do membrane-based organelles
contribute to and coordinate the formation and distribution of protein aggregates is not well
understood. In this study, we investigated the role of organelles in the formation, dissolution and
distribution of protein aggregates. We obtained evidence that cytosolic unfolded proteins do not
aggregate spontaneously; instead, active translation and nascent polypeptides are required to initiate
protein aggregation on the surface of ER, an organelle harboring a majority of translation sites. In
addition to ER, mitochondria play important roles in the dynamics and asymmetric segregation of
aggregates during mitosis.
SUNDAY-ORAL PRESENTATIONS
Minisymposium 4: Endocytic Trafficking
M30
Chemical labelling of primary endocytic vesicles quantifes flux through clathrin
coated pits, caveolae, and alternate endocytic pathways.
E. Shvets1, V. Bitsikas1, I. Correa2, G. Howard1, C. Hansen3, B. Nichols1;
1
MRC-LMB, Cambridge, England, 2New England Biolabs, Ipswich, MA, 3UCSD, La Jolla, CA
Several different endocytic pathways have been proposed to function in mammalian cells. Clathrincoated pits are well characterised, but the identity, mechanism and function of alternative pathways
have been less clear.
We applied chemical labelling of plasma membrane proteins to simultaneously define the origin and
cargo composition of all primary endocytic vesicles. This was complemented by labelling of specific
proteins with a reducible SNAP-tag substrate. These approaches provide high temporal resolution and
stringent discrimination between surface-connected and intracellular membranes. Our data generate a
complete and quantitative inventory of the different types of endocytic vesicle acting in unperturbed
mammalian cells. Experiments employing mutated clathrin adaptors and loss of adaptor function allow
analysis of the cargo load of endocytic vesicles when sorting of high affinity cargoes into clathrin coated
pits is perturbed. These experiments reveal remarkably different effects on different classes of
endocytic cargo.
Around 2% of endocytic vesicles arise from budding of caveolae from the plasma membrane, and these
vesicles deliver cargo to endosomal compartments. Caveolae have a unique role in endocytosis, as both
light and electron microscopy show that the caveolar bulb dynamically sorts plasma membrane
components. The caveolar bulb acts as a molecular device that presents a different paradigm for sorting
to the clathrin coated pit. These data provide new insight into the activity and diversity of endocytic
pathways in mammalian cells.
M31
KIF13B enhances the endocytosis of LRP1 by recruiting LRP1 to caveolae.
Y. Kanai1, D. Wang1, N. Hirokawa1;
1
Cell Biology and Anatomy, Univ Tokyo Grad Sch Med, Tokyo, Japan
Multifunctional low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) recognizes and
internalizes a large number of diverse ligands, including LDL and factor VIII. However, little is known
about the regulation of LRP1 endocytosis. Here, we show that a microtubule-based motor protein,
KIF13B, in an unexpected and unconventional function, enhances caveolin-dependent endocytosis of
LRP1. KIF13B was highly expressed in the liver and was localized on the sinusoidal plasma membrane of
hepatocytes. KIF13B KO mice showed elevated levels of serum cholesterol and factor VIII, and KO MEFs
showed decreased uptake of LDL. Exogenous KIF13B, initially localized on the plasma membrane with
caveolae, was translocated to the vesicles in the cytoplasm with LRP1 and caveolin-1. KIF13B bound to
hDLG1 and utrophin, which, in turn, bound to LRP1 and caveolae, respectively. These linkages were
SUNDAY-ORAL PRESENTATIONS
required for the KIF13B-enhanced endocytosis of LRP1. Thus, we propose that KIF13B, working as a
scaffold, recruits LRP1 to caveolae via LRP1-hDLG1-KIF13B-utrophin-caveolae linkage and enhances the
endocytosis of LRP1.
M32
Building endocytic pits for clathrin-independent uptake into cells.
L. Johannes1;
1
Chemical Biology of Membranes and Therapeutic Delivery unit, INSERM U1143-CNRS UMR 3666,
Institut Curie, Centre de Recherche, Paris cedex 05, France
Several endocytic processes do not require the activity of clathrin, and it has been a major question in
membrane biology to know how the plasma membrane is bent and cargo proteins are recruited in these
cases. Based on studies on Shiga and cholera toxins, polyoma viruses, and cellular galectins, we have
formulated the hypothesis that sugar-binding subunits of these cellular and pathogenic factors interact
with glycosphingolipids in a way such as to endow lectin-glycosphingolipid complexes with curvature
active properties that induce the clathrin-independent formation of endocytic pits. The signal that is
thereby sent to the cytosol is a mechanical one: the formation of a highly curved membrane domain.
We have now set out to address specific aspects of this hypothesis: mechanisms that lead to the
generation of spontaneous curvature and to the efficient clustering of lectin-glycosphingolipid
assemblies; recognition by cellular factors of the highly curved membrane domain for its further
processing into cells by scission and targeting to intracellular compartments; link between cellular
uptake via clathrin-independent carriers and intracellular distribution pathways; nanodomain
construction at the plasma membrane for the generation of compositional distinct clathrin-independent
carriers. An update will be provided with a specific focus on recently completed but yet unpublished
studies.
M33
Release of lateral compression of polymerization-loaded ESCRT-III spiral springs
induces membrane curvature.
A. Roux1;
1
Biochemistry, Univ Geneva, Geneva, Switzerland
ESCRT-III was genetically and biochemically implicated in membrane deformation and scission of
membrane in many membrane remodeling events such as intra-lumenal vesicle formation in multivesicular bodies, abscission in cytokinesis and virion budding from the plasma membrane. However, the
mechanism by which it breaks and deforms the membrane are still under debate, as the molecular
structure of the proteins in the complex do not suggest a mechanism. Here we show that the yeast
ESCRT- III protein Snf7 when reconstituted onto supported bilayer forms circular patches that extend by
a growing front wave at their rim. The center of the patch is saturated with Snf7 that cannot polymerize
anymore. By using High Speed AFM and electron microscopy, we show that these patches are formed of
Snf7 spirals that grow into a tight hexagonal array. By using physical modeling, we predict the stop of
SUNDAY-ORAL PRESENTATIONS
the growth to be connected to lateral compression of the spirals. Indeed, when grown on free lipid
membrane such as giant unilamellar vesicles, Snf7 coat can provoke membrane deformation compatible
with a model where Snf7 spirals act as 2D spiral springs loaded by polymerization and lateral packing,
and which release can cause membrane budding.
M34
Quantitative 4D visualization of clathrin-coated vesicle formation in mammalian
cells by correlated light and electron microscopy.
O. Avinoam1, M. Schorb1, J. Briggs1, M. Kaksonen2;
1
The European Molecular Biology Laboratory (EMBL), Heidelberg, Germany, 2European Molecular
Biology Laboratory (EMBL), Heidelberg, Germany
Clathrin-mediated endocytosis (CME) is a conserved form of selective plasma membrane internalization
in eukaryotic cells that plays a fundamental role in numerous physiological and pathological processes.
Many components of the multi-protein machinery driving endocytosis have been identified in mammals.
However, a comprehensive view of membrane shape transitions during coated vesicle formation is still
missing. Clathrin coated vesicle (CCV) budding may occur either through increase in curvature of a preassembled coat, or through coat growth, with the curvature of the emerging bud remaining constant.
These two models predict different membrane intermediates. Furthermore, for a coated membrane
area to change in curvature, the overlying coat must constantly restructure. To distinguish between
these models we visualized clathrin-coated pits (CCPs) by correlating fluorescence microscopy and
electron tomography in genome-edited mammalian cells expressing fluorescently tagged clathrin and
dynamin at physiological levels. We correlated clathrin fluorescence to endocytic sites ranging from flat
to highly curved membrane patches and to coated vesicles. We focused on productive endocytic events
by analyzing sites on the non-adherent surface of the plasma membrane that were able to recruit cargo.
We quantified the curvature and coated surface area of over 200 CCPs and CCVs and found that the
curvature of the membrane at the tip of the invagination increases during endocytosis while the area of
coated membrane remains constant. These results imply that endocytosis occurs predominantly by
inward deformation of flat clathrin lattices. In addition, the population labeled by both clathrin and
dynamin consisted exclusively of deep invaginations that showed membrane constriction at their base,
consistent with the known role of dynamin in membrane scission at late stages of CME. To test whether
the clathrin coat is remodeled during CME we preformed dual color FRAP analysis of single endocytic
sites containing clathrin-RFP and dynamin-GFP. Our preliminary results show that clathrin fluorescence
largely recovers even in the presence of dynamin suggesting that clathrin is exchanged even at late
stages of CME. Taken together, these results suggest that the coat is a dynamic structure that reshapes
during endocytosis. Our quantitative 4D description of membrane shape transitions during CME in vivo
provides strong evidence for budding via continuous increase in membrane curvature of a constant precoated membrane area.
SUNDAY-ORAL PRESENTATIONS
M35
Epsin deficiency impairs endocytosis by stalling the actin-dependent
invagination of edocytic clathrin coated pits.
M. Messa1, R. Fernandez-Busnadiego2, E.W. Sun3, H. Chen4, H. Czapla1, K. Wrasman5, Y. Wu3,6,7, G. Ko3, T.
Ross8, B. Wendland5, P. De Camilli2;
1
Cell Biology, Yale University School of Medicine/HHMI, New Haven, CT, 2Department of Cell Biology,
Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair,
Yale University School of Medicine, New Haven, CT, 3Department of Cell Biology, Yale University, New
Haven, CT, 4Oklahoma Med Res Fund, Oklahoma City, OK, 5Department of Biology, Johns Hopkins
University, Baltimore, MD, 6department of cell biology, 1Howard Hughes Medical Institute, New Haven,
CT, 7CNNR program, Yale University, New Haven, CT, 8Division of Hematology-Oncology, University of
Texas Southwestern Medical Center, Dallas, TX
Epsin is an evolutionarily conserved endocytic clathrin adaptor, which is encoded by three different
genes (EPN1, EPN2 and EPN3) in mammals. Both housekeeping roles in clathrin-mediated endocytosis
and a specific role in the clathrin-dependent internalization of ubiquitinated cargos have been attributed
to epsin. However, its most critical function(s) in metazoan cells remain(s) elusive. To elucidate such
function(s), we generated embryonic fibroblasts from conditional epsin triple knock out (TKO) mice.
These cells displayed a dramatic increase in cell size and in the number and/or size of nuclei,
demonstrating a role of epsin in cell division, as previously reported for other endocytic factors including
clathrin. Additionally, a global defect in clathrin-mediated endocytosis with the accumulation of shallow
and U-shaped pits was observed in TKO cells, supporting a role of epsin in clathrin coat invagination.
These changes correlated with the accumulation of actin foci at the cell surface, typically in close
proximity of arrested coated pits. A similar actin defect was previously observed in Hip1R knockdown
cells. Accordingly, we found that Hip1R, a factor known to directly link the clathrin coat to actin, was no
longer recruited to endocytic clathrin coated pits both in intact cells and in a cell free system. We also
found that the ENTH domain of epsin binds Hip1R, while its unfolded “tail” binds actin directly. These
findings, which point to a role of epsin in providing a link between endocytic clathrin coats and actin
leading to deep invagination, reconcile observations on epsin function previously made in yeast and
dictyostelium, with the function of epsin in metazoan cells. Finally, we report here a low affinity
interaction, but potentiated by the presence of PI(4,5)P2, between the SNARE motif of
synaptobrevin2/VAMP2 and the ENTH domain of epsin. Such interaction, which is conserved from yeast
to mammals, may help couple clathrin coat nucleation to incorporation of a SNARE in the nascent bud.
Collectively, our results demonstrate a housekeeping role of epsin in clathrin-mediated endocytosis, in
addition to cargo specific functions as suggested by previous studies. A key function of this protein is to
help generate the force that leads to the invagination of clathrin coated pits as a premise to their fission.
SUNDAY-ORAL PRESENTATIONS
M36
FCHo2 regulates protein assembly and efficiency of clathrin-mediated
endocytosis.
E. Evergren1, H. McMahon1;
1
Medical Research Council Laboratory of Molecular Biology, Cambridge, England
Clathrin-mediated endocytosis is important for receptor uptake, synaptic vesicle recycling, virus
internalisation and tissue morphogenesis. Many of the proteins interactions involved in clathrin coated
vesicle formation are mediated by short linear interactions motifs (SLiMs). The presence of multiple
motifs within intrinsically disordered regions (IDRs) and the self-polymerization of clathrin in turn
provide an ability to build large complexes necessary for vesicle formation. This implies a dynamic
process involving the assembly but also exchange of interacting partners with a functional efficiency, as
assessed by uptake of ligands and receptors, based on the stoichiometry of components within the
network. Whilst this is an established principle underpinning signal transduction it is so far unexplored in
understanding clathrin coat formation. We hypothesize that endocytosis can be altered either by effects
on recruitment or on the stability of recruited complexes. In this study we focus on FCHo2 and
comprehensively define the motifs regulating its association with interacting proteins, discovering three
novel motifs which mediate the binding of clathrin, AP2, Eps15R, Eps15, epsin1, CALM and intersectin-1.
We then study the impact of disrupting these interactions on important subcomplexes en route to
functional endocytic uptake of transferrin. Disruption has two important phenotypic outcomes:
separation of protein subcomplexes from each other, or coalescing subcomplexes into larger complexes
that do not mature. Adaptor proteins AP2 and FCHo2 bind to overlapping motifs in Eps15 via weak
affinity interactions, and interactions between Eps15-Eps15R-FCHo2-AP2 function cooperatively to
strengthen the complex in network modules. By changing their concentration a significant impact was
found both in complex formation, colocalisation in cells, and the efficiency of ligand uptake. On the basis
of our findings, we propose a model where colocalisation and protein complex formation of high-avidity
low-affinity interactions between clathrin, FCHo2, AP2, Eps15 and Eps15R are fundamental to
establishing efficient maturation of clathrin-coated pits. These cooperative interactions are sensitive to
protein concentration. In cell-lines with different endogenous expression levels this was reflected in
efficiency of transferrin uptake. Our study highlights the importance of assessing the impact of targeting
a single endocytic adaptor protein on network organisation and protein complex formation rather than
a single endpoint phenotype. It is well known in cell signalling that construction of higher order
complexes is instrumental in building dynamic and yet robust processes. Here we show that similar
principles also apply to the efficient assembly of clathrin coats.
SUNDAY-ORAL PRESENTATIONS
Minisymposium 5: Nanoscale to Systems Level Analysis of Cells
Making Decisions
M37
Signaling reactions on membrane surfaces: the roles of space, force, and time.
J.T. Groves1,2;
1
Chemistry, Univ California-Berkeley/HHMI, Berkeley, CA, 2Mechanobiology Institute, Singapore,
Singapore
The cell membrane provides a dynamic and highly interactive surface environment on which signaling
processes take place. I will describe several physical experiments, performed both in reconstituted
systems an in living cells, that probe the role of the membrane in coordinating signaling reactions. An
emerging theme is that the membrane is not just the stage on which signaling reactions take place.
Instead, we find the membrane to be intimately involved in modulating the structure, assembly, and
functionality of proteins during the signaling process. Specific attention will be directed towards
proximal signaling events during T cell antigen recognition.
M38
Natural history of the T cell receptor from receptor in T cells to ligand in B cells.
M.L. Dustin1, D. Depoil1, K. Attridge1, K. Choudhuri2;
1
NDORMS, University Oxford/Kennedy Inst Rheumatol, Headington, England, 2Skirball Institute, New
York University School of Medicine, New York, NY
T cells are sensitive to single antigenic peptides embedded in major histocompatibility complex proteins
on an antigen presenting cell. We have visualized this process in the interface between live T cells and
supported lipid bilayers (SLB). We can demonstrate that single peptide-MHC trapping events
correspond to sites of TCR clustering, ZAP-70 recruitment and CD45 exclusion. However, detection of
these events requires averaging over dozens of engaged peptide-MHC. The single molecule triggering
process does not lead to extensive central clustering of TCR, which is increased in a linear manner by
increasing the density of peptide-MHC in the SLB. The central TCR clustering corresponds in part to the
budding of TCR enriched extracellular vesicles (EV) from an annular compartment near the center of this
immunological synapse (Choudhuri K, Llodra J, Roth EW, Tsai J, Gordo S, Wucherpfennig KW, Kam LC,
Stokes DL, Dustin ML. Polarized release of T-cell-receptor-enriched microvesicles at the immunological
synapse. Nature. 2014;507(7490):118-23.). EV formation terminates TCR signaling in the T cell, but allow
transfer of polyvalent TCR positive particles that can activate signaling in T cells. Further analysis of
microvesicles from human T cells revealed that they are tethered to the cell and are dragged by the cell
as the immunological synapse breaks symmetry and becomes a kinapse. We found that the unusual GPI
anchored type II transmembrane protein BST-2/tetherin is highly accumulated in the EV rich central
compartment of activated human T cells. BST-2/tetherin is known to tether retroviral particles to
surface of activated T cells, but is absent from quiescent T cells. However, quiescent human T cells still
have their EV tethered to the surface of the T cell. Furthermore, knockdown of BST-2/tetherin or
SUNDAY-ORAL PRESENTATIONS
cleavage of the GPI anchor failed to reduce EV tethering to migrating activated T cells. Thus, the linkage
of EV to migrating T cells is circumstantially associated with BST-2/tetherin, but other mechanisms of EV
tethering are sufficient in the absence or reduced expression of BST-2/tetherin. Nonetheless, the
accumulation of BST-2/tetherin in the center of the immunological synapse opens the possibility that T
cell receptor may have a secondary signaling role in T cell by contributing to activation of NFkappaB
down-stream of BST-2/tetherin aggregation by EV enriched in T cell receptor continue to engaged
peptide-MHC.
M39
Distinct nano-scale organization of paired receptors at Natural Killer cell surfaces
revealed by super-resolution microscopy.
A. Oszmiana1, D. Williamson1, S. Cordoba2, D. Davis1;
1
Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester,
United Kingdom, 2UCL Cancer Institute, University College London, London, United Kingdom
Human Natural Killer (NK) cells are regulated by a variety of germ-line encoded activating and inhibitory
receptors. Broadly, activating receptors detect ligands that are expressed or up-regulated on cancerous
or infected cells, while inhibitory receptors bind self-molecules to induce tolerance against healthy cells.
Highly homologous pairs of activating and inhibitory receptors are also expressed on NK cells, including
Killer Ig-like Receptors KIR2DL1 and KIR2DS1, which bind the same ligands, class I MHC proteins. With a
view to understanding signal integration between these paired receptors, we used two super-resolution
microscopy techniques - Ground State Depletion (GSD) and Stimulated Emission Depletion (STED)
microscopy - to determine how KIR2DL1 and KIR2DS1 are organized on a nanometre-scale. Firstly,
neither receptor was distributed randomly; both were observed to constitutively assemble in
nanometre-scale clusters. 39 ± 13% of KIR2DS1 molecules were found in clusters, distributed at 3.5 ± 0.7
clusters/µm2, while only 27 ± 9% of KIR2DL1 accumulated in clusters, at 8 ± 2 clusters/µm2. Importantly,
the activating receptor KIR2DS1 formed nano-clusters 2.4-fold larger (diameter 120 ± 13 nm) than its
inhibitory counterpart KIR2DL1. The density of KIR2DS1 and KIR2DL1 within clusters was 7.5 and 4.5 fold
greater than the average membrane density, respectively. Interestingly, clusters of KIR2DL1 and
KIR2DS1 were significantly segregated from each other (with a negative Pearson’s correlation
coefficient). Thus, these data establish that paired NK cell receptors have a distinct nano-metre scale
organisation at the cell surface which likely influences signalling; their segregation rules out models of
signal integration which requires paired receptors to be juxtaposed to each other.
SUNDAY-ORAL PRESENTATIONS
M40
Mechanisms and functional consequences of T cell microclustersMechanisms
and functional consequences of T cell microclusters.
X. Su1, J. Ditlev2, E. Hui3, J. Rumpf3, S. Banjade2, J. Taunton3, M.K. Rosen4,5, R. Vale3;
1
Department of Cell and Molecular Pharmacology, University of California, San Francisco, San Francisco,
CA, 2University of Texas Southwestern Medical Center, Dallas, TX, 3University of California, San
Francisco, San Francisco, CA, 4Department of Biophysics, University of Texas Southwestern Medical
Center, Dallas, TX, 5Howard Hughes Medical Institute, Dallas, TX
The assembly of higher-order protein structures has been suggested to promote signaling whereas a
clear cause-and-effect relationship has been difficult to establish, largely due to limited knowledge
about how these structures are formed. To investigate the mechanisms and functional properties of
membrane-bound signaling clusters, we developed an in vitro reconstitution system in which signaling
clusters are assembled from individual purified components on synthetic supported lipid bilayers. We
found multivalent protein-protein interactions can drive the cluster formation of LAT, a signaling hub
protein that recruits multiple downstream effectors for TCR signaling. LAT clusters enrich Sos1(RasGEF),
PLCg1(Phospholipase), and Nck1(connecting to actin polymerization). Clustering renders resistance to
dephosphorylation of LAT by protein tyrosine phosphatase, which suggests a mechanism of how
clustering might maintain TCR signaling. In addition, we found two adaptor proteins Grb2 and Gads are
redundant and competitive for clustering LAT. Finally, through reconstituting a signaling pathway from
TCR triggering to LAT clustering, and to actin polymerization, we showed LAT cluster nucleates actin
filaments and promotes membrane-proximal actin network formation. Together, we propose TCR
triggering induces a phase transition of LAT complex that promotes downstream signaling.
M41
Regulation of T cell receptor signaling to NF-κB by microtubule transport of
signalosomes.
M.K. Traver1, S. Paul2, W. Losert3, H. Shroff4, B.C. Schaefer1;
1
Microbiology and Immunology, Uniformed Services University, Bethesda, MD, 2Department of Internal
Medicine, University of Toledo Medical Center, Toledo, OH, 3Dept of Physics, University of Maryland,
College Park, MD, 4Section on High Resolution Optical Imaging, NIH/NIBIB, Bethesda, MD
T cell receptor (TCR) activation of the transcription factor NF-κB is a crucial determinant of T cell
proliferation and differentiation. Our recent work has demonstrated that a signalosome containing the
proteins p62, Bcl10, and Malt1 is of central importance in the TCR-to-NF-κB cascade. Intriguingly, this
signalosome is both the site of terminal activation of NF-κB and a site of modulation of activating signals
by selective autophagy of Bcl10. Here, using confocal and super-resolution microscopy, we demonstrate
that NF-κB-activating signalosomes are transported and aggregated over a period of 1 - 2 hr in a
microtubule dependent manner. Blockade of microtubule polymerization prevents coalescence of
signalosomes into large cytosolic aggregates, but not initial signalosome formation. We have
furthermore determined by several criteria that these microtubule-dependent signalosome clusters
SUNDAY-ORAL PRESENTATIONS
have the properties of aggresomes. Interestingly, the aggresomal clusters appear to continue signaling
to NF-κB, suggesting that they are not a depot of misfolded protein aggregates undergoing immediate
destruction. Rather, our data suggest that formation and maintenance of these aggresomal signalosome
clusters represents a cellular mechanism controlling the magnitude and/or kinetics of NF-κB activation
in effector T cells. More broadly, our data suggest that aggresomes may have a previously unrecognized
role in direct regulation of specific signaling cascades.
M42
Microfluidic and mass cytometric analyses of single human hematopoietic stem
cells demonstrate distinct proliferation and survival responses activated by
differentially signaling growth factors.
D.J. Knapp1, N. Aghaeepour2, P.H. Miller1, G.M. Rabu1, P.A. Beer1, M. Ricicova3, V. Lecault3, D. Da Costa3,
M. VanInsberghe3, J. Piret3, S.C. Bendall4, G.P. Nolan2, C. Hansen3, C.J. Eaves1;
1
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, 2Baxter Laboratory for Stem Cell
Biology Department of Microbiology and Immunology, Stanford University, Palo Alto, CA, 3University of
British Columbia, Vancouver, BC, 4Pathology, Stanford University, Palo Alto, CA
Objective: Understanding the molecular control of human hematopoietic stem cell (HSC) behavior is
critical for the rational design of next generation HSC expansion protocols and improved therapies for
leukemia. Here, we measured early growth factor (GF)-induced signaling events at the single-cell level in
>10% pure human HSC populations and compared the patterns obtained with corresponding survival
and proliferation responses.
Methods: HSCs were isolated from cord blood by FACS and quantified by limiting dilution analysis (LDA)
for their ability to consecutively repopulate primary and secondary NOD/SCID-IL2Rγc-/- (NSG) mice for
30 weeks each time, before and after culture of the input cells for 21 days in serum-free medium
containing 5 GFs (SCF, FLT3L, IL3, IL6, GCSF). HSC viability and proliferation was assessed by tracking
single CD34+38-45RA-90+49f+ cells in either multi-layer poly(dimethylsiloxane) microfluidic chips
imaged every 20-30 minutes, or 72-well Terasaki plates scored visually every day. Mass cytometric
analyses were performed simultaneously on multiple subsets of CD34+ cells fixed after 5-120 min of GF
exposure. Results were obtained for 43 parameters from analyses of 1.6 million cells (4 experiments, 13
surface markers, 18 active signaling marks, 6 transcription factors, 2 survival marks, 3 cell cycle markers,
and DNA content).
Results: LDA showed maintenance of input HSC numbers and quality in 21-day culture containing 5 GFs.
Single-cell cultures showed FLT3L, SCF, and IL3 each promoted HSC survival, although ≥2 GFs were
required for full rescue. Either SCF or IL3 were required for proliferation. Interestingly, FLT3L had no
mitogenic activity alone and did not provide any synergistic proliferative benefit. 5 GFs together rapidly
induced significant responses (within 15 min) in the HSC subset for most of the intracellular nodes
measured, although these had returned to ~input levels within 2 hrs despite continuing GF exposure.
Effects on strongly activated nodes (e.g. ERK1/2, CREB, AKT, STAT1/3/5) were tightly temporally
correlated, and weakly activated nodes (e.g. β-Catenin, MAPKAPK2, p38) less so. SCF and FLT3L primarily
SUNDAY-ORAL PRESENTATIONS
activated MAPK/AKT-related pathways but with variable differences in duration/strength. GCSF and IL6
primarily activated STAT3 and IL3 primarily activated STAT5.
Conclusions: Proliferation and survival of human HSCs are differentially supported by components of a 5
GF cocktail that maintains functional HSC numbers for 3 weeks in vitro. We find evidence of both
rheostat-like and independent switch mechanisms controlling HSC survival/proliferation suggesting
control of HSC biology can be significantly influenced by differential activation of multiple signaling
circuits.
M43
Cdc42 Activation is Necessary for Sustained Oscillations of Ca2+ and PIP2 in
RBL Mast Cells Stimulated by Antigen.
D.A. Holowka1, M.M. Wilkes1, B. Baird1;
1
Chemistry and Chemical Biology, Cornell University, Ithaca, NY
Antigen stimulation of mast cells via FcεRI, the high-affinity receptor for IgE, triggers a signaling cascade
that requires Ca2+ mobilization for exocytosis of secretory granules during the allergic response. To
characterize the role of Rho GTPases in FcεRI signaling, we utilized a mutant RBL cell line, B6A4C1, that is
deficient in antigen-stimulated Cdc42 activation important for these processes. B6A4C1 cells exhibit
severely attenuated Ca2+ oscillations in response to antigen, and these are restored to wild type RBL2H3 levels by expression of constitutively active Cdc42 G12V or by a GEF for Cdc42, DOCK7. Ca2+
oscillations are not restored when the C-terminal di-arginine motif of active Cdc42 is mutated to diglutamine, demonstrating a role for these basic residues in this Rho family function. Antigen-stimulated
FcεRI endocytosis, which occurs independently of stimulated Ca2+ influx, is also defective in B6A4C1
cells, and Cdc42 G12V reconstitutes this response as well. Thus, activation of Cdc42 occurs prior to and
is critical for antigen-stimulated pathways leading separately to both Ca2+ mobilization and receptor
endocytosis. Accounting for these downstream functional consequences, we show that Cdc42 G12V
reconstitutes antigen-stimulated oscillations of phosphatidylinositol 4,5-bisphosphate (PIP2) at the
plasma membrane in B6A4C1 cells, pointing to Cdc42 participation in the regulation of stimulated PIP2
synthesis as a key functional role for this Rho family GTPase in mast cells.
M44
Distinct Actin Filament Networks Modulate Different Cell Signalling Pathways
Involved in Cell Proliferation and Senescence.
G. Schevzov1, B. Wang1, J.W. Hook1, C. Tonthat1, I. Pleines2, B. Kile2, E.C. Hardeman1, P.W. Gunning1;
1
School of Medical Sciences, UNSW, Sydney, Australia, 2Water and Eliza Hall Institute, Melbourne VIC,
Australia
The spatial and temporal control of signalling pathways that regulate cellular processes, including
proliferation and senescence, is orchestrated by a complex network of interactions involving scaffolding
proteins and kinases. The actin cytoskeleton is known to play a critical role in integrating such signalling
pathways. Within a cell functionally diverse actin filament networks are present. Currently, the
SUNDAY-ORAL PRESENTATIONS
molecular mechanism(s) by which these actin networks participate in signalling pathways is not wellunderstood principally due to a lack of tools that discriminate between them. Tropomyosins (Tm), a
family of actin filament-associated proteins, can impart unique cellular functions to the actin
cytoskeleton. Our investigations using mouse embryonic fibroblasts (MEFs) isolated from Tm isoformspecific knockout (KO) and transgenic mice have revealed significant alterations in cell proliferation and
life span. Ablation of Tm5NM1 results in reduced cell proliferation in direct contrast to its
overexpression. This result is Tm5NM1-specific since ablation of Tm4 shows no impact on proliferation.
We show that Tm5NM1-mediated proliferation occurs via the MEK/ERK pathway. Furthermore we
demonstrate that in the absence of Tm5NM1 the formation of pERK and its nuclear shuttling protein
Importin 7 is reduced resulting in the observed impaired cell proliferation. In contrast, an in vitro life
span assay demonstrates that Tm4 KO MEFs can readily escape senescence unlike the Tm5NM1 KO cells.
The Tm4 KO MEFs show dysregulation of the PKC and JAK-STAT pathways. These studies provide the first
evidence that specific Tm-containing actin filaments can influence signalling pathways. Taken together
these findings allow us to begin to understand how signalling pathways are regulated within the cell in
space and time via different actin filament networks marked by specific Tm isoforms.
M45
A Novel Role for Wiskott - Aldrich syndrome Protein (WASP) in the Regulation of
Macrophage Nanotubes.
K. McCoy-Simandle1, D. Cox1,2;
1
Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, 2Albert Einstein College
of Medicine, Bronx, NY
Macrophages are known to interact with tumor cells, pathogens, lymphocytes endothelial cells, and
many other cell types via small molecule intermediaries. However, traditional soluble means of
communication do not account for all macrophage interactions suggesting other means of
communication. Recently discovered tunneling nanotubes (TNTs) are membranous channels that
connect different cells together. These connections are thin and may be up to several cell diameters
long. These structures occur in many immune cells in vitro and in vivo allowing for transfer of signals,
vesicles, and organelles. Even pathogens can use these TNTs to transfer from cell to cell. Due to their
length and the ability to form networks of TNTs, cell signals can be propagated over vast distances
quickly and without interaction with extracellular fluid. Because these structures have no known
markers and are sensitive to light exposure, shearing force, and chemical fixation, little is known about
the formation of these structures in macrophages. In this study, TNTs are characterized in macrophages
by in vitro live cell imaging which minimizes TNT breakage. Live visualization of the membrane was
achieved by either staining with the fluorescent membrane label, FM1-43FX, or by expression of a
fluorescent protein targeted to the plasma membrane using a CAAX motif. Interestingly, macrophages
polarized to either the pro-inflammatory or pro-tumorgenic states, using LPS or IL-4, show increased
number of TNTs. Formation of TNTs was also investigated when actin-polymerization, PI3 kinase or Src
kinase pathways were blocked. Moreover, it was determined that Wiskott-Aldrich syndrome protein
(WASP) regulates formation and stabilization of TNTs. Depletion of WASP by shRNA in a macrophage
cell line greatly reduces formation of these structures. Furthermore, while phosphorylation of WASP
SUNDAY-ORAL PRESENTATIONS
was minimally required for the formation of these structures, it was important for TNT stability. These
results reveal a novel role of WASP in macrophage TNT formation. Interestingly, they also suggest that
some of the immune deficiencies observed in patients with Wiskott-Aldrich syndrome may be due to the
decreased number of TNTs. More research will need to be done to find potential ways to manipulate
TNTs therapeutically.
Minisymposium 6: Reduce, Reuse, Recycle - The Many Strategies of
Microbial Pathogens
M46
Mechanism of protein incorporation into envelop of budding HIV particle.
P. Sengupta1, S. van Engelenburg2,3, M. Johnson4, J. Lippincott-Schwartz2,3;
1
Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and
Human Development, NIH, Bethesda, MD, 2Physiology Course at Marine Biological Laboratory, Woods
Hole, MA, 3Cell Biology and Metabolism Program, NICHD, NIH, Bethesda, MD, 4University of Missouri,
Columbia, MO
Enveloped retroviruses such as HIV incorporate a patch of the host cell plasma membrane, viz. the
envelope, as the outermost layer of the virus particle during the budding process. Selective enrichment
of a specific set of host cell proteins and viral glycoproteins within this envelope is critical for the virus to
enter the host cell and avoid the host immune system. Even though the distinct composition of viral
envelope has been demonstrated biochemically, the underlying physical mechanism of such selective
incorporation of proteins has remained elusive. The assembly of the retroviruses is initiated by the
oligomerization of the structural protein Gag at the plasma membrane. Such specialized composition
can arise either from assembly of viral Gag at specialized microdomains of the host cell plasma
membrane, or can be created by differentiation of the membrane by the protein platform generated by
the oligomerizing Gag. In order to identify which of these two mechanisms drive HIV envelope
formation, we used high resolution TIRF microscopy to visualize the temporal and spatial remodeling of
the host cell plasma membrane at single HIV viral assembly site. We find that the enrichment of host cell
proteins within the nascent viral envelop occurs only after completion of the oligomerization of Gag.
Furthermore, we find that proteins with affinity for ordered environment, such as GPI-anchored CD59,
are enriched in the viral envelop. In contrast, proteins that prefer disordered membrane are excluded
from the patch of membrane juxtaposed to the Gag platform. The curvature of the Gag platform also
seems to play a role in the differentiation of the membrane patch into the viral envelop, with peripheral
proteins with large cytoplasmic domains being actively excluded from the viral membrane patch. It is
energetically unfavorable for such proteins to fit in the concave curvature of luminal side of the viral
envelope. Taken together, these data indicate that the oligomerization of HIV Gag at the plasma
membrane creates a specialized microenvironment and provides the physical forces that drive the
differentiation of the local plasma membrane into viral envelop with distinct protein composition.
SUNDAY-ORAL PRESENTATIONS
M47
F-actin and Golgi rearrangement around the Chlamydia trachomatis inclusion is
mediated by the Arf-recruiting bacterial protein InaC.
M. Kokes1, R.H. Valdivia2;
1
Molecular Genetics and Microbiology, Cell and Molecular Biology, Duke University, Durham, NC,
2
Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC
Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen, and is the leading
cause of preventable blindness worldwide. As an obligate intracellular pathogen, Chlamydia resides
within a membrane-bound vacuole (inclusion) within host cells. A filamentous actin structure surrounds
the inclusion, and disruption of actin polymerization in host cells leads to increased exposure to
cytosolic innate immune receptors and increased expression of pro-inflammatory cytokines, suggesting
that F-actin maintains Chlamydia in a ‘hidden’ state within cells.
To identify Chlamydia factors involved in cytoskeletal cage assembly at inclusions, we screened an
arrayed library of chemically-mutagenized strains and identified one mutant lacking F-actin at inclusions.
Using genetic tools recently developed for Chlamydia, we show that a secreted bacterial protein
associated with the cytosolic surface of the inclusion membrane, renamed InaC, is necessary for actin
assembly at the inclusion. Surprisingly, InaC is dispensable for suppression of host cytokine production.
To further define roles of InaC in modifying the host intracellular environment, we used a combination
of immunopricipitation and mass spectroscopy to identify host proteins that interact with InaC. We
show that InaC interacts with and recruits host Arf GTPases – important regulators of trafficking and
organization at the Golgi – to the inclusion. Remarkably, during Chlamydia infection, the Golgi ribbon
fragments into ministacks which rearrange around the periphery of the inclusion. We find Golgi redistribution around the inclusion to be mediated by InaC in an F-actin-dependent manner. Sphingolipids
normally trafficked from the Golgi to the plasma membrane are intercepted by Chlamydia, and host
sphingolipids are incorporated into bacteria and the inclusion membrane. Golgi re-distribution around
the inclusion has previously been proposed to enhance bacterial acquisition of host sphingolipids.
However, we find that InaC-deficient Chlamydia lacking actin assembly at the inclusion acquire normal
levels of sphingolipids, suggesting an alternative role for InaC-mediated Golgi re-distribution around the
inclusion.
M48
Repurposing Flagellar Components in the Toxoplasma Conoid.
N.S. Morrissette1;
1
University California-Irvine, Irvine, CA
Apicomplexan parasites include the agents of human malaria, cryptosporidiosis and toxoplasmosis and
are also serious pathogens of agricultural livestock. These organisms have a complex life cycle during
which they are obligate parasites in a variety of intracellular niches. Although most apicomplexan
lineages have motile asexual stage forms, only male gametes form flagella to drive movement. Asexual
stage apicomplexans use apical complex organelles to invade host cells. A morphologically similar apical
SUNDAY-ORAL PRESENTATIONS
1.
2.
3.
4.
5.
complex is also found in related free-living alveolate lineages that feed by myzocytosis, a process that
involves suction of prey cytoplasm by the predator. A subset of apicomplexans and related alveolate
lineages construct a microtubule-containing conoid at the cell apex which is thought to aid in host cell
invasion or myzocytosis. Studies by others have demonstrated that dinoflagellate and chromerid
organisms that contain both apical flagella and a conoid link these adjacent structures through
filamentous connections.(1,2) We and others have established that the conoid of the apicomplexan
Toxoplasma gondii contains flagellar apparatus components.(3,4,5) As the conoid and flagellum are
mutually exclusive structures during the Toxoplasma life cycle, this suggests that these proteins were
retained to preserve important functions or repurposed to serve new roles in the apicomplexan conoid.
We are currently using mass spectroscopy of a purified sample to identify additional flagellar proteins
that also localize to the Toxoplasma conoid. We predict that these components are critically important
to essential processes including regulated secretion, replication and motility and were therefore
retained after loss of the flagella in asexual stage forms.
Portman, N. et al. 2014. Evidence of intraflagellar transport and apical complex formation in a free-living
relative of the apicomplexa. Eukaryotic Cell 13:10-20.
Okamoto, N. and Keeling, P. J. 2014. The 3D Structure of the Apical Complex and Association with the
Flagellar Apparatus Revealed by Serial TEM Tomography in Psammosa pacifica, a Distant Relative of the
Apicomplexa. PLoS ONE 9: e84653.
de Leon, J. C. et al. 2013. A SAS-6-like protein suggests that the Toxoplasma conoid complex evolved
from flagellar components. Eukaryotic Cell 12:1009-1019.
Francia, M. E. et al. 2012. Cell division in Apicomplexan parasites is organized by a homolog of the
striated rootlet fiber of algal flagella. PLoS biology 10:e1001444.
Katris, N. J. et al. 2014. The apical complex provides a regulated gateway for secretion of invasion factors
in Toxoplasma. PLoS pathogens 10:e1004074.
M49
Activation of Focal Adhesion Kinase by Salmonella Suppresses Autophagy via an
Akt/mTOR Signaling Pathway and Promotes Bacterial Survival in Macrophages.
K.A. Owen1, C.B. Meyer1, A.H. Bouton2, J. Casanova1;
1
Cell Biology, University of Virginia, Charlottesville, VA, 2Microbiology, University of Virginia,
Charlottesville, VA
Autophagy has emerged as an important antimicrobial host defense mechanism that not only
orchestrates the systemic immune response, but also functions in a cell autonomous manner to directly
eliminate invading pathogens. Pathogenic bacteria such as Salmonella have evolved adaptations to
protect themselves from autophagic elimination. Here we show that signaling through the non-receptor
tyrosine kinase focal adhesion kinase (FAK) is actively manipulated by the Salmonella SPI-2 system in
macrophages to promote intracellular survival. In wild-type macrophages, FAK is recruited to the surface
of the Salmonella-containing vacuole (SCV), leading to amplified signaling through the Akt-mTOR axis
and inhibition of the autophagic response. In FAK-deficient macrophages, Akt/mTOR signaling is
attenuated and autophagic capture of intracellular bacteria is enhanced, resulting in reduced bacterial
survival. We further demonstrate that enhanced autophagy in FAK-/- macrophages requires the activity
SUNDAY-ORAL PRESENTATIONS
of Atg5 and ULK1 in a process that is distinct from LC3-assisted phagocytosis (LAP). In vivo, selective
knockout of FAK in macrophages resulted in more rapid clearance of bacteria from tissues after oral
infection with S. typhimurium. Clearance was correlated with reduced infiltration of inflammatory cell
types into infected tissues and reduced tissue damage. Together, these data demonstrate that FAK is
specifically targeted by S. typhimurium as a novel means of suppressing autophagy in macrophages,
thereby enhancing their intracellular survival.
M50
A molecular mechanism for endosomal avoidance by the pathogen Legionella
pneumophila.
A. Gaspar1, M. Lucas2, A. Rojas2, A. Hierro2, M. Machner1;
1
NIH, Bethesda, MD, 2CIC bioGUNE, Bilbao, Spain
Endosomal maturation is an innate immune mechanism that targets invading microbes for degradation
thus protecting human cells from infection by pathogens. Legionella pneumophila, the causative agent
of Legionnaires’ pneumonia, efficiently bypasses the microbicidal endosomal compartment by an
unknown mechanism. We now discovered that the effector protein VipD, once injected by L.
pneumophila into the infected cell, specifically localizes to early endosomes and alters their protein and
lipid composition, thus rendering them fusion-incompetent. Remarkably, the activity of VipD is strictly
dependent on the presence of active Rab5, a key regulator of endosomes, explaining why other host cell
organelles that lack Rab5 are protected from the damaging effects of VipD so that they can now support
intracellular L. pneumophila replication.
M51
AMPylation of Rho GTPases disrupts multiple host signaling processes.
A.R. Woolery1, X. Yu2, J. LaBaer2, K. Orth1;
1
Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, 2Biodesign Institure,
Arizona State University, Tempe, AZ
Rho GTPases are frequent targets of virulence factors as they are keystone signaling molecules in many
processes. Modifications of their switch-1 loop are known to be a potent method of disabling the actin
cytoskeletal machinery, but Rho GTPases are also important for many other signaling pathways. We
attempted to broaden the understanding of the consequences of switch-1 loop modification in
downstream signaling using AMPylation by the Vibrio parahaemolyticus type three secreted effector
VopS. Using infection models, biochemistry and versatile self-assembled microarrays, several novel
effects of Rho GTPase AMPylation were identified. We found that multiple signaling interactions of
GTPases were inhibited, including NFkB, MAP kinase, the Inhibitor of Apoptosis proteins (IAP) and the
phagocytic NADPH oxidase system (NOX2.) Phosphorylation of IkB**a **and JNK kinase was inhibited
in a VopS-dependent manner during infection with Vibrio parahaemolyticus. VopS also sequestered p65
in the cytosol during infection, while removal of VopS allowed robust translocation. AMPylation also
prevented the generation of superoxide by the phagocytic NADPH oxidase complex. Furthermore, the
SUNDAY-ORAL PRESENTATIONS
interaction of GTPases with the E3 ubiquitin ligases cIAP1 and XIAP was hindered, leading to decreased
degradation of Rac and RhoA during infection. Finally, we screened for novel Rac1 interactions using a
nucleic acid programmable protein array (NAPPA) and discovered that Rac1 binds to the protein C1QA, a
protein known to promote immune signaling in the cytosol. Interestingly, this interaction was disrupted
by AMPylation. In addition to these findings, we also adapted NAPPA to use fluorescent probes to
screen for novel substrates of protein transferases, including AMPylation. We conclude that
modification of the switch-1 loop by VopS and other toxic proteins is a multifaceted virulence
mechanism that counters several host immunity strategies.
M52
Deciphering the molecular mechanisms of Listeria transcytosis across the
intestinal epithelium in intestinal organoids.
C. Fevre1,2, N. Barilone1,2, M. Lecuit1,2;
1
Institut Pasteur, Paris, France, 2Inserm, Paris, France
The human intestinal mucosa is a physical barrier that delineates the frontier between the external
environment and the host. It can be crossed by luminal antigens and enteroinvasive microbes. Listeria
monocytogenes (Lm), a Gram-positive foodborne bacterial pathogen responsible for human listeriosis,
crosses the intestinal barrier across goblet cells to access the lamina propria and disseminate
systemically. This translocation requires the interaction between Lm surface protein InlA and its speciesspecific epithelial receptor E-cadherin, but is independent of LLO (involved in Lm escape from the
vacuole) and ActA (involved in Lm intracellular motility), suggesting that Lm is transferred across goblet
cells by transcytosis. Beside E-cadherin and microtubules, the host factors involved in Lm transcytosis
across goblet cells are unknown. Lm transcytosis is not observed in in vitro cultured epithelial cells,
which are either too polarized to express apically accessible E-cadherin, or unpolarized and devoid of
bona fide apical basal vesicular trafficking. To identify the host factors involved in Lm transcytosis across
goblet cells, we have used intestinal organoids (miniguts), which recapitulate faithfully ex vivo intestinal
epithelium differentiation into intestinal epithelial cell subtypes, including goblet cells, and which are
genetically amenable. Using high-resolution confocal microscopy to follow the fate of microinjected Lm
inside minigut lumen, we have shown that, similar to what is observed in vivo, Lm is translocated
through goblet cells, in a microtubule- and InlA-dependent manner but InlB- LLO- and ActA-independent
manner, validating the intestinal organoid as a suitable model to identify the host factors involved in this
process. Our working hypothesis is that Lm takes advantage of E-cadherin recycling from the apical to
basolateral pole of enterocytes. In such a scenario, inhibition of E-cadherin recycling would interfere
with Lm transcytosis. To test this hypothesis, inducible organoid mutants expressing inactive forms of a
series of proteins involved in E-cadherin recycling, such as Rab GTPases and exocyst components have
been generated. Our preliminary results show that their inhibition prevents Lm transcytosis, and
experiments are now under way to fully decipher the molecular mechanisms of Lm transcytosis across
the intestinal epithelium. Intestinal organoids stand as a very promising model for the spatial and
temporal studies of the cell biology of host-pathogen interactions, as it preserves cell differentiation,
polarization and tissue architecture, which are exploited by pathogens to invade the host.
SUNDAY-ORAL PRESENTATIONS
Minisymposium 7: Synergy between Biophysical and Biochemical
Cues in Stem Cell Function
M53
Synergy of biochemical and biophysical cues rejuvenates the aged stem cell
population which restores strength to aged muscles.
H.M. Blau1, B. Cosgrove2, P. Gilbert3;
1
Baxter Laboratory, Microbiology and Immunology Department, Stanford University, Stanford, CA,
2
Department of Biomedical Engineering, Cornell University, Ithaca, NY, 3Institute of Biomaterials and
Biomedical Engineering, University of Toronto, Toronto, ON
The aged suffer from progressive muscle weakness and regenerative failure. We demonstrate that
muscle regeneration is impaired with aging due in part to a cell-autonomous functional decline in
skeletal muscle stem cells (MuSCs). Two-thirds of aged MuSCs are intrinsically defective relative to
young MuSCs, with reduced capacity to repair myofibers and repopulate the stem cell reservoir in vivo
following transplantation due to a higher incidence of cells that express senescence markers and that
have elevated p38α/β MAPK activity. We show that these limitations cannot be overcome by
transplantation into the microenvironment of young recipient muscles. In contract, subjecting the aged
MuSC population to transient inhibition of p38α/β in conjunction with culture on soft hydrogel
substrates rapidly expands the residual functional aged MuSC population, rejuvenating its potential for
regeneration, serial transplantation, and strengthening damaged muscles of aged mice. These findings
reveal a synergy between biophysical and biochemical cues that provides a paradigm for a localized
autologous muscle stem cell therapy in aged individuals.
M54
Muscle extracellular matrix composition modulates the differentiation/selfrenewal balance in skeletal muscle progenitor cells.
K. Thomas1, A.J. Engler2;
1
Biomedical Science, University of California, San Diego, La Jolla, CA, 2Bioengineering, University of
California, San Diego, La Jolla, CA
Duchenne Muscular Dystrophy (DMD) leads to sarcolemmal instability and skeletal muscle
deterioration. While hyperactivation of CD45+NCAM+ skeletal muscle progenitors (SMPs) to repair
degeneration depletes them at an elevated rate, exogenous SMP supplementation can reverse muscle
deterioration. SMPs are limited in both number and expansion capabilities, but they can temporarily
retain their ability to proliferate in vitro and engraft in vivo when cultured in niche of physiological (~11
kiloPascal, kPa). However, a more complete niche including growth factors, tissue-matched substrate
stiffness, and muscle-mimetic extracellular matrix (ECM) proteins may maintain the SMP phenotype. To
establish which matrix components comprise normal and DMD muscle so that they can be mimicked in
vitro, we compared ECM composition using liquid chromatography coupled with tandem mass
SUNDAY-ORAL PRESENTATIONS
spectroscopy (LC-MS/MS). DMD muscle up-regulated collagens I, II, III and VI and proteins involved in
collagen fibrillogenesis (eg, decorin and mimecan), whereas basement membrane proteins, including
laminins, nidogen, perlecan, and collagen type IV, were expressed more in normal muscle.
Polyacrylamide (PA) gels of physiological (11 kPa) or DMD stiffness (34 kPa) were created with ECM
protein combinations mimicking either healthy or DMD ECM to create permissive or non-permissive
environments, respectively. C2C12 mouse myoblasts and freshly isolated human SMPs were then
culture-expanded and subsequently differentiated in myogenic medium (5% horse serum, 10 µg/ml
insulin) to assess the ability of these niches to promote expansion without phenotype loss. Experiments
suggest that population-doubling times of human SMPs and C2C12s remain consistent in all niches, but
surprisingly differentiation into myosin heavy chain (MHC)-positive multinucleated myotubes remains
possible at later passages for SMPs. These data are consistent with C2C12s, which on single ECM protein
coatings and permissive stiffness (11 kPa) progressively downregulate Myf5 as myogenic differentiation
occurs. Together these data suggest that ECM composition in conjunction with growth factors and ECM
stiffness can expand SMPs without limiting their self-renewal and differentiation capacity, thus making it
possible to use them in therapeutic applications for muscle.
M55
Uncovering Cellular and Signaling Mechanisms of Skin Regeneration using Twophoton Microscopy.
V. Greco1;
1
Genetic Department and Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT
My laboratory’s goal is to understand how stem cells and their environment, also called niche,
contribute to organ regeneration, and how dysregulation of growth signals leads to cancer. The major
challenge in studying these questions has been the inability to follow the same cells in vivo and to
understand how their interactions with neighboring cells contribute to tissue growth. To overcome this
challenge, my lab has established the ability to visualize and manipulate stem cells and their
environment in an intact mammal using two-photon microscopy and laser ablation. Using the hair
follicle as a stem cell model system, we have thus far 1) elucidated the cellular behaviors driving hair
regeneration, including novel migratory epithelial behaviors which exemplify the power of live imaging
over static analysis. 2) Demonstrated that the niche is required for tissue regeneration whereas stem
cells are dispensable. These unexpected findings indicate that an intact niche provides a robust
compensatory mechanism, whereby other cell types can adopt specialized stem cell function to drive
tissue regeneration. 3) Shown that stem cells are not all equal in fate and their fate decision depends on
the position they inhabit in their environment. This discovery revealed the fundamental importance of
the environment in stem cell fate and overall tissue regeneration. 4) Demonstrated that an
evolutionarily conserved pathway, Wnt/ β-catenin, promotes growth non cell-autonomously. These
findings provide a novel understanding of β-catenin-mediated tissue growth and it identifies a novel
signaling mechanism by which cells can co-opt their neighbors to participate in tissue growth. 5)
Identified mechanisms of skin tumor regression via activation of Retinoic Acid (RA) by utilizing a unique
skin tumor capable of self-regressing, called Keratoacanthoma. Importantly, we show that applied RA
can also induce regression of the malignant Squamous Cell Carcinoma. Taken together, by visualizing
SUNDAY-ORAL PRESENTATIONS
stem cells in vivo and identifying signaling pathways that regulate normal skin and skin cancer growth,
we have made key contributions to our understanding of organ growth and cancer regulation. Our work
has provided important insights into tissue regeneration, which relies upon the coordinated activation
of resident stem cells and their environment, and into key signaling pathways controlling dynamic stem
cell behaviors and decisions.
M56
Requirement for fibronectin matrix assembly in the chondrogenic differentiation
of mesenchymal stem cells.
J. Schwarzbauer1, P. Singh1, L. Steirer1, M. Ramos1;
1
Dept. of Molecular Biology, Princeton University, Princeton, NJ
Mesenchymal cell condensation is the initiating event in formation of the vertebrate skeleton. Cell
condensation is followed by induction of chondrogenic gene expression and cell differentiation into
chondrocytes that form a cartilage template for bone development. Gene mutations that perturb
chondrogenesis cause chondrodysplasias and other skeletal defects; many of these mutations affect
extracellular matrix (ECM) protein structure or function. Using mesenchymal stem cells (MSCs) in an in
vitro chondrogenesis assay, we found that knockdown of diastrophic dysplasia sulfate transporter
(DTDST), which is required for normal cartilage development, blocked cell condensation. DTDST imports
sulfate for modification of glycosaminoglycan chains on proteoglycans. Inhibition of glycosaminoglycan
attachment to proteoglycans with β-xylosidase also prevented cell condensation. MSCs initiate assembly
of a fibrillar fibronectin matrix prior to cell condensation and the density of the matrix increases during
and after condensation. We previously showed that DTDST-dependent sulfation of glycosaminoglycan
chains on cell surface proteoglycans is an essential step in fibronectin matrix assembly. Roles for
glycosaminoglycan sulfation and fibronectin matrix in condensation were indicated by a significant
reduction in both matrix and condensation with DTDST knockdown. Two approaches were used to
directly target fibronectin matrix assembly during condensation: knockdown of fibronectin with siRNAs,
and inhibition of fibronectin matrix assembly by a fibronectin-binding bacterial peptide (FUD). Both
treatments blocked condensation. FUD inhibition of fibronectin matrix assembly also affected type I
procollagen processing and incorporation of type I collagen into the matrix. Our data show that cell
condensation and chondrogenic differentiation by MSCs depend on fibronectin matrix assembly and
suggest the possibility that certain skeletal defects in DTD patients might derive from deficiencies in
fibronectin matrix. These findings further suggest that the matrix assembly process might be an
important factor in directing stem cell differentiation in other tissues.
SUNDAY-ORAL PRESENTATIONS
M57
Nitric oxide plays a role in transmitting biochemical signal from the extracellular
matrix for mammary epithelial morphogenesis.
S. Furuta1, M.J. Bissell1;
1
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA
Nitric oxide (NO) is a short-lived, gaseous signaling molecule produced in response to biochemical or
mechanical stress in diverse tissues and organisms. Despite its evolutionary conservation and ubiquitous
expression, the bioactivities of NO have been primarily studied in specialized tissues, including
mammalian neurons, muscles, endothelial or immune cells, where NO triggers cell membrane
hyperpolarization and relaxation or exerts cytotoxic action. In the past decade, however, different
functions of NO in other tissues and organisms have been gradually unraveled, including embryogenesis
and morphogenesis of Xenopus, pond snail and Drosophila. Nevertheless, whether NO is involved in the
establishment of a mammalian tissue is yet to be determined. We found that non-malignant mammary
epithelial cells (MECs) produced NO in response to the extracellular matrix (ECM) proteins laminin-5 and
-1 and that NO production was essential for their ability to form growth-arrested polarized acini (the
functional unit of mammary gland) in three-dimensional (3D) laminin-rich (lr) ECM cultures. While this
mechanism was abrogated in breast cancer cells that failed to form acini in 3D cultures, induced NO
production by the use of an NO donor allowed them to restore the formation of normal-like polarized
colonies. We further found that such an essential role of NO in mammary acinar formation was
attributed to its ability to trigger cell membrane hyperpolarization and hence enhance tight junctions
through co-localization of E-cadherin and cortical actin. These observations, for the first time, uncover
that NO is produced by MECs in response to a biochemical stimulus of the ECM and plays a critical role
in mammary acinar morphogenesis, whereas the lack of NO production is relevant to the malignant
phenotype of breast cancer cells.
M58
Tissue surface mechanics drive mesenchymal-to-epithelial transition in
embryonic cell aggregates.
H.Y. Kim1, L.A. Davidson1,2;
1
Bioengineering, University of Pittsburgh, Pittsburgh, PA, 2Developmental Biology, and Computational
and Systems Biology, University of Pittsburgh, Pittsburgh, PA
Cells change their morphology and function as they adapt to new environments during tissue renewal,
cancer progression, and embryonic development. Cells may undergo an epithelial-to-mesenchymal
transition (EMT) from stably connected epithelial tissues that scatter as migratory cells into surrounding
tissue; conversely, migratory mesenchymal cells can undergo a mesenchymal-to-epithelial transition
(MET) as they aggregate and build tightly connected epithelial tissues. METs play a critical role in
reprograming stem cells, assembling primary epithelium in mouse embryos, constructing basic kidney
and somite units, and forming secondary tumors, however, little is known about what environmental
cues that induce MET or the intracellular pathways that regulate MET.
SUNDAY-ORAL PRESENTATIONS
Here we describe a case of MET that emerges within embryonic mesenchymal cell aggregates isolated
from early Xenopus embryos. Mesenchymal cell aggregates have pluripotent potential analogous to
embryonic stem cells. MET occurs at the surface of the tissue over a few hours as patches of cells
develop epithelial-like boundaries and localize epithelial markers apically (aPKC) and at cell-cell junctions
(ZO-1) as they lower expression and assembly of fibronectin extracellular matrix. Within 24 hours of
aggregation, MET-generated epithelial cells cover the entire aggregates even as mesenchymal cells
remain deeper within the tissue. Live-cell confocal imaging of MET with GFP-ZO1 show MET occurs
stochastically in small patches which then connect and expand to cover the large surface of the
aggregate over time.
To investigate the role of surface mechanics we measured tissue mechanical properties before, during,
and after MET using micro-aspiration, and then perturbed cell contractility and cell-cell adhesion to test
their roles in establishing the tissue surface mechanical conditions needed for MET. Inhibiting cell
contractility (Y27632, Blebbistatin) or decreasing cell-cell adhesion (ΔE-C-cadherin) abolished the MET
within embryonic aggregates. Moreover, increasing cell contractility with either Calyculin A or the
constitutively active RhoGEF-H1 significantly accelerated progressive METs. By controlling tissue
mechanics that drive MET we have demonstrated a novel physical mechanism in regulating cell
differentiation critical to tissue renewal, embryonic development, and human disease.
M59
A force balance explains local and global cell movements during early zebrafish
development.
J. Chai1, A.L. Hamilton2, C.D. Buckley1, M. Krieg2, I.H. Riedel-Kruse3, A.R. Dunn1;
1
Chemical Engineering, Stanford University, Stanford, CA, 2Molecular and Cellular Physiology, Stanford
University, Stanford, CA, 3Bioengineering, Stanford University, Stanford, CA
Biological tissues undergo radical changes in shape during development, growth, and repair. Little is
known about how 100-1000s of cells generate these intricately choreographed transformations. Here
we examine the origins of collective cell movement during early zebrafish development, a powerful
model system for addressing this topic. The first morphogenetic movement in zebrafish is epiboly, in
which the blastoderm spreads uniformly across the yolk and converges at the vegetal pole. In this study,
we show that tension within the actin band, a dense mesh of actin and myosin that assembles near the
blastoderm margin, coordinates blastoderm migration on both the organismal and cellular length scales.
We find that gentle mechanical deformation of the developing embryo causes shape-dependent
alterations in blastoderm migration. In addition, we observe realignment of the anterior-posterior (AP)
axis away from the animal-vegetal axis toward the new long axis of the embryo. Chemical disruption of
the actin band restores uniform blastoderm migration, eliminates AP axis reorientation, and increases
cellular disorder at the blastoderm margin. An analytical model and resulting simulation based upon
forces generated by the actin band recapitulates the experimental observations. Taken together, our
findings suggest that a relatively simple physical mechanism, in this case tension generated by the actin
band, can lead to long-range coordination of cell movements that would be difficult to achieve by
biochemical signaling alone. Further, we show that AP axis specification, a fundamental step in the
SUNDAY-ORAL PRESENTATIONS
development of all embryos, is exquisitely sensitive to mechanical cues. These observations support the
possibility that mechanical forces may play a ubiquitous role in
M60
Biophysical Properties of the Cellular Microenvironment Influence Hepatocyte
Differentiation.
J.C. Tung1, A.A. Grimm2, H. Willenbring3, V.M. Weaver4;
1
Department of Surgery and Center for Bioengineering and Tissue Regeneration, University of California,
San Francisco, San Francisco, CA, 2Department of Pediatrics and Eli and Edythe Broad Center of
Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco,
CA, 3Department of Surgery and Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell
Research, University of California, San Francisco, San Francisco, CA, 4Center for Tissue Engineering and
Regenerative Medicine, University of California, San Francisco, San Francisco, CA
Tissue-engineered liver constructs possess the potential to not only serve as cell-based therapeutics for
treating liver disease, but they may also serve as model systems for drug toxicity analysis or the study of
disease progression. Unfortunately, the utility of these liver constructs is limited, as current protocols
for the differentiation of hepatocytes result in immature hepatocytes that fail to replicate the
physiologic function of fully-differentiated adult hepatocytes. Furthermore, current in vitro culture
methods fail to maintain hepatocyte function over extended periods of time. One possible means for
addressing these issues is through manipulation of the biophysical microenvironment, which has been
shown to play a significant role in the differentiation and functionality of numerous cell types. While
previous studies have varied extracellular matrix coatings and substrate elastic modulus in order to
optimize hepatocyte differentiation, little is known about the mechanism by which biophysical
properties influence hepatic cell fate decisions. An understanding of this mechanism is key before the
biophysical microenvironment can be properly engineered to generate functional liver constructs. In this
study, we characterized changes in the biophysical microenvironment during the course of hepatocyte
differentiation and began to identify a mechanism by which these properties influence hepatic fate
determination. We used a multi-stage protocol for the differentiation of embryonic stem cell-derived
hepatocytes during which cells were directed from a definitive endoderm stage, to specified hepatic
lineage, to hepatoblast formation, to a final hepatocyte-like maturation state. Flow cytometry analysis
demonstrated differentiation stage-specific integrin expression profiles during hepatic differentiation. In
turn, these lineage-specific integrin expression profiles drove specific cell adhesion behaviors (cell
attachment, cell spreading, actin organization, focal adhesion formation) in response to changes in
substrate stiffness and extracellular matrix composition. Furthermore, changes in the biophysical
microenvironment significantly influenced the process of hepatocyte differentiation. These findings
demonstrate that biophysical properties, such as substrate elastic modulus and extracellular matrix
composition, play a vital role in hepatocyte differentiation. Additional studies are now underway to
further identify the mechanism by which biophysical properties influence hepatic differentiation and to
harness this insight in order to generate physiologically relevant tissue-engineered liver constructs for
cell-based therapeutics, drug toxicity assays, and models of liver disease.
SUNDAY-ORAL PRESENTATIONS
M61
De novo formation of insulin-producing “neo-ß-cell islets” from intestinal crypts.
Y. Chen1, S. Finkbeiner2, C. Li3, J. Spence4,5, B. Stanger1;
1
Department of Medicine, University of Pennsylvania, Philadelphia, PA, 2Internal Medicine, University of
Michigan, Ann Arbor, MI, 3Pediatrics, University of Pennsylvania, Philadelphia, PA, 4Department of
Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, 5Department of Cell and
Developmental Biology, University of Michigan Medical School, Ann Arbor, MI
The ability to interconvert terminally differentiated cells could serve as a powerful tool for cell-based
treatment of degenerative diseases, including diabetes mellitus. To determine if any adult tissues are
competent to activate an islet β-cell program, we performed an in vivo screen by expressing three β-cell
“reprogramming factors” in a wide spectrum of tissues. We report that transient intestinal expression of
these factors – Pdx1, MafA, and Ngn3 (PMN) – promoted rapid conversion of intestinal crypt cells into
endocrine cells, which coalesced into “neo-islets” below the crypt base. Neo-islet cells expressed insulin
and had ultrastructural features of β-cells. Importantly, intestinal neo-islets were glucose responsive and
able to ameliorate hyperglycemia in diabetic mice. Moreover, PMN expression in human ES-derived
intestinal “organoids” stimulated the conversion of intestinal epithelial cells into β-like cells. Our results
thus demonstrate that the intestine is an accessible and abundant source of functional insulin-producing
cells.
This work was supported by grants from NIH/NIDDK (R01-DK083355 and DP2-DK083111 to B.Z.S., K01DK091415 to J.S., T32-DK094775 to S.R.F.), the Penn Institute for Diabetes Obesity and Metabolism, the
University of Michigan Center for Organogenesis, the University of Michigan Biological Sciences Scholar
Program, the Pew Charitable Trusts, and the Abramson Family Cancer Research Institute.
Education Minisymposium: Envisioning Educational Change in the
Digital Age
M62
Inquiry-based Research Strategies, Student Independence and Time Help
Transform an Undergraduate Biology Laboratory Experience into a Research
Environment.
J. McLaughlin1, M. Coyle1, R. Baker2;
1
Biology, Pennsylvania State University - Lehigh Valley, Center Valley, PA, 2Education, Pennsylvania State
University, State College, PA
Transformative changes were implemented in an undergraduate sophomore level introductory biology
lab experience. The lab syllabus was overhauled to include several student centered laboratory
experiences that each span the course of up to 6 weeks and utilize higher-level, inquiry-based strategies.
The goal is to move students from mere “exposure” to scientific investigation to critical thinking,
independent researchers who set out to answer real-world questions using primary literature, course
SUNDAY-ORAL PRESENTATIONS
content, and the scientific process itself. A four-step pedagogical framework was developed and applied
for each lab make-over wherein: 1) Instructors teach students essential experimental techniques via prelab videos and hands-on training; 2) Instructors ask a guided-question, then have students work in small
groups to research, select, and read, three primary literature articles related to the guided-question in
order to devise a more specific, self-directed research question and accompanying experiment. 3)
Students carry-out their autonomous experiment by performing learned techniques and collecting data
in a laboratory notebook using an “open laboratory approach.” 4) Students interpret their data and
write a formal scientific paper to present their research study and findings. Importantly, throughout this
framework, instructors act as research chaperones, guiding student scientists through each step of the
pedagogical framework. Assessed herein was a lab wherein students were taught state-of-the-art cell
culture techniques, cell passage and cell counting, and then asked to design and carry-out an original
experiment based an over-arching guided-question pertaining to cell viability (life vs. death) in cell
culture. Identical assessments were given three times over a three year period that focused on
evaluation and synthesis of the research process, analysis and application of research knowledge and
skills, and synthesis of research, published and their own, into a critical paper. Key findings indicate
benefits and effects on communication, content knowledge, new techniques, technology experience,
critical thinking, time management, organization, data collection, interpreting data, scientific writing,
experimental design, and future interest in undergraduate research.
M63
Spreading Vision and Change Through Long-Term Mentorship Opportunities: the
ASCB Mentorship in Active Learning and Teaching (MALT) program.
M.J. Wolyniak1, D.E. Allen2, J.K. Hood-DeGrenier3, G.E. Plopper4, A.J. Prunuske5, S.M. Wick6, A.N.
Becalska7, J. Dyer8, L.A. Gurski9, M. Kokes10, S. Murugesan11, W. Xie12;
1
Biology Department, Hampden-Sydney College, Hampden-Sydney, VA, 2University of Delaware,
Newark, DE, 3Worcester State University, Wellesley, MA, 4Rensselaer Polytech Inst, Troy, NY, 5Medicine,
University of Minnesota-Duluth, Duluth, MN, 6University of Minnesota, Saint Paul, MN, 7Biology,
Brandeis University, Waltham, MA, 8MIT, Cambridge, MA, 9Urology, Univ Pittsburgh Med School,
Pittsburgh, PA, 10Biology, Duke University, Durham, NC, 11NIH\NHLBI, Bethesda, MD, 12Institute of
Medical Biology, Singapore, Singapore
Extensive pedagogical research recognizes active learning strategies as the most effective way to teach
STEM disciplines, yet a disparity exists between these ideas and their widespread and effective practice.
To promote reform in undergraduate STEM education, the 2013 Vision and Change in Biology
Undergraduate Education working group challenged professional scientific societies to take action to
help their members implement lasting changes in their approaches to teaching. In response to this call
to action, the ASCB has created the Mentoring in Active Learning and Teaching (MALT) program, which
is designed to give society members access to a long-term hands-on experience implementing active
learning in their classrooms. This small grant program allows individuals at any point in their career path
to work with faculty who are experienced in employing active learning practices in their classrooms,
providing mentees with hands-on experience and long-term mentorship in active learning best
practices. A pilot version of MALT began in late 2013, matching 6 experienced active learning-based
SUNDAY-ORAL PRESENTATIONS
teachers with enthusiastic mentees responding to a call put out in ASCB communications. The mentees
have thus far engaged in a range of activities with their mentors: giving department seminars;
collaborating on course development; and developing multi-session active-learning modules.
Preliminary assessment through interviews with mentees suggests a rise in their confidence in
developing and implementing active learning strategies in their classes. As the program grows, we
intend to assess the degree of use of active learning materials with a newly developed, validated tool
called the Classroom Observation Protocol for Undergraduate STEM (COPUS) (Smith et al., 2013).
COPUS utilizes a coding system to observe and record the actions of instructors and students at given
points of time in a class period. The ASCB intends to partner with the Genetics Society of America (GSA)
and the American Society of Plant Biologists (ASPB) to create an expanded version of MALT that will best
serve the increased need for active learning mentorship.
M64
“How we learn may not always be good for us” – Analytics of students’ study
strategies, educational resource choices, and learning success in a medical
histology course.
L.W. Holaday1, D. Selvig1, J. Purkiss2, M. Hortsch3;
1
Medical School, University of Michigan, Ann Arbor, MI, 2Learning Health Sciences, University of
Michigan, Ann Arbor, MI, 3Cell and Developmental Biology, University of Michigan, Ann Arbor, MI
Histology is a traditional core basic science component of many biomedical education programs. It links
the structure of cells and tissues at the microscopic level with their biological functions. It often presents
a didactic challenge for students, as many are unfamiliar with analyzing microscopic images. Identifying
successful learning strategies and recognizing students who are likely to struggle with histology would
allow for early supportive and effective intervention. This project set out to find common characteristics
among students, who either did well or were academically challenged by histology and to exam the
effectiveness of traditional versus novel electronic teaching approaches. To evaluate students’ learning
strategies and resource usage and to identify student characteristics that correlate with learning success
in histology, three first year medical school classes at the University of Michigan (>440 students) were
surveyed about their educational background, attitudes towards learning histology, and their use of
histology learning strategies and tools. These characteristics were correlated with the students’ final
cumulative examination results in the M1 histology component. This analysis demonstrated a strong
preference of students studying histology to use electronic over traditional learning resources. In
particular, students’ use of scheduled didactic opportunities like faculty-guided lectures and labs
decreased over the progression of the course. Students also exhibited a strong inclination to study
histology individually rather than in study groups. Students who reported previous experience with
histology or pathology and/or hold science or biomedical science college degrees usually did well in
histology examinations. Learning success in histology was also positively associated with students’
perception that histology is important for their professional career. Other positive indicators are inperson participation in teacher-guided learning experiences, specifically lectures and laboratory
sessions. In contrast, students who relied on watching histology lectures by video rather than going to
SUNDAY-ORAL PRESENTATIONS
lectures in person performed significantly worse. Parameters that influence the didactic efficiency of
lectures versus video recording are currently under investigation. The characteristics and learning
strategies of students doing well in this very visual study subject will be of help for identifying students
early, who might be at risk of failing a histology course. Furthermore, the results of this study indicate
that although students often prefer electronic learning tools over traditional didactic approaches, the
effectiveness of a teaching strategy or a resource is often determined by the way it is offered to and
used by them.
M65
Scichats @ Salk connects students and scientists for informal video chat
conversations.
A.L. Buchwalter1, N. Swanberg2, D. Mapston3, E. Potter3;
1
Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, 2Del Mar
Hills Academy, Del Mar, CA, 3Education Outreach, The Salk Institute for Biological Studies, La Jolla, CA
SciChats is a video-chat-based science outreach program that pairs a group of elementary school
students (at Del Mar Hills Academy, Del Mar, CA) with a volunteer scientist (at The Salk Institute for
Biological Studies, La Jolla, CA) for lunch-hour "chats" about what it's like to be a scientist. Scichats’ dual
mission is to function as an informal learning experience for students and an immersive communication
exercise for scientists. To promote student engagement and to encourage the best content from the
participant scientists, SciChats is structured as a game. The students vote to decide which scientist did
the best job of explaining what they do, and the winning and runner-up scientists visit the school and
give a seminar for the students, parents, and teachers. One week before each scheduled chat session,
the participant scientist fills out a profile describing their research interests, including an interesting
piece of media related to their work. Examples of media used include a video tour of a laboratory and a
movie of neurons firing in the brain of a running mouse. This profile is posted on the SciChats blog,
where the students view the profile and decide whether to sign up for the chat. On the day of the video
chat, the scientist and the teacher start a Skype video call. The scientist uses screen-sharing to give a
short presentation (~15 minutes) to the students during lunch. The scientist discusses how they became
interested in science, what they work on, and why they think it’s important. The scientist will often
revisit the media that they included in their profile and talk about it in more depth. The students are
then given the floor to ask questions. Each video presentation is recorded and posted on the SciChats
blog along with a transcript of the question and answer session. After each video chat concludes, the
students are given a survey to rate the scientist. The scores are recorded and the scientist with the
highest score wins. SciChats kicked off in Spring 2014 with 7 chats each featuring a different Salk
scientist. Our goals for the next run of SciChats are to (1) build a website that effectively serves the
needs of participant scientists, students, and teachers; (2) maintain high levels of student engagement
and encourage the best science content by improving the game aspect of SciChats; and (3) develop
more polished videos of scientist presentations supplemented with discussion materials for use as an
interactive classroom activity. Our long-term goal is to scale up SciChats to include additional San Diegoarea schools, including schools with fewer technology and science education resources.
SUNDAY-ORAL PRESENTATIONS
M66
Flipping the Classroom to Make Time for Active Learning.
A.J. Prunuske1, J. Batzli2, E. Howell2, S.M. Miller3;
1
University of Minnesota, Duluth, MN, 2University of Wisconsin, Madison, WI, 3University WisconsinMadison, Madison, WI
Flipping the classroom is a model where students read or watch a video prior to class creating time inclass for students to participate in student-centered learning activities like those promoted in the Vision
and Change report. We integrated the flipped classroom model into a subset of class periods in an
introductory biology course at a research university and used a mixed method approach to assess the
effect on student learning. The majority of the students viewed the online lectures before coming to
class, reported stopping the lectures to reflect on the information, and agreed that the online lectures
helped them to complete the in-class activity. Students who viewed the online lectures prior to class
performed better on clicker questions designed to test lower-order cognitive skills. The in-class
activities gave students practice applying the information in groups and provided the instructor with
feedback about students’ understanding of the material. As compared to previous years, the addition of
the lectures did not significantly increases the number of hours the students reported spending on the
course and students valued the online lectures as a learning tool. On the basis of the results of this
study, we support creating hybrid course models that allow students to learn the fundamental
information outside of class time, thereby creating time during the class period to be dedicated toward
the conceptual understanding of the material.
M67
Genome Solver: Training Faculty to Deliver Bioinformatics Content to Life
Science Students.
A.G. Rosenwald1, G. Arora1, R. Madupu2, J.A. Roecklein-Canfield3, J. Russell4;
1
Department of Biology, Georgetown University, Washington, DC, 2J. Craig Venter Institute, Rockville,
MD, 3Department of Chemistry, Simmons College, Boston, MA, 4Center for New Designs in Learning and
Scholarship, Georgetown University, Washington, DC
Bioinformatics is becoming a routine part of biological research, yet students entering the workforce
may not have had much training in this relatively new discipline. This stems in part from the fact that
many life science faculty have little training and so are reluctant to teach this material. To address this
deficit, we have developed the Genome Solver Project. Over the past 3 years, we have trained more
than 120 faculty members at a variety of different institutions, including community colleges and
minority serving institutions, to use basic, open-source bioinformatics tools, within a context involving
good pedagogical practices. In addition, we are now in the process of developing a community science
project using the rich DNA sequence data sources available in public databases to examine horizontal
gene transfer between bacteriophages and bacteria to demonstrate how to use bioinformatics for
original undergraduate research experiences.
SUNDAY-ORAL PRESENTATIONS
M68
College students’ understanding of cellular architecture: A qualitative case study.
C.A. Saunders1, A.R. Taylor2;
1
Leadership, Policy, Adult, and Higher Education, North Carolina State University, Raleigh, NC, 2Watson
College of Education, University of North Carolina Wilmington, Wilmington, NC
Misconceptions of cellular structure and function are well-documented at all levels of education. We
present the results of a qualitative study where non-science majors utilize biological images from The
Cell: An Image Library to construct a deeper understanding of the diversity of cellular structure and the
correlation of a cell’s structure to its function. In addition to learning science content, students also
developed increased self-confidence and a greater overall appreciation for cells.
M69
Molecular Flipbook: A new 3D animation software for cell and molecular
biologists.
M. Pan1, R. Riyo1, G. McGill2, J.H. Iwasa1;
1
Biochemistry, University of Utah, Salt Lake City, UT, 2BCMP, Harvard Medical School, Boston, MA
Researchers currently lack software tools to readily create accurate and dynamic visual models of the
molecular processes they study. Three-dimensional (3D) animation software offers an attractive solution
to this need, and there has been rapid growth in the use of 3D molecular animations to model and
communicate scientific concepts to broad audiences. Use of these powerful visualizations has been
limited to a small number of specialists, however, due to the steep learning curve for most 3D graphics
applications. The Molecular Flipbook project seeks to empower researchers to create dynamic
molecular visualizations. These animated models are invaluable visual tools that are able to synthesize
diverse data and will play important roles in scientific research, communication and education across
broad biological disciplines. The Molecular Flipbook project is made up of two major components. The
first component is the 3D Molecular Toolkit, a free, open-source software package that will allow users
to start creating molecular animations after viewing a short tutorial. The second component is
Molecular FlipBook’s online database that enables users to share their animations and animation source
files with others.
M70
iBiology: open-access online resources for the classroom.
S. Goodwin1, R.D. Vale2,3;
1
ASCB, Bethesda, MD, 2Department of Cell and Molecular Pharmacology, University of California, San
Francisco, San Francisco, CA, 3The Howard Hughes Medical Institute, San Francisco, CA
iBiology is a free open-access collection of videos by scientists. Educators have used iBiology videos to
enhance their curriculum in a variety of ways. In this session, I will give an overview of iBiology's
SUNDAY-ORAL PRESENTATIONS
educational resources and share examples of how these videos have been leveraged to demonstrate
and engage students with the process of science.
Keith R. Porter Lecture
G3
Rigidity Sensing by Sarcomeric Contractions Controlled by Tumor Suppressors
and Promotors.
M.P. Sheetz1,2;
1
Mechanobiology Institute, National University of Singapore, Singapore, NY, 2Biological Sciences,
Columbia University, New York, NY
Control of cell growth, death or differentiation involves the integration of microenvironmental signals
through cell motile processes to produce the desired cellular responses. Abnormal microenvironment
sensing is a hallmark of cancer. In the case of cell-matrix interactions, it seems that there are several
different levels of mechanosensing, including early testing of matrix stability and rigidity sensing. Using
lipid-linked matrix ligands, we found that cells would not spread on the surface unless the liganded
integrins were clustered and barriers were placed in the lipid bilayers to enable cells to generate force
on the ligands. Upon activation of cell spreading, the flattening of the cells removes the folds in the
membranes that causes a rise in membrane tension and activates contraction to sense rigidity. The
process of rigidity sensing involves pulling to a constant displacement of about 130 nm in a local region
of the cell. In recent studies, we find that the rigidity sensing apparatus appears to be sarcomere-like in
that it drives local contractions in a region about the size of a sarcomere. Further, the z-line protein, αactinin, is at the edge of the contracted pillars and myosin II bipolar filaments are in the middle
(unpublished observations). The local contractions are controlled by tropomyosin (a tumor suppressor)
and the length and duration of the contractions are controlled by different membrane tyrosine kinases
(oncogenes). Once a rigid surface is sensed, the cell will assemble a matrix adhesion through another
force-dependent process and grow. This indicates that the process of cell- matrix adhesion formation
involves multiple, sequential if/then decisions based upon the physical as well as biochemical properties
of the matrix. The presence of many different matrix components and cell geometries gives rise to the
multi-faceted mechanisms of microenvironmental control of growth, death, or differentiation of normal
and cancer cells.
MONDAY-ORAL PRESENTATIONS
ORAL PRESENTATIONS- Monday, December 08
Symposium 3: Cell Structure and Signaling across Scales
S6
Looking at tumor invasion and angiogenesis from a biomechanical, dynamical
and correlative point of view.
J.G. Goetz1;
1
Tumor Biomechanics, INSERM U1109, MN3T, Strasbourg, France
Three mains reasons explain why most of the critical events driving normal and pathological scenarios
had been less investigated: they occur rarely in space and time, they are highly dynamic, they differ
when studied in situ in an entire living organism. Here, we have decided to develop imaging approaches
with high temporal resolution, in living organisms, and with a correlative imaging framework (from light
to electron microscopy) that would allow us to dissect these singular events with the highest resolution
possible. Those techniques allowed us to highlight the importance of the mechanical remodeling of the
stroma in tumor progression. We showed that caveolin-1 (Cav1) promotes Rho- and force-dependent
contraction, matrix alignment, and microenvironment stiffening. Fibroblastic expression of Cav1 thereby
remodels peri- and intratumoral microenvironments to facilitate both tumor invasion and metastatic
potency. We are currently developping an intravital and correlative imaging approach in two model
organisms (mouse and zebrafish) in order to capture cancer cells at crucial steps of metastasis
formation. We also used whole-embryo live microscopy in the zebrafish embryo for testing the
biomechanical input of hemodynamics to angiogenesis. Using a correlative combination of live-cell
imaging at high temporal resolutions and electron microscopy coupled to electron tomography, we
dissected the mechano-detection mechanisms at play at the cellular scale allowing the developing
endothelium to sense flow-mediated forces. This allowed us to study, in its most details, the behavior,
the nature and the architecture of endothelial primary cilia and how it is capable of relaying the
biomechanical information carried by blood flow.
In conclusion, using two distinct events representative of normal and pathological situations and
appropriated imaging techniques, our studies highlight how cells sense static extracellular forces
mediated by the extracellular matrix and dynamic forces resulting from fluid flows.
MONDAY-ORAL PRESENTATIONS
S7
Structural Studies into the Mechanistic Origin of Microtubule Dynamic Instability.
G. Alushin1, G. Lander2, E. Kellogg3, R. Zhang4, D. Baker5, E. Nogales6;
1
Cell Biology and Physiology Center, NHLBI, Bethesda, United States, 2Integrative Structural and
Computational Biology, Scripps, San Diego, United States, 3UC Berkeley - HHMI, Berkeley, United States,
4
LSD, LBNL, Berkeley, United States, 5University of Washington, Seattle, United States, 6University of
California, Berkeley, Berkeley, CA
Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule
function. This behavior is driven by GTP hydrolysis in the microtubule lattice, and is inhibited by
anticancer agents like Taxol. Using cryo-electron microscopy (cryo-EM) we have provided new insight
into the mechanism of dynamic instability, based on high-resolution structures of dynamic microtubules
and microtubules stabilized by GMPCPP or Taxol. Our studies show that hydrolysis leads to a
compaction around the E-site nucleotide at longitudinal interfaces, as well as a conformational change
that is most obvious in the α-tubulin subunits. We propose that upon hydrolysis and phosphate release,
compaction of the E-site leads to an energetically unfavorable longitudinal translation of the α-tubulin
intermediate domain, including the H7 helix. The observed rearrangements of tubulin upon hydrolysis
are reminiscent of the conformational trajectory towards a depolymerized, bent tubulin structure, as
observed by crystallographic studies of inhibited tubulin, but of smaller magnitude. This difference is
likely due to lattice constraints that maintain a strained, straight conformation compatible with the
microtubule lattice. Thus, we propose that hydrolysis leads to conformational strain that would be
released by bending during depolymerization. More recently, and using new detector technology that is
revolutionizing the cryo-EM field, we have been able to improve the resolution of our microtubule
structures to 4 Å and beyond, so that atomic models can be generated directly from our density maps.
We are applying this methodology to characterize the interaction of microtubules with proteins that
regulate dynamic instability.
S8
Connectomics.
J. Lichtman1;
1
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA
Connectional maps of the brain may have value in developing models of both how the brain normally
works and how it fails when subsets of neurons or synapses are missing or misconnected. Such maps
might also provide information about how brain circuits develop and age. I am eager to obtain such
maps in neonatal animals because of a longstanding interest in the ways neuronal circuits are modified
during early postnatal life. Some work in my laboratory has focused on obtaining complete wiring
diagrams of the projections of peripheral motor and autonomic axons in young and adult muscles. This
MONDAY-ORAL PRESENTATIONS
work has been aided by the development of transgenic mice in which individual axon projections are
traced by virtue of their expression of a unique ratio of several different fluorescent proteins
(“Brainbow”). We think this effort provides insights into the way mammalian nervous systems mold
themselves in response to experience. In contrast to the peripheral nervous system, the high density of
neuropil in the brain is overwhelming. One strategy to see through the dense wiring is to use new
generations of Brainbow technology that allow more selective imaging of particular classes of central
neurons. But in order to see the full details of central circuits other technologies may have to be
employed. My colleagues and I have developed an automated electron microscopy approach in which
we collect tapes containing of tens of thousands of 30 nm thick brain sections and then use automated
computational methods to image them at high resolution. The image data is then amenable to
automatic segmentation by new computational algorithms. We are hopeful that this imaging pipeline
will make large scale connectomic analysis of brain circuits more routine.
Symposium 4: Machinery of the Cell
S9
Kinesin Superfamily Molecular Motors, KIFs and Intracellular Transport : from
Regulation of Learning/Memory and Development to Diseases.
N. Hirokawa1;
1
Cell Biology and Anatomy, Univ Tokyo Grad Sch Med, Tokyo, Japan
The intracellular transport is fundamental for cell morphogenesis and functioning. To elucidate this
mechanism we have identified and characterized kinesin superfamily proteins, KIFs. KIFs transport
various cargoes such as mitochondria (KIF1Balpha/KIF5s), synaptic vesicle precursor (KIF1A/KIF1Bbeta),
NMDA type (KIF17) and AMPA type (KIF5s) glutamate receptors and mRNAs with a large protein
complex (KIF5s) in neurons and other cells along microtubules. Concerning regulation of transport cells
use adaptor proteins for recognition of cargos and phosphorylation and hydrolysis of G-proteins for
unloading cargoes. Our recent molecular genetic studies revealed KIF5A transports GABA A receptors in
dendrites through the interaction with GABA Receptor Associated Protein, and play a fundamental role
for the inhibitory neuronal transmission. The deletion of KIF5A caused epilepsy. KIF13A transports
serotonin receptor 5-HT1A in neurons to the plasma membrane. Mice deficient in KIF13A exhibit
elevated anxiety behavioral phenotype because of a reduction in 5HT1A receptor transport. KIF19A
regulates ciliary length by depolymerizing microtubules at the tips of cilia. KIF19A deficient mice
displayed hydrocephalus and female infertility phenotypes due to abnormally elongated cilia that
cannot generate proper fluid flow. Further, KIFs’ unexpected functions have been uncovered. KIF2A is
fundamental for neuronal migration and brain wiring by depolymerizing microtubules in growth cones
and suppressing unnecessary elongation of axon collaterals. KIF4 controls the activity-dependent
survival of neurons by regulating PARP-1 activity during brain development. KIF26A controls
development of enteric nervous system as a key suppressor of GDNF/Ret signaling cascade. KIF13B,
working as a scaffold, recruits LRP1 to caveolae via LRP1-hDLG1-KIF13B-utrophin-caveolae linkage and
MONDAY-ORAL PRESENTATIONS
enhances the endocytosis of LRP1. Deletion of KIF13B causes hypercholesterolemia. Beta cell injury due
to oxidative stress is a typical etiology of diabetes caused by nutritional excess. KIF12-knockout mice
suffer from hypoinsulinemic glucose intolerance due to increased beta cell oxidative stress. KIF12 was
revealed to be involved in an antioxidant signaling cascade as a scaffold for the transcription factor SP1
and Hsc 70. Thus, KIFs not only regulate intracellular transports, fundamental for cellular functions, but
also control very important phenomena in life such as regulation of memory and learning (KIF17) and
development including brain wiring (KIF2A), activity dependent neuronal survival (KIF4), determination
of left-right asymmetry (KIF3) and suppression of tumorigenesis (KIF3) and their functional impediments
cause certain diseases.
S10
Life at the Cell's Edge*.
S. Mayor1;
1
National Centre for Biological Sciences (NCBS), TIFR, Bangalore, India
At the cell’s edge is the plasma membrane, a flimsy four, nanometer thick material where proteins are
‘dissolved’ in a fluid-like lipid bilayer, encapsulating the cell within. Cells sense their extracellular milieu
via a wide repertoire of membrane receptors embedded in this lipid layer, continuously communicating
vital information via these information-transducers. The membrane has a complex protein and lipid
composition, maintained by many active energy-consuming processes such as synthesis, transport and
exo-, endocytosis. Although, when reconstituted outside the cell the lipid and protein constituents form
a thermodynamic mixture at physiological temperatures, the same constituents in a living cell exhibit
remarkable lateral heterogeneities in their organization. These lateral heterogeneities facilitate the
establishment of very specific local environments in the vicinity of these information-transducing
receptors, necessary for their function. In turn, this allows a cell to respond to and interpret its physical
and chemical environment. This raises a very fundamental question- how can the living cell surface,
envisaged as a 2D equilibrium fluid bilayer, regulate its local membrane composition and shape?
A combination of quantitative in vivo and in vitro experiments and theory together suggest that the
membrane is not a simple equilibrium fluid, but is an active actin-membrane composite. In this picture,
‘active’ actin filaments apply tangential stresses to the cytoplasmic aspect of the cell membrane and
provide local control on membrane protein and lipid composition. Dynamic actin filaments embedded in
a more stable cortical actin mesh undergo ATP-dependent movement driven by treadmilling and
engagement of motors. These ‘active‘ actin filaments self-organize into sub-micron scale aster-like
patterns. The ability to couple membrane components to active filaments provides a new paradigm to
organize and control membrane molecules locally, especially if the receptors can regulate the
production of actin filaments and motor activity. If membrane proteins or lipids are coupled to this type
of dynamic actin, they form dynamically reorganizing nanoscale cluster-enriched regions. By virtue of
the specific membrane components that are coupled to dynamic actin, this confers special properties to
regions enriched (or depleted) in these clusters. For example, we show that the coupling of long acyl
MONDAY-ORAL PRESENTATIONS
chain containing inner-leaflet lipids to actin can help create local liquid ordered domains, or rafts. This
active composite model naturally exhibits regulated local membrane composition and domain
organization, and has implications for how a living cell membrane may function.
*in close collaboration with Madan Rao, NCBS and RRI, Bangalore.
ePoster Talks Session 7: Spindle and Kinetochore Dynamics
E43
Feedback control of chromosome separation by a midzone Aurora B gradient.
O. Afonso1, I. Matos1,2, A. Pereira1, P. Aguiar1,3, H. Maiato4,5;
1
Chromosome Instability and Dynamics Laboratory, IBMC, Porto, Portugal, 2Laboratory of Mammalian
Cell Biology and Development, Howard Hughes Medical Institute, The Rockefeller University, New York,
NY, 3Center for Mathematics, Universidade do Porto, Porto, Portugal, 4Chromosome Instability
Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal,
5
Cell Division Unit, Department of Experimental Biology, Faculdade de Medicina, Universidade do Porto,
Porto, Portugal
Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids
prior to nuclear envelope reassembly (NER). However, how these two processes are coordinated
remains unknown. Using live-cell imaging, RNAi, pharmacological inhibitions and laser microsurgery in
Drosophila S2 cells, we identified a conserved feedback control mechanism that delays chromosome
decondensation and NER in response to incomplete chromosome separation during anaphase. A
midzone-associated Aurora B gradient was found to monitor chromosome position along the division
axis and prevent premature chromosome decondensation by retaining Condensin I. PP1/PP2A
phosphatases counteract this gradient to trigger chromosome decondensation and NER. Thus, the
Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome
decondensation and NER until effective separation of sister chromatids. This promotes the correction
and re-integration of lagging chromosomes in the main nuclei prior to completion of NER.
MONDAY-ORAL PRESENTATIONS
E44
Three-dimensional reconstructions of the first mitotic spindle reveals a novel
mechanism for spindle assembly in C. elegans.
S. Redemann1, A. Kratz2, D. Needleman3, S. Prohaska2, T. Müller-Reichert4;
1
TU Dresden/MTZ, Dresden, Germany, 2Zuse Institute, Berlin, Germany, 3Harvard School of Engineering
and Applied Sciences, Cambridge, MA, 4Medical Faculty "Carl Gustav Carus", TU Dresden, Dresden,
Germany
Upon entering mitosis, the microtubule cytoskeleton of any eukaryotic organism undergoes a
fundamental reorganization to form a bipolar spindle, which first aligns the condensed chromosomes on
the metaphase plate and then segregates them to the resulting daughter cells. Despite the fact, that
bipolar spindles are composed of the same building blocks and conduct the same function, there is a
huge variety in spindle size, assembly and architecture across eukaryotic species. In order to understand
spindle architecture, it is essential to understand the detailed 3D ultrastructure of a mitotic spindle and
to quantitatively study the key features. We generated full 3D reconstructions of the first mitotic spindle
in C. elegans during metaphase and anaphase using serial-section electron tomography. We combined
this detailed structural data with dynamic data from light microscopy. Using this approach we were able
to detect individual microtubules attached to the holocentric kinetochores. This analysis provided us
with detailed information on the number of microtubules attached per chromosome, as well as the
position of attachment sites on the chromosomes. To our surprise the percentage of kinetochore
microtubules within the spindle is very small, only about 5%. In contrast to our expectations, our results
argue that kinetochore microtubules do not originate at the centrosomes, but polymerize from
kinetochores, most likely with the minus ends of the microtubules facing towards the centrosome. This
suggests a yet unknown mechanism in C. elegans, in which microtubules nucleated from centrosomes
interact with microtubules originating from kinetochores to form the bipolar mitotic spindle.
E45
Essential role of Golgi-derived microtubules in Golgi positioning and function at
the mitotic entry.
M. Fomicheva1,2, D. Yampolsky 1, K. Frye1, V. Magidson3, E. Kolobova1, E. Smirnova2, A. Khodjakov3, X.
Zhu1, I. Kaverina4;
1
Vanderbilt University medical center, Nashville, TN, 2Lomonosov Moscow State University, Moscow,
Russia, 3Wadsworth Center, Albany, NY, 4Cell and Developmental Biology, Vanderbilt University Medical
Center, Nashville, TN
In interphase vertebrate cells, the microtubule (MT) minus end-directed motor dynein localizes to Golgi
membranes and organizes them into a single complex in the proximity of the centrosome. This
mechanism is thought to facilitate proper partitioning of the Golgi membranes in mitosis, and Golgi
complex reassembly in the daughter cells. Yet, the exact mechanism(s) responsible for segregation of
MONDAY-ORAL PRESENTATIONS
the Golgi during cell division remain unclear. It has been proposed that the Golgi membranes split into
two groups and move apart with the separating centrosomes during transition from G2 to mitosis. This
view has been recently challenged by the finding that dynein dissociates from the Golgi in late G2,
breaking the connection to the centrosome. To discriminate these conflicting ideas, we followed the
movements of the centrosome and Golgi in human cells via high-resolution multi-dimensional
microscopy, and assessed the distribution of the Golgi membranes by a newly-developed computational
approach. We found that late in G2, Golgi stacks redistributed independently of the centrosome
separation. However, Golgi stacks did not scatter randomly in the cytoplasm, but slid alongside the
nuclear envelope approaching even distribution around the nucleus. Furthermore, we detected
extensive nucleation of MTs at the Golgi at this cell cycle stage. Specific elimination of Golgi-associated
MT nucleation by a number of alternative approaches prevented Golgi redistribution around the
nucleus, indicating that Golgi-derived MTs were essential for this process. We also found that Golgi
redistribution required recruitment of dynein to the nuclear envelope, which normally occurs in late
G2/prophase. Together, our data indicate that redistribution of the Golgi stacks at the G2-mitosis
transition is driven by nuclear envelope-localized dynein that acts along the Golgi-derived MTs. A
corollary of this mechanism is that Golgi-derived MTs are responsible for equal segregation of Golgi
membranes between the daughter cells. Indeed, we found that Golgi distribution in proliferating cell
populations became irregular upon depletion of Golgi-derived MTs. Additionally, we propose that the
described mechanism of Golgi redistribution in prophase serves to properly position Golgi-associated
membrane coat protein complexes (COPI), which are essential for nuclear envelope breakdown.
E46
Multiple Pathways for Spindle Assembly in Mouse Oocytes.
L. Bury1, P.A. Coelho1, M. Zernicka-Goetz2, D. Glover1;
1
Department of Genetics, University of Cambridge, Cambridge, United Kingdom, 2Department of
Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom
Mammalian oocytes are naturally acentriolar, and assemble their spindles by self-organization of
cytoplasmic microtubule organizing centers (MTOCs) that congress at the center of the oocyte at the
time of nuclear envelope breakdown. Although Ran-GTP contributes to the generation of sufficient
microtubule density, it appears that bipolar spindles are still able to form in the absence of a stable RanGTP gradient, implying the existence of other factors that might support spindle assembly in the
maturing oocyte. A Recent study investigating acentriolar spindle formation in the early mouse embryo
has discovered that centriolar proteins, namely Plk4 and Cep152, which are present despite the absence
of centrosomes, are crucial for microtubule nucleation and spindle assembly. However, the mechanism
of acentrosomal spindle formation in the mouse oocyte is thus far unknown. It has been hypothesized
that the long duration of metaphase (6-8 hours) would allow time for multiple spindle assembly factors
to form a robust microtubule network, even in the absence of a Ran-GTP gradient. On the other hand,
the fact that enucleation of oocytes abrogates bipolar spindle formation, has supported the idea that
indeed other nuclear factors might exist that are responsible for regulating microtubule nucleation.
MONDAY-ORAL PRESENTATIONS
Consistent with studies in Xenopus egg extracts that point to an important role of Aurora A in
chromosomal MT nucleation, we find using small molecule inhibitors that Aurora A but not Auroras B/C
contribute to bipolar spindle assembly in meiosis I. Using a combination of pathway-specific inhibitors
and dominant negative constructs, we find that Plk4 also contributes to this process. These two protein
kinases appear to act in addition to Ran-GTP to locally stabilize microtubules surrounding chromatin,
and synergism between these pathways is required to build the bipolar meiotic spindle. Interestingly,
these proteins and several of their partners are concentrated in the nucleus and accumulate around
chromatin at the time of the initial MT nucleation following nuclear envelope breakdown. Inhibition of
any of these pathways strongly reduces the early stages of MT nucleation, but does not interfere with
the actin-dependent migration of chromatin to the oocyte cortex. Eventually, cytoplasmic MTOCs
nucleate small aster-like spindles and meiotic exit is able to occur.
E47
Laser microsurgery reveals conserved viscoelastic behavior of the kinetochore.
G. Cojoc1, E. Roscioli2, L. Zhang3, E. Civelekoglu-Scholey4, A. García-Ulloa5, J. Shah6, M. Berns7, D. Cimini2,
I.M. Tolic5,8, J. Gregan3,9;
1
Max Planck Instute of Molecular Cell Biology and Genetics, Dresden, Germany, 2Biological Sciences,
Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, 3Department of Chromosome Biology,
Max F. Perutz Laboratories, University of Vienna, Vienna, Austria, 4Molecular and Cellular Biology,
University of California - Davis, Davis, CA, 5Max Planck Institute of Molecular Cell Biology and Genetics,
Dresden, Germany, 6Harvard Medical School, Boston, MA, 7University of California, San Diego, La Jolla,
CA, 8Dept. Molecular Biology, Ruder Boškovic Institute, Zagreb, Croatia, 9Department of Genetics,
Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia
Accurate chromosome segregation depends on proper attachment of kinetochores to microtubules.
Mounting evidence suggests that the mechanical properties of the kinetochore are fundamentally
important for faithful segregation of chromosomes. We developed an assay where we use merotelic
kinetochore as a model for studying mechanical properties of the kinetochore in vivo. A merotelic
kinetochore is attached to microtubules emanating from both spindle poles and during anaphase,
pulling forces exerted by microtubules lead to lateral stretching of such kinetochore. In our assay, we
use laser ablations to sever microtubules attached to a stretched merotelic kinetochore, thus releasing
the forces acting on this kinetochore. The mechanical properties of the kinetochore can then be inferred
from the change of the kinetochore shape after microtubule severing. We used Hec1/Ndc80 and
CenpA/Cnp1 fused to GFP to visualize outer and inner kinetochore domains, respectively, in mammalian
PtK1 cells and in the fission yeast Schizosaccharomyces pombe. In both model systems, kinetochores
shortened after severing microtubules. An initial rapid shortening was followed by a period of slow
relaxation. Interestingly, the inner kinetochore relaxed faster than the outer kinetochore. Whereas yeast
kinetochores typically regained their unstretched size, many PtK1 kinetochores remained stretched over
a longer period of time after microtubule severing. Our analysis of the time dependent kinetochore
MONDAY-ORAL PRESENTATIONS
shortening reveals a viscoelastic behavior of the kinetochore that is evolutionarily conserved between
yeast and mammalian cells.
E48
Lateral and end-on kinetochore attachments are required for chromosome
segregation in Drosophila oocytes.
S.J. Radford1, K.S. McKim1;
1
Waksman Institute, Piscataway, NJ
Proper chromosome segregation is achieved through the regulated interaction of chromosomes with a
bipolar array of microtubules that constitute the spindle. The meiotic spindle in the oocytes of many
organisms, including humans and Drosophila, is built in the absence of the classical microtubuleorganizing centers known as centrosomes. In the presence of centrosomes, chromosome segregation
depends on interactions between the kinetochore, a protein complex that assembles at the centromere,
and microtubules that connect to the centrosomes. However, it has been suggested that kinetochoremicrotubule interactions may not participate in chromosome congression or segregation in
acentrosomal oocytes. To determine the role of kinetochores during spindle assembly and chromosome
orientation in Drosophila oocytes, we used a technique in which RNAi knockdown of kinetochore
components is confined to oocytes after meiotic entry. Our results suggest that the proportion of
kinetochore-dependent microtubules in the spindle changes as oocytes progress from prometaphase to
metaphase, such that kinetochores are essential for metaphase spindle stability. In addition, we found
that both lateral and end-on kinetochore-microtubule interactions are required for the proper
orientation of centromeres toward spindle poles, although lateral interactions are sufficient for
prometaphase chromosome movements. Surprisingly, we also found that the kinetochore may play a
role in sister centromere co-orientation, perhaps by affecting sister chromatid cohesion. These results
support a model in which oocyte chromosomes initially interact with microtubules laterally at
kinetochores while orientation is established, then end-on kinetochore-microtubule interactions are
required to direct chromosome segregation.
E49
Spatio-temporal Model for Silencing of the Mitotic Spindle Assembly Checkpoint.
J. Chen1, J. Liu2;
1
NIH, Bethesda, MD, 2National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda,
MD
The spindle assembly checkpoint arrests mitotic progression until each kinetochore secures a stable
attachment to the spindle. Despite fluctuating noise, this checkpoint remains robust and remarkably
sensitive to even a single unattached kinetochore among many attached kinetochores; moreover, the
checkpoint is silenced only after the final kinetochore-spindle attachment. Experimental observations
MONDAY-ORAL PRESENTATIONS
showed that checkpoint components stream from attached kinetochores along microtubules toward
spindle poles. Here, we incorporate this streaming behavior into a theoretical model that accounts for
the robustness of checkpoint silencing. Poleward streams are integrated at spindle poles, but are
diverted by any unattached kinetochore; consequently, accumulation of checkpoint components at
spindle poles increases markedly only when every kinetochore is properly attached. This step-change
robustly triggers checkpoint silencing after, and only after, the final kinetochore-spindle attachment.
Our model offers a conceptual framework that highlights the role of spatiotemporal regulation in mitotic
spindle checkpoint signaling and fidelity of chromosome segregation.
ePoster Talks Session 8: Cell Dysfunction in Cancer and Other
Diseases
E50
Regulation of Cadherin-Mediated Cell Adhesion and Lumen Formation by the
Tyrosine Phosphatase, PTPN14.
P. Dimarco1, A. Gladden1;
1
Univ Texas M.D. Anderson Cancer Ctr, Houston, TX
Cell-cell adhesion is mediated through dynamic regulation of cell junctions including adherens junctions
(AJ). The AJ comprises the E-cadherin/β-catenin protein complex that is assembled in the Golgi and
transported to cell-cell contacts. Multiple regulatory mechanisms, including tyrosine phosphorylation,
influence how the E-cadherin/β-catenin complex promotes adhesion. The non-receptor tyrosine
phosphatase, PTPN14, is a member of the Band 4.1, Ezrin, Radixin, Moesin (FERM) family of proteins
that plays a critical role in linking membrane proteins to the cell cytoskeleton, thereby aiding in the
communication of extracellular signals to the cytoskeleton. PTPN14 is mutated or deleted in several
human cancers, including colorectal cancer, suggesting that PTPN14 activity may be involved in tumor
development and/or progression. Previous work identified β-catenin as a PTPN14 substrate,, but how
PTPN14 regulates β-catenin-mediated adhesion or signaling is not known. The observations that PTPN14
is altered in cancer, coupled with its potential role in AJ regulation, led us to examine the function of
PTPN14 in the non-malignant intestinal cell line, Caco-2BBE. We found that endogenous PTPN14 colocalizes with E-cadherin during initial cell junction formation and with both E-cadherin and the tight
junction (TJ) protein ZO-1 in both mature cell contacts and in 3D cysts. PTPN14 knockdown (kdPTPN14)
disrupted ZO-1 localization, actin cytoskeleton organization and formation of functional AJ and TJ.
Furthermore, kdPTPN14 cells were unable to form lumens in 3D cultures. To determine how PTPN14
regulates cell contacts and lumen formation, we examined the levels of phosphorylation at β-catenin
tyrosine residues known to be phosphorylated by upstream kinases. Notably, kdPTPN14 cells displayed
increased levels of β-catenin Y654 phosphorylation, but no change in Y142 or Y489 phosphorylation. Src
kinase phosphorylates β-catenin Y654, triggering disruption of the β-catenin/E-cadherin complex. Livecell imaging revealed that whereas control cells displayed primarily intramembranous movement of Ecadherin at cell:cell contacts, PTPN14 depleted cells showed a significant increase in E-cadherin
MONDAY-ORAL PRESENTATIONS
movement on and off of the membrane at cell:cell contacts. FRAP analysis confirmed increased Ecadherin movement and recovery after photobleaching, indicating that PTPN14 stabilizes E-cadherin
complexes at cell:cell contacts. E-cadherin movement can be restored by expression of full-length
PTPN14 or treatment with a Src inhibitor, thus providing evidence that PTPN14 activity opposes Srcmediated disruption of the AJ. These results suggest that PTPN14 promotes cell adhesion, tissue
organization and could be utilized therapeutically to prevent tumor progression.
E51
Apical mistrafficking of epiregulin can be a driver event in epithelial cancers.
B. Singh1, G. Bogatcheva1, R.J. Coffey1;
1
Medicine (GI), Vanderbilt University Medical Center, Nashville, TN
The EGF receptor (EGFR) ligand epiregulin (EREG) is delivered preferentially to the basolateral cell
surface of polarized MDCK cells. Recently, we showed that EREG basolateral trafficking is regulated by a
conserved tyrosine residue within a YXXΦ motif (Y156ERV) in its cytoplasmic domain. Interestingly, a
Y156A substitution led to apical mistrafficking of EREG and transformation of polarized MDCK cells
(PNAS 110: 8960-5, 2013). We have identified EREG mutations (R147stop) in human tumors that is
predicted to disrupt the basolateral sorting motif of EREG; we now report that EREG mistrafficks to the
apical surface in MDCK cells expressing the this mutation. To test the hypothesis that apical
mistrafficking of EREG can be a driver event in epithelial cancers rather than a mere passenger, we have
generated MDCK cells stably expressing, inducible (Tet-ON) wild-type and Y156A mutant EREG.
Uninduced clones form normal appearing cysts in 3D Matrigel cultures, regardless of EREG status. Upon
induction, however, only (Y156A)EREG-expressing cells form abnormal cysts with ectopic lumens and
inward growth that correlates with transformation in Matrigel cultures, providing support for our
hypothesis. Furthermore, we show that EGFR activity is required for EREG mistrafficking-inducd ectopic
lumen formation as preincubation with the irreversible EGFR kinase inhibitor, EKI-785 abrogates
formation of lateral lumens. Using EREG cytoplasmic domain as a bait in a yeast-2-hybrid screen, we
have identified interacting proteins that could modulate polarized sorting of EREG. We have
subsequently confirmed these interactions by co-immunoprecipitation. Under a separate inducible
control (Cumate switch), we have now expressed these interacting proteins simultaneously with Tetinducible EREG constructs, to test if their interaction alters EREG localization and subsequent ectopic
lumen formation. Results from these experiments will be presented at the meeting. In summary,
dynamic regulation of EREG localization (by interacting proteins) can mediate transformation induced by
apical mistrafficking of EREG.
MONDAY-ORAL PRESENTATIONS
E52
Retention of Somatic Mutations in Cancers by Gain in pH Sensing.
K.A. White1, Z.A. Szpiech2, D. Garrido Ruiz3, N.B. Strauli2, M.P. Jacobson3, R. Hernandez2, D.L. Barber1;
1
Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, 2Department of
Bioengineering and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA,
3
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA
A critical unresolved question in cancer is how somatic mutations are selectively retained and what
selective pressures direct this process. One common feature of cancers is constitutively increased
intracellular pH (pHi) that enables diverse cancer cell behaviors, including increased cell proliferation
and migration as well as metabolic adaptation and decreased apoptosis. We propose that increased pHi
provides a selective pressure for the retention of somatic mutations in some human cancers and that
these mutations provide an adaptive advantage to the increased pHi of cancer cells. Here, we focus on
Arg>His mutations, predicting that replacing a non-titratable arginine (pKa 12) with a titratable histidine
(pKa 6.5) may confer pH sensitive function (pH sensing) to the mutant protein. To determine whether
Arg>His mutations are selectively retained, we performed bioinformatics analyses of COSMIC and
tumor/normal paired sequencing datasets. These analyses revealed that Arg>His mutations are
significantly overrepresented in a subset of cancers including stomach, prostate, colorectal, and glioma.
Arg>His mutations are enriched even after accounting for codon bias and CpG site frequency, suggesting
selective pressure may drive retention. Next, we investigated whether Arg>His mutations confer an
adaptive advantage to cancer cells. We identified Arg>His somatic cancer mutations in p53 and EGFR
and tested mutants for a gain in pH sensing. First, we show that the most highly recurrent p53 mutation
in cancer, p53-R273H, has pH-sensitive promoter binding with decreased DNA binding at the increased
pHi of cancer cells, while wild-type p53 promoter binding is pH insensitive. We predict that Arg273
interacts with the phosphate backbone of DNA, and His273 is less able to form stable interactions with
negatively charged DNA at higher pHi. Second, we investigated two Arg>His mutations in EGFR that are
found in mesotheliomas and lung cancer: R776H and R831H. Using both in vitro and cell-based assays,
we found that the kinase activity of these mutants is pH sensitive with increased activity at higher pH,
while wild-type EGFR kinase activity is pH insensitive. Additionally, molecular dynamics simulations
suggest potential mechanisms for pH sensing by EGFR-R776H, including critical conformational changes
in the αC helix of the kinase domain resulting from changes in histidine protonation. These data suggest
that Arg>His somatic mutations can confer adaptive pH sensing to p53 and EGFR. Together, our studies
integrate whole-genome bioinformatics, biochemical, cellular, and structural analyses to show that the
increased pHi of cancer cells may be a selective pressure driving retention of Arg>His mutations that
may confer adaptive pH sensing to tumorigenic proteins.
MONDAY-ORAL PRESENTATIONS
E53
Electrophilic Nitroalkenes Cause Degradation of NFκB RelA in Triple Negative
Breast Cancer Cells.
C. Woodcock1, S.R. Woodcock1, N. Davidson2, B.A. Freeman1, Y. Huang2;
1
Pharmacology and Chemical Biology, Univ Pittsburgh, Pittsburgh, PA, 2University of Pittsburgh Cancer
Institute, Pittsburgh, PA
Triple negative breast cancer (TNBC), which lacks estrogen receptor (ER), progesterone receptor (PR)
and Her2/Neu, is an aggressive and therapy-resistant metastatic cancer that accounts for up to 20% of
breast cancer incidence in the US. Women with TNBC are at higher risk for early relapse within 5 years
of treatment, and recurrent tumors become more aggressive and invasive. Current chemotherapy for
TNBC is limited because of ill-defined targets and poor efficacy; therefore, there is an unmet need for
novel therapeutic agents with improved efficacy that can also prevent disease recurrence. Electrophilic
fatty acids are endogenously-generated signaling mediators that modulate cell differentiation and
proliferation via post-translational modification of functionally-significant nucleophilic amino acids (Cys,
His) of transcriptional regulatory proteins such as NFkB, PPARr, and Keap1/Nrf2. Preliminary studies
have revealed that an exemplary electrophilic fatty acid nitroalkene, 10-nitro-oleic acid (OA-NO2),
displays potential therapeutic value in TNBC cells, by preferentially reducing the growth and viability of
two TNBC human breast ductal epithelial cell lines (MDA-MB-231, MDA-MB-468), but not normal
MCF10A cells. Essential to proliferation and survival, NFkB plays an important role in TNBC
development, and its signaling actions are constitutively activated in ER-negative breast cancer cell lines
and primary tumors. Notably, the protein level of NFkB RelA/p65 subunit was diminished in TNBC cells
upon OA-NO2 treatment. Therefore, it is hypothesized that electrophilic fatty acids reduce TNBC cell
proliferation and survival through down-regulation of NFkB expression and signaling. We have
demonstrated that fatty acid nitroalkenes induce caspase-3 activation and down-regulate transcript
levels of NFkB –regulated genes, cyclin D1 and the pro-survival genes, bcl-xL and survivin, in TNBC cells.
Moreover, nitro-fatty acids promote p65/RelA protein polyubiquitination via nitroalkylation of p65/RelA
in TNBC cells. Overall, this study will help to extend our current knowledge of diverse electrophile
functions with proteins, and also serve as a prelude to the clinical study of electrophilic nitrated lipids as
therapeutic agents that may display a high selectivity for killing TNBC cells/tumors.
E54
Transendothelial migration of uveal melanoma cells.
M.D. Onken1, J. Li1, J.A. Cooper1;
1
Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO
Uveal melanoma (UM) is cancer arising from the pigmented layers of the eye that is highly metastatic
and only spreads through the bloodstream. Almost half of UM patients develop distant metastatic
disease, most often in the liver, even after the tumor-bearing eye is completely removed. The key to
understanding and targeting UM metastasis is in understanding how circulating tumors cells enter and
MONDAY-ORAL PRESENTATIONS
exit the bloodstream and then invade and colonize distant organs. We use primary human dermal
microvascular endothelial (HDMVEC) monolayers grown on polyacrylamide soft substrates that mimic
the physiological stiffness of normal tissue as a model of the blood vessel wall. To these monolayers, we
add UM cells and follow their transendothelial migration. We found that UM cells transmigrate via a
unique route that is distinct from the transendothelial migration common to immune cells. During this
multistep process, UM cells identify HDMVEC cell-cell junctions and then intercalate between adjacent
HDMVECs, taking on a flattened morphology while maintaining contacts with the adjacent endothelial
cells. UM cells remain intercalated for an extended period in a state reminiscent of vasculogenic
mimicry. After intercalation, UM cells extend invasive projections beneath adjacent HDMVECs, and use
these to migrate beneath the monolayer. Immunofluorescence in fixed monolayers revealed cortical
actin networks with strong cortactin staining within these invasive projections. We used UM cells
expressing F-tractin, a fluorescent fusion protein that specifically binds filamentous actin without
disrupting actin function, to image of actin dynamics in live cells. Time-lapse confocal images revealed
complex actin-rich projections invading and sampling the interface between the endothelial monolayer
and the substrate. Dynamic actin cytoskeleton reorganization was also apparent as cells migrated under
the monolayer. We used blocking antibodies to identify cell adhesion molecules that were involved in
UM transmigration. We found that blocking VCAM1 inhibited early intercalation but enhanced overall
migration, suggesting VCAM1 might be necessary for initiation and maintenance of intercalation. BAP1
is the major metastasis suppressor in human UM tumors. We knocked down BAP1 in our uveal
melanoma cell lines, and found that loss of BAP1 had no significant effect on initiation or intercalation of
UM cells. However, overall migration was significantly enhanced by BAP1 knockdown. Our studies show
that UM transendothelial migration occurs in two phases: vasculogenic intercalation that depends upon
VCAM1 adhesion, and subsequent invasion under the monolayer, which is regulated by BAP1.
E55
Tumor cell motility in microenvironment context: From intravital microscopy to
systems view.
B. Gligorijevic1,2, J.S. Condeelis3, A. Bergman1;
1
Systems and Computational, Albert Einstein College of Medicine, Bronx, NY, 2Department of
Bioengineering, Temple University, Philadelphia, NY, 3Anatomy and Structural Biology, Albert Einstein
College of Medicine, Bronx, NY
While it has been established that a number of microenvironment components affect the likelihood of
metastasis, the influence of various determinants between microenvironment and tumor cell
phenotypes is poorly understood. Here we have examined factors affecting microenvironment control
over two different tumor cell motility phenotypes required for metastasis. By high-resolution
multiphoton microscopy of mammary carcinoma in mice, we detected two phenotypes of motile tumor
cells, different in locomotion speed. Slow- and fast-locomotion occurred at different spatio-temporal
coordinates and only 3/184 regions showed both behaviors. Slower tumor cells exhibited invadopodia,
small, cortactin-rich protrusions with capability of degrading extracellular matrix. To understand how
MONDAY-ORAL PRESENTATIONS
the tumor microenvironment controls invadopodium formation and tumor cell locomotion in general,
we systematically analyzed components of the microenvironment previously associated with cell
invasion and migration. No single microenvironmental property was able to predict the locations of
tumor cell phenotypes in the tumor when used in isolation or combined linearly. To solve this, we
combined multiphoton microscopy with the Support Vector Machine (SVM) algorithm to classify
phenotypes in a nonlinear fashion. This approach identified conditions that promoted either motility
phenotype. We then demonstrated that varying one of the conditions can either change the number of
tumor cells with invadopodia or switch tumor cell behavior in a context-dependent manner. In addition,
to establish the link between motility phenotypes and cell fates, we photoconverted and monitored the
fate of tumor cells in different microenvironments, finding that only tumor cells in the invadopodiumrich microenvironments, but not in microenvironments where fast-locomotion occurred, have degraded
surrounding extracellular matrix and disseminated. The number of invadopodia positively correlated
with ECM degradation, while the MMP-inhibitor inhibiting metalloproteases inhibited eliminated
degradation and stopped lung metastasis, consistent with a direct link among invadopodia, ECM
degradation and metastasis. In summary, we have detected and characterized two phenotypes of
motile tumor cells in vivo, which occurred in spatially distinct microenvironments of primary tumors. We
show how machine-learning analysis can classify heterogeneous microenvironments in vivo to enable
prediction of motility phenotypes and tumor cell fate. The ability to predict the locations of tumor cell
behavior leading to metastasis in mouse models of breast cancer models has the potential for
identifying may lead towards understanding the mechanisms behind the heterogeneity of response to
treatment.
E56
Mitochondrial DNA deletion breakpoints are strongly enriched for G-quadruplexforming and direct repeat sequences.
B.A. Kaufman1, D.W. Dong1,2;
1
Department of Animal Biology School of Veterinary Medicine, University of Pennsylvania, Philadelphia,
PA, 2Penn Genome Frontiers Institute, University of Pennsylvania, Philadelphia, PA
Mitochondrial DNA (mtDNA) deletions are associated with the aging process and are a major cause of
human mtDNA-related diseases. The sequence distribution of mtDNA deletion breakpoints around this
circular genome is non-random. Although various mechanisms have been proposed recently, the role of
sequence in deletion genesis remains inconclusive. In this study, we used novel and rigorous statistical
measures to identify which of the proposed mtDNA sequence motifs are associated with mtDNA
deletions.
To that end, we generated lists of six types of mtDNA motifs, including: G-quadruplex (GQ) forming
sequences, stem-loop/cruciform (SC) structures, and several repeat sequences (direct, inverted,
complementary, and inverted complementary). Those motif lists were then compared against the lists of
mtDNA deletions found in healthy tissues or tissues of six clinical categories of diseases. For the full set
of mtDNA deletions and for each disease category, we calculated the percentage of mtDNA deletions
MONDAY-ORAL PRESENTATIONS
whose ends lie within 20 nucleic acid bases of either a repeat sequence pair (for each type of repeat
motif), or two motif locations (for GQ or SC motifs). These values were compared to those calculated for
controls, which were generated by rotating the corresponding motif set to every location along the
circular human mtDNA to generate a finite p-value.
In the full mtDNA deletion set, a strong enrichment was detected for both ends being in close proximity
to G-quadruplex motifs (p-values less than 10e-12). Similarly, we found both ends of mtDNA deletions to
be enriched near those of a direct repeat (p-values also less than 10e-12). The mtDNA deletions
enriched for these motifs preferentially showed enrichment at both breakpoints simultaneously. None
of the other types of motifs produced enrichment of mtDNA deletions. Furthermore, we found that both
G-quadruplex and direct repeat motifs were enriched in deletions from Parkinson’s disease and healthy
tissues. Direct repeat motifs also produced enrichment for tumor and inclusion body myositis. Gquadruplex motifs also produced enrichment for Kearns-Sayre syndrome, Pearsons syndrome, and
progressive external opthalmoplegia.
In conclusion, both G-quadruplex and direct repeats are associated with mtDNA deletions. For different
diseases, different deletion formation mechanisms are potentially at work.
ePoster Talks Session 9: Nuclear Organization, Structure, and
Dynamics
E57
Exploring the Complete Spectrum of CRM1 Cargoes.
K. Kirli1, S. Karaca2, H. Urlaub2, D. Görlich1;
1
Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany, 2Bioanalytical
Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
CRM1 is an essential, highly conserved RanGTPase-driven exportin. It mediates biosynthetic nuclear
export, counteracts leakage of cytoplasmic proteins into the nucleus, and mediates many instances of
regulated nuclear export. CRM1 recognizes its cargoes through nuclear export signals (NESs), which are
10-15 residues long and contain 4-5 hydrophobic residues (Φ residues) that dock into complementary
binding pockets of the exportin. So far, more than 250 CRM1 cargoes have been identified from various
species; however, this probably represents just a small fraction of the actual cargo pool. To explore the
complete spectrum of CRM1 dependent cargoes, we developed an optimized CRM1 affinity
chromatography method. Indeed this strategy revealed hundreds of new CRM1 cargo candidates each
from S. cerevisiae, Xenopus laevis and human cells. These candidates might be the direct CRM1 binders
or the associated partners of CRM1 cargoes. Analysis of primary protein structure, in principle, could
reveal direct CRM1 cargoes by identifying a functional NES. However, it is still a major challenge to
predict NESs with high sensitivity and accuracy. We now present a new bioinformatics NES prediction
method that is based on (i) the recent crystal structures of different NES peptides in complex with
CRM1. (ii) It considers new experimental data correlating systematic mutations in NES sequences with
MONDAY-ORAL PRESENTATIONS
export activity and CRM1-binding strength, which suggest that a greater variety of residues are allowed
at Φ positions and that the inter Φ spacers impose more constraints than previously thought. (iii) It also
considers that functional NESs must be solvent exposed; it thus includes a filtering of initial hits with
domain and disorder prediction tools. We validated this prediction tool with already known NESs as
controls and also verified cargoes with so far unidentified NESs. With that, we were able to predict NESs
of human eIF2β and S. pombe Rna1p, and these predictions perfectly matched subsequent experimental
validations.
E58
Super-resolution microscopy study of the pre-ribosomal subunit nuclear export
mechanism.
J. Kelich1, A. Goryaynov1, W. Yang1;
1
Biology, Temple University, Philadelphia, PA
The existence of the nuclear envelope (NE) in eukaryotic cells provides a barrier separating nascent preribosomal subunits assembled in the nucleus from matured subunits functioning to translate proteins in
cytoplasm. Nuclear pore complexes (NPCs) act as the sole gatekeeper for large and small pre-ribosomal
subunits (pre-60s and pre-40s) exiting the nucleus. Hindered by the diffraction limit and insufficient
temporal resolution of conventional light microscopy, the precise transport mechanism including the
transport kinetics and the spatial transport routes for both large and small pre-ribosomal subunits
through NPCs remains largely unknown. Here we combined an innovative super-resolution singlemolecule microscopic approach, termed as single-point edge-excitation sub-diffraction (SPEED)
microscopy, with förster resonance energy transfer (FRET) to obtain real-time single-molecule
trajectories as well as the three-dimensional transport pathways for both pre-60s and pre-40s in live
HeLa cells. Our results reveal that both pre-ribosomal subunits conduct a fast-slow-fast diffusion pattern
through the three sub-regions of the NPC (nuclear side, central channel, and cytoplasmic side) during
their export time of ~14 ms for pre-60S and ~8 ms for pre-40S. Remarkably, approximately two thirds of
all NPC-interacting pre-60s and pre-40s subunits successfully exit the nucleus and enter the cytoplasm
resulting in much higher transport efficiencies than protein cargos and mRNAs.
E59
Abnormal processing of prelamin A in progeria affects nuclear movement and
cell migration.
W. Chang1, Y. Wang1,2, H.J. Worman1,2, G.G. Gundersen1;
1
Department of Pathology and Cell Biology, Columbia University, New York, NY, 2Department of
Medicine, Columbia University, New York, NY
Hutchinson-Gilford progeria syndrome (HPGS) is a rare genetic disease characterized by appearance of
aging phenotypes in childhood. HPGS is caused by mutations in the LMNA gene that generate a
MONDAY-ORAL PRESENTATIONS
truncated prelamin A (progerin) that cannot be processed and remains farnesylated. Lamin A anchors
LINC complex nesprin and SUN proteins and we found by fluorescence recovery after photobleaching
that progerin expression reduced the mobility of nesprin-1, nesprin-2, and SUN2, but not that of
nesprin-3, nesprin-4 or SUN1. Actin-dependent nuclear movement in NIH3T3 fibroblasts polarizing for
migration requires the formation of nesprin-2G and SUN2 transmembrane actin-associated nuclear
(TAN) lines that are anchored by lamin A (Luxton et al., Science, 2010; Folker et al, PNAS, 2011). We
found that nuclear movement was inhibited in both fibroblasts from patients with HGPS and NIH3T3
fibroblasts expressing progerin. Importantly, both the mobility and nuclear movement defects were
rescued by treating cells with farnesyltransferase inhibitors (FTI-276 and FTI-277) and cells expressing a
CAAX mutant progerin that could not be farnesylated, exhibited normal nuclear movement. Two factors
contributed to defective nuclear movement in progerin-expressing cells. First, TAN lines, which normally
are fixed in place on the nucleus, moved over the nuclear surface, indicating a failure to anchor TAN
lines. Unlike wild type lamin A, GFP-progerin accumulated in the TAN lines and moved with them,
probably due to the association of progerin with the membrane rather than the lamina. Second, the
velocity of rearward actin cable movement was dramatically reduced from 0.28 μm/min in controls to
0.08 μm/min in progerin-expressing cells. ZMPSTE24 is the metallopeptidase that specifically cleaves
prelamin A. Mutations in ZMPSTE24 lead to an inability to process prelamin A and cause progeria and
restrictive dermopathy. We found that nuclear movement was also impaired in embryonic fibroblasts
from Zmpste24-null mice and this defect was rescued by FTI-277 treatment. Zmpste24-null cells showed
defects in cell spreading and failed to polarize and initiate cell migration. We conclude that both the
abnormal processing of prelamin A and accumulation of progerin result in defective nuclear movement
and cell migration in fibroblasts and suggest that these defects many contribute to the early aging
symptoms of HGPS.
E60
Visualization of large scale genomic data in single cells uncovers the functional
organization of chromosomes.
T.R. Luperchio1, X. Wong1, M. Hallstead2, R. Ach3, K. Pekrun3, N. Yamada3, K.L. Reddy1;
1
Biological Chemistry and Center for Epigenetics, Johns Hopkins University School of Medicine,
Baltimore, MD, 2UC Davis, Davis, CA, 3Agilent Technologies, Agilent Laboratories, Santa Clara, CA
Nuclear structure and scaffolding have been implicated in expression and regulation of the genome.
Discrete domains of chromatin exist within the nuclear volume, and are suggested to be organized by
patterns of gene activity. The nuclear periphery, which consists of the inner nuclear membrane and
associated proteins, forms a sub-nuclear compartment that is mostly associated with transcriptionally
repressed chromatin and low gene expression. Previous studies from our lab and others have shown
that repositioning genes to the nuclear periphery is sufficient to induce transcriptional repression; these
regions of chromatin that come in molecular contact with the nuclear periphery are called Lamin
Associated Domains (LADs). Our current work highlights the relationship of LADs, the epigenome and
three-dimensional architecture in development and potential disease phenotypes. LADs are dynamic
MONDAY-ORAL PRESENTATIONS
and serve as a mechanism for regulation of specific gene sets during cellular programs and these
dynamics are both seen in populations and single cells. We hypothesized that LAD patterns are reflective
of and influenced by higher order chromatin structure and that higher order chromatin structure and
epigenetic signatures are closely and functionally linked. Visual studies of LAD architecture in single cells
using novel oligonucleotide DNA probes underscores these relationships, and also defines a novel
compartment at the nuclear periphery where Lamina Associated Domains are restricted.
E61
Nucleocytoplasmic Partitioning and Dynamics of a Vertebrate Proteome.
M. Wühr1,2, T. Güttler1, L. Peshkin2,3, G.C. McAlister1, A.C. Groen2, R.R. Rad1, T.J. Mitchison2, S.P. Gygi1,
M.W. Kirschner2,3;
1
Department of Cell Biology, Harvard Medical School, Boston, MA, 2Department of Systems Biology,
Harvard Medical School, Boston, MA, 3Systems Biology, Harvard Medical School, Boston, MA
Despite the nucleus’ central role in multi-cellular biology, how the cell’s proteome is partitioned
between nucleus and cytoplasm is still poorly understood. This is mostly due to the difficulty of
separating nuclear and cytoplasmic content and the challenge to comprehensively measure relative
protein abundance and dynamics. Here, we quantify the nucleocytoplasmic distribution for more than
9000 proteins, with two different methods of quantitative proteomics, using the giant Xenopus laevis
oocytes, which allow nuclear isolation via microdisection. We find a trimodal distribution, where most
proteins localize exclusively to the nucleus or the cytoplasm, while a third subset of proteins is nearly
equidistributed. By measuring the physiological protein size in undiluted cytoplasm we show that nearly
all partitioned proteins have a physiological size larger than ~100kDa, while physiologically smaller
proteins are typically equipartitioned. This suggests that protein assembly plays an important and so far
underappreciated role in proteins' retention within a membrane bound organelle. Unexpectedly, we
also find multiple examples of large, equipartitioned complexes. By following subcellular protein
dynamics upon nuclear export perturbation with Leptomycin B, we provide evidence that these
complexes are equipartitioned by active bi-directional nuclear transport. Surprisingly, this perturbation
also re-localizes many kinases towards the nucleus, suggesting an intriguing explanation for the efficacy
of this drug class in the treatment of various cancers. Thus, we present the first resource for the
quantitative nucleocytoplasmic partitioning of a vertebrate proteome, measure its dynamics upon
perturbation, shed new light on the mechanisms of subcellular protein localization, and suggest novel
mechanisms of action for a cancer therapeutic.
MONDAY-ORAL PRESENTATIONS
E62
Intranuclear trafficking and export of single ribosomal subunits in mammalian
cells.
L. Büttner1, J.P. Siebrasse1, C. Montellese2, U. Kutay2, U. Kubitscheck1;
1
Institute for Physical and Theoretical Chemistry, Rheinische Friedrich-Wilhelms-University, Bonn,
Germany, 2Institute of Biochemistry, ETH Zürich, Zürich, Switzerland
In eukaryotes, the assembly of ribosomes requires more than 200 RNA and protein cofactors. The exact
role of many of these factors is not fully understood. Also, the dynamics of the ribosomal subunit
synthesis and their complex intracellular processing, e.g. the intranuclear transport and export process
of single subunits is largely unknown. The major difficulty to study these questions is the visualization of
ribosomal subunits without interfering with their native behavior. Recently, we established a proteinbased mRNA-labeling approach using an endogenous mRNA binding protein in the salivary gland cells of
Chironomus tentans [1]. Now, we used a similar approach to fluorescently label ribosomal subunits in
mammalian cells. Here we use Dim2/Pno1, a protein known to bind cotranscriptionally to the pre-40SrRNA and to remain bound to it during maturation and export [2]. We created stable cell lines
expressing either Dim2-eGFP or -Snap fusion proteins under control of a tetracycline-repressor. For
labeling the nuclear envelope we microinjected fluorescent NTF2, a transport factor being enriched at
nuclear pores. Very sparse fluorescence labelling of the Snap-Tag was performed using nanomolar
concentrations of SiR-Snap [3], what allowed extended intracellular visualization and tracking of single
Dim2 and Dim2-labelled ribosomal subunits. The trajectories of single native ribosomal subunits could
conveniently be followed using fast and sensitive fluorescence microscopy with HILO illumination. Since
Dim2 accompanies the rRNA from the transcription site to the cytoplasm the route of transport,
including the NPC passage, could directly be monitored in living cells with high space and time
resolution. We found that the particles remained bound in the nucleolus for extended time periods
before they diffused rapidly through the nucleoplasm and were exported across the nuclear envelope.
Binding durations, diffusion constants and translocation times could quantitatively be determined.
[1] Siebrasse et al. (2012), Proc Natl Acad Sci USA 109: 9426-31
[2] Campbell & Karbstein (2011), PLoS One 6, e16194
[3] Lukinavičius et al. (2013), Nat Chem 5, 132-139
MONDAY-ORAL PRESENTATIONS
E63
How forces generated in the cytoplasm are dissipated across the nucleoskeleton
to move nuclei.
C.R. Bone1, E.C. Tapley1, P. Kuehnert1, M. Gorjanacz2,3, D.A. Starr1;
1
MCB, University of California Davis, Davis, CA, 2European Molecular Biology Laboratory (EMBL),
Heidelberg, Germany, 3Therapeutic Research Group Oncology, Bayer HealthCare Pharmaceuticals,
Berlin, Germany
Nuclear migration is a critical aspect of many developmental and cellular processes including
fertilization, cell polarization, and differentiation. Disruption of proteins at the nuclear membrane
responsible for nuclear migration leads to a class of diseases known as laminopathies, which includes
Hutchinson-Gilford Progeria Syndrome and Emery-Dreifuss Muscular Dystrophy. For nuclear migration
to occur, a bridge termed the LINC complex (for linker of the nucleoskeleton and the cytoskeleton),
forms across the nuclear envelope. In C. elegans, the nuclear envelope bridge that functions in nuclear
migration is made up of the KASH protein, UNC-83, which is recruited to the outer nuclear membrane by
the SUN protein, UNC-84, in the inner nuclear membrane. This complex is thought to transduce forces
generated by microtubule motors in the cytoplasm, across the nuclear envelope, and to the
nucleoskeleton where the force is dissipated throughout the nucleus. We show that C. elegans lamin,
LMN-1, is required for nuclear migration and interacts with the amino terminus of the SUN protein,
UNC-84. This interaction is weakened by the UNC-84(P91S) mutation previously found to partially
disrupt nuclear migration. Live imaging of nuclear migration in UNC-84(P91S) animals shows many nuclei
migrate normally. However, other nuclei fail to initiate migration, while a third class of nuclei initiate
migration before subsequently failing prior to completion. Our data also suggest a role for the
NET5/Samp1/Ima1 homolog during nuclear migration in C. elegans. Interestingly, unlike in mammalian
cells, C. elegans SAMP-1 does not require lamin for nuclear envelope localization. Additionally, RNAi
experiments demonstrated SAMP-1 plays roles in organizing the mitotic spindle in the early embryo. Our
working model is that the N-terminus of UNC-84 directly binds lamin to allow the transfer of force from
the LINC complex to the nucleoskeleton where it is dissipated by a complex of proteins. Other inner
nuclear membrane proteins, including SAMP-1, also function in these processes.
MONDAY-ORAL PRESENTATIONS
ePoster Talks Session 10: Cytoskeleton Organization, Mechanics, and
Motors
E64
Pavarotti/MKLP1 regulates microtubule sliding and neurite outgrowth in
Drosophila neurons.
U. del Castillo1, W. Lu1, M. Winding1, M. Lakonishok1, V.I. Gelfand1;
1
Department of Cell and Molecular Biology, Northwestern University, Feinberg School of Medicine,
Chicago, IL
Sliding of microtubules against each other, driven by kinesin-1, generates the mechanical force required
for initial neurite extension in Drosophila neurons. This sliding is only observed in young neurons actively
forming neurites and dramatically downregulated in older neurons. The downregulation of sliding is not
caused by the global shut-down of kinesin-1, as the ability of kinesin-1 to transport membrane
organelles is not diminished in mature neurons, suggesting that microtubule sliding is regulated by a
dedicated mechanism. Here we identified a “mitotic” kinesin Pavarotti (Pav-KLP) as an inhibitor of
microtubule sliding. Depletion of Pav-KLP from neurons strongly stimulated the microtubule sliding and
neurite growth, while ectopic overexpression of Pav-KLP in the cytoplasm blocked both sliding and
neurite extension. Furthermore, postmitotic depletion of Pav-KLP in Drosophila neurons in vivo reduced
embryonic/larval viability, with only a few animals surviving to the third instar stage. In agreement with
data observed in cultured neurons, surviving elav>Pav RNAi larvae showed overextended neurite
outgrowth and mistargeting of motor neuron axons. Taken together, our results identify a new role for a
“mitotic” kinesin as a negative regulator of neurite formation, and suggest an important parallel
between microtubule-microtubule sliding in the mitotic spindle and sliding of interphase microtubules
during formation of cellular processes.
E65
Coronin 7 and its role in organizing the actin cytoskeleton.
K. Bhattacharya1, K. Swaminathan2, A. Noegel1,3,4,5, R. Rastetter6;
1
Institute for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany, 2Beatson Institute
for Cancer Research, University of Glasgow, Glasgow, Scotland, 3Cologne Center for Genomics (CCG),
Cologne, Germany, 4Center for Molecular Medicine Cologne (CMMC), Cologne, Germany, 5Cluster of
Excellence, Cellular Stress Responses in Aging-Associated Disease, Cologne, Germany, 6Department of
Anatomy and Developmental Biology, Monash Univ, Melbourne, Australia
Coronins are F-actin binding proteins which belong to an evolutionary conserved family of WD40-repeat
proteins. They are widely expressed in cells and tissues and are involved in signal transduction,
transcriptional regulation, remodeling of the cytoskeleton and regulation of vesicular trafficking. Here,
we focus on mammalian Coronin 7 (CRN7), the only `long´ coronin known in mammals, having two
MONDAY-ORAL PRESENTATIONS
WD40-repeat domains which form a highly symmetrical tandem β-propeller. CRN7 localizes to the Golgi
apparatus where it has a role in protein trafficking and maintenance of Golgi morphology. However, the
role of CRN7 in cytoskeleton remodeling has not been addressed so far. Therefore, we have generated a
CRN7 knockout (KO) mouse model and established primary fibroblast cultures to address the cellular
functions and regulatory mechanisms involved with respect to the dynamics of the actin cytoskeleton.
Analysis of CRN7 KO cells in vitro revealed that primary CRN7 KO fibroblasts exhibited a disrupted Golgi
architecture and altered Golgi-centrosome positioning compared to the wild type (WT) counterpart.
Importantly, fibroblasts deficient of CRN7 exhibited a faster cell polarization, an increased cell migration
and an accelerated wound closure. Additionally, we detected a higher F-actin content in the KO
fibroblasts. Re-expression of CRN7 reversed the Golgi-related phenotypic deficits in the KO fibroblasts.
In line with the recently published data that a Cdc42- and Rac-interactive binding (CRIB) domain
mediates functions of coronin (corA) in Dictyostelium discoideum, we likewise study here the
significance of the CRIB motif present in CRN7 on both β-propellers. We found that CRN7 has a higher
affinity for GDP-bound GTPases than for the corresponding GTP-bound GTPase and therefore, we draw
our focus on investigating the downstream effectors of CRN7-mediated regulation of small RhoGTPases.
Taken together, this study will provide an insight into the role of mammalian CRN7 as an organizer of
actin-dependent cellular events.
E66
PLP forms novel centriole satellites and is critical for embryonic development.
D.A. Lerit1, J.S. Poulton2, H.A. Jordan1, M. Peifer2, N.M. Rusan1;
1
Cell Biology and Physiology Center, NHLBI, NIH, Bethesda, MD, 2Department of Biology, University
North Carolina, Chapel Hill, NC
Centrosomes serve as the microtubule-organizing centers (MTOC) of most eukaryotic cells. Functional
changes in centrosome behavior and activity are linked to oscillations in pericentriolar material (PCM)
levels during the cell cycle. Elucidating how PCM dynamics are regulated is critical to understanding
centrosome function throughout the cell cycle and how centrosome dysfunction drives pathologies,
such as microcephaly and cancer. Pericentrin is a key regulator of the PCM. Intriguingly, we have
identified a novel, cell cycle-dependent structural reorganization of Pericentrin-like-protein (PLP) in the
early Drosophila embryo. We find PLP localizes to radially extended “satellites” specifically during
interphase that partially colocalize with Centrosomin (Cnn) flares. Unlike flares, however, PLP satellites
are not ejected from the centrosome, suggesting they may serve as structural components of the
interphase centrosome. Clonal mutant analysis confirms that PLP satellites function primarily to
organize the interphase PCM structure. Moreover, we find the Pericentrin/AKAP-450 centriole-targeting
(PACT) domain is dispensable for PLP targeting to satellites or centrioles, but is required to maintain
proper PLP satellite and PCM organization. Further, we demonstrate that PLP is critical for efficient
microtubule (MT) organization, genome stability, and embryo viability. Collectively, our data argue that
MONDAY-ORAL PRESENTATIONS
proper organization of the interphase PCM by PLP is essential for normal cell division and early
development.
E67
A new microtubule end-binding protein, NOCA-1, coordinates with patronin to
control assembly of non-centrosomal microtubule arrays in C. elegans.
S. Wang1, K. Oegema1, A. Desai1;
1
Ludwig Institute for Cancer Research, La Jolla, CA
In contrast to the centrosomal microtubule arrays in dividing cells, many differentiated cells assemble
non-centrosomal microtubule arrays adapted for specific cellular functions. In an RNAi screen in C.
elegans, we identified NOCA-1 as a novel protein that does not contribute to centrosome-driven
embryonic cell divisions but is required to form microtubule arrays in the germline that are essential for
organismal fertility. The noca-1 gene encodes 8 isoforms expressed in a variety of tissues. Two distinct
long isoforms control assembly of microtubule arrays in the germline and embryonic epidermis,
respectively. In contrast, a short isoform functions in parallel to the microtubule minus end-binding
protein Patronin (PTRN-1) to control assembly of microtubule arrays in the post-embryonic epidermis.
Evidence for the redundant activity of NOCA-1 and PTRN-1 includes synthetic lethality and early larval
stage dye permeability of noca-1Δ;ptrn-1Δ double mutants, both of which are rescued by selectively
expressing PTRN-1 in the post-embryonic epidermis. In support of the genetic interaction, NOCA-1 cosediments with taxol-stabilized microtubules from worm lysates and purified recombinant NOCA-1, like
PTRN-1, binds to microtubule ends in a microtubule-anchoring assay. Live imaging of the epidermal
syncytium where NOCA-1 and PTRN-1 function redundantly revealed an array of evenly spaced
microtubule bundles that run circumferentially around the animal. This array, comprised of both stable
and dynamic microtubules, is subtly affected in single noca-1∆ and ptrn-1∆ mutants but nearly
completely eliminated in noca-1Δ;ptrn-1Δ worms, suggesting that this specialized non-centrosomal
array supports epidermal integrity during animal growth. We conclude that NOCA-1 represents a new
class of microtubule end-binding proteins with essential functions of its own, as well as parallel
functions with Patronin, in the assembly of non-centrosomal microtubule arrays in multiple C. elegans
tissues.
E68
Vimentin filament precursors exchange subunit in an ATP-dependent manner.
A. Robert1, M. Rossow1, C. Hookway2, V.I. Gelfand2;
1
Department of Cell and Molecular Biology, Northwestern University, Chicago, IL, 2Department of Cell
and Molecular Biology, Northwestern University, Feinberg School of Medicine, Chicago, IL
Unit-length filaments (ULFs) are non-polar oligomers formed by the lateral association of eight
tetramers of vimentin polypeptides. These oligomers are the basic building blocks that form
MONDAY-ORAL PRESENTATIONS
intermediate filaments by longitudinal annealing. Previous studies have shown that mature
intermediate filaments can exchange segments along their length probably through severing and
annealing. The long time scale for this process is indicative of the stability of the vimentin polymer as
compared to the highly dynamic turnover of microtubules and actin filaments. Here we studied dynamic
properties of ULFs using the vimentin mutant Y117L, which does laterally associate into ULFs but fails to
longitudinally anneal and thus does not form vimentin filaments. By monitoring the fluorescence of
Y117L mutant tagged with the photoconvertible protein EOS3.2, we found that ULFs are highly dynamic.
Subunit exchange between ULFs occurred within seconds and is limited by diffusion of vimentin
polypeptides in the cytoplasm rather than their association and dissociation from ULFs. Biochemical
analysis and microscopy data demonstrate that cells expressing vimentin Y117L mutant contain a large
pool of soluble vimentin that is in the rapid equilibrium with ULFs. Furthermore, our study
demonstrated that subunit exchange between ULFs requires ATP; ATP depletion causes a dramatic
reduction of the soluble vimentin pool. Therefore, this study shows that vimentin filament precursors
can be present in two distinct states, the ULF vimentin oligomer and a soluble pool. The fast transition
between these two states is an ATP-dependent active process that might play a critical role in the
regulation of the first steps of the filament assembly in the cell.
E69
Arp2/3 complex-dependent assembly of filopodia by the formin FMNL3
contributes to cell-cell adhesion.
L.E. Young1, T.J. Gauvin2, E.G. Heimsath3, H. Higgs4;
1
Dartmouth College, Hanover, NH, 2Dartmouth-Geisel Sch Med, Hanover, NH, 3NIH/NIDCD, Bethesda,
MD, 4Department of Biochemistry, Geisel School of Medicine at Dartmouth College, Hanover, NH
Filopodia are finger-like, actin-dependent membrane protrusions that play fundamental roles in
directional cell migration, cell-cell adhesion and chemosensing. The mechanism for filopodial assembly
remains to be established, with two predominant mutually exclusive models: 1) the tip nucleation model
describes that filopodia are created de novo by a molecule that nucleates new actin filaments; or 2) the
convergent model describes that filopodia are re-modeled from Arp2/3 complex-nucleated dendritic
networks, such as lamellipodia. Previously, we have shown that two formin proteins, FMNL3 and mDia2,
are potent filopodial inducers, but whether they act by the tip nucleation or convergent model was
uncertain. Here, we show that both FMNL3- and mDia2-induced filopodia are largely Arp2/3 complexdependent in multiple cell types (non-adherent and adherent). Interestingly, a small percentage of these
filopodia are Arp2/3 complex-independent, suggesting that FMNL3 and mDia2 may use actin filaments
generated by alternative nucleators in certain situations. Biochemical assays show that, under
conditions that mimic mammalian cytosol, both FMNL3 and mDia2 are poor nucleators, further
suggesting the need of a separate nucleation factor during filopodial assembly. To address the
physiological function of FMNL3, we show that the full-length protein (both endogenous and GFPtagged proteins) localizes predominately at the plasma membrane, where it enriches in filopodia,
membrane ruffles, and at cell-cell contact sites. Interestingly, FMNL3-enriched filopodia are 10-fold
MONDAY-ORAL PRESENTATIONS
more stable when at the cell-cell interface in comparison to when interacting with the cell-free coverslip
surface. A small proportion of FMNL3 localizes to dynamic vesicular structures in the cytoplasm; and
these structures can migrate to and fuse with the plasma membrane at cell-cell contact sites.
Suppression of FMNL3 causes defects in filopodial assembly and cell-cell adhesion. To summarize, our
results show that FMNL3 and mDia2 assemble filopodia in an Arp2/3 complex-dependent manner.
FMNL3-dependent filopodia in particular are specialized for cell-cell adhesion.
E70
The yeast kinesin Kip2p promotes microtubule growth through polymerase and
anti-catastrophe activities.
A. Hibbel1, A. Bogdanova1, D. Liakopoulos2, J. Howard3;
1
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, 2CRBM-CNRS,
Montpellier, France, 3Molecular Biophysics and Biochemistry, Yale University, New Haven, CT
The lengths of individual microtubules need to be tightly controlled for cytoskeletal functions such as
mitotic spindle positioning. Length control requires a precise balance between microtubule growth and
shrinkage, but how this is achieved is poorly understood. In budding yeast, the orphan kinesin Kip2p is
suggested to promote microtubule growth, as deletion of this kinesin results in shorter, less abundant
cytoplasmic microtubules. However, how Kip2p promotes microtubule growth is not known.
We studied the effect of Kip2p on microtubule dynamics in vitro, using reconstitution assays combined
with total internal reflection fluorescence (TIRF) and differential interference contrast (DIC) microscopy.
In the presence of ATP, Kip2p increases the growth rate and inhibits the catastrophe rate of
microtubules grown from either pig or yeast tubulin. These activities increase the mean length of
dynamic microtubules, thereby promoting microtubule growth.
To investigate the molecular mechanism by which Kip2p controls microtubule dynamics, we determined
biophysical properties of Kip2p on stabilized and dynamic microtubules in single-molecule experiments.
In the presence of ATP, Kip2p is a highly processive motor that transports tubulin to the microtubule
plus-end. In the presence of the non-hydrolyzable ATP analog AMP-PNP, Kip2p is stationary on the
lattice and microtubule growth is no longer promoted. The flux and end-residence time of Kip2p on
dynamic microtubules indicate that the mechanism by which Kip2p promotes microtubule growth is
primarily by tubulin subunit addition while Kip2p is at the microtubule plus-end. The targeting of tubulin
to the end by Kip2p plays a relatively small role in growth promotion.
Our results provide evidence that a kinesin motor can act directly as a microtubule polymerase and anticatastrophe factor in the absence of accessory proteins.
MONDAY-ORAL PRESENTATIONS
ePoster Talks Session 11: Organelle Dynamics and Crosstalk in
Health and Disease
E71
Novel mitochondria-microtubule crosstalk regulates mitochondrial biogenesis
and morphology.
V. Jayashankar1, J. Aiken2, S. Rafelski3, J.K. Moore4;
1
University California-Irvine, Irvine, CA, 2Univ Colorado Sch Med, Aurora, CO, 3Developmental and Cell
Biology, University California-Irvine, Irvine, CA, 4Cell and Development, University of Colorado School of
Medicine, Aurora, CO
Mitochondrial homeostasis requires a complex coordination of biogenesis, organization, function,
dynamics, and inheritance. Here we identify novel regulation of mitochondrial homeostasis mediated by
cross talk with tubulin proteins. We previously conducted a genome wide screen to identify genetic
interactions for mutants of yeast α- and β-tubulins that lack negatively charged carboxy-terminal tail
regions (CTTs), which project from the microtubule surface. We uncovered a number of mitochondrial
genes, including genes involved in biogenesis, morphology, and inheritance. This suggested a role for
tubulin CTTs in regulating mitochondrial homeostasis; especially surprising because the actin network in
yeast drives mitochondrial transport. To test the hypothesis that tubulin CTTs are important for
mitochondrial homeostasis, we imaged live mutant yeast cells using spinning-disk confocal microscopy
and quantified the mitochondrial networks in 3D using our MitoGraph image processing method. We
found that mutants lacking α-CTT contain fewer, less branched mitochondria resulting in a 20%
decreased mitochondrial to cell volume ratio compared to WT cells. This suggests that α-CTTs are
required to maintain mitochondrial content, most likely by regulating mitochondrial biogenesis
pathways. α-CTT mutant cells also display wider, swollen mitochondrial tubules in respiring cells. This
suggests that α-CTT mutant cells may have altered internal ultrastructure organization, which we are
verifying by electron microscopy. These phenotypes are unique to α-CTT – mutants lacking β-CTT
contain normal mitochondrial content, but have misshapen and occasionally aggregated tubules.
Furthermore, cell growth rate and mitochondrial membrane potential are not affected in respiring α-CTT
mutant cells, indicating that mitochondria remain functional, despite defects in content and
morphology. To define the role of α-CTT in mitochondrial biogenesis and morphology, we examined the
sequence features of α-CTT that are necessary for function. We find that α-CTT function can be rescued
by mutants that lack negatively charged residues in α-CTT or by replacing α-CTT with the longer, and
more negatively charged, β-CTT. These data suggest that α-CTT function depends on the length of the
tail, but not its charge. We are currently investigating the molecular mechanisms behind tubulin-CTT
regulation of mitochondrial biogenesis and morphology.
MONDAY-ORAL PRESENTATIONS
E72
The molecular link between SERT and ERp44 in gestational diabetes mellitus
(GDM)-associated pregnancy.
Y. Li1, F. Kilic1;
1
Biochemistry and Molecular Biology, UAMS, Little Rock, AR
The molecular link between the serotonin (5-HT) transporter (SERT) and ERp44, an endoplasmic
reticulum (ER) protein, in gestational diabetes mellitus (GDM)-associated pregnancy is not known. We
show in the present study that ERp44 links to SERT and down-regulates the 5-HT uptake rates of
trophoblasts in GDM-associated placentas through two negative feedback mechanisms. Earlier, we
reported that ERp44 binds to Cys200 and Cys209 residues on SERT to facilitate the disulfide bridge
formation. But in this study we explore that in GDM, the 5-HT uptake rate of trophoblasts becomes
significantly down-regulated due to the reduced number of SERT molecules on their surface compared
to the trophoblasts of healthy placenta. Interestingly, in healthy placenta, insulin signaling breaks the
interaction between SERT-ERp44, allowing SERT to be translocated to the plasma membrane; this
process is reversed by insulin receptor blocker AGL2263. Therefore, the first negative feedback
mechanism that ERp44 links to SERT is through GDM-associated defective insulin signaling; which arrests
SERT at intracellular compartments and down-regulates the 5-HT uptake rates of trophoblasts.
Furthermore, in the trophoblast cells of GDM, SERT appears to be differentially glycosylated. The second
extracellular loop of SERT has two N-linked glycosylation sites, Asn208 and Asn217; and has Cys200 and
Cys209 where ERp44 binds to SERT. While ERp44 is constitutively bound to Cys200 and Cys209 residues,
the glycolytic enzymes cannot recognize Asp208 (between the two Cys residues) on SERT in order to
promote its glycosylation. Therefore, the second negative feedback mechanism that ERp44 links to SERT
is through the differential glycosylation of SERT in GDM-associated placental cells. These data show that
post-translational modifications of SERT, in placental trophoblast cells, are impaired due to the lack of
insulin signaling in GDM-associated pregnancy. In this regard, therapies designed to promote the
expression of SERT on the trophoblast surface may represent a novel approach to alleviating GDMassociated down-regulation of 5-HT uptake rates by taking advantage of an endogenous protective
mechanism already in place to reduce plasma 5-HT.
MONDAY-ORAL PRESENTATIONS
E73
A conserved RNA binding protein Celf1 is essential for vertebrate lens
development and fiber cell differentiation.
A. Siddam1, C. Gautier-Courteille2, V. Legagneux2, A. Kakrana3, A. Méreau2, C.A. Dang1, J. Viet2, D.
Scheiblin1, L. Perez-Campos4, D.C. Beebe5, J. Gross4, L. Paillard2, S.A. Lachke1,3;
1
Biological Sciences, University of Delaware, Newark, DE, 2Institut de Génétique et Développement de
Rennes, Université de Rennes, Rennes, France, 3Center for Bioinformatics and Computational Biology,
University of Delaware, Newark, DE, 4Department of Molecular Biosciences, University of Texas, Austin,
TX, 5Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St.
Louis, MO
The ocular lens is a transparent tissue made of epithelial and fiber cells that focuses light on the retina
to enable high-resolution vision. Loss of lens transparency results in cataract - the leading cause of
blindness affecting 40 million worldwide. While the significance of signaling molecules and transcription
factors in lens development, homeostasis, and cataractogenesis is well characterized, that of posttranscriptional regulators is unclear. Post-transcriptional regulation - controlled by RNA binding proteins
(RBPs) among other molecules and defined as regulation of RNA transcript processing, localization,
stability, degradation, and translation - is essential for cell function. Yet, while the human genome
encodes 1500 RBPs, less than 20 are associated with development or disease. To identify new regulators
associated with lens differentiation and cataract, we developed a novel systems tool iSyTE (integrated
Systems Tool for Eye gene discovery) and reported that deficiency of an RNA granule (RG) protein Tdrd7
results in juvenile cataracts in human and mouse (Lachke 2011 Science 331:1571). Here, we report a
new iSyTE-identified RG-RBP Celf1 (Cugbp1) that functions in the post-transcriptional control of gene
expression in vertebrate lens fiber cell differentiation. Celf1 has three RNA Recognition Motifs that bind
to target RNAs to control mRNA decay, pre-mRNA alternative splicing or translation. We find that Celf1
exhibits a highly fiber cell-enriched expression pattern that is conserved in fish, frog and mouse. To
investigate its function in fiber cells, we generated and characterized both germline and lens-specific
Celf1 deletion mouse mutants. We find that Celf1-/- mice develop fiber cell defects during
embryogenesis that result in severe cataracts at birth. Significantly, Celf1 knockdown in Zebrafish and
Xenopus also causes lens defects. Moreover, Celf1 deficient Zebrafish and mouse mutants exhibit fiber
cell nuclear degradation defects emphasizing its functional conservation across diverse vertebrate
species. Interestingly, microarray-based expression profiling of Celf1-/- mouse lens reveals downregulation of Dnase2b, an enzyme necessary for nuclear degradation in fiber cell differentiation.
Furthermore, Celf1 deficiency results in mis-regulation of the cyclin-dependent kinase inhibitors p21 and
p27 and of the F-Actin cross-linking protein Actn2. Finally, we find that Celf1 directly binds to Dnase2b
and p27 transcripts and regulates their stability or translation into protein, respectively. In sum, we
describe a new function for Celf1-mediated control mechanisms that critically function in vertebrate
lens development by regulating diverse cellular properties such as fiber cell differentiation, cell cycle
control and cell cytoskeleton.
MONDAY-ORAL PRESENTATIONS
E74
Real-time visualization of intracellular potassium dynamics in mouse
macrophages during inflammasome-associated processes.
J.R. Yaron1,2, L. Zhang1, X. Kong1, C.P. Ziegler1, F. Su1, S. Gangaraju1, Y. Tian1, H.L. Glenn1, D.R. Meldrum1;
1
Center for Biosignatures Discovery Automation, The Biodesign Institute at Arizona State University,
Tempe, AZ, 2School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ
Inflammasomes are caspase-1-activating, multiprotein platforms that mediate processing and secretion
of pro-inflammatory cytokines and cell death in macrophages in response to host danger signals and
pathogen invasion. It is well recognized that potassium efflux from the cell is a critical step for caspase-1
activation by inflammasomes in response to most stimuli. However, existing methods for directly
investigating potassium in live cells are limited and most studies focus on bulk cell biochemical
measurements or indirect indicators of pathway inhibition. Using novel intracellular potassium sensors
and laser scanning confocal microscopy we demonstrate visualization of real-time potassium dynamics
in live mouse macrophages. Specifically, we describe the kinetics of potassium efflux via the P2X7
purinergic receptor stimulated by exogenous ATP (active pathway) as well as the potassium ionophore
nigericin (passive pathway). Further, we describe the mobilization of the mitochondrial potassium pool
in response to exogenous ATP, suggesting a previously unobserved effect of P2X7 engagement beyond
effecting solely cytosolic potassium efflux. These observations support a broader inflammasomeassociated potassium response and demonstrate the applicability of an improved intracellular potassium
sensor.
E75
Blocking iron release alters the behavior of transferrin-containing endosomes in
epithelial cells.
A. Das1, M.M. Barroso1, A.B. Mason2, M.J. Gastinger3;
1
Cardiovascular Sciences, Albany Med Coll, Albany, NY, 2Biochemistry, University of Vermont School of
Medicine, Burlington, VT, 3Bitplane Inc., South Windsor, CT
Intracellular iron transport occurs by the binding of two iron molecules to transferrin, which is then
internalized upon binding to the transferrin receptor and subsequent clathrin-mediated endocytosis.
Upon endosomal acidification, iron is released from the transferrin-transferrin receptor complex and is
finally taken up by the mitochondria, which is the principal site of iron-sulfur clusters synthesis and iron
incorporation into haeme group. However, the mechanism at the basis of the endosomal iron transfer
to mitochondria remains poorly understood. Recently, it has been established that iron can be
transported via a direct interaction (“kiss and run”) between transferrin-containing endosomes and
mitochondria. To characterize these “kiss and run” events we have employed time-lapse live-cell TIRF
microscopy to simultaneously track fluorescently labeled transferrin-containing endosomes and
MONDAY-ORAL PRESENTATIONS
mitochondria in epithelial cells. Spatio-temporal endosomal parameters such as endosomal speed and
distance from mitochondria have been derived using the image analysis software Imaris. We found that
a transferrin-containing endosome generally shows significantly reduced speed during its interaction
with the mitochondrion (the “kiss” phase) and increased speed during the “run” step of the “kiss and
run” event. The role of iron release from transferrin on the behavior of transferrin-containing
endosomes and on their ability to interact with mitochondria was investigated using a single point
transferrin mutant, which cannot release iron during endocytic acidification. Blocking iron release
increased the half-life of the “kiss” phase of transferrin-containing endosome-mitochondria interactions
to 416ms compared to 216ms when using wild-type transferrin. Interestingly, we also found that
blocking iron release leads to significantly higher endosomal speeds of mutant transferrin containing
endosomes (mean speed = 0.58µm/s) than that of wild-type transferrin endosomes (mean speed =
0.46µm/s). These results suggest that blocking iron release affects endosome-mitochondria interactions
resulting in longer interactions with mitochondria as well as increased endosomal speed of transferrincontaining endosomes. In order to address the mechanism underlying the effect of iron release on
endosomal speed, we will investigate the role of intra-endosomal milieu such as pH, or extra-endosomal
cytoskeletal elements such as actin and tubulin. In the future, we also plan to study the functional
relevance of transferrin-containing endosomes and mitochondrial “kiss and run” interactions in cancer
cell lines owing to their naturally enhanced iron requirements, altered endocytic pathways, and
mitochondrial metabolisms.
E76
Inhibition of ChLoride Intracellular Channel (CLIC) Proteins by IAA-94 Induce
Reactive Oxygen Species Release from Cardiac Mitochondria.
J. Farber1, D. Ponnalagu1, S. Sukur1, W. Xin1, S. Gururaja Rao1, H. Singh1;
1
Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, PA
Reactive oxygen species (ROS) are known to play a key role in modulating cell signaling pathways and,
when produced at high levels, they induce the development of disease states such as atherosclerosis,
ischemic injury, cardiac hypertrophy and hypertension and cell death. In mammalian cells, mitochondria
are a major source of ROS. A small amount of leaked electrons from the generally efficient electron
transport chain (ETC) participate in reduction of oxygen to superoxide mostly in complex III and, due to
the back flow of electrons, in complex I. In cardioprotection, modulation of ROS production is controlled
by activation of mitochondrial ion channels. Even though ChLoride Intracellular Ion Channel (CLIC)
proteins are predicted to localize to the mitochondria, there is no evidence of involvement of CLICs in
ROS modulation in cardiac mitochondria. In this study, we focus on the role of CLICs in mitochondrial
ROS production. CLIC proteins are present in mammals (CLIC1-6) and are regulated by redox potential.
R(+)-Indanyloxyacetic acid 94 (IAA-94), a known CLIC blocker is shown to play an inhibitory role in
cardioprotection. Here, using specific antibodies, we demonstrate the expression and localization of
CLICs in mitochondria of neonatal and adult cardiomyocytes isolated from rats and in cardiac tubes of
Drosophila by Western blots and immunocytochemistry. To establish the role of CLICs in mitochondrial
MONDAY-ORAL PRESENTATIONS
ROS production, we measured ROS production from isolated cardiac mitochondria using amplex red dye
(Invitrogen). We found that, in the presence of the complex II/III substrate (3 mM, succinate) and
complex I substrate (5mM, glutamate/malate), addition of 100 μM IAA-94 resulted in a robust release of
ROS from mitochondria. The EC50 for IAA-94 was found to be 16.5 +¬ 0.15 μM (n=3) for ROS produced
in presence of succinate. After an initial fast release (ROS spike) there was an 82.5% (n=3) reduction in
rate of ROS production with succinate as a substrate; however there was an 18.1% increase (n=3) in rate
of ROS production with glutamate- malate as a substrate for 100 μM IAA-94. We also reconstituted
mitochondrial membrane proteins in a planar lipid bilayer, and discovered that IAA-94 partially blocked
channel activity that could arise from CLIC-like proteins. Furthermore, we found that CLIC5 is localized to
the mitochondria of neonatal and adult cardiomyocytes. The molecular identity of an inner
mitochondrial anion channel (iMAC) has not yet been established, and our results indicate that CLIC5
could be a putative candidate for an iMAC. The differential effects of mitochondrial ROS production by
blocking CLICs, and its impact on cardiac mitochondrial physiology may help to determine specific
therapeutic strategies for cardioprotection.
E77
Investigation of Caprin2 function in lens fiber cell homeostasis.
S. Dash1, C.A. Dang2, D.C. Beebe3, S.A. Lachke2,4;
1
UDel, Newark, DE, 2Biological Sciences, University of Delaware, Newark, DE, 3Department of
Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO, 4Center
for Bioinformatics and Computational Biology, University of Delaware, Newark, DE
The eye disease cataract is the leading cause of blindness worldwide and is caused by the loss of lens
transparency. In order to prevent or delay cataract, it is essential to understand the molecular
mechanisms that contribute to the formation and maintenance of lens transparency. We recently
developed a bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) that identifies
genes highly enriched in mouse lens development and uses this information to effectively predict
candidates whose deficiency associates with mammalian cataracts. Using iSyTE we recently identified
two post-transcriptional regulatory proteins (Tdrd7 and Celf1) that are components of cytoplasmic RNA
granules, and are essential for vertebrate lens development. This is demonstrated by the severe lens
defects including cataracts that are caused by their deficiency in multiple animal models, in turn
suggesting a conserved function for RNA granule component proteins in lens development across
diverse species. Here we investigate the function of a third conserved RNA granule component protein
Caprin2 that is newly identified by iSyTE to be associated with lens biology. Caprin2 protein has multiple
conserved domains including the coiled-coil and RGG domains as well as the C1q domain, which is found
in TNF super-family of proteins to facilitate protein-protein interactions. Interestingly, Caprin2
expression has been recently shown to be responsive to induction by FGF8 in chicken lens fiber cells,
supporting a potential role in the lens. As predicted by iSyTE, we confirmed by performing qRT-PCR, in
situ hybridization, western blotting and immunostaining that Caprin2 mRNA and protein exhibits highly
lens-enriched expression in mouse embryonic and postnatal lens. To functionally characterize this
MONDAY-ORAL PRESENTATIONS
protein, we crossed mice obtained from EUCOMM that carried Caprin2 conditional mutant alleles
(Caprin2tm2a(EUCOMM)Wtsi) with an established lens Cre deleter line Pax6GFPCre to generate lensspecific Caprin2 conditional knockout (cKO) mutants. Phenotypic characterization has revealed that
Caprin2 cKO mutants exhibit, albeit with incomplete penetrance, lens and eye defects, including a
developmental defect called Peter’s Anomaly. Scanning electron microscopy demonstrates that Caprin2
cKO mutants have a mild but consistent abnormality in lens fiber cells that contribute to the bulk of the
lens tissue. Interestingly, Caprin2 has been associated with terminal differentiation of erythroblasts from
a highly proliferative state into cells devoid of nuclei, similar to the transition of lens epithelial cells into
terminally differentiated fiber cells that undergo nuclear degradation for lens transparency. In sum, our
data has revealed a new function for Caprin2 in mammalian lens fiber cell.
ePoster Talks Session 12: Membrane Traffic: Dynamics and
Regulation 2
E78
Observation of Individual Secretory Granules in Living Mast Cells Using Confocal
Microscopy.
S. Tanaka1, Y. Takakuwa1;
1
Department of Biochemistry, School of Medicine, Tokyo Women's Medical University, Tokyo, Japan
Mast cells, which are distributed in the mucous membranes and epithelial tissue of the body surface,
cause various allergic symptoms by releasing inflammatory mediators such as histamine from their
intracellular secretory granules (SGs) in response to allergic stimuli (degranulation). It is hoped that the
development of treatments that inhibit degranulation will enable the mitigation of allergy symptoms. To
do so, it is necessary to observe the intracellular dynamics of these SGs to elucidate the process of their
maturation, intracellular transport, and degranulation. However, it has been difficult to observe
individual and distinct SGs by the conventional microscopic methods (which is by tagging SG marker
proteins with fluorescent proteins) since marked fluorescent dispersion makes the target outlines
unclear. Here, to overcome this problem, we observed individual SGs by overexpressing green
fluorescent protein (GFP) in the cytoplasm of mast cells and using confocal microscopy to photograph
the “absence of GFP” representing SGs that arose as a result of GFP not penetrating the granular lumen.
First, we demonstrated that the accuracy of this new method is higher than that of the conventional
observation. To validate the principle, we observed polystyrene beads labeled with a red fluorescent dye
in a GFP solution. When we constructed 3D images of the beads from sequential tomography images,
the dispersion of light caused the images derived from the red fluorescence to be greatly distorted in
the direction of the z-axis, while the images derived from the “absence of GFP” were spherical and
corresponded to the theoretical volume of the beads. Next, in a similar manner, we overexpressed GFP
in the cytoplasm of the rat mast cell line RBL-2H3 and constructed 3D images. From these images, we
obtained information on the construction of individual SGs (number of particles, size, volume, and
arrangement), as well as on the cell as a whole. We demonstrated that the cells in these images indeed
MONDAY-ORAL PRESENTATIONS
showed SGs by co-localization of red fluorescent protein-fused SG marker proteins (Neuropeptide Y or
VAMP7) co-expressed in the cells. Furthermore, by inducing degranulation, we obtained information on
the structure of exocytotic SGs in the cells. In this presentation, based on the structural characteristics of
the SGs that we have thus obtained, we will discuss the process of the maturation, intracellular
transport, and degranulation of SGs.
E79
Monoubiquitination regulates Golgi membrane dynamics in the cell cycle.
X. Zhang1, S. Huang1, Y. Wang1;
1
Dept. of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI
Partitioning of the Golgi membrane into daughter cells during mammalian cell division occurs through a
unique disassembly and reassembly process. Several converging lines of evidence have suggested that
monoubiquitination plays an essential role in the regulation of post-mitotic Golgi membrane fusion.
Monoubiquitination, as a regulatory signal, occurs during mitotic Golgi disassembly and is required for
subsequent Golgi reassembly. The AAA ATPase p97 and its adapter protein p47 are involved in
membrane fusion during post-mitotic Golgi reassembly. The p97/p47 complex binds to monoubiquitin
through the UBA domain of p47, and this interaction is required for p97-mediated Golgi membrane
fusion. Proteasome activity is not involved in either Golgi disassembly or reassembly. We have identified
a Golgi-localized ubiquitin E3 ligase HACE1 which is involved in mitotic Golgi disassembly; reduced
HACE1 activity is human cancer such as Wilms’ tumor results in fragmented Golgi. We have also
discovered the p97/p47 binding protein VCIP135 as a deubiquitinating enzyme whose activity is required
for post-mitotic Golgi reassembly. VCIP135 activity is regulated by phosphorylation in the cell cycle; it is
inactivated by phosphorylation in metaphase and activated upon dephosphorylation in telophase.
Recently, we have identified the substrates on the Golgi membranes whose ubiquitination and
deubiquitination are regulated by HACE1 and VCIP135 in the cell cycle. These data suggest that cycles of
addition and removal of ubiquitin to and from substrates regulate Golgi membrane dynamics during cell
division.
E80
Rab4 orchestrates a small GTPase cascade for recruitment of adaptor proteins at
the early endosome.
R. D'Souza1, R. Semus1, E. Billings1, C. Meyer1, K. Conger1, J. Casanova1;
1
Cell Biology, University of Virginia, Charlottesville, VA
Early, sorting endosomes are a major crossroad of membrane traffic, at the intersection of endocytic
and exocytic pathways. The sorting of endosomal cargo for delivery to different subcellular destinations
is mediated by a number of distinct coat protein complexes, including AP-1, AP-3 and GGAs.
Ultrastructural studies suggest that these coats assemble onto tubular subdomains of the endosomal
MONDAY-ORAL PRESENTATIONS
membrane, but the mechanisms of coat recruitment and assembly at this site remains poorly
understood. The early endosome is in a constant state of flux where subdomains form and get replaced
by new ones, as the early endosome matures into late endosomes. These endosomal subdomains are
enriched in small GTPases of the Rab family which act as master regulators controlling protein and lipid
composition, to aid carrier formation. Here we report, Rab4 marks one subdomain early in the
endosomal pathway and orchestrates a GTPase cascade which results in the sequential recruitment of
the Arf-like protein Arl1, the Arf-specific guanine nucleotide exchange factors BIG1 and BIG2, and the
class I Arfs, Arf1 and Arf3. Knockdown of Arf1, or inhibition of BIG1/2 activity with Brefeldin A results in
the loss of AP-1, AP-3 and GGA-3, but not Arl1, from endosomal membranes and the formation of
elongated tubules. In contrast, depletion of Arl1 randomizes the distribution of Rab4 on endosomal
membranes, inhibits the formation of tubular subdomains and blocks recruitment of BIG1/2, Arfs and
adaptor complexes to the endosome. Together these findings indicate that Arl1 links Rab4-dependent
formation of endosomal sorting domains with downstream assembly of adaptor protein complexes that
constitute the endosomal sorting machinery.
E81
Structural determinants and functional consequences of transmembrane protein
partitioning to membrane rafts.
I. Levental1;
1
Integrative Biology and Pharmacology, University of Texas - Houston Medical School, Houston, TX
The plasma membrane is the physical and functional interface between a cell and its surroundings, and
is therefore responsible for a myriad of parallel processing tasks, each of which must be tightly regulated
to avoid aberrant signaling. To achieve this complexity, the functionality of the plasma membrane is
enhanced by its subdivision into functional lateral domains, including lipid rafts - ordered lipid and
protein assemblies whose formation is driven by lipid interactions, and which recruit specific membrane
proteins by virtue of their unique physicochemical environment. This selective protein recruitment
underlies the functionality of membrane domains; however, the structural determinants of protein
partitioning to membrane rafts remain almost wholly unexplored. We have developed and
characterized a robust experimental system for isolating and observing intact plasma membranes,
allowing the first direct, quantitative measurements of protein affinity for raft domains. Our
observations confirm that protein transmembrane domains (TMDs) contain the necessary determinants
for raft affinity, as chimeric combinations between TMDs and intra/extracellular protein domains retain
the raft affinity of the isolated TMDs. To explore the structural bases for these observations, we tested
raft partitioning for a panel of TMD variants and confirmed the long-standing theoretical prediction that
longer TMDs would prefer the thicker hydrophobic core of membrane rafts, and thus that TMD length is
a key determinant of raft partitioning. We have extended these experimental observations by
Molecular Dynamics modeling of protein TMDs in a simulated membrane environment, generating
testable predictions for protein-protein and protein-lipid interactions. Using these predictions, we have
identified a novel PxxxG motif in transmembrane a-helices that appears to be both necessary and
MONDAY-ORAL PRESENTATIONS
sufficient for raft targeting by the transmembrane domain of a specific immune system adapter protein
– Linker for Activation of T-cells (LAT). This motif - related to the common GxxxG motif that mediates
helix-helix oligomerization - drives lateral clustering of LAT to promote raft association. We have
generalized these findings by bioinformatic approaches to generate predictive heuristics for protein
affinity for membrane rafts. Finally, we are exploring the functional consequence of raft affinity, by the
raft partitioning of LAT constructs with their efficiency in rescuing activation of leukocytes rendered
unresponsive to antigen by a knockdown of endogenous LAT. In summary, our results identify proteins
that rely on raft association for their function, define the physicochemical nature of this association, and
clarify the mechanisms by which PM organization regulates cell physiology.
E82
ER-coordinated activities of Rab22a and Rab5a drive uptake, endosomal
compaction and intracellular processing of Borrelia burgdorferi by primary
human macrophages.
S. Linder1, X. Naj1;
1
Inst. for Medical Microbiology, University Medical Center Eppendorf, Hamburg, Germany
Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting primarily
skin, joints and nervous system. Macrophages and dendritic cells counteract Borrelia dissemination in
the human body through capturing, internalization and subsequent degradation of spirochetes. Here,
we show that B. burgdorferi internalization by primary human macrophages involves compaction of
spiral-shaped borreliae into endosomal vesicles. Compaction of borreliae is an active process that is
driven by Rab22a and Rab5a, as shown by use of respective mutant constructs and siRNA-mediated
depletion. RabGTPase-dependent trafficking is also important for maturation of borreliae-containing
endosomes into LAMP1-positive and degradative lysosomes. Live cell imaging further shows that uptake
of Borrelia is a multistep process, where Rab22a-positive endosomes enwrap elongated spirochetes,
while endosomal compaction is mediated by contact with Rab5-positive vesicles at sites of membrane
extrusion from endosomes. These sites are apparently coordinated by contact with the endoplasmic
reticulum, as visualized by the ER marker Sec61beta. Collectively, these data identify Rab22a and Rab5a
as crucial regulators of B. burgdorferi intracellular processing by primary macrophages. We also
demonstrate that RabGTPase-driven endosomal compaction of borreliae is a crucial step in the vesicular
cascade that ultimately leads to elimination of spirochetes. Moreover, Rab22a and Rab5a exert their
influence through discrete vesicular entitites, whose activities are coordinated at ER contact sites.
MONDAY-ORAL PRESENTATIONS
E83
Starvation-induced phosphorylation of the exchange factor DENND3 by Unc-51like kinases activates Rab12 inducing macroautophagy.
J. Xu1, P.S. McPherson1;
1
McGill University, Montreal, QC
Unc-51-like kinases (ULKs) are the most upstream kinases in the initiation of autophagy, yet the
molecular mechanisms underlying ULK function in autophagy are essentially unknown. By investigating
two poorly characterized proteins, the guanine nucleotide exchange factor (GEF) DENN domaincontaining protein 3 (DENND3) and its substrate, the small GTPase Rab12, we have now discovered a
novel mechanism accounting for ULK function in autophagy. We demonstrate that both DENND3 and
Rab12 play positive roles in starvation-induced autophagy. Furthermore, under starvation, ULK1/2
phosphorylates DENND3 at two sites, activating Rab12 through upregulation of DENND3 GEF activity. By
binding to LC3 and associating with autophagosomes, active Rab12 facilitates autophagosome
trafficking. Together, our data reveal a novel pathway from starvation-induced ULK signaling through
DENND3 to Rab12-mediated membrane trafficking required for autophagy.
E84
Rab3/rabphilin is present on a subset of vesicles predestined to dock to RIM
clusters at the plasma membrane.
N.R. Gandasi1, S. Barg1;
1
Institute of Medical Cell Biology, Biomedical Center, Uppsala University, Uppsala, Sweden
Insulin is released by regulated exocytosis, which requires secretory vesicles to be docked at the plasma
membrane. The number of release ready vesicles at the plasma membrane is therefore rate limiting for
hormone secretion. Stable docking is preceded by a loosely tethered state, and we showed recently that
this transition occurs within seconds after arrival of the vesicle by recruitment of syntaxin and munc18
to the docking site. The molecular nature of the tethered state is not known and it remains elusive
whether the vesicles tether to a preexisiting receptor complex in the plasma membrane or attach to
random sites. To answer this we quantified GTP-binding rab proteins and their effectors at the docking
site by imaging GFP-tagged proteins using TIRF microscopy. Clusters of the rab3 interacting protein RIM
and rabphilin existed at docking sites prior to vesicle tethering and docking. A further increase in RIM
fluorescence was seen at vesicles during their maturation into the releasable pool, confirming a role of
RIM in priming. Vesicles that successfully docked carried rab3 and rabphilin whereas those that only
temporarily tethered did not. In contrast, rab27 and its effector granuphilin were present on both types
of vesicles. These results suggest that rab3 and rabphilin act on the incoming granules as signals to
initiate the docking process. Since RIM is thus far the only protein found to be enriched at the docking
site it may act as a docking receptor for the incoming vesicle.
MONDAY-ORAL PRESENTATIONS
Minisymposium 8: New Ways for Probing and Interrogating Cells
M71
A statistical framework to analyze molecule colocalization.
T. Lagache1, N. Sauvonnet1, L. Danglot2, J. Olivo-Marin1;
1
Cell Biology and Infection, Institut Pasteur, Paris, France, 2Membrane Traffic in Health and Disease,
INSERM ERL U950, UMR 7592, Institut Jacques Monod, CNRS, Univ Paris Diderot, Paris Cedex 13, France
The quantitative analysis of molecule interactions in bioimaging is key for understanding the molecular
orchestration of cellular processes and is generally achieved through the study of the spatial
colocalization between different populations of molecules.
Most colocalization methods are based on pixel overlap between the previously denoised signal that is
emitted from two (or more) different fluorescent labels, and use a global image correlation such as
Pearson's or Manders' coefficients. These data, however, cannot be linked to physical parameters such
as the real percentage of colocalizing molecules or the average colocalization distance. In addition,
randomly distributed molecules can partially overlap, and it is hard to measure the statistical
significance of the computed correlation indices. Here, we present a novel statistical method to analyze
molecule colocalization that is based on the automatic detection of molecule fluorescent spots, followed
by their representation as Point Processes and the statistical analysis of their spatial distribution. This
allows to :
1.
Test robustly and rapidly whether two molecule distributions colocalize in space, thanks to a
closed-form analysis with no need for simulations;
2. Measure statistically the real percentage of colocalizing molecules and the interaction mean
distance.
Using our method and Total Internal Reflexion Fluorescence (TIRF) microscopy, we first analyse the
spatial colocaliszation between different endocytic cargos and cellular molecules at the cell membrane.
Moreover, by coupling our statistical method to single-particle tracking techniques, we propose an
automated framework to study the dynamical interactions of molecules and the endocytosis
orchestration.
M72
Force Scaling in Stress Fibers.
L. Kurzawa1, V. Timothee1, B. Fogelson2, F. Senger1, A. Mogilner3, L. Blanchoin1, M. Théry4;
1
iRTSV, CEA, Grenoble, France, 2Mathematics, UC Davis, Davis, CA, 3Department of Biology, Courant
Institute of Mathematical Sciences, New York, NY, 4iRTSV, CEA, Paris, France
Cells have the remarkable ability to sense geometrical and physical cues from their environment and
adapt their architecture accordingly. This process requires a tight regulation of the permanent
MONDAY-ORAL PRESENTATIONS
remodeling of the acto-myosin network, that can both transmit and generate intra-cellular forces.
Despite numerous works on the molecular composition of stress fibers, little is known about the
mechanism determining the magnitude of force production in these structures. In addition, the
relationship between actin cytoskeleton dynamics and intra-cellular force production has yet to be
investigated. Here we studied the scaling of contractile force magnitude in stress fibers and investigated
the role of actin network dynamics and architecture in this process.
We used micropatterned substrates to control the length and spatial organization of stress fibers in
adherent cells and measured the traction forces they produced on deformable substrates. Thereby, we
demonstrated that forces scaling exhibit a biphasic behavior. Force magnitude first increased with the
length of the stress fibers and then dropped above a critical length. Strikingly, very long cells appeared
capable to produce only weak forces. This typical force scaling appeared independent of substrate
elasticity and conserved in various cell types having distinct intrinsic cell size limitation.
Monitoring stress fiber relaxation upon laser nano-surgery, we showed that stress fibers were
connected to the surrounding actin meshwork all along their length. A theoretical model accounting for
the biphasic behavior of force scaling established friction between actin stress fibers and their
surrounding cytoskeleton as being a key parameter in the regulation of force production by the cells. It
contributed to dissipation of myosin work outside of the fiber. Efficiency of force transmission to focal
adhesions appeared regulated by myosin motors distribution and dynamics along the stress fiber,
dissipation of stress in adjacent meshwork and connection strength between these stress fibers and
focal adhesions. All parameters were further validated experimentally using drugs or actin-binding
protein down-regulation.
M73
Hydraulic fracture during epithelial stretching.
L. Casares Garcia1,2, R. Vincent1, D. Zalbidea1, N. Campillo2, D. Navajas1,2,3, M. Arroyo4, X. Trepat1,2,5;
1
Institute for Bioengineering of Catalonia (IBEC), Barcelona, Spain, 2University of Barcelona, Barcelona,
Spain, 3Ciber Enfermedades Respiratorias, Bunyola, Spain, 4LaCàN, Universitat Politècnica de CatalunyaBarcelonaTech, Barcelona, Spain, 5Institució Catalana de Recerca i Estudis Avançats (ICREA),, Barcelona,
Spain
The epithelium is a cohesive cell sheet adhered on a soft hydrogel matrix that covers free surfaces and
cavities throughout the body. During development and adult life, the epithelium performs a broad
diversity of functions including morphogenesis, wound healing, tissue compartmentalization, and
protection against environmental pathogens. Epithelial sheets carry out these functions in a dynamic
mechanical environment characterized by elevated levels of cell and tissue stretching. Failure to
withstand stretching leads to epithelial fracture, which is associated with impaired development and
severe pathological conditions. Epithelial fracture is commonly attributed to excessive tension in key
stress bearing elements of the epithelium such as the cytoskeleton, the plasma membrane, and cell-cell
junctions. We developed a new experimental approach to study fracture dynamics of micropatterned
MONDAY-ORAL PRESENTATIONS
epithelial monolayers adhered to soft hydrogel substrates. Using this approach, we demonstrate
epithelial cracks caused by tissue stretching but independent of epithelial tension. We show that the
origin of these cracks is hydraulic; they form to release a transient buildup of pressure in the hydrogel
substrate during stretch/unstretch maneuvers. Shortly after stretch cessation, transepithelial pressure
equilibrates and cracks seal through an acto-myosin dependent mechanism. We demonstrate this
behavior in a variety of synthetic and physiological hydrogel substrates including polyacrylamide,
matrigel, and decellularized animal tissue. The theory of poroelasticity successfully captures the
observed phenomenology and allows us to predict the size and healing dynamics of epithelial cracks as a
function of the stiffness, geometry, and composition of the hydrogel substrate. Thus, coupling between
tissue stretching and matrix hydraulics determines epithelial integrity in a tension-independent manner.
M74
From Microdishes to microniches.3D microenvironmental control around single
cells. Application to single cell apico basal polarization and lumenogenesis
control.
V. Viasnoff1,2, Q. Li1, J. Thiery3,4, H. Yu1,5,6, Z. Weng7;
1
MBI/DBS, Mechanobiology Institute, Singapore, Singapore, 2UMI3639, CNRS, Singapore, Singapore,
3
Biochemistry, NUS, Singapore, Singapore, 4IMCB, Astar, Singapore, Singapore, 5School of Medicine,
NUS, Singapore, Singapore, 6IMB, Astar, Singapore, Singapore, 7Mechanobiology Institute, Singapore,
Singapore
The influence of the microenvironment on cell behavior and fate is increasingly recognized as of key
importance. It follows that new techniques to control the 3D environment around cells are key to
understand the processes by which cells probe and respond to their microniches. We present here an
approach that allow to transform microwells into artificial microniches where the chemical coating,
rheological properties and topographical properties can be differentially controlled on the top, sides and
bottom of the wells and assembled in a combinatorial way. This technique is also compatible with high
and super resolution imaging that allows to probe the dynamics of cell cytoskeleton and regulatory
proteins with unprecedented resolution down to the single molecule level in 3D. We exemplify how
theses bone fide artificial microniches can be used to induce full apico-basal polarization of single
epithelial cells and as well as to control intercellular stresses driving the anisotropic growth of
intercellular lumens. Other on going applications will also be briefly discussed.
Ref: Li et al, Nature Cell Biology, in revision
MONDAY-ORAL PRESENTATIONS
M75
Localized light-induced protein dimerization in living cells using a photocaged
dimerizer.
E.R. Ballister1, C. Aonbangkhen2, A.M. Mayo1, M.A. Lampson1, D.M. Chenoweth2;
1
Department of Biology, University of Pennsylvania, Philadelphia, PA, 2Chemistry, University of
Pennsylvania, Philadelphia, PA
Regulated protein localization is critical for many cellular processes. We present a new technique to
rapidly and reversibly control protein localization in living human cells with subcellular spatial resolution
using a cell permeable, photoactivatable chemical inducer of dimerization. This novel dimerizer consists
of a Halotag ligand linked to a photocaged derivative of trimethoprim (TMP). The Halotag ligand
covalently binds to Halotag fusion proteins, which serve as anchor domains for protein relocalization
experiments. Photocaged TMP remains inert until illuminated with ~400 nm light, at which point the
unmasked TMP non-covalently binds E. coli dihydrofolate reductase (EcDHFR), thereby dimerizing
EcDHFR and Halotag fusion proteins. Using this system, we demonstrate light-induced recruitment of a
cytosolic protein to individual centromeres, kinetochores, mitochondria, and centrosomes in living
human cells. Several systems for light-induced dimerization utilizing naturally light-sensitive proteins
have been developed, but none of these systems have been successfully applied at centromeres or
kinetochores, complex chromatin structures which are crucial for cell division. Although the
photochemical uncaging reaction is irreversible, dimerization can be reversed by addition of free TMP.
This work represents a new strategy for controlling protein localization with light using a modular
photocaged dimerizer covalently pre-localized to an anchoring protein. This technique should be readily
and widely applicable at a variety of cellular locations to address a range of biological questions.
M76
MechanOptoGenetics: a new way to uncode mechanotransduction.
M. Balland1, O. Destaing2, C. Albiges-Rizo2, A. Kerjouan2, K. Hennig3, E. Vitiello4;
1
Materials, Optics and Instrumental Techniques for the Life Sciences (MOTIV), Laboratory of
Interdisciplinary Physics (LiPhy), Grenoble, France, 2Differentiation and Cell Transformation, Institute
Albert Bonniot (IAB) Inserm U823, Grenoble, France, 3Physics, LIPHY, Saint Martin d'Heres, France,
4
UJF/UMR 5588, Laboratoire Interdisciplinaire de Physique, Grenoble, France
Mechanotransduction refers to cell ability to sense the physical properties of their microenvironment, a
critical feature for tissue homeostasis. The signal transduction occuring in this cellular haptic sensory
perception has been shown to exist in both directions from the mechanical cues to the biochemical
signals and vice versa, the overall process implying dynamic feedback loops. Research in
mechanotransduction has often coupled genetic approaches with force measurements. Classical genetic
approaches have pointed out the major role of the cell contractile machinery elements (adhesive
proteins, cell cytoskeleton) in that process without giving access to the dynamics of
mechanotransduction. Our approach, inspired by electronic science, consider the cell as an active and
MONDAY-ORAL PRESENTATIONS
dynamic system which filters and transforms signals in both directions using nano-scale protein based
transducers. Our goal is to probe the dynamics of mechanotransduction by establishing the transfer
function between mechanical and biochemical cues using a combination of optogenetic (input
generator) and time resolved Traction Force Microscopy (functional output). Here we report an
optogenetic approach based on light-induced Cry2-CIBN dimerization to probe to internal dynamics of
mechanotransduction by playing with light on the activation of the proto-oncogene Src and on RhoGEF2,
an upstream activator of Rho. We coupled the optogenetic cell targets with Fast Fourier Traction
Cytometry which enables us to measure the first mechano-genetic transfer function of those cell
dynamics regulators. We strongly believe that generic principals governing cell mechanics will emerge
from this approach that could be apply to cellular control in both pathological and healthy conditions
and that MechanOptoGenetics will be a new way to interrogate mechanotransduction processes.
M77
Dynamic micropatterning of cells on nanostructured surfaces using a cell-friendly
photoresist.
J. Doh1, S. Kweon2, K. Song1;
1
Department of Mechanical Engineering, POSTECH, Pohang, Korea, 2I-Bio, POSTECH, Pohang, Korea
Nanoscale topographical features of the extracellular matrix (ECM) influence the behavior of cells
including cell differentiation, migration, and proliferation. While how nanoscale topographical structures
affect various cellular behaviors has been active area of research, dynamics of cells on nanostructured
surfaces has not been systematically examined. Here, we developed a new micropatterning method to
control cellular dynamics on nanostructured surfaces using a cell-friendly photoresist poly(2,2dimethoxy nitro-benzyl methacrylate–r-methyl methacrylate –r-poly(ethylene glycol) methacrylate)
(PDMP), and studied how nanotopogrphy influences spreading dynamics of cells. Surfaces containing
nanoscale ridge/groove structures were fabricated by replicating a mold using a UV-curable resion
polyurethane acrylate (PUA). the PUA substrates were coated with gelatin and subsequently spincoated
with PDMP. Then, Microscope projection photolithography (MPP) was performed by illuminating light
through a photomask inserted in a field diaphragm of the microscope. UV-exposed areas of PDMP thin
films were selectively removed and nanostructured surfaces coated with gelatin were exposed. Cells
seeded onto the patterned surfaces were selectively attached onto the UV-exposed area, resulting in
single cell array formation. Finally, PDMP thin films surrounding cells were removed by briefly
illuminating UV without photomask to trigger spreading of cells. And spreading dynamics of HeLa cells
on nanostructured surfaces were monitored by interference reflection microscopy (IRM). In addition to
the simple groove/ridge structures, spreading dynamics of complex nanostructured surfaces were
quantitatively analyzed. This method will be useful to understand cellular behaviors under complex
microenvironments.
MONDAY-ORAL PRESENTATIONS
M78
Micro-Cavity based Force Sensors for Cell-biological Studies.
N.M. Kronenberg1, P. Liehm1, A. Steude1, M.C. Gather1;
1
School of Physic and Astronomy, University of Saint Andrews, Saint Andrews, Scotland
Mechanics is increasingly recognized as an important factor in numerous biological processes.
Monitoring the mechanical properties of cells and tissue is considered a key factor to the understanding
of a range of fundamental biological processes and may enable more accurate diagnosis in a range of
diseases.
Today, Traction Force Microscopy (TFM) is the most widely-used method to investigate cellular forces
exerted on 2D substrates. Until recently, it was assumed that vertical forces can be neglected in the
analysis of cell mechanics. However so-called 2.5D TFM studies have now shown that the out-of-plane
and in-plane forces exerted by cells are of the same order of magnitude. Although 2.5D TFM has
contributed to our understanding of vertical forces for different cell types, it suffers from several
immanent limitations: The need to perform 3D confocal laser scanning for bead tracking and associated
photo-toxic effects limit temporal resolution and restrict the field of view when performing time-lapse
measurements. The need to take zero-force images at the end of each experiment complicates the
measurement further.
Here, we present a completely new approach to measure cellular forces which overcomes the
limitations of 2.5D TFM by interferometrically detecting deformations of an elastic probe rather than by
tracking bead displacement. The centrepiece of our innovation is a novel optical micro-cavity sensor that
enables fast force mapping across a large field of view by analysing changes in resonance wavelength.
Our approach avoids any phototoxic effects and therefore allows the measurement of cellular forces at
high frame rates over hours or days. Being based on wide-field imaging, our new method measures the
deformation at each point of the image simultaneously and with diffraction limited lateral resolution.
Vertical displacements are detected with accuracy far beyond conventional confocal microscopy (5 nm
or better). Force maps can be recorded without the need for zero-force images, increasing throughput,
eliminating the need to detach non-migrating cells after force mapping and allowing measurements of
multiple cells on one substrate. Additionally, the optics needed for the readout of the new biosensor can
be readily integrated with a conventional inverted microscope.
In this presentation we will discuss the fabrication of our micro-cavity sensors and provide detailed
investigations of force and spatial resolution of the device by means of AFM indentation analysis. Cell
mechanical measurements of different cell lines, including human neuronal cells and primary immune
cells, will be presented and links between recorded force patterns and subcellular structures labelled by
fluorescence staining will be discussed.
MONDAY-ORAL PRESENTATIONS
M79
Optogenetic control of symmetry breaking in Saccharomyces cerevisiae.
K. Witte1, D. Strickland1, M. Glotzer1;
1
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL
Polarization induces the shift from molecular symmetry to asymmetry and is critical to many cellular
events including embryogenesis, motility, and cytokinesis. Polarization is inherently dynamic thereby
enabling rapid responses to spatial and temporal cues. Optogenetic tools allow functional probing of
cellular polarization and spatial control of signaling molecules. Here, we use the optogenetic tool TULIPs
to investigate one of the canonical polarization systems: bud site selection in Saccharomyces cerevisiae.
While wild type cells use landmark directed cues to define the polarization axis, cells lacking these
spatial cues can spontaneously define an axis in a process known as symmetry breaking. The current
model of symmetry breaking suggests that a stochastic accumulation of the active form of the Rho
family GTPase, Cdc42, induces a positive feedback loop mediated by a polarity complex containing the
Cdc42 GEF, Cdc24, the p21-activated kinase, Cla4, and a scaffold protein, Bem1. Though this model is
consistent with a large body of data, it relies on a number of assumptions and it has not been directly
tested.
Using TULIPs, we found that local accumulation of either Bem1 or Cdc24 is sufficient to induce
recruitment of polarity regulators and effectors, and to promote bud emergence. The ability of Cdc24 to
induce polarization depends on its GEF activity. These results are consistent with existing models.
However, we find that the polarity proteins do not always function in a potent positive feedback loop.
Specifically, membrane recruitment of Cdc24 induces Cdc42 activation early in the cell cycle, but Bem1
does not accumulate until late G1. Although Bem1 recruitment can induce Cdc42 activation in late G1, it
is unable to to do so during early G1. These results indicate that Cdc42 activation does not invariably
induce Bem1-mediated positive feedback. Furthermore, we demonstrate that the switch to Bem1mediated positive feedback activation requires Cdk1 kinase activity.
Optogenetics allows facile control of competing sites of polarization. We find that Cdc24-generated sites
in early G1 can be self sustaining, but that they are readily squelched by a second site in the same cell.
Conversely, in late G1, once Bem1 has been recruited, a single site of Cdc24 recruitment cannot
outcompete an existing site. Collectively, our study reveals unexpected behaviors of the yeast Cdc42
activation module and demonstrates the utility of optogenetics in dissecting cellular signaling.
MONDAY-ORAL PRESENTATIONS
M80
Super-active TALEN with improved stability at 37 degree Celsius enables highly
efficient and homogeneous gene knockout in mammalian embryos.
K. Ikeda1, Y. Terahara1, K. Sumiyama1, N. Miyashita1, Y. Okada1;
1
RIKEN Quantitative Biology Center (QBiC), Osaka, Japan
Gene editing in vivo has become possible by the development of artificial nucleases that can be
designed to cut the genome DNA selectively at the target site in the genome. The DNA binding domain
of a plant pathogen protein called Transcription Activator-Like Effector (TALE) has been used as the
designable sequence specific DNA-binding domain. The fusion protein of TALE and the nuclease domain
of Fok I endonuclease, called TALE nuclease or TALEN, has been proven to be useful for the genome
editing in lower vertebrates such as zebrafish and Xenopus. In mammalian cells and embyos, however,
TALEN often shows poor activity, which limited its applications. We have surmised that the TALE protein
might not be stable at 37 oC, because the physiological temperature for the plant pathogen is around 25
o
C. Through the analysis of the crystal structure of TALE protein, we have chosen two amino acid
residues that might be pivotal for the stable alignment of the linear solenoid structure of TALE. Several
mutations were introduced, and some successfully showed significantly higher activity at 37 oC both in
vitro and in vivo. All-atom molecular dynamics simulations confirmed the stabilization of the
conformation. The mutated residues apparently suppressed the intramolecular fluctuations. We have,
therefore, named this mutant TALEN as “super-active” TALEN and examined its activity in mouse
embryos. We have designed TALENs, both wild type and super-active mutant, for the mouse tyrosinase
gene. The mRNAs for the TALENs were introduced to the pronuclear stage mouse embryos. The wild
type TALENs failed to introduce mutations, and the coat color of the babies were agouti. Contrastingly,
more than half of the babies were albino with super-active TALENs, which means both alleles of the
tyrosinase gene were mutated throughout the body. Interestingly, CRISPR/Cas9 mediated gene editing
of tyrosinase gene in mouse pronuclear stage embryo yielded even higher rate of coat color mutants.
However, the babies were mostly mosaic of agouti and white, indicating that only some population of
cells got biallelic mutation. Consistently, the genome analyses detected only less than four different
alleles in each baby with super-active TALEN, while more than eight alleles were often detected in a
single CRISPR/Cas9 baby. These results suggest that super-active TALEN shows its activity at two-cell
stage, earlier than CRISPR/Cas9, suggesting that super-active TALEN might serve as an effective tool for
the genome editing in mammalian cells and embryos.
MONDAY-ORAL PRESENTATIONS
M81
Reconstructing alveolar epithelial development using single-cell RNA-seq.
D.G. Brownfield1,2,3, B. Treutlein4, M. Krasnow5, S. Quake4, H. Espinoza1, T. Desai6, A. Wu4, N.F. Neff7, G.
Mantalas4;
1
Biochemistry, Stanford University, Stanford, CA, 2Life Sciences Division, Lawrence Berkeley National
Laboratory, Berkeley, CA, 3Department of Bioengineering, University of California, Berkeley, Berkeley,
CA, 4Bioengineering, Stanford University, Stanford, CA, 5Stanford Univ Sch Med/HHMI, Stanford, CA,
6
Internal Medicine, Stanford School of Medicine, Stanford, CA, 7Stanford Univ Sch Med, Stanford, CA
Although aspects of alveolar development have been studied by marker expression analysis and fatemapping, the mechanisms that control the progression of lung progenitors along distinct lineages into
mature alveolar cell types remain incompletely known, in part because of the limited number of lineage
markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on
cell populations. With single-cell transcriptome analysis we were able to directly measure the various
cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNAseq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the
transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung
epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide
approach that did not require a priori knowledge of the underlying cell types or the previous purification
of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type
diversity in the distal lung and led to the discovery of many previously unknown cell-type markers,
including transcriptional regulators that discriminate between the different populations. We
reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar
lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics
approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell
types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.
Minisymposium 9: Optical Microscopy and Superresolution Imaging
M82
Molecular insights into the regulation of T cell signalling with single molecule
localisation microscopy.
K. Gaus1;
1
ARC Centre of Excellence in Advanced Molecular Imaging, University of New South Wales, Sydney,
Australia
T cell activation begins with the formation of signalling complexes at the cell surface involving the T cell
antigen receptor (TCR), the Src family kinase Lck and the adaptor protein, linker for activation of T cells
(LAT). How early TCR signalling events are regulated to prevent inopportune signalling in resting T cells
MONDAY-ORAL PRESENTATIONS
but efficient activation upon receptor ligation is poorly understood. We have established single
molecule localization microscopy to determine how TCR engagement reorganizes signalling proteins on
the molecular scale. Imaging single molecules in intact cells has provided new insights into the
mechanisms of Lck clustering (Rossy et al. Nat Immunol 2013) and LAT recruitment (Williamson et al.
Nat Immunol 2011) upon TCR activation. We are now extending this work to elucidate how the
membrane environment (Owen et al. Nat Commun 2012) and topography (Owen et al. Biophys J 2013)
affects protein interactions to better understand the molecular principles of TCR signalling regulation.
M83
Red fluorescent proteins: chromophore formation and advanced applications.
V.V. Verkhusha1;
1
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY
In the past few years a number of red-shifted fluorescent proteins (RFPs) that emit orange, red and farred fluorescence has been developed. These advanced RFPs provide new possibilities to study biological
processes at the levels ranging from single molecules to whole organisms. We describe the relationship
between the properties of RFPs of different phenotypes and their applications to various fluorescence
imaging techniques. Existing and emerging imaging approaches are discussed for conventional RFPs, farred FPs, RFPs with a large Stokes shift, irreversibly photoactivatable and reversibly photoswitchable
RFPs. The progress in the development of RFPs has been accompanied with detailed studies of
chromophore chemistry. An understanding of the molecular mechanisms involved in the chromophore
formation and phototransformation enables scientists to design RFPs with the desired properties to
advance imaging applications.
M84
iPALM and FRET reveal the mechanism of vinculin activation and nano-scale
spatial organization in focal adhesions.
L. Case1, M. Baird2, G. Shtengel3, S. Campbell4, H. Hess3, M.W. Davidson5, C. Waterman6;
1
Cell Biology and Physiology Center, NHLBI/NIH, Bethesda, MD, 2Florida State University, Tallahassee, FL,
3
Janelia Farm, HHMI, Ashburn, VA, 4Biochemistry and Biophysics, UNC, Chapel Hill, NC, 5National High
Magnetic Field Laboratory and Department of Biological Science, Florida State University, Tallahassee,
FL, 6National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD
Focal adhesions (FAs) are multi-protein complexes that link the extracellular matrix (ECM) to the actin
cytoskeleton to mediate cell adhesion, migration, mechanosensing and signaling. Previously,
interfermoteric photoactivatable localization microscopy (“iPALM”), which uses single molecule
localization to measure lateral position with ~20nm accuracy and interferometry to measure the axial
position with ~10nm accuracy, revealed that FAs have a conserved laminar structure, with an integrin
signaling layer (ISL) ~10-20 nm from the plasma membrane, an actin regulatory layer (ARL) ~100 nm
MONDAY-ORAL PRESENTATIONS
from the membrane that extends into the stress fiber, and a force transduction layer (FTL) that spans
the intervening space. However, it is unknown how the layered organization of proteins within focal
adhesions contributes to the regulation of protein activity and function. Vinculin (Vcl) is a protein that
undergoes a conformational change at focal adhesions to interact with multiple binding partners and
regulate diverse cellular functions including protrusion, migration, mechanosensing and survival. Vcl has
over 10 binding partners distributed throughout the FA including paxillin in the ISL, talin (Tln) in the FTL,
and actin in the ARL. We hypothesized that the spatial compartmentalization of different binding
partners into distinct FA layers contributes to Vcl activation, regulation and functional specificity at focal
adhesions. To test this, we used point mutants to perturb specific protein interactions and assayed Vcl
nanoscale localization and Vcl activation state at FAs. We used iPALM to image the 3D localization of
vinculin molecules with nanometer accuracy as well as a FRET biosensor to assay vinculin
conformational changes. By systematically perturbing specific protein interactions, we found that
vinculin is recruited to the ISL in an inactive conformation by its interaction with paxillin. Subsequently,
vinculin activation is mediated by talin and actin binding, and talin binding promotes vinculin association
with the higher FTL and ARL. Active vinculin localizes ~10nm higher than inactive vinculin in FA. Vcl
exhibits a gradient of low-to-high activation from the front-to-back of FA, and Vcl exhibits increased
localization with the ARL in the back of FA. Finally, while Vcl is not required for the proper nanoscale
localization of paxillin or actin, Vcl is required for Tln to be maintained in the maximally vertically
extended conformation. By performing complimentary experiments with iPALM and FRET, we have
been able to study the mechanism of vinculin activation on a structural level in a cellular setting. We
propose that movement up the laminar FA structure facilitates Vcl activation and mechanical
reinforcement of FA.
M85
Single-molecule tracking of Smoothened reveals binding in the primary cilium
that is altered by pathway agonists.
L.E. Weiss1, L. Milenkovic2, J. Yoon1, S.J. Sahl1, M.P. Scott2, W.E. Moerner1;
1
Chemistry, Stanford University, Stanford, CA, 2Developmental Biology, Stanford University, Stanford, CA
Hedgehog (Hh) signaling plays an essential role in cell division and differentiation in embryonic and adult
stem cells. Disruption of the pathway can often lead to fatal defects in embryos, as well as a wide variety
of cancer types later in life. The pathway is initially switched on by the binding of the Hh ligand to the
transmembrane protein Patched. Shortly after, anther transmembrane protein, Smoothened (Smo), is
derepressed and relocalizes to a small microtubule-structured organelle that protrudes from the cell's
surface called the primary cilium. Once there, it influences members of the Gli family of transcription
factors that then upregulate target genes in the nucleus. Although the ciliary structure is known to be
very complex, standard fluorescence microscopy measurements have been hindered by the small
dimensions (~400 nanometers in diameter and 2-5 microns long) which are on the order of the
diffraction limit. To overcome this barrier, we have used highly sensitive, single-molecule microscopy to
obtain the trajectories of individual Smo proteins on the surface of cilia with high temporal and spatial
MONDAY-ORAL PRESENTATIONS
resolution (10 millisecond and 30 nanometers, respectively). By analyzing their movements, we have
observed three distinctive modes of motion: diffusion, directed motion, and binding, the last of which is
altered in the presence of natural and synthetic small-molecule agonists.
M86
Multi-protein assemblies underlie the mesoscale organization of the plasma
membrane.
S.K. Saka1, A. Honigmann2, C. Eggeling3, S.W. Hell2, T. Lang4, S.O. Rizzoli1;
1
Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Goettingen,
Germany, 2Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry,
Goettingen, Germany, 3MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine,
Oxford, UK, 4Department of Membrane Biochemistry, Life and Medical Sciences (LIMES) Institute, Bonn,
Germany
Most proteins have uneven distributions in the plasma membrane (Lang and Rizzoli, Physiology, 2010).
This may be caused by mechanisms specific to each protein, or may be a consequence of a general
pattern that affects the distribution of all membrane proteins. To find out if a general mechanism exists
behind this observation, we introduced several imaging approaches that aim to investigate all proteins
in the plasma membrane simultaneously rather than focusing on interactions of individual protein
species. This was achieved by large-scale metabolic labeling of proteins in mammalian cells through
extended incorporation of non-canonical amino acid analogues, followed by fluorescent tagging via click
chemistry (Dieterich et al., Nature Neuroscience, 2010). By combining this direct labeling method with
super-resolution stimulated emission depletion (STED) microscopy, we studied the protein patterning in
plasma membranes of living cells, as well as in membrane sheets. We found that a general mosaic-like
pattern governs the proteins organization (Saka et al., Nature Communications, 2014). Multiple proteins
were heterogeneously gathered into protein-rich domains surrounded by a protein-poor background.
We termed these long-lived high-abundance domains “protein assemblies” and examined the
contributions of different factors to their formation and maintenance. We identified cholesterol as the
main organizer of the assembly pattern and the actin cytoskeleton as a secondary factor that borders
and separates the assemblies. To understand the relation of this mesoscale arrangement to the
nanoclusters of individual protein species, we analyzed distributions of specific proteins with respect to
the protein assemblies. All of the specific proteins we analyzed were enriched in the assemblies, but
they displayed differential enrichment profiles. Many proteins were preferentially located in particular
areas within the assemblies, such as their edges or centers. Functionally related protein groups showed
similar preferences, suggesting that functional protein-protein interactions create specialized
subdomains within the assemblies. We conclude that the assemblies constitute a fundamental principle
of the mesoscale membrane organization, which affects the nanoscale patterning of most membrane
proteins, and possibly also their activity.
MONDAY-ORAL PRESENTATIONS
M87
Video-rate super resolution microscopy in living cells.
F. Huang1, C.E. Laplante2, Y. Lin3, T.D. Pollard1,2,4, J. Bewersdorf1;
1
Department of Cell Biology, Yale University School of Medicine, New Haven, CT, 2Department of
Molecular, Cellular and Developmental Biology, Yale University School of Medicine, New Haven, CT,
3
Department of Biomedical Engineering, Yale University School of Medicine, New Haven, CT,
4
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven,
CT
Super resolution microscopy based on single molecule localization relies on precise and accurate
localization of large numbers of single molecules. However, the necessity of accumulating large numbers
of localization estimates limits the time resolution typically to seconds to minutes1,2.
Two major limitations are the acquisition speed and the photon budget. An EMCCD camera is usually
used in such experiments. Replacing it with a recently introduced sCMOS camera results in leaps in both
acquisition speed and effective quantum efficiency. However, the intrinsic pixel-dependent Gaussian
noise of the sCMOS cameras introduces localization artifacts and greatly reduces the reliability of the
results.
Here, we present a set of specially designed statistics-based algorithms that characterize an sCMOS
camera for the first time and allow for unbiased and precise localization analysis. Using this method we
demonstrate Cramer-Rao lower bound-limited single molecule localization with an sCMOS camera.
Combining the novel algorithm with a recently developed multi-emitter fitting algorithm3 and optimized
imaging condition, we show that this technique shortens the typical acquisition time for fixed samples
by up to two orders of magnitude without compromising the field of view. Furthermore, we
demonstrate localization-based super-resolution microscopy in live cells by monitoring dynamics of
protein clusters, vesicles and organelles at a temporal resolution from 2 to 30 frames per second4.
These methods allowed us to investigate cytokinetic apparatus in live fission yeast at 20-30 nm
resolution. In general, the significantly improved temporal resolution allows super resolution imaging of
a large range of dynamic events in living cells.
Patterson, G., Davidson, M., Manley, S. & Lippincott-Schwartz, J. Superresolution imaging using singlemolecule localization. Annu. Rev. Phys. Chem. 61, 345–67 (2010).
Gould, T. J., Hess, S. T. & Bewersdorf, J. Optical nanoscopy: from acquisition to analysis. Annu. Rev.
Biomed. Eng. 14, 231–54 (2012).
Huang, F., Schwartz, S. L., Byars, J. M. & Lidke, K. A. Simultaneous multiple-emitter fitting for single
molecule super-resolution imaging. Biomed. Opt. Express 2, 1377–93 (2011).
Huang, F. et al. Video-rate nanoscopy using sCMOS camera-specific single-molecule localization
algorithms. Nat. Methods 10, 653–8 (2013).
MONDAY-ORAL PRESENTATIONS
M88
Continuous throughput and long-term observation of single-molecule FRET
without immobilization.
S. Tyagi1, V. Vandelinder2, N. Banterle1, G. Fuertes1, S. Milles 1, M. Agez1, E. Lemke1;
1
Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg,
Germany, 2Sandia National Laboratories, Albuquerque, NM
Single-molecule FRET (smFRET) studies through Total internal reflection fluorescence microscopy
(TIRFM) is a powerful method to extract and reveal hidden heterogeneity in biological systems from
long-term FRET trajectories of individual single molecules. TIRFM popularity also owes to the possibility
of adding exogeneous reagent and following the biological processes over long-time. The high signal to
noise (SNR) necessary for observing fluorescence from single emitters is achieved by tethering single
molecules within the shallow evanescent field (less than 200 nm) generated at the interface of coverslip
and buffer. This requirement of tethering molecules to coverslip restricts the applicability of TIRFM to a
limited set of biomolecules as complex immobilization procedures cannot be generally applied and
sometimes perturb the native state of the biological sample. Here we present a versatile technology,
termed “SWIFT” (Single molecules Without Immobilization For TIRFM) that enables us to perform
multisecond observation of freely diffusing single molecules with high SNR while bypassing the need for
immobilization procedures. SWIFT is a microfluidic-nanofluidic hybrid platform where nanochannels are
actively and reversibly generated by collapsing 1 µm deep flow channels using pressurized nitrogen gas
in a control channel, which additionally increases photo stability of fluorophores by removing oxygen.
Biomolecules continuously flow through 12 parallel 100 nm deep channels, which keep them within the
evanescent field. The microfluidic device is fabricated with soft lithography and molding techniques out
of an elastomeric material, which enables to exploit the full potential of microfluidics including low-cost,
ease of use and fast solution exchange. We show that several second long trajectories of thousands of
molecules can be recorded with millisecond time resolution, illustrating the potential for long-term
automated and high throughput experiments. We demonstrate the power of our method by studying a
variety of complex nucleic acid and protein systems, including DNA, Holliday junctions, nucleosomes and
human transglutaminase 2.
Reference:
Tyagi, S., Vandelinder, V., et al. Nature Methods (2014)
MONDAY-ORAL PRESENTATIONS
M89
Lattice light sheet microscopy: imaging molecules, cells, and embryos at high
spatiotemporal resolution.
W.R. Legant1, B. Chen2, K. Wang1, L. Shao1, E. Betzig1;
1
HHMI/Janelia Farm Research Campus, Ashburn, VA, 2Research Center for Applied Sciences, Academia
Sinica, Taipei, Taiwan
By definition, living specimens are animate. Therefore, a full understanding of dynamic biological
systems will only be obtained by observing them with enough 4D spatio-temporal resolution and for a
sufficient duration, to capture the phenomena of interest. Unfortunately, conventional widefield or
confocal microscopes are either too slow, lack the spatial resolution, or induce too much photodamage
to meet these requirements. To address these limitations, we have developed a new approach for subcellular light-sheet microscopy capable of imaging fast three-dimensional (3D) dynamic processes in vivo
at signal to noise levels typically obtained only in total internal reflection fluorescence (TIRF)
illumination. By utilizing 2D optical lattices, we generate a thin (~400 nm full width half maximum)
plane of light coincident with the focus of a high NA detection objective. Using this technique, we
demonstrate substantial advantages in speed, sensitivity and reduced phototoxicity compared to
conventional point scanning and spinning disc confocal microscopes as well as light-sheet microscopes
utilizing single Gaussian or Bessel beams. We leverage these advantages to image samples ranging over
three orders of magnitude in length scale from single molecules to whole embryos. Specific examples to
be presented include: 3D single molecule tracking of fluorescently tagged transcription factors in
densely labeled embryonic stem cell spheroids, 3D imaging and quantification of microtubule growth
phases and organelle dynamics throughout the course of cell division, rapid 3D imaging of Tetrahymena
motility at over 3 volumes per second, and 3D protein localization throughout the course of dorsal
closure in Drosophila embryos. By combining lattice light sheet microscopy with novel fluorescent
probes, we will also demonstrate 3D super-resolution localization microscopy with unprecedentedly rich
detail over large fields of view and in thick 3D samples such as dividing cells and small embryos.
M90
Novel spectral imaging and analysis to unravel the organelle interactome.
A.M. Valm1, S. Cohen1, W.R. Legant2, M.W. Davidson3, E. Betzig2, J. Lippincott-Schwartz4,5;
1
Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and
Human Development, NIH, Bethesda, MD, 2HHMI/Janelia Farm Research Campus, Ashburn, VA,
3
National High Magnetic Field Laboratory and Department of Biological Science, Florida State University,
Tallahassee, FL, 4Physiology Course at Marine Biological Laboratory, Woods Hole, MA, 5Cell Biology and
Metabolism Program, NICHD, NIH, Bethesda, MD
The organization of the eukaryotic cell into membrane-bound compartments allows for regulation of
cellular processes. However, the morphodynamic activities of spatially distinct organelles must be
coordinated so that cellular processes can be carried out. Inter-organelle membrane contacts have
MONDAY-ORAL PRESENTATIONS
emerged as key sites for exchange of metabolites and signaling molecules, as well as playing roles in
organelle division and maturation. Much progress has been made recently in elucidating the functions of
pairwise organelle interactions, including the interaction between mitochondria and the endoplasmic
reticulum (ER). However, it is likely that important cellular activities are coordinated between three or
more organelles over time. For example, lipid droplets have been shown to interact with multiple
organelles, including the ER, mitochondria, peroxisomes and the endocytic system, presumably in order
to facilitate exchange of lipids in response to changing metabolic needs. A systems-level understanding
of the dynamic organelle interactions within eukaryotic cells, i.e., the organelle interactome, remains
unattained due to the inability to label and distinguish more than a few different fluorescent proteins in
a single sample. Here, we present a novel cell labeling, image acquisition and analysis approach to study
the spatial distribution of multiple organelles within single eukaryotic cells. Cells are transfected with
multiple fluorescent fusion proteins targeted to various organelles or with organelle-specific fluorescent
chemical dyes to label 6 or more different subcellular compartments. Live-cell, time-lapse images are
acquired of labeled cells using commercially available imaging systems, then linear unmixing algorithms
are applied to the recorded image data to deconvolve spectrally overlapping fluorophores. A
computational image analysis procedure is then used to track the observed inter-organelle contacts
through time. We further adapted a lattice light sheet imaging system for spectral imaging by
introducing multiple laser lines coupled with fast acousto-optical beam splitting to record spectral
images in the excitation regime. We developed a novel excitation-side unmixing algorithm and applied it
to the recorded data to reconstruct full isotropic three dimensional time lapse images of live cells
labeled with 6 different organelle markers. We imaged cells that simultaneously express fluorescent
fusion protein markers for peroxisomes, lysosomes, mitochondria, the ER and the Golgi apparatus, as
well as a vital chemical stain to label lipid droplets. We applied our organelle interactome analysis to
test the hypothesis that there are subpopulations of lipid droplets within mammalian cells that differ in
their organelle interactions.
Minisymposium 10: Organelle-Based Pathways in Cellular
Homeostasis and Signaling
M91
Autophagosome Formation and the role of WIPI2.
S. Tooze1, H. Dooley1, M. Razi1, M. Wilson2;
1
Cancer Research UK, London Research Institute, London, England, 2Babraham Institute, Cambridge, UK
Mammalian cell homeostasis during starvation depends on initiation of autophagy by endoplasmic
reticulum- localized phosphatidylinositol 3-phosphate (PtdIns(3)P) synthesis. Formation of doublemembrane autophagosomes that engulf cytosolic components requires the LC3-conjugating Atg12–516L1 complex. The molecular mechanisms of Atg12– 5-16L1 recruitment and significance of PtdIns(3)P
synthesis at autophagosome formation sites are unknown. By identifying interacting partners of WIPIs,
WD-repeat PtdIns(3)P effector proteins, we found that Atg16L1 directly binds WIPI2b. Mutation
MONDAY-ORAL PRESENTATIONS
experiments and ectopic localization of WIPI2b to plasma membrane show that WIPI2b is a PtdIns(3)P
effector upstream of Atg16L1 and is required for LC3 conjugation and starvation-induced autophagy
through recruitment of the Atg12–5-16L1 complex. Atg16L1 mutants, which do not bind WIPI2b but
bind FIP200, cannot rescue starvation-induced autophagy in Atg16L1-deficient MEFs. WIPI2b is also
required for autophagic clearance of pathogenic bacteria. WIPI2b binds the membrane surrounding
Salmonella and recruits the Atg12–5-16L1 complex, initiating LC3 conjugation, autophagosomal
membrane formation, and engulfment of Salmonella.
M92
TRIM family proteins regulate selective autophagy and act as a new class of
autophagic receptors.
M. Mandell1, T. Kimura1, V. Deretic1;
1
Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, NM
Tripartite-motif containing proteins (TRIMs) constitute a large family with effects on cell cycle control,
innate immunity, inflammation, and antiviral defense. We screened the TRIM family for roles in
autophagy and found that more than half of TRIMs modulated autophagy. For most TRIMs tested to
date (TRIM5α, TRIM6, TRIM17, TRIM22, and TRIM49), this is accomplished by TRIMs serving as
platforms for the assembly of autophagy initiation factors ULK1 and Beclin 1 in their activated states,
although TRIMs such as TRIM55 also employ additional and/or alternative mechanisms to promote
autophagy induction. We next considered whether TRIMs played additional roles as selective autophagy
receptors, this being of interest due to a present dearth of the known selective autophagy receptors in
mammalian cells. All TRIMs tested interacted with mammalian Atg8s (mAtg8s), a class of proteins
functionally associated with autophagosome membranes. To find a putative autophagic cargo for a
TRIM, we used TRIM5α, as it has a well-known physiologically relevant target: retroviral capsid. We
found that knock-down of autophagy factors in rhesus cells increased retroviral infection with a virus
(HIV) known to be restricted by rhesus TRIM5α but had no effect on a retrovirus (SIV) not recognized by
rhesus TRIM5α. We furthermore found that autophagic degradation of retroviral capsid by TRIM5α
required TRIM5α’s binding to mAtg8s, thus establishing TRIM5α as an autophagic receptor. Whereas the
target of TRIM5α is an exogenous protein (retroviral capsid), we have identified several endogenous
proteins that interact with and are delivered for autophagic degradation by specific TRIMs. Thus, the
function of TRIMs as autophagic receptor—regulators is conserved among the family. Based on our
findings and the sheer number of TRIMs (>70 genes in the human genome), we propose that TRIMs
direct and conduct selective autophagy of diverse substrates in metazoan cells.
MONDAY-ORAL PRESENTATIONS
M93
Molecular mechanisms of nutrient sensing at the lysosome.
B. Castellano1, R. van der Welle1, R. Lawrence1, R. Zoncu1;
1
Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA
The lysosome is emerging as a key player in nutrient sensing and metabolic regulation, due in part to its
ability to govern the activity of the master growth regulatory kinase, mechanistic Target of Rapamycin
Complex 1 (mTORC1). Through mechanisms that are only partly understood, amino acids within the
lysosomal lumen induce the recruitment of mTORC1 to the lysosomal surface, where mTORC1 becomes
activated. Combining imaging approach in live cells with biochemical assays in vitro, we are investigating
how amino acid signals originating within the lysosome lead to activation of the mTORC1 scaffolding
complex, composed of the Rag GTPases and Ragulator, on the cytoplasmic face of this organelle. We
have identified specific domains and residues within the vacuolar H+ ATPase (V-ATPase) that may endow
this enzyme with the ability to sense amino acids. We are presently investigating how amino acidinduced conformational changes within the V-ATPase are communicated to the other components of
the mTORC1 scaffolding complex, and how the catalytic activity of the V-ATPase may specify the timing
and strength of amino acid-induced mTORC1 signaling.
M94
Wilson disease protein ATP7B utilizes lysosomal exocytosis to maintain copper
homeostasis.
E. Polishchuk1, M. Concilli1, S. Iacobacci1, G. Chesi1, N. Pastore1, P. Piccolo1, S. Paladino2, S. van
IJzendoorn3, J. Chan4, C. Chang4, A. Amoresano2, F. Pane2, P. Pucci2, A. Tarallo1, G. Parenti1, N. BrunettiPierri1, C. Settembre1, A. Ballabio1, R. Polishchuk1;
1
Telethon Institute of Genetics and Medicine, Naples, Italy, 2Federico II University, Naples, Italy,
3
University Medical Center, Groningen, Netherlands, 4Howard Hughes Medical Institute, University of
California, Berkeley, CA
Copper is an essential yet toxic metal as its overload causes Wilson disease, a genetic liver disorder due
to mutations in copper transporter ATP7B. To remove excess copper into the bile ATP7B traffics towards
canalicular/apical area of hepatocytes. However the trafficking mechanisms of ATP7B remain poorly
understood. Here we show that in response to elevated copper ATP7B moves from the Golgi to
lysosomes and imports metal into their lumen. ATP7B also enables lysosomes to undergo apical
exocytosis and therefore to release stored copper into bile. This exocytic process is triggered by the
copper-dependent interaction between ATP7B and p62 subunit of dynactin that allows lysosomes to
move along the microtubule tracks towards the canalicular pole of hepatocytes. Transcriptional
activation of lysosomal exocytosis significantly increases ATP7B delivery to the canalicular membrane
and copper clearance from the hepatocytes and allows rescue of the most frequent Wilson diseasecausing ATP7B mutant to appropriate functional site. Our findings indicate that lysosomes serve as an
important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral
MONDAY-ORAL PRESENTATIONS
process in copper excretion and hence can be targeted for novel therapeutic approaches to combat
Wilson disease.
M95
The leukodystrophy protein FAM126A/Hyccin regulates PI4P synthesis at the
plasma membrane.
J.M. Baskin1, X. Wu2, R. Christiano2, M. Oh3, E. Gazzerro4, S. Assereto4, C. Minetto4, M. Simons5, T.C.
Walther2, K.M. Reinisch2, P. De Camilli1;
1
Department of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience,
Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT, 2Department of Cell
Biology, Yale University School of Medicine, New Haven, CT, 3Dept Cell Biology, Yale University School of
Medicine/HHMI, New Haven, CT, 4Giannina Gaslini Institute, Genova, Italy, 5Max Planck Institute for
Experimental Medicine, Göttingen, Germany
Myelin comprises concentric, tightly apposed layers of lipid-rich glial cell plasma membranes that
surround, insulate, and trophically support axons in the vertebrate nervous system. Genetic defects in
myelin formation and maintenance cause leukodystrophies, a large, heterogeneous group of brain white
matter diseases whose mechanistic underpinnings are poorly understood. Hypomyelination and
Congenital Cataract (HCC), one of these disorders, is caused by mutations in the FAM126A gene, which
encodes FAM126A/hyccin, a protein of previously unknown function. Here, we show that FAM126A
regulates the synthesis of phosphatidylinositol 4-phosphate (PI4P), a key determinant of plasma
membrane identity. We demonstrate that FAM126A is an intrinsic component of the
phosphatidylinositol 4-kinase complex at the plasma membrane, known to comprise PI 4-kinase IIIα
(PI4KIIIα) and two non-catalytic subunits, the peripheral membrane protein EFR3 and the putative
scaffold TTC7. FAM126A directly binds to TTC7. A crystal structure of the FAM126A–TTC7 complex
reveals an almost all-α-helical heterodimer with an unusually large protein-protein interface consistent
with a high-affinity interaction and a conserved surface that may mediate binding to the kinase.
Functional analysis of fibroblasts from HCC patients reveals reduced plasma membrane PI4P levels
relative to controls. We propose that defects in PI4KIIIα-mediated PI4P production in oligodendrocytes
may be involved in HCC disease pathogenesis. The specialized function of oligodendrocytes in myelin
formation is mediated by their property to massively expand their plasma membrane, and this property
is highly dependent on the generation of PI4P and its downstream metabolites PI(4,5)P2 and PI(3,4,5)P3.
We find that FAM126A is highly expressed in oligodendrocytes, and studies of FAM126A knockout mice
support a primary role of this protein in such cells. Thus, our results imply a critical role of FAM126A and
PI4P generation in supporting myelin formation, a process of essential importance not only in
development but also during remyelination following injury, as occurs in multiple sclerosis and other
demyelinating diseases.
MONDAY-ORAL PRESENTATIONS
M96
Regulated by mitochondrial fusion dynamics, lipid droplets serve as central fatty
acid conduit to supply mitochondria with fatty acids for oxidation during nutrient
stress.
A. Rambold1, S. Cohen2, J. Lippincott-Schwartz2;
1
Max-Planck-Institute-IE, Freiburg, Germany, 2Cell Biology and Metabolism Program, Eunice Kennedy
Shriver National Institute of Child Health and Human Development, NIH, Bethesda, MD
Cells must adapt their metabolism to changing nutrient and environmental conditions in order to
survive. One key adaptation occurs during nutrient deprivation when cells reprogram their metabolism
from glucose to mitochondrial fatty acid oxidation for maintaining cellular energy levels. Although it is
well known that endogenous fatty acids act as major energy substrate during nutrient stress, from
where and how such fatty acids are mobilized and selectively trafficked to mitochondria without causing
damage to membranes and acting as toxic bioactive lipids is a major question. Using a novel imaging
based approach to visualize fatty acid trafficking in live cells in combination with metabolic profiling
experiments, we revealed that lipid droplets, specialized lipid storage organelles, serve as selective
conduit to direct fatty acids into mitochondria for oxidation. We found that fatty acid recycling by bulk
autophagy of cellular membranes helped replenish lipid droplets with fatty acids, increasing lipid droplet
number over time. From lipid droplets, fatty acids were subsequently liberated by cytoplasmic lipases,
and efficiently transferred into mitochondria. Surprisingly, we found that this transfer step required
mitochondria to both be localized near lipid droplets and highly fused. Mitochondrial fusion increased
the connectivity to lipid droplets and promoted mitochondrial fatty acid distribution and oxidation.
When mitochondrial fusion was prevented in cells lacking the major fusion proteins mitofusin 1 or optic
atrophy protein 1, fatty acids neither homogeneously distributed within mitochondria nor became
efficiently metabolized. Instead, these morphodynamic defects caused massive alterations in cellular
fatty acid routing. Not only were non-metabolized fatty acids redirected to and stored in lipid droplets,
they became excessively expulsed from cells. Together this study highlights the importance of
coordinated organelle dynamics to direct cellular fatty acid flow and oxidation during nutrient stress.
Given that fatty acids and their derivatives play important roles in regulating cellular signaling cascades
in metabolism and inflammatory responses, future work studying the multi-organelle controlled fatty
acid flux system described here might be of high potential for contributing to our understanding of the
pathophysiology of lipid-associated diseases, such as obesity, diabetes, cancer and inflammatory
disorders.
MONDAY-ORAL PRESENTATIONS
M97
Glucose Regulates Mitochondrial Motility via Milton Modification by O-GlcNAc
Transferase.
G. Pekkurnaz1, J.C. Trinidad2, X. Wang3, D. Kong4, T.L. Schwarz1,5;
1
The F.M. Kirby Neurobiology Center, Boston Children's Hospital, Boston, MA, 2Department of
Chemistry, Indiana University, Bloomington, IN, 3Department of Neurosurgery, Stanford University, Palo
Alto, CA, 4Department of Neuroscience, Tufts University School of Medicine, Boston, MA, 5Department
of Neurobiology, Harvard Medical School, Boston, MA
Cellular homeostasis critically depends on the ability of cells to monitor nutrient levels that can be used
for ATP generation by mitochondria. Among the many specialized cell types, neurons are particularly
dependent on mitochondria due to their complex morphology and regional energy needs. Hence,
abnormalities in mitochondrial distribution have been implicated in many neuropathologies including
Alzheimer’s, Parkinson’s, and Huntington’s diseases. Here, we report a molecular mechanism by which
nutrient availability in the form of extracellular glucose regulates mitochondrial motility in neurons. We
show that this mechanism requires the activity of O-GlcNAc Transferase (OGT), an enzyme whose
activity depends on glucose availability, and that activation of OGT diminishes mitochondrial motility.
We establish the mitochondrial motor adaptor protein Milton as a required substrate for OGT to arrest
mitochondrial motility by mapping and mutating the key O-GlcNAcylated serine residues. We find that
the O-GlcNAcylation state of Milton is altered by extracellular glucose, pharmacological inhibition of OGlcNAcase or UDP-GlcNAc synthesis, and that OGT alters mitochondrial motility in vivo. Our findings
suggest that, by dynamically regulating Milton O-GlcNAcylation, OGT tailors mitochondrial dynamics in
neurons based on nutrient availability. Although in this study we took advantage of the polarized and
regular arrays of microtubules in axons, the components of this pathway are present in all metazoan
cells, and glucose, via OGT, may therefore be a widespread regulator of mitochondrial movement.
M98
Variable stoichiometry among core ribosomal proteins.
N. Slavov1,2, A. van Oudenaarden3;
1
Massachusetts Institute of Technology, Cambridge, MA, 2Systems Biology, Harvard University,
Cambridge, MA, 3Hubrecht Institute, Utrecht, Netherlands
Understanding the regulation and structure of the eukaryotic ribosome is essential to understanding
protein synthesis and its deregulation in disease. While ribosomes are believed to have a fixed
stoichiometry among their core ribosomal proteins (RPs), some experiments suggest a more variable
composition. Reconciling these views requires direct and precise quantification of RPs. We used massspectrometry to directly quantify RPs across monosomes and polysomes of budding yeast and mouse
embryonic stem cells (ESC). Our data show that the stoichiometry among core RPs in wild-type yeast
cells and ESC depends both on the growth conditions and on the number of ribosomes bound per
mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to
MONDAY-ORAL PRESENTATIONS
the enrichment of the corresponding RP in ribosomes bound to multiple mRNAs. Together, these
findings support the existence of ribosomes with distinct protein composition and physiological
function.
http://biorxiv.org/content/early/2014/05/26/005553
M99
Atypical mitochondrial fragmentation upon Listeria infection.
F. Stavru1, P. Cossart1;
1
Cell Biology and Infection, UIBC Pasteur Institute, INSERM U604, INRA USC2020, Paris, France
Mitochondrial function is tightly connected to mitochondrial dynamics and a variety of physiological and
pathological conditions have been shown to induce either fusion or fission of the mitochondrial
network. Strikingly, functional mitochondrial dynamics appears to be important for Listeria infection. L.
monocytogenes rapidly causes a transient fragmentation of the mitochondrial network at early time
points of infection through its pore-forming toxin listeriolysin O (LLO). The rapid mitochondrial
fragmentation observed upon LLO treatment does not rely on a block in mitochondrial fusion, but rather
occurs through an activation of fission. In contrast to the classical mitochondrial fragmentation, the
Listeria- and LLO-induced fragmentation is Drp1 independent. However, as during the classical
fragmentation process, actin dynamics contributes significantly to LLO-induced mitochondrial
fragmentation. Interestingly, the mitochondrial fragmentation sites were marked by the ER in a Drp1independent manner, suggesting that molecules other than Drp1 can terminate mitochondrial
abscission downstream of the ER. Together, we have highlighted a novel mechanism of mitochondrial
fragmentation that will be further discussed during the presentation.
Minisymposium 11: Organization, Quality Control and Remodeling of
the Nuclear Boundary
M100
The budding yeast polo kinase, Cdc5, regulates domain-specific expansion of the
nuclear envelope.
O. Cohen-Fix1, A. Walters1, C. May1, E. Dauster1, B. Cinquin2, E. Smith2, C. Larabell2;
1
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health,
Bethesda, MD, 2Department of Anatomy, School of Medicine, University of California, San Francisco, San
Francisco, CA
The nuclear envelope (NE) is a double membrane that encircles the cell's chromosomes. In most
organisms, the NE breaks down during mitosis. It then reforms at the end of mitosis and expands during
interphase. Fungi, such as budding yeast, undergo closed mitosis, where the NE remains intact. In these
MONDAY-ORAL PRESENTATIONS
organisms the mitotic nucleus must elongate, often through NE expansion, to allow chromosome
segregation. Where examined, nuclear size increases proportionately with cells size, but how NE
expansion is controlled is not known. An interesting situation arises when budding yeast delay in
mitosis. We previously found that NE expansion continues unabated despite the block to chromosome
segregation. The nucleus, however, does not expand symmetrically. Rather, a sub-domain of the NE,
adjacent to the peripheral nucleolus, forms an extension, or flare, while the rest of the NE remains
adjacent to the DNA mass.
What confines NE expansion to the flare, and how is flare formation related to normal NE expansion? To
address this we screened for mutants that form a round, rather than flared, nucleus during a mitotic
delay. One such mutant was in the budding yeast polo kinase homolog, CDC5. While polo kinase is
known to have roles in mitosis, a role in NE expansion has yet to be described. Moreover, we found that
this function of Cdc5 is independent of its known roles in regulating mitotic progression. One can
imagine two ways by which a round mitotic nucleus can form: either NE expansion is blocked, or the NE
expands but it does so isometrically. To distinguish between these possibilities we quantified NE
expansion using soft X-ray tomography. We found that in the cdc5 mutant the NE expands isometrically,
suggesting that polo kinase is needed to confine NE expansion to a sub-domain of the NE, adjacent to
the nucleolus.
Finally, we examined whether Cdc5 plays a role in the spatial regulation of NE expansion during an
unperturbed cell cycle. We found that immediately prior to mitosis, nuclei form a "mini-flare" in the NE
adjacent to the nucleolus. The formation of this mini-flare is also dependent on Cdc5. We imagine that
during cell cycle progression, the rate of NE expansion may exceed the pace at which chromosomes
progress towards mitosis. Under these conditions, Cdc5 may phosphorylate one or more proteins that
sequester the excess membrane to the NE adjacent to the nucleolus, forming a mini-flare, thus
potentially avoiding a disruption to NE-associated processes that occur outside the nucleolus. If there
are no further perturbations to cell cycle progression the nucleus will elongate and the mini-flare will be
absorbed. If mitosis is delayed, the mini-flare will continue to expand, forming a flare. Thus, Cdc5
controls the asymmetric expansion of the NE.
M101
Mitotic control of the nuclear membrane.
M. Makarova1, S. Oliferenko1;
1
King's College of London, London, England
The nuclear membrane has to remodel during mitosis to accommodate the mitotic spindle assembly,
chromosome partitioning and formation of the daughter nuclei. This can be accomplished through a
variety of strategies, with “open” and “closed” modes of mitosis positioned at the opposite ends of the
spectrum. In closed mitosis, the nuclear envelope remains intact throughout nuclear division. During
open mitosis, the envelope of the original nucleus breaks down in prophase and reassembles around the
segregated daughter genomes. We have shown that within the fission yeast clade, the mitotic control of
MONDAY-ORAL PRESENTATIONS
the nuclear surface area may determine the choice between the nuclear envelope breakdown and a
fully closed division. I will present our recent work on the mechanistic basis of this divergence and argue
that comparative cell biology studies using two fission yeast species could provide unique insights into
physiology and evolution of mitosis.
M102
Atomic Scale Dynamic Behavior of the Nuclear Transport Selectivity Barrier.
L. Hough1, K. Dutta2, A. Kamal3, D. Temel4, S. Sparks4, J. Tetenbaum-Novatt3, D. Cowburn4, M. Rout5;
1
University of Colorado-Boulder, Boulder, CO, 2New York Structural Biology Center, New York, NY, 3The
Rockefeller University, New York, NY, 4Albert Einstein College of Medicine, Bronx, NY, 5Laboratory of
Cellular and Structural Biology, The Rockefeller University, New York, NY
The Nuclear Pore Complex (NPC) mediates all transport between the nucleus and cytoplasm. The
channel of the NPC is lined with “FG Nups”, a family of intrinsically disordered proteins characterized by
phenylalanine-glycine repeat motifs. FG nups form the exquisitely selective filter of the NPC; nonbinding proteins are excluded while the binding of transport factors to the FG Nups facilitates their
passage through the NPC. Like other intrinsically disordered proteins, the FG Nups appear to be very
sensitive to their environment, showing vastly different behavior in different experimental conditions; in
vitro, the observed behavior of the FG Nups varies from rigid gels to flexible random-coil polymers. By
combining NMR spectroscopy with experiments designed to recapitulate essential elements of
nucleocytoplasmic transport, we have provided a unique insight into the dynamic nature of NPC gating
at the atomic scale. We probed the behavior of model FG Nups of different types, within a variety of
environments and when bound to transport factors. A straightforward mechanism is suggested by our
data, without the need to invoke additional emergent features. Two key properties seem evident: first,
the particular IDP nature of the FG repeats in forming a barrier selecting against the passage of all but
transport factors; and second, the selective nature of high frequency transport factor interactions with
the phenylalanine-glycine motifs in overcoming this barrier to permit their rapid passage. Our results
have important implications for the various current models regarding the molecular mechanisms of
nucleocytoplasmic transport and behavior of weak cellular interactions generally. (LH, KD equal
contributors; DC, MPR corresponding authors.)
M103
A genetic system to probe how intrinsically disordered FG domains form the
permeability barrier of NPCs.
M. Ridders1, D. Görlich1;
1
Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
The permeability barrier of nuclear pore complexes (NPCs) controls all exchange between cell nucleus
and cytoplasm. It suppresses passage of inert macromolecules, but allows facilitated translocation of
MONDAY-ORAL PRESENTATIONS
large cargoes when these are bound to appropriate nuclear transport receptors (NTRs). FG repeat
domains are critical for this selectivity. In the yeast S. cerevisiae, they occur in 11 different “FG Nups”,
and comprise up to 800 residues and 50 FG (Phe-Gly) motifs. NTRs bind to FG motifs and this interaction
is essential for their privileged passage. The selective phase model attributed another essential property
to the barrier forming FG domains, namely cohesiveness. Cohesive FG domains can interact
multivalently with each other and form sieve-like hydrogels that represent selective diffusion barriers.
Given their fundamental function, FG domains appear surprisingly diverse. They differ not only in length,
number and type of FG motifs, but also drastically in the sequences of their inter-FG spacers, and
consequently also in cohesiveness. It had been unclear how much of this diversity is required and which
FG domain features play a really essential role. To address these issues, we tested the consequences of
mutations, deletions, or exchange of individual FG domains for cell viability. We observed that S.
cerevisiae tolerates more FG domain deletions than previously thought. However, the simultaneous
deletion of the FG domains from Nup100 and Nup116 was lethal. The Nup100 and Nup116 FG domains
are highly cohesive and feature very NQ-rich inter FG spacers. We found that none of the other FG
domains could functionally replace them, even when fused to the Nup100 or Nup116 anchor points.
This and a subsequent comprehensive mutational analysis of the Nup100 FG domain demonstrate that
FG domain function cannot be reduced to NTR-binding alone. Instead, it supports the assumption that
also FG domain cohesion is critical for the NPC barrier.
M104
Karyopherin-centric control of nuclear pores based on multivalent binding with
FG Nucleoporins.
L.E. Kapinos1, R.L. Schoch1, K.D. Schleicher1, R.S. Wagner1, R.Y. Lim1;
1
Biozentrum, University of Basel, Basel, Switzerland
Intrinsically disordered Phe-Gly nucleoporins (FG Nups) within nuclear pore complexes (NPCs) exert
multivalent interactions with transport receptors (Karyopherins or Kaps) that orchestrate
nucleocytoplasmic transport. Current FG-centric views reason that selective Kap translocation is
promoted by alterations in the barrier-like FG Nup conformations. However, the strong binding of Kaps
with the FG Nups due to avidity contradicts rapid Kap translocation in vivo, thereby underscoring how
mechanistic and kinetic views of NPC functionality remain at odds. Here, I will discuss our recent efforts
to understand how multivalent Kap-FG Nup binding impacts on transport selectivity and speed. This
includes (i) studying the crosstalk between equilibrium affinity and kinetic rates of Karyopherinβ1
(Kapβ1) binding with conformational changes in the FG Nups in situ; and, (ii) assaying Kap-facilitated
transport when binding FG Nups in vitro. A characteristic feature of the FG Nups is their ability to
accommodate large numbers of Kapβ1 molecules at physiological concentrations by conformational
changes. However, whereas most bound Kapβ1 molecules become long-lived constituents inside a FG
Nup layer due to strong binding avidity, Kapβ1 molecules located at the layer periphery are weakly
bound and dominate fast transport kinetics due to limited binding with the pre-occupied FG Nups.
Hence, the number of free FG-repeats in a FG Nup layer, and the strength of Kapβ1 binding are directly
MONDAY-ORAL PRESENTATIONS
correlated to the amount of Kapβ1 in solution. As proof-of-principle, we find that varying Kapβ1
concentration can control Kapβ1-facilitated motion on a FG Nup layer. This evokes differential behavior
ranging from highly constrained to two-dimensional diffusion (i.e., Reduction of dimensionality) at
physiological Kapβ1 concentrations. In contrast to FG-centric views, I will discuss how our findings show
support for an emergent Kap-centric mechanism that may underlie barrier functionality and selective
transport control in NPCs.
1. R.L. Schoch, L.E. Kapinos, and R.Y.H. Lim, Nuclear transport receptor binding avidity triggers a selfhealing collapse transition in FG-nucleoporin molecular brushes. PNAS 109 16911 (2012)
2. L.E. Kapinos, R.L. Schoch, R.S. Wagner, K.D. Schleicher and R.Y.H. Lim, Karyopherin-centric control of
nuclear pores based on molecular occupancy and kinetic analysis of multivalent binding with FGNucleoporins. Biophys. J., 106 1751 (2014)
3. K.D. Schleicher, S.L. Dettmer, L.E. Kapinos, S. Pagliara, U.F. Keyser, S. Jeney and R.Y.H. Lim, Selective
transport control on molecular velcro made from intrinsically disordered proteins. Nature
Nanotechnology, 9 525 (2014)
M105
Crystal structure of Nup62·58·54 nucleoporin complex.
H. Chug1, S. Trakhanov1, B.B. Hulsmann1, D. Görlich1;
1
Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
Nucleoporins (Nups) are the building blocks of a highly sophisticated translocation machinery in the cell
called the Nuclear Pore Complex (NPC), which allows the controlled exchange of macromolecules across
the nuclear envelope (NE). Many Nups interact tightly to form subcomplexes that arrange in octahedral
symmetry to impart a rigid NPC scaffold. The Nup62·58·54 subcomplex is located near the central NPC
translocation channel. It comprises three subunits, Nup62, Nup58 and Nup54, all predicted to contain
coiled coil (CC) domains. The complex is essential, highly conserved in evolution, a major anchor site for
NPC barrier-forming FG domains and attached to the NPC via Nup93 (Nic96 in S.cerevisiae). The
molecular structure of Nup62·58·54 subcomplex is hotly debated, because attempts to crystallize a
complete complex have so far failed and only fragments with apparently non-physiological
oligomerization propensity and stoichiometry have been crystallized instead (see Science 2007, 315:
1729; Cell 2011, 147:590; PNAS 2013, 110: 5858 and MBoC 2014, 25:1484 for an opposing perspective).
On the basis of the fragment structures, it had been proposed that the subunits of Nup62·58·54 complex
would slide against each and thereby dilate or narrow the central NPC channel. In order to crystallize a
complete xlNup62·58·54 complex, we mapped its domain boundaries and reached a stable, heterotrimeric complex that can also incorporate efficiently into the NEs of in vitro assembled nuclei,
suggesting a biologically relevant complex. In order stabilize the flexible parts and hinge regions of the
complex that interfered with its crystallization, we screened many single-domain antibodies
(Nanobodies, Nbs), and found a few that bound the trimeric state and a single one that conferred
crystallizability. We also identified the Nup93 binding sites on Nup62·58·54 complex. Crystals obtained
from the Nb-bound Nup62·58·54 complex diffracted to 3.2Å. The Nup62·58·54 complex structure
MONDAY-ORAL PRESENTATIONS
revealed a very extensive hetero-trimerisation interface including two hetero-trimeric coiled coil regions
that are connected by a sharp kink and pack against each other through another extensive hydrophobic
interface. This structure of the biologically relevant Nup62 58 54 complex rules out any sliding between
the subunits. The structure includes a number of striking features, whose functional significance is
currently being tested.
M106
Correlative light-electron microscopy reveals the assembly process of the
nuclear pore complex in interphase.
S. Otsuka1, K.H. Bui2, M. Schorb2, J. Hossain1, M. Eltsov1, M. Beck2, J. Ellenberg1;
1
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany,
2
Structural and Computational Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg,
Germany
The nuclear pore complex (NPC) mediates selective transport across the nuclear envelope (NE) and plays
crucial roles in several additional cellular functions. The NPC is a very large protein complex with a mass
of approximately 110 MDa, composed of multiple copies of over 30 different proteins termed
nucleoporins (Nups). In higher eukaryotes NPCs are assembled in two cell cycle stages, during nuclear
assembly after mitosis and nuclear growth in interphase. After mitosis the double nuclear membrane
and the NPC channel assemble concomitantly. By contrast, NPC assembly in interphase requires
insertion into the closed NE and thus a fusion between outer and inner nuclear membranes, and its
mechanism is poorly understood. Studying interphase NPC assembly has been challenging since
assembly events are rare and single pore resolution imaging is needed to distinguish newly-assembling
NPCs from already-formed NPCs. Here, by using correlative live imaging and electron tomography, we
found novel structural intermediates of interphase NPC assembly in the NE. To confirm that they
represent assembly intermediates, we show that they are specifically enriched in Nups by immuno-EM
labeling and that their abundance at different cell cycle stages matches the increased number of fully
assembled NPCs observed subsequently. In addition, structural analysis of the three-dimensional EM
tomograms revealed that the intermediates exhibit an 8-fold symmetry resembling the nuclear ring of
the mature NPC. Quantitative structural comparison of the NPC intermediates in temporally ordered
assembly stages allows us to propose a novel mechanism for NPC biogenesis in the intact nuclei of
interphase cells.
MONDAY-ORAL PRESENTATIONS
M107
Surveillance of nuclear pore complex assembly by the ESCRT machinery.
B.M. Webster1, P. Colombi1, J. Jager1, P. Lusk1;
1
Department of Cell Biology, Yale University School of Medicine, New Haven, CT
The assembly of Nuclear Pore Complexes (NPCs) during interphase is thought to occur through the
stepwise recruitment of the ~30 nucleoporins (nups) to the nuclear envelope, which occurs
concomitantly with the fusion of the inner and outer nuclear membranes. Given that a single defective
NPC could compromise the barrier function of the nuclear envelope, it is essential to understand how
NPC quality is ensured. Working in budding yeast, we will show evidence for a surveillance mechanism
that oversees NPC assembly. Our data are consistent with a model in which surveillance is achieved
through the recognition of early NPC assembly intermediates by integral inner nuclear membrane
proteins of the Lap2, emerin, MAN1 family, Heh1 and Heh2. When NPC assembly is compromised, Heh2
recruits the Endosomal Sorting Required for Transport (ESCRT) –III subunit, which, along with the AAAATPase Vps4, acts to potentially clear and target the degradation of misassembled nups. Under
conditions in which surveillance is compromised, defective NPCs accumulate in a compartment that we
term the SINC for Storage of Improperly assembled Nuclear pore Complexes. Consistent with the idea
that the SINC is a repository for malfunctioning NPCs, it is most prevalent in ‘old’ mother cells and is
almost never transmitted to daughter cells through mitotic divisions. Thus, there are several
mechanisms that ensure NPC quality to support proper nuclear compartmentalization. Moreover, our
data highlight an exciting functional relationship between two ancient membrane-bending/associated
machineries.
M108
Quality control of inner nuclear membrane proteins by the Asi complex.
O. Foresti1, V. Rodriguez Vaello1, C. Funaya2, P. Carvalho1;
1
Cell and Developmental Biology Programme, Center for Genomic Regulation (CRG), Barcelona, Spain,
2
Electron Microscopy Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg,
Germany
Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a quality control system called
ER-associated protein degradation (or ERAD). This pathway also mediates the turnover of some folded
ER proteins, thereby affecting other ER-related functions such as sterol homeostasis. However, how
misfolded membrane proteins in the inner nuclear membrane (INM), a specialized ER subdomain with
restricted access, are degraded is not known. Here, a quantitative proteomics approach led to the
identification of a novel ERAD branch required for protein quality control in the INM. We show that this
branch is defined by the integral membrane proteins Asi1, Asi2 and Asi3 (which assemble into the Asi
complex at the INM) and requires the Cdc48 ATPase. Among the substrates of the Asi complex are both
misfolded proteins and functional regulators of sterol biosynthesis, a feature common to all ERAD
MONDAY-ORAL PRESENTATIONS
branches. Our findings highlight that the quality control of membrane proteins is spatially segregated
between INM and the rest of the ER membrane.
M109
The role of SUN proteins in nuclear envelope spacing of force-bearing cells.
N. Cain1, E.C. Tapley1, K. McDonald2, B. Cain3, D.A. Starr1;
1
MCB, University of California Davis, Davis, CA, 2EML, University of California Berkeley, Berkeley, CA,
3
Physics, University of California Davis, Davis, CA
The nuclear envelope is made up of two membranes, the inner and outer nuclear membranes,
separated by a uniform distance of 40-50 nm, known as the perinuclear space. The mechanism by which
even spacing of the two nuclear membranes is maintained has been poorly understood. Sad1p-UNC-84
(SUN) proteins, which interact with Klarsicht, ANC-1, and SYNE homology (KASH) proteins to connect the
nucleoskeleton to the cytoskeleton, have been implicated as molecular rulers that set the nuclear
envelope space. This hypothesis was based on the observation that HeLa cells depleted of SUN proteins
show blebbing of the outer nuclear membrane away from the inner nuclear membrane. To test the
hypothesis that the C. elegans SUN protein UNC-84 spans and regulates the perinuclear space, we
examined the morphology of the nuclear envelope by electron microscopy in unc-84(null) mutant
animals. In most unc-84(null) mutant tissues, nuclear envelope morphology was normal. Therefore, SUN
proteins are not necessary to regulate nuclear envelope spacing. However, we did observe extreme
separation of the outer nuclear membrane from the inner nuclear membrane in unc-84(null) L1 body
wall muscle nuclei. These mutant animals also displayed irregular locomotion in liquid and perhaps serve
as a model for human laminopathies due to defects in LINC complex proteins that have reduced muscle
function. Our data suggest a correlation between nuclear envelope morphology and muscle function.
Surprisingly, UNC-84 protein with a 300 amino acid deletion in the luminal domain was able to form
functional nuclear envelope bridges, but did not have a noticeable effect on nuclear envelope spacing.
We propose that SUN proteins are only required to maintain the nuclear envelope space in cells that are
under strain. Rather than dictating the perinuclear space, SUN proteins have appeared to evolve to
reach across it.
M110
Nuclear actin counters gravity during cell growth.
M. Feric1, C. Brangwynne2;
1
Chemical Engineering, Princeton University, Princeton, NJ, 2Chemical and Biological Engineering,
Princeton University, Princeton, NJ
Mechanics is highly influential in determining cell behavior from cell division to cell motility. However,
the exact role of mechanics in controlling cell growth and cell size still remains unclear. The oocytes or
immature eggs of the frog X. laevis are known to grow and reach extraordinarily large sizes of
MONDAY-ORAL PRESENTATIONS
approximately 1 mm in diameter. During this process of growth, the oocytes actively maintain
significantly high concentrations of nuclear actin. We have recently shown that this nuclear actin
meshwork stabilizes the liquid-like nuclear bodies from rapid gravitational sedimentation and fusion.
However, the mechanical properties of nuclear actin remain poorly characterized, despite their
apparent importance in kinetically stabilizing the nucleus. Here, we use approaches in active and passive
microrheology, in addition to confocal and two-photon excitation microscopy and quantitative image
analysis, to probe the local mechanics and microstructure of this nuclear actin network. We find that
actin forms a surprisingly soft, viscoelastic scaffold with a mesh size of ~0.5 microns. Furthermore, the
distribution of nuclear bodies within the actin network shows signatures of gravitational creep during
growth. Upon application of forces of ~1 pN, nuclear actin exhibits shear-thickening behavior, which
could serve a protective role in response to shocks. However, significantly higher forces ultimately lead
to mechanical failure and rupture of the actin network. These measurements elucidate mechanical and
geometric aspects of cell organization, which suggests that biophysical constraints can play an important
role in cell growth and size control.
Minisymposium 12: Role of ECM/Cell Junction/Polarity Proteins in
Tissue Morphogenesis
M111
Critical roles for the apical extracellular matrix and EGF-Ras signaling in building,
maintaining and remodeling tiny tubes in C. elegans.
J. Parry1, J. Cohen1, M.V. Sundaram1;
1
Department of Genetics, University of Pennsylvania, Philadelphia, PA
Unicellular tubes are formed from single cells that create an internal lumen. Such tubes are found in the
microvasculature of mammals and in simple tubular organs of invertebrates. We use the C. elegans
renal-like excretory organ as a model system to study unicellular tube development. The excretory
organ is composed of three tandemly-connected unicellular tubes, the canal, duct, and pore, which form
through de novo epithelialization coupled either to hollowing or wrapping modes of single-cell
tubulogenesis. Through forward genetic screening, we found several families of apical transmembrane
or secreted proteins (including leucine-rich repeat proteins, zona pellucida domain proteins and
lipocalins) that are required for lumen integrity and junction maintenance. Our results point to a critical
role for the apical extracellular matrix in maintaining the integrity of narrow bore tubes.
We also showed that EGF-Ras signaling plays multiple roles in building and remodeling the excretory
organ; one important role is to permit G1 pore tube delamination. During normal development, the
initial pore cell, G1, delaminates from the organ to become a neuroblast and is replaced by a new cell,
G2. G1 delamination and G2 intercalation involve cytoskeletal remodeling, interconversion of
autocellular and intercellular junctions and migration over a luminal matrix, followed by G1 junction
loss. EGF-Ras signaling acts cell non-autonomously in the duct cell to permit G1 junction loss and
MONDAY-ORAL PRESENTATIONS
delamination. In humans, aberrant EGF-Ras signaling promotes tumorigenesis and metastasis. Our
results demonstrate that Ras signaling in cells that remain in an epithelium can create a
microenvironment that is permissive for neighboring cells to detach.
M112
Regulation of epithelial morphorgenesis by a tight junction (TJ)-apical complex.
S. Tsukita1, T. Yano2, H. Kanoh2, A. Tamura1;
1
Graduate School of Frontier Bioscience and Graduate School of Medicine, Osaka University, Osaka,
Japan, 2Osaka University, Suita, Osaka, Japan
Epithelial cells adhere to each other by tight junctions (TJs) to form cell sheets, which is a critical step in
epithelial morphogenesis. We recently discovered that a network of microtubules exists just below the
apical membrane of the epithelial cell sheet. Because this apical microtubule network appears to be
connected to TJs and associated with actin and keratin filaments, we defined the TJ and its associated
membrane and apical structures as the “TJ-apical complex.” The TJ-apical microtubules represent one of
the layered cytoskeletal networks within the TJ-apical complex. Gel overlay assays of isolated TJ
fractions on microtubules in the presence of taxol revealed four TJ-associated microtubule-binding
proteins (TJ-MAPs): TJ-MAP1, TJ-MAP2 (cingulin), TJ-MAP3, and TJ-MAP4. A knockdown (KD) analysis for
cingulin revealed that cingulin-KD partially disrupted the association between microtubules and TJs.
More recently, we found a similar effect of knocking out TJ-MAP3. We further found that TJ-MAP3
critically influences the TJ-actin organization by regulating the phosphorylation of myosin, suggesting a
functional link between the microtubules and the actomyosin ring at TJs. In addition, we found that TJMAP3 contributes to the apical constriction mediated by Rho/ROCK signaling and functionally regulated
by microtubules.
Based on these findings, we propose the novel idea that the planar network of apical microtubules is
linked to circumferential actin rings at the TJ-apical complexes. Future studies will explore the role of
the TJ-apical complex in the general context of all cytoskeletons, including those based on microtubules,
actin, and keratin filaments. We propose that the TJ-apical complex is critical for organizing the TJ and
apical membrane systems, including the location and function of membrane proteins that play essential
roles in epithelial cell sheet functions, and thus in the regulation of biological systems.
M113
UPK3a coordinates Par complex and ezrin function during the polarization and
morphogenesis of urinary tract-associated epithelial cells.
G. Apodaca1, S. Mitra1, L.I. Gallo1, W.G. Ruiz1;
1
Medicine and Cell Biology, University of Pittsburgh, Pittsburgh, PA
Mutations in human UPK3a, a transmembrane protein that is expressed early in urinary tract
development, leads to congenital anomalies of the kidney and lower urinary tract (CAKUT), but why is
MONDAY-ORAL PRESENTATIONS
unknown. We previously reported that the UPK3a-like protein Upk3l is expressed at the apical surfaces
of the epithelial cells lining the zebrafish pronephros, which functions as an early urinary tract during
larval development. Embryos injected with upk3l-targeted morpholinos showed decreased pronephros
function, which was attributed to defects in pronephric tubule epithelial cell morphogenesis and
polarization including: loss of their apical brush border and associated phospho-ERM proteins, apical
redistribution of the basolateral Na+/K+-ATPase, and altered or diminished expression of the apical
polarity complex proteins Prkcz (aPKC zeta isoform) and Pard3 (Par3). In our current studies we
observed that Upk3l formed genetic interactions with Prkcz and the morphogenic protein Ezrb (ezrin),
that morphants with diminished expression of Upk3l, Prkcz, Pard3, or Ezrb had a similar phenotype, and
that Upk3l apparently acted upstream of Prkcz, which functioned prior to Ezrb. Human UPK3a coexpressed with its heterodimer partner UPK1b rescued the Upk3l morphant phenotype, indicating that
both proteins share conserved motifs. Furthermore, co-immunoprecipitation analysis demonstrated
that Upk3l and UPK3a/UPK1b interacted with ezrin and aPKCzeta, and that UPK3a/UPK1b also bound
Par3. GST pull-down assays further confirmed that these interactions were dependent on the C-terminal
cytoplasmic domain of UPK3a. To further explore the possibility that UPK3a may function by recruiting
aPKCzeta and ezrin to the apical pole of UPK3a-expressing urinary tract-associated epithelial cells
(UEECs), we co-expressed UPK3a/UPK1b in MDCK cells, which do not normally express these proteins.
This ectopic expression triggered the formation of longer microvilli, and increased the localization of
Par3, aPKCzeta, and activated, phospho-ezrin at the apical domain of the cells. Our studies support a
model in which UPK3a recruits the Par-complex and ezrin to the apical pole of UEECs. The Par complex
may then facilitate activation of ezrin (possibly via aPKCzeta-dependent phosphorylation), which would
stimulate brush border formation. By altering these critical steps during UEEC polarization and
morphogenesis, mutations in UPK3a may lead to CAKUT.
M114
PROBING COLLECTIVE MIGRATION OF A COMPLEX MULTI-CELLULAR
EMBRYONIC TISSUE THROUGH NOVEL 3D BIOETCHING.
M. Hazar1, Y. Kim2, L.A. Davidson3,4, P.R. LeDuc1, W.C. Messner5;
1
Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA, 2Mechanical Engineering, Georgia
Institute of Technology, Atlanta, GA, 3Bioengineering, University of Pittsburgh, Pittsburgh, PA,
4
Developmental Biology, and Computational and Systems Biology, University of Pittsburgh, Pittsburgh,
PA, 5Mechanical Engineering Department, Tufts University, Medford, MA
Embryonic development consists of a complex series of cell signaling, cell migration and cell
differentiation processes that are coordinated during morphogenesis. Collective cell sheet migration is
an important process that sculpts the shape of an organism and its internal tissues during early
development. Studies of these collective cell movements have focused on the behavior of epithelial
monolayers of cultured cells such as Madin Darby canine kidney MDCK cells. By contrast, most
embryonic development and tissue self-assembly requires the integration of cell movements within
multiple cell layers composed of different cell types. Although the role of cell mechanics in tissue self-
MONDAY-ORAL PRESENTATIONS
assembly has been demonstrated, little is known about the mechanical response of the multi-layer
tissues to environmental cues. One of the reasons for this knowledge gap is the lack of the technologies
to analyze the individual responses of epithelial and mesenchymal cell sheets in a multi-cell layer
tissues. To investigate the processes that guide collective movements of multiple cell layers our group
has focused on developing a novel microfluidic technique capable of producing complex patterns of
multicellular structures. We call this technique "3D tissue-etching” by analogy with silicon
micromachining techniques used to fabricate 3D microelectromechanical structures (MEMS) by
successive steps to remove material from a monolithic solid. We use tissue etching to shape a complex
multi-layered embryonic tissue and explore the dynamic collective responses of epithelial and
mesenchymal cells in a single tissue. We use a custom-designed microfluidic control system to deliver a
range of tissue etching reagents (detergents, chelators, proteases, etc.) over tissues microsurgically
isolated from embryos of the African Claw-toed frog, Xenopus laevis. Using etching we produce freeedges of epithelial cells over mesenchymal cells and free-edges of mesenchymal cells. This allows us to
study multi-layer coordination of epithelial and mesenchymal cell layers, as well as acute mechanical
and behavioral responses of intact epithelial and mesenchymal cell sheets to the removal of neighboring
or overlying tissues. The ability to control the forms of multicellular tissues will have high impact in
tissue engineering and regeneration applications in bioengineering and medicine as well as in the
synthesis of highly complex 3D integrated multicellular biosystems.
M115
The Adhesion GPCR GPR126 has distinct, domain-dependent functions in
Schwann cell development and myelination mediated by interactions with
Laminin-211.
S.C. Petersen1, R. Luo2, I. Liebscher3, M. Feltri4, T. Schöneberg3, X. Piao2, K. Monk1;
1
Developmental Biology, Washington University School of Medicine, St. Louis, MO, 2Pediatrics, Boston
Children's Hospital, Boston, MA, 3Biochemistry, University of Leipzig, Leipzig, Germany, 4Biochemistry
and Neurology, University at Buffalo, Buffalo, NY
Myelin ensheathes axons to allow rapid propagation of action potentials and proper nervous system
function. In the peripheral nervous system, myelin is formed by Schwann cells, which radially sort axons
into a 1:1 relationship before wrapping an axonal segment to form the myelin sheath. For proper
development and myelination, Schwann cells must synthesize and secrete extracellular matrix (ECM)
proteins to form a functional basal lamina (BL). Schwann cell myelination also requires the orphan
Adhesion G protein-coupled receptor GPR126, which undergoes autoproteolytic cleavage into an Nterminal fragment (NTF) and a 7-transmembrane C-terminal fragment (CTF). Here, we show that
GPR126 has domain-specific functions in Schwann cell development whereby the NTF is necessary for
radial sorting while the CTF promotes wrapping through cAMP elevation. These biphasic roles of GPR126
are governed by interactions with the ECM protein and BL constituent Laminin-211, which we define as
a novel ligand for GPR126. Our work suggests a model in which Laminin-211 mediates a switch in
MONDAY-ORAL PRESENTATIONS
GPR126 signaling states between cAMP suppression and elevation to control different stages of
Schwann cell development
M116
The many roles of the cytoskeleton during seamless tube morphogenesis.
J. Schottenfeld-Roames1,2, J. Rosa1, A. Ghabrial1;
1
Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA,
2
Biology, Swarthmore College, Swarthmore, PA
The Drosophila tracheal system is composed of a network of tubes that forms by specialized tip cells
leading the migration of new branches and mediating their interconnection. Some of these tip cells
become specialized “terminal cells” that go on to form branched seamless tubes, unicellular tubes that
lack epithelial junctions, that ramify extensively on target tissues.In the mammalian vascular system,
endothelial cells form seamless tubes during angiogenesis.Very little is known about the apical-basal
properties of seamless tubes and the genes required to establish and maintain this polarity. We
investigated terminal cell polarity and found that the lumenal membrane is “apical,” since puncta of the
definitive apical membrane marker, Crumbs, decorate the seamless tube. Actin:GFP outlines this apical
membrane and also highlights elaborate filopodia at the tips of growing branches. Microtubules also
show striking polarity in seamless tubes: g-tubulin, a primary component of the microtubule organizing
center, lines the lumenal membrane and is enriched at the tube tip. Since g-tubulin marks the site of
microtubule nucleation, minus-ends of terminal cell microtubules are directed towards the lumen, thus
leading us to hypothesize that minus-end directed transport along microtubules is required for tube
formation. Stable microtubules appear in parallel arrays to the tube, with thickened microtubule
bundles extending past the tube tip, likely laying the foundation for future membrane addition. In
support of this hypothesis, terminal cells mutant for components of the dynein motor complex (Dynein
heavy chain 64c, dynein light intermediate chain, Gluedp150, and lissencephaly-1) generate extensions
that fail to form seamless tubes.In the absence of tubes, microtubule organization remained intact,
indicating a direct role for minus-end directed microtubule transport in seamless tube formation.
We have also found a role for the actin cytoskeleton in regulating seamless tube shape. Terminal cells
compromised for early endocytosis display small cysts throughout their seamless tubes, a phenotype we
attribute to the accumulation of the apical membrane proteins Crumbs and p-Moesin in these mutant
backgrounds. Overexpression of either Crumbs, or phospho-mimetic Moesin, induced lumenal cysts and
mutations in crumbs or Moesin were able to suppress the cystic phenotype in terminal cells mutant for
Syx7, a regulator of early endocytosis. Thus we propose that defects in early endocytosis lead to high
steady state levels of Crumbs, resulting in increased apical p-Moe activity, which in turn leads to cysts as
a result of a stabilized actin cortex. Accordingly, terminal cells lacking Cofilin, the actin-severing protein,
also display cysts.
MONDAY-ORAL PRESENTATIONS
M117
A role for dynein light chain in germline stem cell maintenance in C. elegans.
X. Wang1, D. Rasoloson2, E. Voronina3;
1
DBS, Univ Montana, Missoula, MT, 2MBG, Johns Hopkins University School of Medicine/HHMI,
Baltimore, MD, 3Univ Montana, Missoula, MT
PUF family RNA binding proteins are conserved stem cell regulators in Drosophila and planaria (Lin and
Spradling, 1997; Salvetti et al., 2005). FBF-1 and FBF-2, two similar PUF family translational repressors,
are required for maintenance of germline stem cell in C. elegans (Crittenden et al., 2002). Here we
report identification of dynein light chain DLC-1 as a specific cofactor of FBF-2, but not FBF-1. In germline
stem cells, FBF-2 localizes to perinuclear germ granules or P granules in C. elegans (Voronina et al.,
2012). This localization is important for FBF-2 binding to target mRNAs and its activity as a translational
repressor. By contrast, FBF-1 does not localize to P granules, and its function does not depend on P
granule integrity. The goal of this study was to investigate mechanisms of FBF-2 localization to P
granules. DLC-1 is an LC8-type light chain, a cargo-binding component of dynein motor complex. Dynein
traffics organelles, proteins, and mRNAs toward the minus ends of microtubules. Knock-down of dlc-1 by
RNAi correlates with loss of FBF-2 perinuclear accumulation even when perinuclear P granules are not
affected and also causes a decrease in FBF-2 activity as a translational repressor. By contrast, FBF-1
localization is not affected by dlc-1 knock-down, and FBF-1 remains able to regulate its targets. In vivo,
FBF-2 is found in a complex with DLC-1 by co-immunoprecipitation. In the in vitro pulldown assay, DLC-1
interacts with FBF-2, but not FBF-1, suggesting that specificity of DLC-1 contribution to FBF-2 function is
based on selectivity of protein-protein interactions. Interaction between DLC-1 and FBF-2 depends on
the regions of sequence divergence between FBF-2 and FBF-1.
We hypothesize that DLC-1 binds FBF-2 and promotes its localization to P granules, which is important
for FBF-2-mediated translational regulation and germline stem cell maintenance. Dynein motor complex
has been implicated in formation, transport, and dynamics of RNA granules in several cell types. Our
findings suggest that LC8-type light chains such as DLC-1 may directly regulate recruitment of specific
components to the RNA granules in stem cells.
M118
Compensatory branching morphogenesis of stalk cells in the Drosophila trachea.
D. Francis1, A. Ghabrial1;
1
Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA
Tubes are essential for nutrient transport and gas exchange in multicellular eukaryotes, but how
connections between different tube types are maintained over time is unknown. In the Drosophila
tracheal system, mutations in oak gall (okg) and conjoined (cnj) confer identical defects, including late
onset blockage near the terminal cell (TC)-stalk cell (SC) junction, and ectopic extension of autocellular,
seamed tubes into the TC. We determined that okg and cnj encode the E and G subunits of the vacuolar
MONDAY-ORAL PRESENTATIONS
ATPase (vATPase), and showed that both V0 and V1 domains are required for TC morphogenesis.
Remarkably, the ectopic seamed tubes running along vATPase-deficient TCs belonged to the neighboring
SCs. All vATPase-deficient tracheal cells had reduced apical domains, and TCs displayed mislocalized
apical proteins. Further, TCs mutant for apical polarity genes, par6 or aPKC, had ectopic seamed tubes.
We thus identify a novel mechanism of compensatory branching in which SCs extend autocellular tubes
into neighboring TCs with undersized apical domains.
M119
Integrin αv is necessary for generation of human epidermis.
E. Duperret1, T. Ridky1;
1
Dermatology, University of Pennsylvania, Philadelphia, PA
Integrin subunits play crucial roles in epithelial adhesion, proliferation, differentiation and wound
healing. However, the roles of many of the 26 integrin subunits in epidermis have not been determined.
We designed a multiplexed screening approach to define roles for each member of the integrin family in
human skin using a 3-dimensional in vitro organotypic skin model. In this screen, we transduced
individual populations of human keratinocytes with shRNAs against each integrin subunit. We then
quantitatively determined the fitness of each cell population in reconstituted human skin. This screen
identified the integrin αv class of heterodimers as essential for generation of human skin tissue: the
population of keratinocytes lacking αv was selected against 125-fold compared to non-silencing control
cells (p
Minisymposium 13: Synthetic and Chemical Biology: Reconstituting
and Probing Cells
M120
Characterization of the mechanical properties of reconstituted contractile actin
networks using single wall carbon nanotubes.
K. Keren1, N. Fakhri2, M. Malik-Garbi1, E. Abu Shah1, C.F. Schmidt2;
1
Physics, Technion-Israel Institute of Technology, Haifa, Israel, 2III. Physikalisches Institut-Biophysik,
Georg-August-Universität, Göttingen, Germany
The actin cytoskeleton plays a major role in large oocytes and egg cells during the initial stages of
development. In particular, the actin cytoskeleton can switch, in a cell-cycle dependent manner, into a
contractile state and exhibit myosin-driven global actin flows which are essential for various processes in
early development, including cortical polarization in the C. elegans zygote and chromosome congression
in the starfish oocyte. We developed a reconstituted model system to study cytoskeletal organization
and emulate these processes in artificial cell-like compartments. Cytoplasmic Xenopus egg extracts are
MONDAY-ORAL PRESENTATIONS
encapsulated within water-in-oil emulsions, and actin nucleators are added to induce the formation of
various cytoskeletal structures. By controlling the localization and concentration of the actin nucleators,
we can tune the properties of the system, and induce myosin-dependent cortical polarization which
appears similar to the initial polarization of the embryo in many species, or bulk actin network
contraction which can drive directional transport as seen during chromosome congression. To further
study these reconstituted cytoskeletal networks, we introduce inert single wall carbon nanotubes
(SWNTs) as probes and utilize their intrinsic ultrastable infrared fluorescence to characterize the
contractile behavior of the system, as a function of its composition, over a wide range of time scales.
Analysis of the movement of individual SWNTs embedded in the actin meshwork provides high
resolution mapping of the contractile actomyosin flows and the mechanical properties of these dynamic
networks. Overall, our reconstituted system provides a powerful platform to study important
cytoskeletal phenomena in a simplified environment detached from the complexity of the living cell.
M121
Actomyosin drives membrane dynamics in an in vitro active composite layer.
D.V. Köster1, K. Husain1, E. Iljazi1, P. Bieling2,3, R.D. Mullins2, M. Rao1,4, S. Mayor1;
1
National Centre for Biological Sciences (NCBS), TIFR, Bangalore, India, 2Department of Cellular and
Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, 3Department of
Bioengineering, University of California, Berkeley, Berkeley, CA, 4Raman Research Institute, Bangalore,
India
The surface of a living cell provides a platform for processes such as receptor signaling, protein sorting,
transport and endocytosis. The regulation of these processes requires the controllable organisation of
membrane components. A recent framework for the organisation of a certain class of plasma membrane
components is based on the active mechanics of acto-myosin juxtaposed to the membrane
(Gowrishankar et al., 2012; Rao & Mayor, 2014). A systematic study of the dynamics and consequences
of this active composite in living cells is challenging. Here we reconstitute an active composite in vitro,
by a stepwise addition of the minimal ingredients: a supported lipid bilayer with an actin-binding
component, short actin filaments and myosin motors. By systematically varying the concentrations of
actin and myosin as well as the level of ATP we find a rich phase diagram of membrane-confined actin
and myosin configurations. By increasing the level of available ATP we induce a constitutively
remodeling state, in which asters composed of short actin filaments form and dissolve. In this state, the
coupling of actin to the bilayer drives the membrane components out of equilibrium, imparting distinct
signatures of activity in a manner entirely consistent with measurements in the living cell. These results
highlight the fundamental basis of the active composite framework and indicate its relevance in the
study of membrane organisation.
MONDAY-ORAL PRESENTATIONS
M122
Regulation of T cell microcluster diffusion by the cortical actin network.
J. Ditlev1, D. Köster2, X. Su3, A. Vega4, S. Banjade1, K. Jaqaman4, R. Vale5, S. Mayor2, M.K. Rosen4,6;
1
University of Texas Southwestern Medical Center, Dallas, TX, 2National Centre for Biological Sciences
(NCBS), TIFR, Bangalore, India, 3Department of Cell and Molecular Pharmacology, University of
California, San Francisco, San Francisco, CA, 4Department of Biophysics, University of Texas
Southwestern Medical Center, Dallas, TX, 5University of California, San Francisco, San Francisco, CA,
6
Howard Hughes Medical Institute, Dallas, TX
Actin retrograde flow and the acto-myosin network are essential for the movement of individual LAT
microclusters into the supramolecular activation cluster in mature T cell synapses. Additionally, LATenriched, membrane-bound microclusters link to the actin cytoskeleton through Nck, WASp and Arp2/3
complex recruitment. However, the mechanism by which the existing cortical acto-myosin network
regulates LAT microcluster movement is unknown. To investigate this mechanism, we have developed
an in vitro reconstitution assay on synthetic supported lipid bilayers in which we anchor a preformed
acto-myosin meshwork to the bilayer using ezrin and induce LAT microcluster formation with Grb2 and
Sos1. When LAT microclusters formed on supported lipid bilayers without actin, diffusion of LAT
microclusters was restricted to a small area of the membrane. However, when LAT microclusters formed
within an existing acto-myosin network, microcluster diffusion across the membrane markedly
increased. We also were able to study how coupling LAT microclusters to the acto-myosin network via
phosphorylated SLP-76, Nck, N-WASp and Arp2/3 complex affected microcluster movement and actin
dynamics. Our unique experimental design and image analysis provide us tools that will be used to
thoroughly investigate the role of the acto-myosin network in LAT microcluster movement and develop
hypotheses that can be tested in activated T cells.
M123
Reconstituting membrane budding with influenza A virus matrix protein.
M.D. Vahey1, D.A. Fletcher2,3,4;
1
Bioengineering, University of California, Berkeley, Berkeley, CA, 2Department of Bioengineering,
University of California, Berkeley, Berkeley, CA, 3Biophysics Graduate Group, University of California,
Berkeley, Berkeley, CA, 4Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley,
CA
Enveloped viruses replicate by budding from the plasma membrane of infected cells, exploiting cellular
components in the process. Unlike many other enveloped viruses (e.g. HIV), which leverage the ESCRT
machinery to exit the cell, influenza A virus appears to bud through an alternate mechanism. Despite
the small number of viral proteins that have been identified as required for influenza A budding, the
sequence of events that organize and shape the membrane during virus assembly remain poorly
understood. In order to understand this process and identify common physical and biochemical
mechanisms underlying budding, we have developed a minimal in vitro system that reconstitutes
MONDAY-ORAL PRESENTATIONS
aspects of viral assembly and budding on giant unilamellar vesicles with controlled membrane
composition. With this system, we have characterized the assembly of the influenza A virus matrix
protein M1, which forms an oligomeric layer immediately adjacent to the viral membrane, provides a
link between the viral envelope proteins the packaged genome, and functions as the primary
determinant of virus shape. By controlling the lipid and protein composition of model membranes, we
find that M1 oligomerization drives sorting of both lipids and membrane-bound proteins, and that M1
can shape the membrane into structures that geometrically and topologically resemble the budding
virus. These results suggest how M1 may act together with the viral envelope proteins to transform
sites in the plasma membrane into new virus particles.
M124
Development of three color variants of super-brilliant luciferase for multi-color,
real time imaging of gene expression and dynamics of organelles and the
cytoskeleton without external illumination.
A. Takai1, R. Haruno2, T. Nagai2,3, Y. Okada1;
1
RIKEN Quantitative Biology Center (QBiC), Osaka, Japan, 2Osaka Univ, Osaka, Japan, 3JST PRESTO, Tokyo,
Japan
Since bioluminescence does not require excitation light, it is free from auto-fluorescence. Therefore, it
has been used for quantitative analysis of gene expression and in vivo imaging. Furthermore, it is free
from potential phototoxicity and is compatible with optogenetic tools. However application of
bioluminescent imaging has been limited mainly by two drawbacks. Firstly, the light output from the
bioluminescent protein or luciferase was much darker than fluorescent proteins (FPs), making it difficult
to study fine structures such as cytoskeletal dynamics with luminescent imaging. Secondly, color variants
of luciferase have been limited compared with FPs, precluding multicolor imaging.
In our recent study, we addressed the first limitation by developing a super brilliant yellow luminescent
protein, Nano-lantern (Saito et al., Nat. Commun. 2012). In this study, we have overcome the second
barrier. We report the development of cyan and orange variants of Nano-lantern, both of which are
even brighter than the original yellow Nano-lantern by 1.5-2.3 times, and bright enough for observation
with the naked eye or with a smartphone camera. Fusions of these multicolor Nano-lanterns with a
variety of subcellular localization tags showed correct localization in both luminescent and fluorescent
imaging, demonstrating their utility as imaging probes. In addition, expansion of the color palette of
Nano-lanterns enabled not only multicolor luminescent imaging of subcellular structures, but also
expression analysis of multiple genes at single cell level in embryonic stem cells, which are known to be
very sensitive to phototoxicity. Furthermore, by combining split luciferase complementation with Ca2+sensing peptide (CaM-M13), we demonstrated simultaneous measurement of Ca2+ dynamics in the
nucleus and mitochondria.
Our multicolor Nano-lanterns are expected to be excellent imaging tools for gene-expression analysis, in
vivo imaging, stem cell study and combinatorial analysis with optogenetics. Considering that the
MONDAY-ORAL PRESENTATIONS
expanded color palette of FPs has driven wider application of fluorescent live imaging, we believe that
our multicolor Nano-lanterns should change the world of luminescent imaging from dim and
monochrome to extremely bright and colorful.
M125
High-resolution mapping of intracellular fluctuations using carbon nanotubes.
N. Fakhri1,2, A.D. Wessel1, C. Willms1, M. Pasquali3, D.R. Klopfenstein1, F.C. MacKintosh4, C.F. Schmidt1;
1
III. Physikalisches Institut-Biophysik, Georg-August-Universität, Göttingen, Germany, 2Department of
Physics, Massachusetts Institute of Technology, Cambridge, MA, 3Department of Chemical Engineering,
Rice University, Houston, TX, 4Department of Physics and Astronomy, Vrije Universiteit, Amsterdam,
Netherlands
Cells are active systems with molecular force generation that drives complex dynamics at the
supramolecular scale. We present a quantitative study of molecular motions in cells over times from
milliseconds to hours. Noninvasive tracking was accomplished by imaging highly stable near-infrared
luminescence of single-walled carbon nanotubes targeted to kinesin-1 motor proteins in COS-7 cells. We
observed a regime of active random "stirring" that constitutes an intermediate mode of transport,
different from both thermal diffusion and directed motor activity. High-frequency motion was found to
be thermally driven. At times greater than 100 milliseconds, nonequilibrium dynamics dominated. In
addition to directed transport along microtubules, we observed strong random dynamics driven by
myosins that result in enhanced nonspecific transport. We present a quantitative model connecting
molecular mechanisms to mesoscopic fluctuations.
M126
A strategy for tissue self-organization that is robust to cellular heterogeneity and
plasticity.
A. Cerchiari1, J. Garbe2, N. Jee3, M. Todhunter3,4, T. Desai5, M.A. LaBarge2, M. Thomson6, Z.J. Gartner3,6;
1
Bioengineering, UC Berkeley - UCSF, San Francisco, CA, 2Lawrence Berkeley National Laboratory,
Berkeley, CA, 3Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA,
4
Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA, 5Bioengineering
and Therapeutic Sciences, University of California, San Francisco, San Francisco, CA, 6Center for Systems
and Synthetic Biology, University of California, San Francisco, San Francisco, CA
A combination of cell-intrinsic and extrinsic cues dictates how cell-cell and cell-ECM adhesions
orchestrate the self-organization of cells into tissues. The balance between these interactions is central
to the outcome of morphogenesis, tissue homeostasis, wound healing, disease progression, and the
reconstructions of human tissues in vitro. Characteristics such as differential expression of cadherins and
regulation of cortical tension are important cell-intrinsic parameters that influence this process.
However, how extrinsic parameters, such as the physicochemical properties of the ECM, modify and
MONDAY-ORAL PRESENTATIONS
relate to intrinsic interactions in the context of tissue self-organization is poorly understood. To clarify
how cell-intrinsic and extrinsic cues combine to define tissue architecture in complex epithelia, we FACSsorted human mammary epithelial cells derived from breast reduction mammoplasties into their luminal
(LEP) and myoepithelial (MEP) lineages, quantified the differential mRNA and protein expression of key
cell-cell and cell-ECM adhesion molecules via qPCR and flow cytometry, quantified their adhesion to
each other as well as to the ECM via contact-angle measurements, translated these measurements into
works of adhesion, and computationally modeled the self-organization process as a function of variable
cell intrinsic and extrinsic parameters using a Metropolis-Hasting algorithm. The mathematical model
predicted that MEP-ECM interactions are the dominant factor directing tissue self-organization, and that
final tissue architecture is robust to perturbations to other parameters contributing to self-organization.
The model also predicted that specific alterations to the balance of parameters directing selforganization could drive catastrophic breakdown of tissue architecture. To test the predictions of our
model, we reconstituted compositionally and geometrically defined aggregates of luminal and basal cells
fully embedded in different ECMs using 3D cell-patterning technologies we have previously developed.
This allowed us to test the consequences of specific perturbations generated with siRNA knockdowns of
proteins affecting the primary cell-cell and cell-ECM interactions. We found that talin1 knockdown
decreased the work of adhesion between MEPs and the ECM and promoted tissue inversion. In contrast,
perturbations such as p120 knockdown in MEPs and/or LEPs decreased work of cell-cell adhesion but
had no consequences on cell positioning through self-organization. Contrary to studies of early
metazoan development, our results suggest that a single interaction between the basal cells and the
ECM overrules cell-cell interactions and directs the self-organization of dynamic and heterogeneous
epithelial tissues such as the human mammary gland.
Kaluza Minisymposium
K1
Organizing gene regulatory events.
E.J. Clowney1, S. Lomvardas2, C. Larabell3;
1
Rockefeller University, New York, NY, 2Columbia University, New York, NY, 3Department of Anatomy,
School of Medicine, University of California, San Francisco, San Francisco, CA
Genomes were originally understood cytologically. Heterochromatin and euchromatin were optical
descriptions of silent and active regions of the genome that were only later biochemically characterized.
Would transcriptional regulation make more sense if considered in its cell biological context? Working
in olfactory sensory neurons, we found that cell-type-specific organization of the genome in nuclear
space governs the gene regulatory events that can occur in particular cells.
In the olfactory systems of many animals, different olfactory neurons express different olfactory
receptor proteins. Each olfactory receptor protein binds certain odor molecules, allowing detection of
different odorants in different cells. In mice, each olfactory sensory neuron (OSN) expresses exactly one
MONDAY-ORAL PRESENTATIONS
allele of one of more than 1000 olfactory receptor genes. Cells seem to choose at random among
available OR alleles, and once a cell has expressed a functional allele, it continues to express this allele as
long as it lives.
Determined promoter: transcription factor interactions are inadequate to explain monogenic olfactory
receptor expression because the promoters of different olfactory receptor genes are indistinguishable in
the information they contain. So, how can a cell set aside certain genes, ruling out their expression, even
in the presence of transcription factors that could drive them?
Using DNA FISH to stain all the olfactory receptor alleles at the same time, we found that in olfactory
neurons, these 2000 alleles form interchromosomal aggregates that exclude non-OR genes and are
incompatible with transcription. The active olfactory receptor allele in each cell escapes this
aggregation, allowing transcription.
In OSNs, heterochromatin (including olfactory receptor gene aggregates) occupies the nuclear core, and
euchromatin the nuclear periphery; this topology is inverted compared to most other cells. In mice,
mutation of the lamin b receptor (LBR) nuclear envelope protein causes central neurons to acquire this
inverted topology. Lamin b receptor is thus thought to tether heterochromatin to the nuclear periphery.
Strikingly, we found that the lamin b receptor protein is naturally downregulated during OSN
development, and ectopic expression of LBR in OSNs disrupts olfactory receptor gene aggregates and
allows transcription of many olfactory receptor genes per cell. Having lost their identities as detectors of
particular odors, these OSNs also target downstream neurons in the olfactory bulb aberrantly.
K2
Mechanistic studies of SecY channel-mediated protein translocation by a novel in
vivo approach.
E. Park1, T. Rapoport2;
1
Department of Cell Biology, Harvard Medical School, Boston, MA, 2Harvard Med Sch/HHMI, Boston, MA
The SecY/Sec61 protein-conducting channel mediates membrane translocation of secretory proteins
and integration of membrane-spanning proteins into the lipid bilayer, which occur in the plasma
membrane of prokaryotes or endoplasmic reticulum membrane of eukaryotes. The channel forms an
hourglass-shaped pore with a constriction in the middle across the membrane. The channel also has a
lateral gate that can open toward the lipid phase. During initiation of translocation, a ribosome-nascent
chain complex binds to the SecY/Sec61 channel, resulting in insertion of the nascent chain. Despite
extensive studies for the last couple of decades, important mechanistic problems in SecY/Sec61mediated protein translocation have been difficult to address largely due to scarcity of biochemical tools
to interrogate this transient process. We developed a novel methodology which allows stable
generation of highly defined SecY-mediated translocation intermediates in intact bacterial cells, thus
capturing the moment of protein translocation under a physiological condition. Using this approach, we
first addressed how the channel transports large polypeptides and yet prevents passage of small
MONDAY-ORAL PRESENTATIONS
molecules or ions. Our data shows that the SecY channel maintains the membrane barrier for small
molecules by surrounding a translocating polypeptide chain with its gasket-like ring of hydrophobic pore
amino acids. Second, by adapting the developed methodology, we isolated a translocation intermediate
complex containing SecY and a translating ribosome from E. coli. Cryo-electron microscopy analysis of
the obtained sample shows that a nascent chain opens the channel through largely rigid body
movements of the two halves of SecY and inserts as a loop into the channel with the hydrophobic region
of the signal sequence intercalated into the open lateral gate.
K3
Cytosolic DNA Sensing by the cGAS-cGAMP Pathway.
J. Wu1, L. Sun1,2, X. Zhang1, H. Shi3, X. Li1, F. Du1,2, X. Chen1,2, C. Chen3, Z.J. Chen1,2;
1
Department of Molecular Biology, The University of Texas Southwestern Medical Center, Dallas, TX,
2
Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Dallas, TX,
3
Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, TX
In eukaryotic cells, DNA is normally confined within the nucleus and mitochondria. The presence of
cytosolic DNA either through infections or cellular damage is a danger signal that triggers the host
immune responses such as the production of type I interferons (IFNs). Cytosolic DNA induces IFNs
through the STING-TBK1-IRF3 axis but the mechanism by which it is sensed remains elusive.
Using a cell-free complementation assay, we identified cyclic GMP-AMP (cGAMP), as a novel eukaryotic
second messenger generated by DNA stimulated or DNA viruses infected cells. cGAMP bound to STING
with high affinity and induced a dramatic conformational change that led to its activation. Through
biochemical purification and quantitative mass spectrometry, we identified the enzyme that synthesizes
cGAMP in a DNA dependent manner. This enzyme, which we named cyclic GMP-AMP synthase (cGAS),
turned out to be the long sought-after cytosolic DNA sensor. Structural and functional studies revealed
that cGAS is activated by DNA-induced dimerization. We further generated and characterized a cGAS
knockout mouse strain, which failed to produce IFNs and other cytokines in response to DNA stimulation
and were more vulnerable to lethal infection by DNA viruses.
Together, our study not only elucidated the mechanism of cytosolic DNA sensing and signaling, but also
discovered a new class of second messengers in eukaryotes. Furthermore, it also established the
enzyme cGAS as a novel therapeutic target for the treatment of autoimmune disease.
K4
Chromosomal copy number changes contribute to multidrug resistance in human
cancer cells.
L. Kabeche1,2, S. Gerber1,2, A. Grassetti1,2;
1
Department of Biochemistry, Geisel School of Medicine at Dartmouth College, Hanover, NH, 2Norris
MONDAY-ORAL PRESENTATIONS
Cotton Cancer Center, Lebanon, NH
Aneuploidy is an important and common feature of cancer, present in over 90% of solid human tumors.
Many cancer cells become chromosomally unstable (CIN) - a state in which cells lose chromosome
segregation fidelity - which can lead to persistent changes in karyotype and tumor heterogeneity.
Importantly, CIN is associated with drug resistance and poor patient prognosis in most cancers.
Acquired drug resistance is a serious contributing factor to the ineffectiveness of current therapies.
However, it still remains unclear how CIN correlates with drug resistance. There are two competing
models of how CIN relates to increased acquired drug resistance: (i) the selective karyotype model,
which suggests that upon treatment, resistant karyotype(s) of the heterogeneous tumor will continue to
proliferate and repopulate the tumor, while non-resistant karyotype(s) will die, and (ii) the intrinsic
karyotype model, which suggests that CIN cells uniquely exhibit more dynamic cell signaling pathways,
which make them more fit to survive chemotherapy treatments. Based on our findings, we hypothesize
that CIN promotes multidrug resistance through the selective karyotype model. Our preliminary results
support this hypothesis, as we see marked karyotype changes in multidrug resistant HeLa (R-HeLa) cells
compared to parental HeLa cells. R-HeLa cells have increased mRNA and protein expression of the
Multidrug resistant 1 (MDR1) ATP dependent efflux pump and exhibit high rates of lagging
chromosomes. Through fluorescence in-situ hybridization and NanoString nCounter karyotyping, we
discovered that R-HeLa cells exhibit stable karyotype changes with a gain of chromosomes 1 and 7, and
a loss of chromosome 5. Interestingly, MDR1 is localized on chromosome 7. To investigate whether a
single gene (MDR1) on a chromosome is sufficient for the selection of whole chromosome gain/loss, we
have used targeted genome editing to insert MDR1 into the AAVS1 locus on chromosome 19 in HeLa
cells. Confounding evidence for the selective karyotype model emanates from multiple karyotypes seen
in resistant cell lines. However, the extent to which these chromosome changes lead to downstream
protein changes or contribute to drug resistance is not understood. Therefore, we have begun to
quantitatively analyze the proteomes and phosphorylation and ubiquitylation events of R-HeLa cell
clones compared to non-resistant HeLa cells. This will allow us to better understand the downstream
effects of karyotype changes in the context of CIN and multidrug resistance. These studies are crucial in
understanding the cellular and molecular mechanisms of multidrug resistance.
K5
Building an organized organism: Physical forces as biological sculptors.
A.E. Shyer1, T. Savin2, T. Tallinen3, N. Kurpios4, T. Huycke5, L. Mahadevan6, C. Tabin7;
1
MCB, Univ California-Berkeley, Berkeley, CA, 2ETH Zurich, Zurich, Switzerland, 3University of Jyväskylä,
Jyväskylä, Finland, 4Cornell University, Ithaca, NY, 5Harvard Medical School, Boston, MA, 6Harvard School
of Engineering and Applied Sciences, Cambridge, MA, 7Genetics, Harvard Medical School, Boston, MA
As tissues form in the embryo, cells must adopt not only the right fate, but also the right collective
shape. The latter phenomenon, called morphogenesis, has been, and continues to be, the focus of my
research. I am particularly interested in the morphogenesis of vertebrate organs, and focused on the
MONDAY-ORAL PRESENTATIONS
intestine during my thesis because of its simplicity of form. I studied how physical forces derived from
differences in growth rates of the components of the tissue drive the formation of loops along its length,
allowing it to fit neatly in the body cavity, and complex topographies on the inside of the tube that
provide the surface area necessary for proper nutrient absorption.
To understand how loops emerge, we observed how the intestine grows in relation to the dorsal
mesentery, a thin elastic tissue that attaches it to the body. We noticed that the tube grows faster than
the attached mesentery, leading to the hypothesis that this differential growth between the two
attached tissues causes the gut tube to loop. We experimentally showed that differential growth drives
loop formation and constructed a quantitative geometric model of the process. The model was based on
the physical and geometric properties of the two attached tissues, and also their relative rates of
growth. Most strikingly, this model could predict the number and radius of loops that would form not
only in the chicken gut (our primary model system), but in the guts of the quail, zebra finch, and mouse,
suggesting that this mechanical mechanism is evolutionarily conserved across vertebrate species.
We hypothesized that differential growth between two attached tissues could also drive the emergence
of villi. Indeed, we found that emerging layers of smooth muscle constrict the adjacent luminal epithelial
layer, forcing a stereotyped folding that gives rise to luminal villi. Once again, we were able to construct
a quantitative geometric model that, with experimentally measured parameters, could recapitulate the
process of villi emergence and pattern across a range of vertebrate species.
As the mechanical mechanisms of loop and villi emergence became clearer, we realized that they could
be used to guide our molecular studies. For instance, given the critical role of differential growth and
tissue stiffness in gut looping, follow-up studies may address how signaling molecules regulate
proliferation rates and how cell biological properties collectively modulate tissue stiffness. More
broadly, this work illustrates that mechanical explanations of morphogenesis can provide a
phenomenological filter to efficiently hypothesize and interpret work conducted at the molecular and
cellular level.
K6
The tails of two histones: It was an old histone, it was a new histone.
V. Tran1, C. Lim1, J. Xie1, J. Buss2, X. Yang2, B. Chen3, E. Betzig3, J. Xiao2, X. Chen1;
1
Johns Hopkins Univ, Baltimore, MD, 2Biophysics and Biological Chemistry, Johns Hopkins Medical
Institution, Baltimore, MD, 3HHMI/Janelia Farm Research Campus, Ashburn, VA
Epigenetic phenomena are heritable features in gene expression or function that does not include
primary DNA sequences, but is retained through successive cell divisions. It is the epigenetic information
that directs cells with identical genomes to become distinct cell types in multicellular organisms,
including humans. One of the best-characterized features of epigenetic is post-translational
modifications on N-terminal histone tails, which can be inherited and confers tissue-specific gene
regulation. My thesis work focuses on the impact of epigenetic inheritance in the adult stem cell system,
MONDAY-ORAL PRESENTATIONS
using the male Drosophila germline as a model. Stem cells are unique in their abilities to both self-renew
and give rise to a variety of differentiated cell types. Mis-determination of stem cell fate and
malfunction of stem cell derivatives are common causes of many human diseases. Using a transgene
system that can distinguish pre-existing histone and newly synthesized histones, I have demonstrated
that pre-existing histones, which carry epigenetic marks on the N-terminal tails, are asymmetrically
retained in the stem cells of the Drosophila male gonad during cell division, but not in cells fated for
differentiation. Furthermore, this asymmetric histone segregation phenomenon does not occur in
differentiating daughter cells. Interestingly, this phenomenon only occurs in canonical histones, which
are histones deposited in bulk during DNA replication, but not for histone variants. These findings
provide the first direct evidence that stem cells may selectively retain preexisting histones that define its
stem cell identity. Our followup work explores the mechanism responsible for retaining pre-existing
histones in the germline stem cells. Through work with our collaborators Dr. Eric Betzig at Janelia Farm
and Dr. Jie Xiao at Johns Hopkins Medical Institution, we have uncovered preliminary evidence that
selection and retention of pre-existing histone occurs in tandem with DNA synthesis. According to our
findings, we propose that preexisting histones are selectively retained in the replication fork onto one
set of sister chromatids while newly synthesized histones are differentially incorporated into the other
during S-phase.
K7
All roads lead to Rome- SRP independent translocation into the endoplasmic
reticulum.
T. Ast1, M. Schuldiner1,2;
1
Molecular Genetics, The Weizmann Institute of Science, Rehovot, Israel, 2Department of Molecular
Genetics, The Weizmann Institute of Science, Rehovot, Israel
Translocation into the endoplasmic reticulum (ER) is an initial and crucial biogenesis step for all secreted
and endomembrane proteins in eukaryotes. Even in the simple eukaryotic model organism,
Saccharomyces cerevisiae, this is no simple task, as over a fifth of its proteome must translocate into the
ER. It's been well established that several ER targeting pathways are present in S. cerevisiae, the best
known of which relies on the signal recognition particle (SRP). We set out to shed new light on
alternative SRP-independent translocation pathways. To do so, we harnessed unbiased and systematic
approaches to further our understanding of the mechanisms by which these pathways function, and
what measures are in place if they are dysfunctional. By combining hydropathy-based analysis and high
throughput microscopy, we uncovered that over 20% of the yeast secretome translocates without the
aid of the SRP. Further investigation of these SRP-independent substrates revealed an additional motif
for ER targeting and uncovered a network of cytosolic proteins that facilitate SRP-independent targeting
and translocation. Finally, by employing a systematic microscopic screen, we revealed that SRPindependent substrates are subject to pre-translocational monitoring that clears the cytosol of proteins
that have failed to translocate in a timely manner. These findings highlight the underappreciated
MONDAY-ORAL PRESENTATIONS
complexity of SRP independent translocation and its central role in enabling the extensive flux of
proteins into the ER.
K8
The Molecular and Cellular Basis of Attractive Salt-taste Coding in Drosophila.
Y.V. Zhang1,2, J. Ni1,2, C. Montell1;
1
Neuroscience Research Institute, University of California , Santa Barbara, CA, 2Department of Biological
Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD
Animals are attracted to low salt-containing foods and reject food with high salt. However, it was
unclear how low salt and high salt taste perceptions were differentially encoded in gustatory receptor
cells, and how they induce distinct behavioral responses.
We found that the fruit fly uses two distinct types of salt GRNs to respond to different concentrations of
salt (NaCl). One type is activated maximally by low salt (50 mM NaCl), and induces attractive feeding
behavior. The other class of GRNs is activated primarily by high salt (500 mM NaCl) and leads to aversive
feeding behavior. These two types of neurons compete with each other to regulate the animal’s
behavioral outputs. The net outcome of the salt behavioral response is determined by the relative
strength of salt-attractive GRNs and salt-aversive GRNs. The identification of salt taste coding
mechanism in GRNs provides a cellular mechanism as to how the animal prefers low-salt while avoiding
high-salt foods.
To unravel the molecular mechanisms underling salt attractive and aversive pathways in the GRNs, we
screened for cation channels required for salt taste discrimination. We discovered that a member of the
ionotropic glutamate receptor (IR) family, IR76b, was required for low salt sensation, and was a Na+ leak
channel. The demonstration that IR76b is a Na+ leak channel suggests an uusual mechanism for
activating a neuron. In contrast to most cells, in which the intracellular Na+ concentration is much lower
than the extracellular concentration, the concentration of Na+ inside and outside Drosophila
chemosensory neurons appears to be similar. Thus, under resting conditions there is very little Na+
conductance through IR76b. However, after exposure to salty food, the extracellular Na+ levels rises,
thereby driving Na+ influx through IR76b. Thus, we uncovered a mechanism for neuronal depolarization
that was mediated by a change in the concentration of an extracellular ion (Na+), rather than activation
of a receptor or ion channel by a specific agonist.
In conclusion, our work not only explains the fundamental question as to how an animal chooses low
salt over high salt, but also unravels an unprecedented mechanism for how the salt responsive gustatory
receptor neurons are activated by salt.
K9
Feedback control of chromosome separation by a midzone Aurora B gradient.
O. Afonso1, I. Matos1,2, A. Pereira1, P. Aguiar1,3, H. Maiato4,5;
1
Chromosome Instability and Dynamics Laboratory, IBMC, Porto, Portugal, 2Laboratory of Mammalian
MONDAY-ORAL PRESENTATIONS
Cell Biology and Development, Howard Hughes Medical Institute, The Rockefeller University, New York,
NY, 3Center for Mathematics, Universidade do Porto, Porto, Portugal, 4Chromosome Instability
Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal,
5
Cell Division Unit, Department of Experimental Biology, Faculdade de Medicina, Universidade do Porto,
Porto, Portugal
Accurate chromosome segregation during mitosis requires the physical separation of sister chromatids
prior to nuclear envelope reassembly (NER). However, how these two processes are coordinated
remains unknown. Using live-cell imaging, RNAi, pharmacological inhibitions and laser microsurgery in
Drosophila S2 cells, we identified a conserved feedback control mechanism that delays chromosome
decondensation and NER in response to incomplete chromosome separation during anaphase. A
midzone-associated Aurora B gradient was found to monitor chromosome position along the division
axis and prevent premature chromosome decondensation by retaining Condensin I. PP1/PP2A
phosphatases counteract this gradient to trigger chromosome decondensation and NER. Thus, the
Aurora B gradient appears to mediate a surveillance mechanism that prevents chromosome
decondensation and NER until effective separation of sister chromatids. This promotes the correction
and re-integration of lagging chromosomes in the main nuclei prior to completion of NER.
K10
Structure, Polymerization, and Dynamics of a Bacteriophage Tubulin.
J.A. Kraemer1,2, M.L. Erb3,4, E.A. Zehr2, J. Pogliano4, D.A. Agard2;
1
Biology, MIT, Cambridge, MA, 2Biochemistry and Biophysics, UCSF, San Francisco, CA, 3FAS Center for
Systems Biology, Harvard University, Cambridge, MA, 4Biology, UCSD, La Jolla, CA
Tubulins are essential for the reproduction of many eukaryotic viruses, but bacteriophage are generally
assumed not to require such cytoskeletal elements. We recently showed a divergent tubulin-like
protein, PhuZ, encoded by bacteriophage 201ϕ2-1, assembles a dynamic spindle that positions phage
DNA at the center of the cell. This is the first example of a prokaryotic spindle that performs a function
analogous to the microtubule based spindles of eukaryotes. Here, we provide molecular-level insight
into its cooperative assembly mechanism PhuZ filaments. We solved the structure of full length PhuZGDP to 1.67 Å resolution, and found an extended C-terminus that we show to be critical for
polymerization. We show a 7.1 Å cryo-EM of the 3-stranded PhuZ filament. This is the first known
example of a 3-stranded tubulin-like filament. The derived pseudo-atomic model and mutagenesis
experiments reveal the importance of the extended C-terminus for lateral interactions as well. Using
right-angle light scattering, we show that PhuZ polymerization is GTP-dependent and that filaments
rapidly disassemble upon the addition of excess GDP, suggestive of the existence of a nucleotide cap.
Using TIRF microscopy, we have demonstrated that PhuZ filaments are, like microtubules, both polar
and dynamically unstable. These results corroborate data from the Pogliano lab showing dynamically
unstable PhuZ filaments coordinated in a bipolar spindle, with the minus ends anchored at the poles
during phage infection. This spindle is highly reminiscent of the eukaryotic microtubule spindle, with
filament growing ends organized from the cell poles. Our findings have revealed a previously unknown
MONDAY-ORAL PRESENTATIONS
role for tubulins in viral function and provide important insights into the structural basis for polymer
formation. Our work suggests the presence of a rich cell biology by which phages ensure proper
assembly and replication and interact with their hosts.
TUESDAY-ORAL PRESENTATIONS
ORAL PRESENTATIONS-Tuesday, December 9
Symposium 5: Life and Death in the Cell
S11
The lysosome as a control center for cellular clearance and energy metabolism.
A. Ballabio1;
1
Telethon Institute of Genetics and Medicine, Naples, Italy
Recent evidence indicates that the importance of the lysosome in cell metabolism and organism
physiology goes far beyond the simple disposal of cellular garbage. This dynamic organelle is situated at
the crossroad of the most important cellular pathways and is involved in sensing, signaling and
transcriptional mechanisms that respond to environmental cues, such as nutrients. A main mediator of
these lysosomal adaptation mechanisms is the TFEB transcription factor. TFEB is a master regulator of
lysosomal biogenesis and autophagy and mediates a lysosome-to-nucleus signaling pathway. This
pathway provides the lysosome with the ability to adapt to extracellular cues and control its own
biogenesis. Modulation of lysosomal function by acting on TFEB has a profound impact on cellular
clearance and energy metabolism and is a promising therapeutic target for a large variety of disease
conditions, such as lysosomal storage disorders and neurodegenerative diseases.
S12
Molecular Dissection of Autophagy in Yeast.
Y. Ohsumi1;
1
Frontier Research Center, Tokyo Institute of Technology, Yokohama, Japan
Autophagy is a bulk protein degradation system well conserved from yeast to higher eukaryotes.
Recently it is getting clear that autophagy is relevant to so many physiological events and diseases.
However, the molecular details of autophagy are still remained to be uncovered. Autophagosome
formation, sequestration process of a portion of cytoplasm or organelles, is the most critical event in
autophagy, which is different from the conventional membrane trafficking. Since discovery of autophagy
in yeast, S. cerevisiae, we have been working to understand the molecular mechanism of the membrane
dynamics of autophagy for these 26 years. Eighteen Atg proteins were identified as essential factors for
starvation-induced autophagy. These proteins are subdivided into six functional groups, including a
protein kinase and its regulators, PI3 kinase complex, Atg9 membrane protein, Atg18-Atg2, and
ubiquitination-like protein- and lipid-conjugation systems. These proteins function spatiotemporally in a
concerted manner for isolation membrane formation. Upon starvation a portion of these Atg proteins
TUESDAY-ORAL PRESENTATIONS
assemble at a peri-vacuolar dot, named the PAS (pre-autophagosomal structure). First Atg1 kinase forms
a pentameric complex with Atg13 and Atg17-Atg29-Atg31 complex to be a scaffold for other functional
units. Next a small number of Atg9 containing vesicles are recruited to the PAS, which indicates that PAS
is not simply assembly of Atg proteins but contains membrane structure. Then PI3 kinase complex, Atg2Atg18 and finally Atg12 conjugation and Atg8 lipidation systems join to the PAS as following the steps.
The PAS consists of considerably large number of Atg proteins, and is highly dynamic structure, changing
interacting partners step by step. Structural basis of these interactions and regulation mediated by
phosphorylation will be presented. As stated above we have been focusing on molecular mechanism of
membrane dynamics. But there are several unsolved questions about physiological roles of autophagy in
yeast. Recent progress in physiological studies will be discussed.
S13
Damage control: how PINK1 and Parkin survey mitochondrial fidelity and respond
with selective autophagy.
R.J. Youle1;
1
National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health, Bethesda,
MD
The products of two genes mutated in autosomal recessive forms of Parkinson’s disease, Pink1 and
Parkin, have been identified to work in the same cell biology pathway that induces autophagy of
damaged mitochondria. PINK1 is a kinase that flags damaged mitochondria for removal. It is
constitutively degraded by the inner mitochondrial membrane protease PARL and maintained at very
low levels in undamaged, healthy mitochondria. When one mitochondrion becomes damaged or
accumulates misfolded proteins, PINK1 is no longer imported and degraded, and instead accumulates on
the outer mitochondrial membrane bound to the TOM complex. From there PINK1 phosphorylates
ubiquitin (and possibly ubiquitin linked to outer mitochondrial membrane proteins) at serine 65 and also
phosphorylates the Parkin UBL domain at serine 65 to activate Parkin E3 ligase activity and recruit Parkin
to mitochondria. Activated Parkin ubiquitinates numerous outer mitochondrial membrane proteins.
These ubiquitin chains on the outer mitochondrial membrane recruit autophagy adaptor proteins and
autophagy machinery to selectively eliminate the damaged mitochondria suggesting that PINK1 and
Parkin mediate a quality control pathway.
How phospho-ubiquitin activates Parkin will be presented as well as the molecular steps acting
downstream of mitochondrial ubiquitination to activate mitophagy. In particular, how autophagy
adaptors p62, NBR1 and NDP52 participate in mitophagy and how the RabGAP TBC1D15 regulates Rab7
following Parkin activation. Lastly animal models of mitochondrial damage yield synthetic phenotypes
with Parkin knock out animals and reveal how the PINK1/Parkin mitophagy pathway may function in
vivo to mitigate parkinsonism.
TUESDAY-ORAL PRESENTATIONS
Symposium 6: Membrane Trafficking
S14
Lipid dynamics in the regulation of membrane traffic and interactions.
P. De Camilli1;
1
Department of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience,
Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT
Bilayer lipids are the building blocks of cellular membranes. In addition, their metabolism regulates 1)
the recruitment to the bilayer of cytosolic proteins, 2) interactions between organelles and 3)
membrane flow along the secretory and endocytic pathways. Major players in this regulation are the 7
phosphoinositides (phosphorylated derivatives of phosphatidylinositol), whose heterogeneous
distribution on different membranes helps generate a code of membrane identity. My talk will address
principles in phosphoinositide signaling, with an emphasis on the metabolism of PI4P and PI(4,5)P2, two
key determinants of plasma membrane identity, and on the impact of the dysfunction of their
metabolism on human diseases. I will also address the newly discovered function of phosphoinositides
in the formation of direct contacts between the endoplasmic reticulum (ER) and other membranes, the
plasma membrane in particular, focusing on the lipid exchange function of these contacts. While the
direct transfer of lipids from their sites of synthesis in the ER to other membranes, including the plasma
membrane, has been known since the 1970s, the role of regulated membrane contacts sites in this
transport has become apparent only in recent years. In this context, I will discuss the properties and
functions of ER-localized proteins which contain lipid transfer modules and whose binding to other
membranes in a phosphoinositide-dependent manner helps control lipid homeostasis within cells.
S15
Mechanisms of membrane shaping by proteins.
M. Kozlov1;
1
Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv Univ, Tel Aviv, Israel
Membranes of intracellular organelles and transport intermediates acquire shapes with large curvatures
and complex morphologies, and undergo persistent remodeling by fission and fusion. We suggest a
unifying mechanistic framework for understanding how specialized peripheral membrane proteins
control the intracellular membrane curvature and dynamics, and address the effects of several specific
proteins. Our consideration is based on two major mechanisms by which proteins shape membranes:
shallow insertion into the membrane matrix of amphipathic or hydrophobic protein domains, and
membrane attachment to the strongly curved and rigid faces of hydrophilic protein scaffolds. We
address the insertion mechanism, by analyzing computationally the generation of membrane curvature
by epsin. We demonstrate that biologically feasible amounts of this and similar proteins bound to the
membrane surface suffice to produce large curvatures of intracellular transport intermediates and
TUESDAY-ORAL PRESENTATIONS
organelles. Considering the scaffolding mechanism, we model computationally the shaping of
endoplasmic reticulum (ER ) membranes by oligomers of reticulon and DP1/Yop1 family proteins. We
demonstrate that membrane molding by these proteins into nearly half-cylindrical shapes underlies
generation of the whole plethora of complex morphologies observed to date in ER of different cells,
which include ER tubules, sheets, inter-tubular three-way junctions, inter-sheet helicoidal connections
and sheet fenestrations. Analyzing the interplay between the insertion and scaffolding mechanisms, we
predict that the two mechanisms act in concert while generating membrane curvature but have
opposite effects on membrane remodeling. Shallow insertions drive membrane fission converting nearly
flat membranes into small vesicles, while crescent-like scaffolds restrict fission by favoring formation of
continuous membrane cylinders. We verify these predictions by addressing the membrane remodeling
by epsin and N-BAR domain proteins, amphiphysin and endophilin.
LGBTQ Diversity Session
G7
High Throughput Proteomics to Define Chlamydia-Host Protein-Protein
Interactions.
J.N. Engel1, C. Elwell1, K. Averette1, A. Frando1, J. Johnson1, O. Rosenberg1, E. Verschueren1, J.
VonDollen1, N. Krogan1, J.D. Dunn2, R.H. Valdivia3, M.N. Starnbach4, A. Olive4;
1
Medicine, University of California, San Francisco, San Francisco, CA, 2Duke University, Durham, NC,
3
Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, 4Harvard Medical
School, Boston, MA
Chlamydiae are obligate intracellular bacterial pathogens that cause human respiratory, genital tract,
and blinding eye infections. Chlamydiae replicate within a membrane-bound compartment, termed an
inclusion, and encode a subset of ~60 effectors that localize within the inclusion membrane, termed
Incs. Incs are ideally poised to interact with and modulate host cell proteins, yet the function and
interacting partners of only a few Incs are known. We have used large-scale affinity purification-mass
spectrometry (AP-MS) combined with sophisticated bioinformatics algorithms to define human host
interacting partners of Incs. high confidence Inc-host cell interactions were identified for ~2/3 of the
expressed Incs to generate an Inc-human interactome map. We have examined in detail the interaction
between IncE and a subset of human sorting nexins, SNX1/2/5/6. These SNXs, along with the cargo
recruitment complex (CRC: VPS26/29/35) consistute the retromer complex, which recycles protein cargo
from endosomes to the TGN. SNX1/2/5/6 bind membrane phosphoinositides (PI) and induce membrane
curvature, leading to membrane tubulation and vesicle formation. Ectopically expressed IncE localized to
SNX-positive compartments, and directed immunoprecipitations (IP) confirmed that IncE co-IPs with
SNX1/2/5/6, but not with the CRC, consistent with the AP-MS data. IncE co-IPs with endogenous SNX5/6
during Chlamydia infection, and ectopic expression of IncE does not affect Chlamydia infection. Confocal
microscopy demonstrates that SNX1/2/5/6 localized to the inclusion membrane as well as to thin fibers
TUESDAY-ORAL PRESENTATIONS
that emanate from mature inclusions. Spinning disc videomicroscopy revealed that these fibers are
microtubule-dependent dynamic structures. Depletion of SNX5/6, while decreasing IncE-positive fiber
formation, increased production of infectious progeny without affecting bacterial entry or inclusion
formation. Thus, the IncE-SNX interaction may restrict production of infectious progeny. Finally, we
found that the C-terminal 24 amino acids of IncE are both necessary and sufficient to bind directly to the
N-terminal PX domain of SNX5 and SNX6 in vitro. Our data supports a model in which the C-terminus of
IncE binds directly to the PX domain of SNX5/6, which in turn recruits its binding partners SNX1/2, but
does not stably recruit the CRC. We are currently investigating how SNX sequestration regulates
production of infectious progency and whether the C-terminus of IncE functions as a PI mimic
(competing for binding) or creates a novel PI-binding interface.
ePoster Talks Session 13: New Ways of Probing, Studying,
Reconstituting, and Interrogating Cells
E85
Nuclear displacement by centrifugal force reveals distinct LINC complexes
engage microtubules and actin filaments to actively maintain nuclear position.
R. Zhu1, G.G. Gundersen1;
1
Department of Pathology and Cell Biology, Columbia University, New York, NY
Nuclei reside at specific sites that are characteristic of cell type. Except in cases where nuclei are actively
moved, it is unclear whether nuclear positioning is an active process and whether the specific position is
important for cell function. To address these issues, we developed a novel technique to displace nuclei
by subjecting adherent cells to centrifugal force. Nuclear displacement was proportional to force from
1,000-20,000 x g and the centrosome, Golgi, ER or mitochondria were not displaced. If wounded
fibroblast monolayers were placed orthogonal to the centrifugal force, nuclei in cells at the wound edge
were displace to the rear on one side, but towards the front on the other. After centrifugation, nuclei
returned to the center from both positions, but at different rates and by different cytoskeletal
mechanisms: front-to-center movement was inhibited by actin and myosin II inhibitors, whereas rear-tocenter movement was inhibited by microtubule and dynein inhibitors and dynein heavy chain
knockdown. Using siRNA to deplete LINC complex components revealed that nesprin-2G was required
for both movements, SUN1 for the microtubule-dependent re-centering and SUN2 for actin-dependent
re-centering. Using small fragments of nesprin-2G to rescue nesprin-2G depleted cells showed that
distinct regions of nesprin-2G rescued the microtubule and actin-dependent movements. Our results
show positioning of apparently stationary nuclei is an active process and suggest that a single nesprin
can form different LINC complexes to engage different cytoskeletal elements to maintain nuclear
position.
TUESDAY-ORAL PRESENTATIONS
E86
Minimal tags for rapid dual-color live cell labeling and super-resolution
microscopy.
I. Nikić1, T. Plass1, O. Schraidt1, J. Szymański1, J. Briggs1, C. Schultz1, E. Lemke2;
1
European Molecular Biology Laboratory (EMBL), Heidelberg, Germany, 2Structural and Computational
Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
The choice of appropriate labeling technique is crucial for optimal super-resolution microscopy (SRM).
Labeling density, probe size and photophysical properties are particularly important and to great extent
determine the power of SRM. In addition, the probe should be attached as close as possible to the
target protein as large linkers can induce artifacts and lower resolution. Many organic fluorophores offer
small size and good photophysical characteristics, especially when compared to widely used GFP
variants, but their direct linking to the target proteins remains challenging. We present a method that
permits direct coupling- with virtually no linker- of SRM compatible dyes site-specifically to the target
proteins by combining Amber codon suppression and click-chemistry technologies. To achieve this, host
cells are modified to incorporate unnatural amino acids (UAAs) in response to a unique codon during
translation. These UAAs carry chemical groups (strained alkynes and alkenes) that can be directly
attached to tetrazine functionalized dyes in a special type of a fully biocompatible click-chemistry
reaction named the inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC). By testing different
tetrazine derivatives of organic dyes, we tuned the SPIEDAC reaction in an orthogonal descendant for
rapid dual-color protein labeling. These two reactions allowed labeling of distinct insulin receptor
populations and Influenza VLPs (virus-like particles) with two different dyes in living mammalian cells, as
shown by SRM. While we demonstrate the applicability of our tools for SRM, generic SPIEDAC ligation
mechanism is transferrable to other techniques and could e.g. be used for making PET/MRI imaging
tracers.
E87
Learning to count: Calculating receptor densities on membranes from super
resolution images.
K. Raghunathan1, A. Ahsan1, U.S. Allam1, M. Nyati1, D. Ray1, S.L. Veatch2;
1
University of Michigan, Ann Arbor, MI, 2Biophysics, University of Michigan, Ann Arbor, MI
Quantifying the number of labeled biomolecules from super resolution images is difficult due to artifacts
from both over-counting and under-counting. We use over-counting artifacts in super-resolution images
to calculate receptor density in cells. This method takes advantage of the observation that the
magnitude of self-clustering due to over-counting is inversely proportional to the density of the labeled
protein, as long as the labeled protein is sampled randomly. Pair autocorrelation functions calculated
from over-counted fluorescence images are correlated (g(r) > 1) at radii on the order of the resolution
limit of the measurement. We also demonstrate the effectiveness of this technique by using single-color
TUESDAY-ORAL PRESENTATIONS
STORM imaging to estimate EGFR density under different conditions. This method faithfully
recapitulated previously reported numbers for EGFR per cell in MCF-7 cell line. Furthermore, the
absolute density of EGFR in different cell lines measured using this method agreed with relative
densities determined using quantitative Western blots. We have further extended this method to
quantitate interactions between labeled proteins in single cells by using two-color super resolution
imaging. Cross correlation between two color channels have been used earlier in various studies as a
measure of the strength of interaction between labeled proteins. Here, we combine cross correlation
measurements with the protein densities calculated from autocorrelation functions to determine the
number of interacting proteins in a single cell. We illustrate how this method complements existing
biochemical techniques by investigating how the interaction of EGFR with the heat shock protein, HSP90
or E3 ubiquitination enzyme, Smurf2 affects its stability. Upon EGF stimulation, the density of Smurf2 on
membrane increased while that of EGFR decreased. siRNA knockdown of Smurf2 increased EGFR
degradation from the membrane upon EGF treatment, suggesting that Smurf2 stabilized activated EGFR.
In support of this, we observed increased the interaction between EGFR and Smurf2 upon EGF
stimulation. These results along with our previous work on HSP90-EGFR interaction show that the
stability of EGFR in the plasma membrane is enforced by toggling its interaction of EGFR with these two
proteins. Monomeric EGFR is stabilized through its interaction with HSP90, while in its activated state, it
gets stabilized through monoubiquitination through interaction with Smurf2. Super-resolution methods
have been used successfully to dissect several biological problems and our method of determining
protein densities will extend the application of super-resolution to complement western blots and coimmunoprecipitation techniques that have traditionally been used to study biochemical pathways.
E88
Ptychography: Label free imaging of the cell cycle and mitosis.
R. Suman1, T.J. Henser-Brownhill1, K. Langley2, P. O'Toole1;
1
University of York, York, England, 2Phasefocus, Sheffield, United Kingdom
The use of fluorescent probes has become commonplace for staining fixed biological specimens;
however when used in living cells they can be toxic and ultimately perturb the natural function of the
cell. With this in mind, there has been an emerging need for label free microscopy. Existing techniques
such as Zernike phase contrast and DIC have limitations and most importantly are not quantitative. Here
we report a novel stain-free, high-contrast and quantitative method for imaging live cells using a
ptychographic microscope (Phasefocus VL21). The technique reconstructs an image from overlapping
diffraction patterns using a ptychographical algorithm (Maiden et al., 2010), to give high contrast,
quantitative information of the sample.
We have previously demonstrated the power of ptychography to quantitatively analyse cells during
mitosis (Marrison et al., 2013). Here we demonstrate further measurement of the mitotic index (MI) of
A549 cells in response to the cell cycle inhibitor, Nocodazole. An advantage to this imaging approach is
the ability to acquire large fields of view in which data from 500+ cells can be analysed in a manner
TUESDAY-ORAL PRESENTATIONS
comparable to flow cytometry. Following the 6hr treatment with 10, 25 and 50ng/ml Nocodazole, the
MI value also increased to 0.05, 0.23 and 0.36 respectively when compared to the control (MI= 0.03).
Furthermore we were able to use ptychography to analyse the cell cycle yielding phase characteristics
related to cell thickness, cell volume and nuclear volume. Following mitosis the volume of the daughter
cell increases linearly during the cell cycle and approximately doubles immediately prior to the next
mitotic event. We also propose that nuclear volume remains constant through G1, but starts to increase
during S phase. There is an increasing need for quantitative label-free imaging methodologies. Here we
demonstrate that ptychography generates high contrast and quantitative images that not only enable
cell cycle analysis using a label free non-invasive approach, but can now be used in studies where cell
volume/mass are underpinning the biological function of the cell. In conjunction with in situ
fluorescence imaging, the quantitative phase information can add additional and vital information that
goes beyond fluorescence imaging itself.
In summary, the work highlighted here demonstrates one potential application of label-free phenotypic
analysis of cells. The method is ideally suited for primary and stem cells where labels are undesirable or
not possible. Furthermore the technique could also be used in many other assay types, such as anticancer therapeutics, cell motility and migration, cell morphology and neurodegeneration.
E89
A sentinel protein assay for the simultaneous quantification of cellular processes.
M. Soste1, R. Hrabakova1, S. Wanka2, A. Melnik1, P. Boersema1, C. von Mering2, P. Picotti1;
1
Institute of Biochemistry, ETH Zürich, Zurich, Switzerland, 2Institute of Molecular Life Sciences,
University of Zürich, Zurich, Switzerland
Here we introduce a novel proteomic screening approach that provides a system-wide, quantitative
snapshot of the activity status of a variety of cellular processes, simultaneously. The approach is based
on the concept of sentinel proteins. Sentinels are biological markers whose change in abundance
characterizes the activation state of a given pathway or functional module in a cell. Sentinels are
proteins, phosphorylation sites or protein degradation products that have been previously validated and
are commonly assayed in molecular biology laboratories, typically one at a time by traditional antibodybased techniques. For example, sentinels include: specific phosphorylation events in the activation loop
of MAP kinases as markers for signalling along different MAPK branches; or induction of the protein
Atg8 (LC3 in mammals) as a marker for the activation of the autophagic response. By combining
previous biochemical knowledge and computational prediction, we selected a set of 309 sentinels to
probe the physiology of S. cerevisiae cells. We assembled an information-rich targeted proteomic
fingerprint assay based on selected reaction monitoring-mass spectrometry (SRM-MS) that measures
the complete sentinel set at high-throughput (1 hour per sample), and reports on the activation state of
188 biological processes. To validate the approach, we applied it to a set of eight well-characterized
environmental perturbations of yeast physiology. We show that it recapitulates many known responses
and enables the identification of novel cellular events, thereby demonstrating that the sentinel
TUESDAY-ORAL PRESENTATIONS
approach could be used to analyse the cellular responses to a large set of uncharacterized
perturbations, such as a collection of drugs.
Our current interest is in applying the approach to characterize at high-throughput the modulation of
cytotoxicity induced by the aggregation-prone protein, alpha-synuclein. Using yeast as a model system,
cytotoxicity induced by alpha-synuclein has been shown to be dose- and time-dependent yet can be
modulated by the co-overexpression of ~100 different protective genes. We are using the sentinel assay
to decipher the effects of these mostly uncharacterized genetic modulators of toxicity. By screening the
activity status of biological processes in yeast rescued by these modulators, we should be able to
identify effectors of the rescue. The findings of this study should highlight the processes most crucial to
rescue from alpha-synuclein toxicity and provide targets for follow-up in higher models.
E90
Development of novel in situ and in vivo RNA detection methods based on FIT
probes.
I. Gaspar1, F. Hövelmann2, A. Ephrussi 1, O. Seitz2;
1
EMBL, Heidelberg, Germany, 2Humboldt University, Berlin, Germany
We have been developing new tools based on fluorogenic forced intercalation (FIT) probes for RNA
detection quantification and interference in biological samples. Upon duplex formation with target
nucleic acids, the base surrogates TO dye increases its quantum yield and brightness substantially (>10
fold). We turned FIT probes brighter by including a second, brighter, slightly red shifted variant dye, JO,
which is hardly responsive. In this setup TO acts as a light harvester that feeds JO - we showed that a
single FIT probe is sufficient to allow detection of oskar mRNA in a rapid, wash free FISH setup using
conventional wide-field microscopy, making it an ideal tool for RNA localization screens.
Locked nucleic acids (LNA) immediately adjacent to the TO base surrogate also enhance brightness –
without significantly affecting responsiveness – by shortening the distance between stacked nucleotides
in duplexes. This unique behavior of FIT probes allows synthesis of nuclease resistant oligos that are
bright and contrasted enough for use in live imaging: as few as two LNA-containing FIT probes targeting
different segments of oskar mRNA are sufficient to monitor oskar RNP motility in living oocytes. Motility
data obtained by using 3-5 different probes in wild-type oocytes recapitulate what has been reported
with the oskMS2-GFP system, indicating that this is a viable, non-transgenic alternative for studying
mRNP biogenesis in vivo.
While normally the aim is to avoid interference with biological processes, nuclease-resistant oligos can
also be targeted against RNA secondary structures and/or protein binding interfaces of interest. FIT
probes, in addition, report successful hybridization, allowing the testing of the functional role of RNA
segments. We find that a FIT probe targeting the 3’ of the proximal stem of the spliced oskar localization
element (SOLE) that is essential for efficient kinesin-dependent transport and oskar localization, causes
TUESDAY-ORAL PRESENTATIONS
motility defects nearly identical to those observed when mutating the SOLE sequence in transgenic
oskar mRNAs.
Finally, we demonstrated that TO fluorescence reaches its maximum at stochiometric probe-to-target
ratio, and equipping FIT probes with a Cy7 presence reporter we could quantify oskar mRNA
concentrations in situ. Although this qFIT method lacks single molecule sensitivity at the moment, it can
measure high RNA concentration - even hundreds of RNA molecules within a confocal unit - providing a
complementary approach to single molecule FISH studies.
E91
Probing cancer cell migration on topographically reconstituted ECM nanofibers.
V.P. Sharma1,2, J.K. Williams3, E. Leung1, M.R. Padgen3, R.J. Eddy1, J. Castracane3, J.S. Condeelis1;
1
Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, 2Gruss Lipper
Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, 3SUNY College of Nanoscale Science
and Engineering, Albany, NY
Intravital imaging has emerged as the dominant imaging modality for the visualization of tumor cell
migration in vivo. Intravital imaging has identified the phenotypes of cell behavior in vivo at single cell
resolution. However, the complexity of the tumor microenvironment makes mechanistic analysis of its
contributions to cell phenotype uncertain. Previously, we utilized micro-patterned extracellular matrix
(ECM) substrates to study cancer cell migration properties in 1D, but due to the flat nature of ECM
substrate on the glass coverslip, we could not faithfully capture the 3D topography of ECM substrate
which cells encounter in vivo. Here, we developed a more realistic in vitro system using reconstituted
ECM with cylindrical substrate topography as found in the tumor microenvironment in vivo and
demonstrate that it supports the phenotypes of cell migration seen in vivo. First, we investigated the in
vivo tumor ECM architecture in two mouse models: PyMT- and breast carcinoma cell-derived mammary
tumors, using second harmonic intravital imaging. Tumor cells were found to move on fibronectin
associated collagen I fibers. We found a wide distribution of collagen I diameters with the major peak
falling in the range, 1.5-2 µm. Based on these in vivo observations, we engineered cylindrical poly(lacticco-glycolic acid) (PLGA) nanofibers matching in vivo dimensions and coated them with fluorescentlylabeled ECM components (collagen I, fibronectin). Remarkably, all of the emergent tumor cell behaviors
seen in vivo were recapitulated on the 1.5-2 micron fibers suggesting that the fiber topography is a key
contributor to cell behavior in vivo. Tumor cells in the presence of macrophages formed linear
assemblies of alternating tumor cells and macrophages, resembling streams found in vivo. Average cell
speed on these fibers reached 1 µm/min, with maximum speed reaching 4 µm/min, consistent with
previous observations made for tumor cell speed in vivo; and two times faster than speeds seen on 2D
surfaces. Surprisingly, we discovered dynamic nuclear deformations during carcinoma cell migration on
nanofibers, similar to nuclear deformation observed in cells squeezing through a narrow opening in vivo,
suggesting that carcinoma cell nucleus is inherently plastic and ECM space constraint is not required for
nuclear deformation.
TUESDAY-ORAL PRESENTATIONS
ePoster Talks Session 14: Genetic and Structural Mechanisms of
Mitotic Fidelity
E92
Asterless licenses daughter centrioles to duplicate for the first time in
Drosophila.
Z.A. Novak1, P.T. Conduit1, A. Wainman1, J.W. Raff1;
1
Sir William Dunn School of Pathology, University of Oxford, Oxford, England
Centrioles form centrosomes and cilia, and defects in any of these three organelles are associated with
human disease. Centrioles duplicate once per cell cycle when a mother centriole assembles an adjacent
daughter during S-phase. Daughter centrioles cannot support the assembly of another daughter until
they mature into mothers during the next cell cycle [1-4], however the molecular nature of this
daughter-to-mother transition has remained mysterious. Using quantitative live imaging in Drosophila
embryos we show that the conserved centriole duplication protein Asterless (Drosophila Cep152) is not
incorporated into daughter centrioles as they assemble during S-phase, but is only incorporated once
mother and daughter separate at the end of mitosis. The initial incorporation of Asterless (Asl) is
irreversible, requires DSas-4, and is essential for daughter centrioles to mature into mothers that can
support centriole duplication. We therefore propose a “dual licensing” model where Asl incorporation
provides a permanent primary license to allow new centrioles to duplicate for the first time, while
centriole disengagement provides a reduplication license to allow mother centrioles to duplicate again.
Our preliminary data suggests that the primary licensing of new centrioles through the incorporation of
Asl is regulated by cell cycle-dependent phosphorylation, which alters the DSas-4 -Asl interaction.
References: 1: Kleylein-Sohn et. al. (2007) Dev Cell (13) 190-202. 2: Cunha-Ferreira et al. (2009) Curr Biol
(19) 43-49. 3: Loncarek et al. (2008) Nat Cell Biol (10) 322-328. 4: Wang et al. (2011) JCB (193) 727-739.
E93
Chemically-induced reversible organelle knockout of centrioles reveals an
essential role in the proliferation of non-transformed cells.
Y. Wong1, J. Anzola2, R. Davis2, M. Yoon2, A. Motamedi2, A. Kroll1, C. Seo2, T. Gahman2, A. Desai3, A.
Shiau2, K. Oegema3;
1
Department of Cellular and Molecular Medicine, Ludwig Institute for Cancer Research, La Jolla, CA,
2
Small Molecule Discovery Program, Ludwig Institute for Cancer Research, La Jolla, CA, 3Ludwig Institute
for Cancer Research, La Jolla, CA
Centrioles are microtubule-based organelles that direct the formation of centrosomes and cilia. Defects
in centrosome structure and number are linked to aneuploidy and cancer, whereas ciliary dysfunction
TUESDAY-ORAL PRESENTATIONS
leads to a host of ciliopathies. Despite their broad impact on cell physiology, centriole function has been
challenging to study in mammalian cells due to the lack of a facile methodology for specifically,
persistently, and reversibly removing this organelle from cells. To facilitate analysis of centrioles, we
developed centrinone – a specific small molecule inhibitor of PLK4, the kinase that initiates centriole
assembly. Treatment with centrinone at sub-micromolar concentrations allows sustained depletion of
centrioles from human and mouse cells, leading to loss of centrosomes and primary cilia. Depletion of
centrioles from >30 human and mouse cell lines partitioned them into two distinct classes. Cell lines
with a defective p53 pathway proliferated indefinitely in the absence of centrioles. G1+S and G2
durations were not altered, but spindle assembly was slowed and cells were more prone to mitotic
defects that reduced population growth rate. Both centrioles and proliferation rate were restored upon
centrinone washout. In cell lines with a wild-type p53 pathway, centriole removal triggered an
irreversible p53-dependent G1 arrest within a few cell divisions. This arrest was not DNA damage
signaling-dependent, and did not correlate with mitotic duration, indicating that the arrest triggered by
centriole loss is distinct from the previously described prometaphase duration sensor. In transformed
cell lines with different basal levels of centrosome amplification prior to centriole depletion, centrinone
washout triggered a wave of de novo assembly and initial overduplication, followed by recovery to the
level of amplification observed prior to centriole removal. Analysis of this recovery process indicated
that overduplication is balanced by removal of cells with extra centrioles through multipolar mitosis and
death. These results suggest that centriole number set points in cancer cell lines result from a dynamic
equilibrium between centriole overduplication and removal of cells with extra centrioles, rather than
being determined by historical overduplication events.
E94
Postmitotic genome surveillance regulates the abscission checkpoint through
Chk1 activity.
D.R. Mackay1, K.S. Ullman1;
1
Oncological Sciences, University of Utah, Salt Lake City, UT
Faithful transmission of genetic information during cell division requires tight coordination between
DNA replication and cell cycle progression. While checkpoints ensure that cells do not progress into
mitosis until the vast majority of the genome has been duplicated, certain regions of the genome are
inherently difficult to replicate and can be present at the time of mitosis. Common fragile sites are
especially prone to such under-replication and this is further exacerbated by conditions of replication
stress. During mitosis, resolution of under-replicated DNA during chromosome segregation results in
DNA damage. Genomic surveillance appears to routinely take place in newly-formed nuclei, facilitating
protection of these DNA lesions by recruitment of 53BP1 and other proteins that shield the DNA until
repair can take place. Although often studied separately, the final steps of cytokinesis are concomitant
with nuclear formation. In particular, daughter cells are physically separated once ESCRT machinery is
recruited to the midbody structure formed by microtubules in the intercellular bridge where it executes
membrane abscission. The kinase Aurora B is recognized to be a master regulator of abscission timing
TUESDAY-ORAL PRESENTATIONS
and is central to a recently appreciated cell cycle checkpoint – the abscission checkpoint. Here, we
report the finding that 53BP1 recruitment to damage foci in newly-formed nuclei begins prior to
abscission and that elevating replication stress both increases this response and triggers the Aurora Bdependent abscission checkpoint. Inhibition of the kinase Chk1 in midbody-stage cells reverses this
delay in abscission; similarly timed inhibition of the kinase ATR has a partial, but significant, effect. We
further have found that this feedback pathway is active even in the absence of extrinsic factors that
increase replication stress, with Chk1 again being particularly important in controlling Aurora B activity
at the midbody. These results provide the first evidence of a signaling pathway upstream of Aurora B in
regulating abscission and reveal an unexpected connection between genomic surveillance in newlyformed nuclei and the final step of cell division.
E95
Survival of proliferative, radio-resistant polyploid cells in Drosophila requires
FANCD2.
H. Bretscher1, D. Fox1;
1
Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC
Maintaining a stable genome prevents damaged DNA, altered cellular function, and ultimately diseases
such as cancer. Genome instability can result from a wide variety of cellular stresses including DNA
damage. In order to prevent propagation of genome instability, diploid mitotic cells employ a DNA
damage checkpoint to ensure that they do not divide with damaged DNA. Entering mitosis with
damaged DNA can result in mutations and chromosome number imbalance (aneuploidy). While the
response to DNA damage is well characterized in diploid mitotic cells, it has been recently observed that
passage through the endocycle, a variant cell cycle in which mitosis is truncated, can alter the response
to DNA damage. As a result of this alteration, endocycling murine trophoblasts and several Drosophila
larval tissues are highly resistant to DNA damage. Unlike diploid cells, these non-proliferative cells
continue to endocycle despite high levels of DNA damage.
While, most endocycled cells are terminally differentiated and do not re-enter the mitotic cell cycle, we
previously established a model to address mechanisms by which the endocycle promotes genome
instability during mitosis. In the Drosophila rectum, we found endocycled papillar cells can re-enter
mitosis as polyploid cells. We show that, like other endocycled cells, papillar cells acquire radioresistance during the endocycle. Rather than undergoing full repair of DNA breaks, papillar cells re-enter
mitosis with broken chromosomes. Despite frequent DNA breaks, lagging DNA during anaphase and
organism-lethal amounts of DNA damage, papillar cells develop normally and undergo surprisingly little
cell death. To survive, we find that papillar cells enlist a non-canonical DNA damage mechanism to
ensure that anaphase is extended to allow for incorporation of broken DNA into daughter nuclei. This
survival depends on ATM and ATR, but not chk1 and chk2 kinase activity or the transcription factor p53.
In addition to ATM and ATR, we find that the DNA repair protein FANCD2 is also required for cytokinesis
delay and survival of damaged polyploid papillar cells. Cells lacking FANCD2 fail to incorporate broken
DNA into daughter cell nuclei prior to the onset of cytokinesis, and subsequently die by mitotic
TUESDAY-ORAL PRESENTATIONS
catastrophe. We speculate that the mechanism used by polyploid papillar cells to survive mitosis with
high levels of DNA damage may contribute to radio-resistance in cancer cells that have undergone an
endocycle.
E96
Characterization of nuclear cyclin D1 interactions with replication regulatory
elements in human cancer cells.
M.M. Martin1, T. Shimura2, C. Redmon3, H. . Fu4, Y. Zhang4, M. Ryan5, K. Kim5, E.M. Epner6, M.I. Aladjem4;
1
Grambling State University, Ruston, LA, 2Environmental Health, National Institute of Public Health,
Wako, Saitama, Japan, 3University of Maryland of Baltimore County, Baltimore, MD, 4Laboratory of
Molecular Pharmacology, National Cancer Institute/National Institutes of Health, Bethesda, MD, 5InSilico
Solutions, Fairfax, VA, 6Hematology/Oncology, Penn State Hershey Medical Center, Hershey, PA
During normal cell cycle progression, cyclin D1 associates with CDK 4 and CDK 6 to regulate cells passage
from G1 to S phase. In cancers cyclin D1 is either mutated, amplified or overexpressed altering cell cycle
progression and may contribute to tumorigenesis. Cyclin D1 overproduction in the nucleus causes
cancer cells to over-replicate portions of their genome and exhibit high levels of genomic instability.
Here, we investigated mutant cyclin D1’s role in the etiology of cancer. To that end, we characterized
cyclin D1 expression patterns, cellular localization along with chromatin modifications, and replication
profiles in cancer cells that exhibited cyclin D1 modifications. Our studies employed chromatin
immunoprecipitation followed by whole-genome sequencing (ChIP-Seq), gene expression analyses,
whole-genome replication profiling using asynchronous, and cell cycle fractionated cancer cells obtained
by centrifugal elutriation. Since mutant cyclin D1 was shown to interact with members of the prereplication complex in mice, we also asked whether replication patterns change in response to
accumulation of nuclear cyclin D1. Cyclin D1 migrated to the nucleus in human hepatocellular carcinoma
cells that were exposed to low doses of irradiation over a long time period (fractionated radiation). We
also found that cyclin D1 was present in the nucleus of primary patient-derived leukemia cells. In those
leukemia cells, the stabilized nuclear cyclin D1 associated with chromatin and exhibited sequencespecific interactions with activated promoters. These studies generated a whole-genome map of cyclin
D1 chromatin binding patterns in cancer cells harboring mutated nuclear cyclin D1, allowing us to
investigate if nuclear cyclin D1 binding sites co-localized with replication initiation sites and chromatin
modifications. Future studies will probe possible alterations of replication patterns in cancer cells
harboring mutated nuclear cyclin D1.
E97
Characterization of the assembly, regulation and structures of two types of
interphase nodes that merge to form cytokinetic nodes in fission yeast.
M. Akamatsu1, K. Pu2, J. Berro3, Y. Lin4, J. Bewersdorf5, T.D. Pollard5,6,7;
1
Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 2Molecular, Cellular, and
TUESDAY-ORAL PRESENTATIONS
Developmental Biology, Yale University, New Haven, CT, 3Molecular Biophysics and Biochemistry, Yale
University, West Haven, CT, 4Department of Biomedical Engineering, Yale University School of Medicine,
New Haven, CT, 5Department of Cell Biology, Yale University School of Medicine, New Haven, CT,
6
Department of Molecular, Cellular and Developmental Biology, Yale University School of Medicine, New
Haven, CT, 7Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine,
New Haven, CT
Fission yeast assemble a cytokinetic contractile ring from discrete, membrane-bound, multiprotein
complexes called nodes. Interactions of myosin-II with actin filaments pull nodes together into a
contractile ring. Nodes originate during interphase, but their origins, physical movements and fates were
unclear.
We discovered that cytokinesis nodes assemble from two types of interphase nodes composed of
different proteins. Type 2 nodes containing protein Blt1p, GTP exchange factor Gef2p, accessory protein
Nod1p and kinesin Klp8p exist throughout the cell cycle and emerge from the contractile ring as it
disassembles. Type 1 nodes with kinase Cdr1p, kinase Cdr2p and anillin Mid1p follow the separating
nuclei and disperse into the cytoplasm during mitosis and reappear in a medial band around each
daughter nucleus after mitosis. Quantitative measurements and computer simulations showed that
these two types of nodes come together by a diffuse-and-capture mechanism: type 2 nodes diffuse to
the equator where they are captured by stationary type 1 nodes.
The Septation Initiation Network (SIN) triggers septation and constriction of the contractile ring in fission
yeast. We used conditional mutations to turn the SIN on and off and established that SIN activity is
sufficient and necessary to disperse the core type 1 node protein Cdr2p into the cytoplasm. This explains
why type 1 nodes assemble only during interphase through early mitosis when SIN activity is low.
Activating the SIN during interphase dispersed Cdr2p from type 1 nodes a few minutes after the SIN
kinase Cdc7p-GFP accumulated at spindle pole bodies. If the SIN was then turned off in interphase cells,
Cdr2p reappeared in nodes in parallel with the decline in SIN activity. Hyperactivating SIN during mitosis
dispersed type 1 nodes earlier than normal, and prolonged SIN activation prevented nodes from
reforming at the end of mitosis. Conversely, inactivating SIN during mitosis prevented Cdr2p nodes from
dispersing into the cytoplasm.
We used live cell FPALM super resolution microscopy to document the structures and motions of
interphase nodes at ~35 nm resolution. Type 1 and 2 node proteins Cdr2p and Blt1p tagged with
mEOS3.2 appeared as discrete structures ~50 nm in diameter, similar to Mid1p and consistent with their
roles as core structural proteins that scaffold interphase nodes. Imaging cells expressing Blt1p-mEOS3.2
at 200 frames per second revealed nondirectional movements of type 2 nodes in the cell cortex with a
diffusion coefficient ~400 nm2/s, while type 1 and 2 nodes near the cell equator moved little over 60 s,
both requirements for successful simulations of our diffuse-and-capture model.
TUESDAY-ORAL PRESENTATIONS
E98
The HERCulean E3 Ligase HERC2 safeguards cellular quiescence by regulating
the RB/E2F signaling network.
D. Siepe1, R. Pferdehirt1, J.R. Lill1, D.S. Kirkpatrick1, P.K. Jackson2;
1
Department of Protein Chemistry, Genentech Inc., South San Francisco, CA, 2Department Microbiology
and Immunology, Stanford University School of Medicine, Stanford, CA
The retinoblastoma tumor suppressor protein (RB) serves as an integral regulatory cell cycle node
balancing proliferation and quiescence. Although multisite phosphorylation of RB by CDKs has been
extensively studied, regulation by other post-translational modifications such as ubiquitin remains
largely enigmatic. Here, we identify the giant (~0.5 MDa) HECT E3 ligase HERC2 as a critical regulator of
the RB/E2F signaling network in cellular quiescence. Transitioning into quiescence, hypophosphorylated
HERC2 associates with hyperphosphorylated RB (p-RB) via it’s RLD3 (RCC like) domain and is
subsequently degraded in a ubiquitin-proteasome dependent manner. Consequently, HERC2 depletion
results in accumulation of RB and p-RB to supraphysiological levels, followed by dysregulation of cell
cycle licensing factors (e.g. E1F1, CDK1, CDC6 and MCM’s) rendering cells unable to properly transition
into quiescence. In this pseudo-quiescent state, global gene expression profiling reveals that HERC2 loss
triggers a fundamental change in the quiescence transcriptional program channeled through the RB/E2F
signaling network. By contrast, increased HERC2 activity correlates with loss of RB/p-RB in quiescence.
Thus, we propose that HERC2 plays a critical previously unrecognized role in safeguarding cellular
quiescence and genome integrity by regulating RB/p-RB.
ePoster Talks Session 15: Cell Motion and Mechanobiology 2
E99
Fibroblasts use Filopodia Extensions to Probe Substrate Rigidity before
Occupying an Area.
S. Wong1, W. Guo1, Y. Wang1;
1
Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA
Rigidity sensing and durotaxis are thought to be important elements in wound healing, tissue formation,
and cancer treatment. It has been challenging, however, to study the underlying mechanism due to
difficulties in capturing cells during the transient response to a rigidity interface. We have addressed this
problem by developing a model experimental system that confines cells to a micropatterned area with a
rigidity border. The system consists of a rigid domain of one large adhesive island, adjacent to a soft
domain of small adhesive islands grafted on non-adhesive soft gels. This configuration allowed us to test
rigidity sensing away from the cell body during probing and spreading. NIH 3T3 cells responded to the
micropatterned rigidity border similarly to cells at a conventional rigidity border, by showing a strong
preference for staying on the rigid side. Furthermore, cells used filopodia extensions to probe substrate
TUESDAY-ORAL PRESENTATIONS
rigidity at a distance in front of the leading edge and regulated their responses based on the strain of
the intervening substrate. Soft substrates inhibited focal adhesion maturation and promoted cell
retraction, while rigid substrates supported stable adhesions and cell spreading. Myosin II was required
for not only the generation of probing forces but also the retraction of probing structures in response to
soft substrates. We suggest that a myosin II-driven, filopodia-based probing mechanism ahead of the
leading edge allows cells to migrate efficiently, by sensing physical characteristics before moving over a
substrate to avoid back-tracking.
E100
Actomyosin contractility is required for Notch-mediated patterning of sensory
organ precursor cells in the Drosophila notum.
G.L. Hunter1, B. Baum2, G. Charras3;
1
MRC Laboratory for Molecular Cell Biology, University College London, London, United Kingdom,
2
Laboratory for Molecular Cell Biology, University College London, London, United Kingdom, 3London
Centre for Nanotechnology, University College London, London, United Kingdom
The differentiation of sensory organ precursor (SOP) cells in the Drosophila notum epithelium is an
excellent model system in which to study long-range lateral inhibition via Notch signaling. Previous
research showed that robust, tissue-level organization of SOPs requires a population of basal, actinbased protrusions. Here, we investigate whether forces generated by the cytoskeleton also play a role
in the process of long-range lateral inhibition. (1) We identify a dynamic pool of non-muscle myosin II
heavy chain, together with phosphorylated myosin regulatory light chain, which localizes to the basal
cytoskeleton of the notum epithelium. (2) Modulation of myosin II activity via the overexpression of
phospho-mimetic or phospho-dead myosin regulatory light chain alters basal protrusion dynamics (e.g.,
retraction and extension rates) and morphology (e.g., branching and lamellopodia) without affecting
their length. (3) Decreased myosin II activity, induced through the expression of dominant negative
constructs, (a motor-less heavy chain or a phospho-dead regulatory light chain), leads to defects in longrange lateral inhibition, indicated by the decreased spacing of SOP and SOP-like cells in the notum. (4)
Moreover, these changes in signaling are not accompanied by striking changes in the localization of
Notch or Delta protein. Together these results suggest that actomyosin-based forces play a role in
activation of Notch signaling. (5) To investigate how myosin II activity affects the Notch signaling
pathway to induce these changes in tissue patterning, we performed live, quantitative analysis of Notch
signaling in the notum, using a novel Notch signaling reporter (He and Perrimon, unpublished). Using
this tool, we can demonstrate that Notch signaling dynamics in the notum are consistent with previous
mathematical models of lateral inhibition, in which cells undergo dynamic changes in their state as they
commit to an epithelial or SOP fate. (6) We find that the expression of phospho-dead regulatory light
chain results in a shift in the kinetics of the Notch signaling response in notum epithelial cells, and
suggests that Notch activation is delayed in the absence of myosin II contractility relative to control.
These data suggest that actomyosin contractility plays a key role in the activation of Notch signaling
during notum patterning. Thus, protrusions may mediate long range, mechanically-induced Notch
TUESDAY-ORAL PRESENTATIONS
signaling in vivo – as previously proposed for Notch-Delta signaling in vitro. This identifies a mechanism
by which cells can integrate mechanical forces and cell signaling to drive developmental processes.
E101
Microtubules Regulate Focal Adhesion Dynamics through MAP4K4.
J. Yue1, X. Wu1;
1
The Ben May Department, The University of Chicago, Chicago, IL
Focal adhesions are dynamic organelles that establish a connection between the ECM (extracellular
matrix) and cytoskeletal networks and serve as points of traction for cells. The disassembly of focal
adhesions allows cell retraction and integrin detachment from the ECM, processes critical for cell
movement. A long-standing and intriguing observation is that growth of MT filaments in migratory cells
can be guided toward focal adhesions, and targeting of MT growing ends (plus ends) to focal adhesion
often precedes focal adhesion turnover. It has been speculated that MTs can serve as tracks to deliver
proteins essential for focal adhesion disassembly. However, the molecular nature of the focal adhesion
“disassembly factor” remains elusive. By quantitative proteomics, we identified mammalian MAP4K4
(mitogen-activated protein kinase kinase kinase kinase 4) as a focal adhesion regulator that associates
with MTs. Conditional knockout (cKO) of MAP4K4 in skin epidermal cells stabilizes focal adhesions and
impairs epidermal migration. Exploring underlying mechanisms, we further show that MAP4K4
associates with MT plus end tracking protein, EB2 (end binding 2) and an Arf6-specific guanine
nucleotide exchange factor, ARF-GEP100 (ADP-Ribosylation Factor-Guanine Nucleotide Exchange
Protein-100 kDa), which is also known as IQSEC1 (IQ motif and SEC7 domain-containing protein 1).
Together, our findings provide new insights for this critical cellular process, suggesting that EB2containing MT plus ends can deliver MAP4K4 toward focal adhesions, where MAP4K4 can in turn
activate Arf6 via IQSEC1 and enhance focal adhesion dissolution.
E102
Mechanotransduction via the nuclear lamina.
D.E. Discher1,2;
1
Molecular and Cell Biophysics Lab, University of Pennsylvania, Philadelphia, PA, 2Chemical and
Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA
How cells respond to physical cues in order to meet and withstand the physical demands of their
immediate surroundings has been of great interest for many years, with current efforts focused on
mechanisms that transduce signals into diverse phenotypes. Pathways that mechano-regulate the entry
of transcription factors into the cell nucleus are emerging, and our most recent studies show that
mechanical properties of the nucleus itself are actively controlled in response to the elasticity of the
extracellular matrix (ECM) in both mature and developing tissue [1]. The mechano-responsive properties
of nuclei are largely determined by the stoichiometry of intermediate filament lamin proteins that line
TUESDAY-ORAL PRESENTATIONS
the inside of the nuclear envelope and that also impact upon transcription factor entry and broader
epigenetic mechanisms. Signaling pathways are emerging for regulation of lamin levels and cell-fate
decisions in response to a combination of ECM mechanics and other molecular cues [2]. Nuclear
mechanics also synergize with niche anchorage and cell motility during development, at least in the
contexts of adult hematopoiesis [3] and tumor growth [4].
References: [1] Swift et al. Science 2013. [2] Buxboim et al Curr Biol 2014. [3] Shin et al. PNAS 2013.
[4] Harada et al J Cell Biol 2014.
E103
Two distinct actin networks mediate traction oscillations to confer
mechanosensitivity of focal adhesions.
Z. Wu1, S.V. Plotnikov1,2, C. Waterman1, J. Liu1;
1
National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, 2Cell and
Systems Biology, University of Toronto, Toronto, ON
Adherent cells actively sense the mechanical stiffness of their extracellular matrix (ECM) by exerting
traction force through focal adhesions (FAs), which are integrin-based protein assemblies. Also, FAs
control cell spreading, proliferation, survival, differentiation, and migration. FA-mediated
mechanosensation underlies cell durotaxis – the tendency of most cell types to migrate toward stiffer
microenvironment. Strikingly, FA-mediated traction forces oscillate in time and space, and this
oscillation governs durotaxis. The interactions underlying this intriguing spatio-temporal pattern of FA
traction force are unknown, as are the contributions of these interactions to this mechanosensation. To
address these questions, we established the first coherent, experimentally validated model of FA
formation. The model integrated the spatiotemporal coordination between a branched actin network
and stress fibers during FA growth. Our model predicted that retrograde flux of branched actin network
contributed to a traction peak near the FA distal tip and that stress fiber-mediated actomyosin
contractility generated a second traction peak near the FA center. Furthermore, a negative feedback
loop involving formin-mediated stress fiber elongation and actomyosin contractility developed and
resulted in oscillation of the center traction peak. This oscillation competed with the distal traction peak,
and the competition underpinned oscillation of the FA traction maximum in time and space. More
importantly, this negative feedback loop broadened the substrate stiffness range, over which the FAs
could accurately adapt with traction force generation. Our findings shed light on the fundamental
mechanism of FA mechanosensation and durotaxis.
TUESDAY-ORAL PRESENTATIONS
E104
Impact of mechanical stress on Multicellular Tumor Spheroids proliferation.
V. Lobjois1, A. Desmaison1, L. Aoun1, B. Ducommun1;
1
CNRS USR3505 ITAV, Toulouse, France
A tumor micro-region consists of a 3D heterogeneous cell population in which cancer cells growth is
influenced by interaction with the microenvironment. The crosstalk between tumor cells and
microenvironmental components, including the extracellular matrix (ECM), fibroblasts, endothelial and
immune cells, is essential for tumor progression and plays a key role in drug resistance. In such a
complex environment, solid tumors are also subjected to mechanical stress that influences their growth
rate and development. Indeed, modification in mechanical homeostasis within tissues are also observed
during tumor growth. However, little is known about the effects of such mechanical stress on tumor cell
biology and their pharmacological consequences. To explore this issue, we investigated the impact of
mechanical stress on cell proliferation in Multicellular tumor spheroids (MCTS) in which cancer cells are
cultured as 3D organized spheres. Such complex multicellular model reproduces the cell-cell and cellmatrix interactions found in solid tumors. Moreover, MCTS can grow up to several hundred micrometers
in diameter, thus progressively displaying a gradient of proliferating cells similar to what is found in
tumor micro-regions. Specifically, in large spheroids, dividing cells are loacted in the outmost layers
while quiescent cells are found in more central hypoxic and nutrient-poor regions. Using this model, we
showed, by using PDMS microdevices, that spheroids can grow under mechanical confinement and that
reciprocal resistance from the PDMS walls opposed to spheroids growth induces mechanical stress. In
these conditions, we demonstrated that growth-induced mechanical stress induces mitotic
accumulation inside spheroids. We then monitored the impact of mechanical stress on the progression
in mitosis using ligth sheet fluorescence microscopy. We developped specific sample holders that
enabled us to explore 3D mitosis dynamics inside spheroids in control and confined conditions. By
measuring the duration of each phase of mitosis, we demonstrated that mechanical stress induces a
prometaphase delay. As growing solid tumors also exert forces on their microenvironment, in order to
evaluate the mechanical properties of growing spheroids, we designed and produced microdevices
arrays of high aspect ratio pillars. Using these microdevices, we investigated the forces exerted by
spheroids from various cell lines. We will present our latest results on the impact of these specific
controlled environments on cell proliferation and 3D organization of spheroids.
•
•
Aoun L, Weiss P, Laborde A, Ducommun B, Lobjois V, Vieu C. Lab Chip. 2014 Jun 2;14(13):2344-53. *: cocorresponding authors
Desmaison A, Frongia C, Grenier K, Ducommun B, Lobjois V. PLoS One. 2013 Dec 3;8(12):
E105
Temporal association of tensin 1 and p130Cas at focal adhesions serves as a
molecular clutch that links inwardly moving actin cytoskeletons to cell migration.
Y. Sawada1, S. Tan1, H. Hirata1, U. Chaitanya1, H. Machiyama2, Z. Zhao3;
1
Mechanobiology Institute, National University of Singapore, Singapore, Singapore, 2Osaka University,
TUESDAY-ORAL PRESENTATIONS
Osaka, Japan, 3Department of Biological Sciences, National University of Singapore, Singapore,
Singapore
p130Cas (Crk-associated substrate; hereafter Cas) has been reported to be involved in various cellular
processes. However, the molecular mechanism of how Cas contributes or relates to cell motility is
poorly understood. We have recently reported that actomyosin contraction displaces Cas molecules
from focal adhesions depending upon their phosphorylation, thereby promoting cell migration
(Machiyama et al., J. Cell Sci. 2014). To further delineate the mechanism underlying the Cas dissociation
from the adhesion complexes, we sought the molecules that link inwardly-moving actin cytoskeletons to
Cas upon its phosphorylation. Here we show that tensin 1 binds to phosphorylated Cas through its
SH2PTB domain, and facilitates the displacement of Cas from focal adhesions. Analysis of the subcellular
distribution reveals that tensin 1 colocalizes with Cas at focal adhesions in a phosphorylation-dependent
manner. In mouse embryonic fibroblasts, silencing tensin 1 or introducing a truncation mutant of tensin
1 (tensin 1-SH2PTB) significantly retards cell migration. TIRF-FRAP and TIRF-fluorescent speckle
microscopy analyses demonstrate that expression of tensin 1-SH2PTB stabilizes the Cas residence at
focal adhesions. Furthermore, in vitro binding experiments indicate that phosphorylated Cas substrate
domain plays a primary role in binding to tensin 1-SH2PTB. These results suggest that tensin 1 transmits
actomyosin contractility to adhesion sites by temporally binding to phosphorylated Cas, providing a
clutch that supports cell migration.
ePoster Talks Session 16: Actin and Actin-Related Motors
E106
Myosin 18A Co-assembles with Myosin II to Drive the Functional Diversity of
Myosin II Bipolar Filaments.
J.R. Beach1, N. Billington2, K. Remmert1, M. Barzik3, S.M. Heissler2, L. Shao4, D. Li4, T.B. Friedman5, E.
Betzig4, J.R. Sellers6, J.A. Hammer1;
1
LCB, NIH/NHLBI, Bethesda, MD, 2NIH/NHLBI, Bethesda, MD, 3NIH/NIDCD, Bethesda, MD, 4HHMI/Janelia
Farm Research Campus, Ashburn, VA, 5NIH/NIDCD, Rockville, MD, 6LMP, NIH/NHLBI, Bethesda, MD
Bipolar filaments of nonmuscle myosin II (NMII) power myriad cellular and developmental processes.
Here we provide evidence that myosin 18Aα (M18Aα) and myosin 18Aβ (M18Aβ), two M18A splice
variants, co-assemble with NMII, opening the door to novel mechanisms of bipolar filament regulation.
Structurally, M18Aα and M18Aβ resemble NMII with extra domains at their N- and C-termini.
Specifically, both isoforms possess a non-helical tailpiece that contains interaction sites for SH3 and PDZ
domain-containing proteins, while M18Aα also possesses a ~300 residue N-terminal extension
containing a KE-rich region, a nucleotide-insensitive actin-binding site, and a PDZ domain. Importantly,
both isoforms contain numerous substitutions in their active site and lack actin-activated ATPase
activity, arguing that they are not motor proteins (Guzik-Lendrum et al., JBC, 2013). Using a combination
TUESDAY-ORAL PRESENTATIONS
of TIRF and structured-illumination microscopy (TIRF-SIM), which we used recently to show that NMIIA,
IIB and IIC co-assemble into heterotypic bipolar filaments in live cells (Beach et al., Current Biology,
2014), we find clear evidence that M18Aα and M18Aβ (both expressed and endogenous molecules) coassemble with NMII in live cells in a variety of structures, including sub-nuclear stress fibers (SNSFs).
Consistently, sedimentation, EM and single-molecule imaging show that purified M18A co-assembles
with NMII in vitro. Incorporation of M18A into NMII bipolar filaments could serve to regulate filament
assembly/disassembly. Indeed, excess M18A disrupts NMII filament assembly both in vitro and in live
cells. M18A levels in vivo are, however, markedly sub-stoichiometric to NMII (ranging from ~1:10 to
~1:100), and preliminary FRAP studies do not show an obvious effect of M18A depletion on NMIIA
filament turnover. These results favor the idea that small numbers of M18A molecules dope NMII
filaments to serve, via their extra N-and C-terminal domains, as adaptors to link the filament to cellular
structures. Consistently, the targeting of M18Aα to SNSFs depends on its PDZ domain, suggesting that
this domain links SNSFs to the nuclear envelope. M18A could also serve to recruit molecules to the NMII
filament. Consistent with this idea, we show M18A’s non-helical tailpiece binds βPIX, a Rac GEF with
roles in adhesion and protrusion, via βPIX’s SH3 domain. Finally, we identify two additional M18A splice
variants (M18Aδ and M18Aγ) that possess unique N- and C-terminal domains. Collectively, our data
argue that diversity of NMII function in higher eukaryotes is driven in part by co-assembly with various
spliced isoforms of M18A, which serve as adaptors to link the filaments to different cellular structures
and signaling molecules without interfering with NMII motor activity.
E107
Myosin X Reveals Dynamics and Function of Basal Filopodia.
D. Courson1, K. Liu1, A. Fanning1, R. Cheney1;
1
Cell Biology and Physiology, University of North Carolina, Chapel Hill, NC
Though often depicted as simple and flat, the basal surface of monolayers of polarized epithelial cells is
actually seething with protrusive activity. Using live-cell, time-lapse TIRF microscopy we visualize the
dynamics of the basal surface of stable and migrating mammalian epithelial monolayers. Previous work
showed that epithelial cells in migrating sheets extend cryptic lamellipodia, in the direction of migration.
In this report we show that polarized mammalian epithelial cells (MDCK and Caco2) also have filopodia
on their basal surface. Basal filopodia are numerous during monolayer formation and maturation, and
probe ahead of the lamellipodia during migration.
Basal filopodia have gone largely unnoticed, however we demonstrate that myosin X (Myo10) marks the
tips of epithelial filopodia of cells in isolation and basal filopodia of cells in polarized sheets. In wound
healing assays Myo10 puncta are planar polarized at the leading edge of cells found throughout the
monolayer, from deep within the sheet out to the leading edge. Basal filopodia and Myo10 puncta
become less prominent as the monolayer becomes quiescent but reappear within minutes of wounding.
Myo10 and filopodia have both recently been shown to play roles in cancer metastasis. Our findings
demonstrate that Myo10 and basal filopodia both directly affect cellular motility. We show that
TUESDAY-ORAL PRESENTATIONS
knocking down Myo10 results in the loss of basal filopodia and ~40% reduction in the rate of collective
migration. A similar decrease in motility is observed by inhibition of filopodia via low dose cytochalasin.
Interestingly, the difference in motility between wild type and Myo10 knockdown cells is lost upon
treatment with low dose cytochalasin, consistent with the hypothesis that the loss of filopodia, rather
than some other Myo10 function, is responsible for the observed motility defect in the knockdown cells.
Ongoing work is examining the mechanism behind Myo10 recruitment, filopodia formation, and their
roles in collective migration.
E108
Controlling Initiation and Termination of Kinesin-1-Driven Transport with MyosinIc and Non-Muscle Tropomyosin.
B.B. McIntosh1,2, E.L. Holzbaur1, E.M. Ostap1,3;
1
Department of Physiology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA,
2
Pennsylvania Muscle Institute, Philadelphia, PA, 3Pennsylvania Muscle Institute, University of
Pennsylvania, Perelman School of Medicine, Philadelphia, PA
Intracellular transport is largely driven by processive actin- and microtubule-based molecular motors.
Non-processive motors have also been localized to trafficking cargos, but their respective roles during
transport are not well understood. Myosin-Ic (Myo1c) is a non-processive actin motor that functions in a
variety of exocytic events, yet the molecular roles of this motor during trafficking are unclear. To
investigate the interplay between myosin-I motors and the canonical long distance transport motor
kinesin-1, we attached both Myo1c and kinesin-1 motors to lipid-coated bead cargo. This is a more
physiological cargo-motor attachment strategy that allows motors to actively reorganize within the
membrane and respond to the cytoskeletal environment. We compared the motility of these cargos in
the absence and presence of Myo1c motors at engineered actin filament-microtubule intersections
attached to the surface of a coverslip. We found that Myo1c influences kinesin-1 run initiation by
significantly increasing the frequency of microtubule-based runs that begin at actin filamentmicrotubule intersections. Myo1c also regulates run termination. Beads with both motors bound
consistently pause at actin-microtubule intersections, remaining tethered for an average of 20 seconds,
with pauses longer than 200 seconds also observed. Actin-binding proteins such as non-muscle
tropomyosin have been proposed to positively or negatively regulate the interactions between specific
myosin motors and actin filament populations in vivo. In vitro, we found that non-muscle tropomyosin-2
(Tm2) abrogates Myo1c-driven actin filament gliding. In the crossed filament assay, we found that Tm2
abolishes Myo1c-specific effects on run initiation and run termination. Together these observations
suggest that within the cell, Myo1c is important for the selective initiation and termination of kinesindriven runs along microtubules at actin filament intersections. The regulation of Myo1c by tropomyosin
provides a mechanism for regional specificity, preventing inappropriate pausing during long distance
transport, while also enabling targeted delivery of cargo to highly exocytic, dynamic actin populations,
beneath the plasma membrane.
TUESDAY-ORAL PRESENTATIONS
E109
Control of tissue mechanical properties by cellular micro-architecture: cell size,
C-cadherin (cdh3) mediated F-actin assembly, and actin bundling.
J.H. Shawky1, L.A. Davidson1,2;
1
Bioengineering, University of Pittsburgh, Pittsburgh, PA, 2Developmental Biology, and Computational
and Systems Biology, University of Pittsburgh, Pittsburgh, PA
To understand the connection between genetics, the environment, and birth defects we are particularly
interested in physical and genetic factors that regulate tissue mechanics in the developing embryo.
Tissues within embryos of the aquatic frog Xenopus laevis increase in stiffness six-fold as they gastrulate
(form the three germ layers) and neurulate (form the central nervous system), however, the origin of
this increase is not well understood.
We compare the mechanics of embryonic tissues to the predictions of the cellular solids model (CSM;
Gibson, L.J. and M.F. Ashby, Cellular solids: structure and properties. 2nd ed 1997, New York: Cambridge
University Press) which has been used to understand the mechanical properties of natural and manmade closed-cell foam materials. The CSM relates bulk stiffness of a foam to the density, or unit-size of
individual cells, their microstructural organization, and their material properties. CSM suggested that
cell size could be responsible for changes we observe in bulk tissue stiffness. We took advantage of the
biology of cell division in Xenopus embryos to produce tissues with controlled cell sizes. To generate
large cells in tissue explants, we arrested the cell cycle using a combination of cell cycle inhibitors,
hydroxyurea and aphidicolin (HUA). Tissues treated with HUA showed a 34% decrease in nuclear density
equating to a 50% increase in cell volume. CSM predicts a 23% decrease in tissue stiffness and we found
a 25% decrease in the measured elastic modulus. Since cell size plays a role in the large increase in
stiffness we sought to test other CSM predictions.
Additional factors contributing to bulk stiffness in closed-cell foams include the thickness and material
stiffness of the "cell-wall". To manipulate these factors we sought to reduce F-actin assembly in the cell
cortex by expressing a dominant negative C-cadherin (extracellular-domain deleted EP(C)-cadherin;
EPΔE) in dorsal tissues. We observed both a reduction in the amounts of cortical F-actin and found
dorsal tissues exhibited a 32% decrease in elastic modulus. Efforts are underway to regulate cortical Factin levels and their degree of cross-linking to test further predictions of the CSM model.
Understanding how cell size, cell cohesion and actin bundling affect tissue stiffness is imperative to
uncovering the programs in development responsible for stiffening the embryo.
TUESDAY-ORAL PRESENTATIONS
E110
Determining the impact of the actin cytoskeleton on the mechanical properties of
cells.
I. Jalilian1, G. Schevzov1, T. Fath1, C. Heu1, L. Bischof2, H. Vindin1, J.R. Stehn1, M. Knothe Tate3, E.C.
Hardeman1, P.W. Gunning1;
1
School of Medical Sciences, UNSW, Sydney, Australia, 2Quantitative Imaging Group, CSIRO, Sydney,
Australia, 3Graduate School of Biomedical Engineering, UNSW, Sydney, Australia
The influence of the actin cytoskeleton on the shape and mechanical properties of cells has been
revealed extensively using anti-actin drugs. However, due to the lack of the specificity of these
compounds for different actin filament populations it has been difficult to delineate the exact role of the
actin cytoskeleton in these cellular features. Actin binding proteins largely affect the organisation and
dynamics of actin in cells; however, the impact of these proteins on the mechanical properties of cells is
still unknown. Tropomyosins (Tms) form a co-polymer with actin filaments and differentially regulate
actin filament stability, function and organisation through their highly regulated expression and sorting
to specific intracellular sites. Hence, Tm isoforms can be used as tools to identify and manipulate
functionally distinct actin filament populations in a cell. In this study we investigated the impact of Tmcontaining actin filaments on the organisation of the actin cytoskeleton and the mechanical properties
of cells.
To investigate the role of different Tms, and hence different actin filaments, on cellular stiffness we
examined rat neuroblastoma cells stably overexpressing TmBr3, Tm3, Tm4 or Tm5NM1 in comparison
with control cells. Analysis of the number and length of actin cables per cell using a morphometric linear
feature detection algorithm revealed significant differences in the length and number of actin cables per
cell relative to that seen in the control cells. The mechanical measurements conducted by indentationtype atomic force microscopy (AFM) showed that TmBr3-, Tm4- and Tm5NM1-overexpressing cells were
significantly stiffer than control cells. In contrast, cell stiffness did not change in Tm3-overexpressing
cells indicating that the observed changes in cell elasticity are not a generic effect of accumulating more
Tm. Moreover, siRNA knockdown of Tm5NM1 in Tm5NM1-overexpressing cells caused the elastic
modulus to revert to that of control cells.
The results show that cell stiffness increases in TmBr3-, Tm4- and Tm5NM1-overexpressing cells. Since,
the overexpression of Tm changes the numbers and length of the actin cables in an isoform-specific
manner that is not directly related to cell stiffness, our data suggest that the mechanical properties of
cells are more dependent on the organisation of the actin filaments than the number and length of actin
filaments. Therefore, we conclude that it is specific aspects of actin organisation which regulate the
mechanical properties of the cell.
TUESDAY-ORAL PRESENTATIONS
E111
Collapsin Response Mediator Protein-1 as a novel factor modulating Arp2/3dependent actin structures.
H. Yu-Kemp1, W.M. Brieher1;
1
Cell and Developmental Biology, University of Illinois-Urbana-Champaign, Urbana, IL
Listeria monocytogenes is a parasite that uses host proteins to assemble an Arp2/3-dependent actin
comet tail for its propulsion. Comet tail assembly is more efficient in cytosol than in defined systems
implying that unknown factors contribute to this reaction. We fractionated bovine brain cytosol and
identified Collapsin Response Mediator Protein-1 (CRMP-1) as such a factor. CRMP-1 has long been
studied as a factor for microtubule dynamics. Here, we show that CRMP-1 contributes to Arp2/3dependent actin dynamics. Imuunodepletion of CRMP-1 from brain cytosol reduced Arp2/3-dependent
actin assembly around Listeria, and the defect could be successfully rescued with recombinant CRMP-1.
In vitro assays confirmed that CRMP-1 enhanced Arp2/3-dependent branching and stimulated ActA- as
well as VCA-activated Arp2/3-dependent actin polymerization. To test the contribution of CRMP-1 to
Arp2/3-dependent polymerization inside cell, we examined the localization of CRMP-1 using
immunostaining and GFP-CRMP-1 overexpression in epithelial cells. CRMP-1 localized to the leading
edge of lamellipodia and cadherin-mediated cell-cell contacts, both of which are known to be Arp2/3dependent structures. Knockdown and overexpression approaches revealed a positive correlation
between the amount of CRMP-1 and the amount of F-actin inside the cell. In wound closure assays,
CRMP-1-depleted cells had twice less protrusions at the wounded edge, with half actin intensity at the
leading edge compared to control cells; while overexpression of CRMP-1 provided the opposite results.
During wound closure, CRMP-1-depleted cells moved erratically and lost their directionality with
unstable membrane protrusions. In contrast, GFP-CRMP-1 overexpressing cells moved unidirectionally,
with the membrane at the leading edge stably protruding forward. Depletion of CRMP-1 also reduced
the amount of cadherin and actin at cell-cell contacts, resulting in weaker cell adhesion for epithelial
cells. Immunobloting results indicated CRMP-1 present in the membrane fraction of cadherin enriched
plasma membrane. This membrane had been shown to be able to assembly actin filaments in an
Arp2/3-dependent manner. An antibody against CRMP-1 blocked this Arp2/3-dependent reaction,
showing the role of CRMP-1 on regulating Arp2/3-dependent actin assembly at cadherin-mediated cellcell contacts. In summary, our results identify CRMP-1 as a novel regulator of the actin cytoskeleton that
specifically contributes to Arp2/3-dependent actin assembly. CRMP family proteins have been studied in
regulating metastasis and neuronal growth cone guidance. Our data provide a mechanism for
understating how CRMP proteins might modulate those biological events through their potential role in
actin cytoskeletal organization.
TUESDAY-ORAL PRESENTATIONS
E112
Two Functionally Distinct Sources of Actin Monomers Supply the Leading Edge
of Lamellipodia.
E.A. Vitriol1, L. McMillen2, S. Gomez3,4, M. Kapustina5, D. Vavylonis2, J.Q. Zheng1;
1
Department of Cell Biology, Emory University, Atlanta, GA, 2Department of Physics, Lehigh University,
Bethlehem, PA, 3Department of Computer Science, University of North Carolina, Chapel Hill, NC,
4
Department of Pharmacology, University of North Carolina, Chapel Hill, NC, 5Department of Cell Biology
and Physiology, University of North Carolina, Chapel Hill, NC
Lamellipodia, the sheet-like protrusions of motile cells, are made of networks of actin filaments (F-actin)
regulated by the ordered assembly from and disassembly into actin monomers (G-actin). Traditionally,
G-actin is thought to exist as a single, contiguous pool. Here, we show there are two functionally and
molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from
the cytosolic pool requires the monomer binding protein thymosin β4 (Tβ4) for its leading edge
localization, is predominantly targeted to formins, and is responsible for creating an elevated G/F-actin
ratio that promotes membrane protrusions. The second source of G-actin comes from recycled
lamellipodia F-actin. Lamellipodia recycling occurs independently of Tβ4 and appears to regulate
lamellipodia homeostasis. Discrete Tβ4-mediated G-actin localization to the leading edge is achieved by
preventing the actin monomers from associating with Arp2/3 polymerization sites found throughout the
lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources
of monomers with discrete functional outputs.
Minisymposium 14: Cell Cycle Signaling and Regulation
M127
Molecular Mechanism of cell cycle coupling to centromeric chromatin
propagation.
A. Stankovic1, J.F. Mata1, L.Y. Guo2, B.E. Black2, L.E. Jansen1;
1
Instituto Gulbenkian de Ciência, Oieras, Portugal, 2Department of Biochemistry and Biophysics,
University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA
The centromere forms the chromosomal anchorage point to the mitotic spindle, driving chromosome
segregation during mitosis. Centromere position is epigenetically defined by nucleosomes containing the
histone H3 variant CENP-A. Faithful CENP-A inheritance and propagation is critical to ensure
maintenance of centromere identity. During S-phase, while DNA is replicated, CENP-A nucleosomes are
inherited and redistributed between the two sister chromatids. CENP-A replenishment is strictly cell
cycle coupled and occurs upon mitotic exit in early G1 phase. Previous studies from our laboratory
showed that Cyclin-dependent kinases (Cdk) control timing of CENP-A assembly by preventing
centromere propagation during S, G2 and mitotic phases thereby restricting centromere propagation to
TUESDAY-ORAL PRESENTATIONS
G1 phase of the cell cycle. Here we report insight into the molecular mechanism underlying Cdk-based
control of centromere inheritance. We have identified putative phosphorylation sites in the largest
member of the Mis18 complex, Mis18BP1 as well as the CENP-A specific chaperone HJURP that control
their localization and activity. In addition, we have mapped a putative cyclin interaction site within
HJURP. Importantly, co-expression of proteins carrying mutations at these cell cycle responsive residues
is sufficient to induce premature CENP-A assembly and fully uncouple cell cycle progression from
nascent CENP-A deposition. This leads us to propose a two-step inhibitory mechanism that explains G1
phase restricted centromere assembly.
M128
Mitotic inhibition of the DNA double-strand break response preserves genomic
integrity.
A. Orthwein1, A. Fradet-Turcotte1, S. Noordermeer1, M. Canny1, C. Brun1, J. Strecker1, C. Escribano-Diaz1,
D. Durocher1;
1
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON
Exogenous and endogenous DNA-damaging agents continuously threaten genome integrity and can
cause cytotoxic DNA double-strand breaks (DSBs). Interphase cells have evolved dynamic and sensitive
mechanisms to detect, signal, and repair DNA DSBs. A central component of this response is mediated
through ubiquitin (Ub) signaling controlled by the E3 ubiquitin ligases RNF8/RNF168. RNF8/RNF168
ubiquitylate chromatin and promotes the recruitment of 53BP1 to DSBs, an important factor in classical
non-homologous end-joining (C-NHEJ). In stark contrast, mitotic cells inactivate the Ub-dependent
signaling and repair of DNA DSBs, a phenomenon first observed 60 years ago, but the significance of this
inhibition remains elusive.
We investigated how mitosis blocks the DNA DSB response and determined the consequences of its
reactivation. We identified a phosphorylation site in RNF8 downstream of its forkhead-associated (FHA)
domain, which prevents the RNF8-MDC1 interaction and the initiation of the ubiquitylation of
chromatin. Mutation of this site to alanine restores RNF8 recruitment to DSBs in mitosis, but fails to
mobilize 53BP1 as monitored by immunofluorescence, suggesting the presence of a second inhibitory
mechanism. We previously characterized an Ub-dependent recruitment (UDR) motif in 53BP1 that
recognizes RNF168-dependent H2A Ub mark on nucleosomes. Interestingly, we observed that mitotic
53BP1 UDR motif is phosphorylated at two sites, which abrogates binding to ubiquitylated nucleosomes.
Mutation of these two sites to alanine restores 53BP1 capacity to read ubiquitylated H2A and allows its
recruitment to DSBs. Reactivation of RNF8 and 53BP1 in mitosis restores DNA repair, but is paradoxically
deleterious. Indeed, restoration of the DNA DSB response in mitosis leads to Aurora B kinase-dependent
sister chromatid fusions that produce dicentric chromosomes and the formation of micronuclei, a
hallmark of aneuploidy.
TUESDAY-ORAL PRESENTATIONS
Our findings reveal the complexity and the importance of regulating the DNA DSB response in mitosis.
Through multiple phosphorylation events, mitotic cells block the Ub-dependent DNA DSB response, thus
minimizing the risk of mitotic sister chromatid fusions.
M129
Oncogenic RAS/MAPK signaling hyper-activates Aurora B kinase and weakens
kinetochore-MT attachments.
J.A. Herman1, P.J. Paddison2, J.G. DeLuca1;
1
Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, 2Human Biology
Division, Fred Hutchinson Cancer Research Center, Seattle, WA
For over a century it has been appreciated that many cancer cells exhibit a high degree of aneuploidy,
which results from the incorrect segregation of whole chromosomes during mitosis. Recent evidence
has also demonstrated that the chromosome segregation errors leading to aneuploidy commonly arise
from defects in kinetochore-microtubule attachment regulation. Thus, successful chromosome
segregation requires that kinetochores not only link the forces from dynamic spindle microtubules to
chromosomes, but also precisely regulate the attachment strength to these microtubules. For example,
in early mitosis, kinetochores prevent the stable binding to ends of microtubules emanating from the
incorrect spindle pole to prevent the accumulation of attachment errors. Such error prevention is
accomplished primarily through Aurora B kinase (ABK) dependent phosphorylation of the kinetochore
factors responsible for binding microtubules and generating stable kinetochore-microtubule
attachments. Despite a growing understanding of how cells become aneuploid and the mechanisms cells
use to prevent this condition, the mechanism by which cancer cells become prone to errors in
kinetochore-microtubule attachment remains poorly understood. To investigate this issue, we
transformed primary RPE cells with various oncogenic stresses. In doing so, we found that oncogenic
RAS/MAPK signaling compromises kinetochore-microtubule attachments through a phosphorylation
cascade of kinetochore targets. Oncogenic RAS/MAPK signaling aberrantly hyper-activates the ABK error
correction system and destabilizes kinetochore-microtubule attachments. This decreased attachment
stability prolongs mitosis and increases chromosome segregation errors, and both defects are largely
rescued by inhibition of RAS/MAPK signaling. This amplification of ABK activity also results in an
increased dependence on recruitment of the ABK-counteracting phosphatase PP2A to kinetochores. In
order to form stable kinetochore-microtubule attachments, RAS/MAPK-transformed cells require the
BubR1-KARD domain, which contributes to PP2A kinetochore recruitment, while non-transformed cells
do not. Together, these findings demonstrate that in at least a subset of cancers, kinetochoremicrotubule attachments become weakened, and suggest that this defect may be exploited to generate
new, highly specific therapeutics.
TUESDAY-ORAL PRESENTATIONS
M130
Aneuploidy in normal organ formation and function: a key role for the endocycle.
K. Schoenfelder1, R. Montague1, H. Bretscher1, B. Stormo1, D. Fox1;
1
Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC
Cell division errors cause detrimental chromosome number imbalances, or aneuploidy. While
aneuploidy is a well-known hallmark of cellular dysfunction or diseases such as cancer, it is poorly
understood why aneuploid cells occur in many healthy tissues. Such healthy aneuploidy is often
accompanied by whole duplications of the genome, or polyploidy. We developed study of a new model
organ system to uncover the impact of polyploidy and accompanying aneuploidy on normal organ
construction and function. Our system is the Drosophila rectal papillae, which are formed by a
previously un-appreciated cell cycle program. Like many polyploid cells, papillar cells first become
polyploid by undergoing an endocycle- a modified cell cycle that eliminates M-phase. However, unlike
in other known endocycled tissues, papillar cells undergo a cell cycle switch and re-enter mitotic cycles,
and thus they divide as polyploid cells. In line with other studies of polyploid division (e.g. the
mammalian liver) we find papillar divisions produce aneuploid cells1.2.
Here, we present our findings regarding the causes and consequences of aneuploidy and polyploidy
during normal papillar organ construction. Unlike most endocycled cells, we show papillar cells undergo
a distinct pre-mitotic endocycle, which is characterized by the retention of centrioles. Pre-mitotic
endocycles cause these centrioles to amplify, and also interfere with centrosome clustering
mechanisms. As a result, papillar cells undergo frequent multipolar divisions. These multipolar divisions
produce daughter cells with a high degree of aneuploidy, with up to 4-fold differences in the numbers of
a particular chromosome. Remarkably, these multipolar division daughters are viable and functional,
and increasing the frequency of such highly aneuploid divisions has no obvious impact on papillar organ
formation or function. As an explanation for why such highly error-prone divisions are part of normal
organ development, we find that converting papillar cells to diploid, or preventing endocycling papillar
cells from switching back to mitosis, dramatically disrupts organ formation. Taken together, our findings
illuminate a need for polyploid mitosis in specific organ construction, despite generating increased
aneuploidy. This aneuploidy is remarkably well-tolerated in this developmental context, which contrasts
some predictions of the role of aneuploidy in a polyploid cell. Our findings thus impact study of variant
cell cycles in an in vivo context, and also provide new understanding of ploidy alterations in tissue
biology.
1.Fox DT, Gall JG, and Spradling AC (2010) Genes Dev 24: 2294-2302 2.Schoenfelder K, Montague R,
Paramore S, Lennox A, Mahowald A, and Fox DT (in press) Development
TUESDAY-ORAL PRESENTATIONS
M131
Phase separated RNA-protein droplets position cyclin transcripts to organize the
cytosol of multinucleate cells.
H. Zhang1, A.A. Bridges1, P. Occhipinti1, A.S. Gladfelter1;
1
Biological Sciences, Dartmouth College, Hanover, NH
Syncytial cells are found throughout the biosphere from filamentous fungi to early animal development,
muscle, placenta and tumor cells. Multinucleate cells face unique organizational problems and in many
cases must functionally compartmentalize their cytosol for diverse processes including nuclear division
and symmetry breaking. We found that in the filamentous fungus Ashbya, in which nuclei cycle
asynchronously despite sharing a cytosol, that cyclin transcripts are clustered and kept near nuclei by
binding to the RNA-binding protein, Whi3. Whi3 has a substantial polyQ tract and this aggregationprone region is essential for clustering transcripts and asynchronous nuclear division. We hypothesized
that Whi3-cyclin complexes form functional, dynamic aggregates that phase separate from bulk cytosol,
potentially to control the location of cyclin protein production. We have reconstituted the cyclin
transcript-polyQ protein complex and found that at physiological concentrations Whi3 and cyclin mRNAs
phase separate and form liquid droplets both in buffer and in concentrated cell extracts. Biophysical
measurements of the droplet properties indicate that they are dynamic, fluid and highly dependent on
having mRNA and full length Whi3 protein. Similarly, in cells the ability of Whi3 protein to bind mRNA is
essential for its heterogeneous localization into dynamic puncta and tubules, as well as nuclear
asynchrony. These results demonstrate a function for RNA-protein granules in regulating variable and
asynchronous nuclear progression and show mRNA is a critical component for the assembly process. We
propose that phase-separated droplets may be widely employed to pattern cytosol for diverse processes
beyond the regulation of mRNA turnover and are likely critical for large, syncytial cell organization.
M132
A microbial route to cell cycle control in the plant superkingdom.
F.R. Cross1, F. Tulin1, K.C. Atkins1, K. Lieberman1;
1
Rockefeller University, New York, NY
We have initiated a screen for mutations specifically disrupting cell cycle control in Chlamydomonas.
Robotically assisted microbiological methods allow efficient recovery of UV-induced temperaturesensitive lethals. From these, candidate cell-cycle-specific mutants were selected by time-lapse
microscopy, based on the criterion of wild-type cell growth rate combined with first-cycle failure of cell
division. tThe mutants were sorted into ~60 complementation groups, and causative mutations
identified by next-generation sequencing of bulked segregant pools. Almost all the genes had clear
Arabidopsis sequelogs, and included genes required for DNA replication and chromosome segregation.
We also identified ts mutations in conserved cell cycle control machinery: the cyclin-dependent kinases
CDKA and CDKB, two subunits of the anaphase-promoting complex, and the mitotic kinases Aurora B
TUESDAY-ORAL PRESENTATIONS
and Mps1. Phenotypic analysis of the mutants provides an outline of global cell cycle control, in which
CdkA is central for cell cycle initiation, and CdkB is critical later, for execution of mitosis. Our model
suggests a possible negative feedback control architecture in which CdkA activates CdkB, and CdkB
represses CdkA. Transcriptome analysis by RNAseq in wild type and mutants indicated a broad program
of transcriptional regulation through the cell cycle, which both regulates and is regulated by core cell
cycle machinery such as CdkA and CdkB. Most of the genes were homologous to genes implicated in cell
cycle control in yeast and animals. However, some have strong sequelogs in Viridiplantae but not in
yeast or animals. For example the BSL protein phosphatase family likely has an uncharacterized essential
role in Arabidopsis; our results in Chlamydomonas demonstrate an essential and specific role for BSL
phosphatase in entry into mitosis. We are extending the screen to a wider range of phenotypes to cover
a broad range of Chlamydomonas cell biology.
M133
Artificial dimerization of INCENP/Sli15 makes the essential biorientation function
of Aurora B/Ipl1 independent of its localization to microtubules and to the inner
centromere.
C. Campbell1, A. Desai2, K. Turnbull2;
1
University of California, San Diego, La Jolla, CA, 2Ludwig Institute for Cancer Research, La Jolla, CA
Accurate segregation of the replicated genome requires biorientation of chromosomes on the
metaphase spindle, which is ensured by the kinase Aurora B/Ipl1, a member of the 4-subunit
chromosomal passenger complex (CPC). In prometaphase, Aurora B localizes between sister
kinetochores at the inner centromere and this localization is dependent on the CPC subunit Survivin. We
had previously shown in budding yeast that disrupting inner centromere CPC localization, by truncating
the N-terminus of the INCENP/Sli15 scaffold of the CPC to eliminate association with Survivin/Bir1, did
not affect chromosome segregation during mitosis and meiosis (Campbell & Desai. Nature 2013
497:118-21). As N-terminally truncated Sli15, in contrast to wildtype Sli15, concentrated prematurely on
pre-anaphase spindles, we proposed that the microtubule-binding activity of Sli15 was necessary for
biorientation in the absence of inner centromere localization. Consistent with this idea, we show here
that further truncation of Sli15 into the central, microtubule-binding region is lethal. Expression of a
fragment (Sli15CT) that contains only the C-terminal 199 amino acids of the 698 aa-long Sli15 results in
chromosome missegregation equivalent to a null mutant. Surprisingly, Glutathione-S-Transferase (GST)mediated dimerization of Sli15CT rescued the lethality observed with Sli15CT and resulted in only mild
chromosome segregation defects. In addition, GST-Sli15CT no longer localized prematurely to preanaphase spindles, indicating that dimerization compensates for microtubule localization. Cumulatively,
these results indicate that the CPC can sense tension and correct misattachments independently of its
concentration on either chromatin or microtubules. These results are consistent with a model in which
physical clustering activates Aurora B/Ipl1 and kinetochore-intrisic sensing of tension is sufficient for
chromosome biorientation.
TUESDAY-ORAL PRESENTATIONS
M134
Mitotic chromosome alignment is dispensable for accurate segregation of the
genome.
C. Fonseca1, L. Reinholdt2, J.K. Stumpff1;
1
Molecular Physiology and Biophysics, University of Vermont, Burlington, VT, 2Genetic Research Science,
Jackson Laboratory, Bar Harbor, ME
Chromosome alignment at the equator of the mitotic spindle is a highly conserved step during cell
division. This process requires spatial control of kinetochore microtubule (k-fibers) dynamics and is
widely believed to promote the equal distribution of replicated sister chromatids during anaphase.
However, this latter assumption has never been experimentally tested due to the difficulty of disrupting
chromosome alignment without also compromising the attachments between kinetochores and spindle
microtubules. We find that depletion of the kinesin-like motor Kif18A from diploid human cells
significantly disrupts chromosome alignment while only modestly affecting mitotic progression.
Surprisingly, loss of Kif18A does not lead to an increase in aneuploidy, suggesting that chromosome
alignment is largely dispensable for accurate chromosome segregation. Instead, we find that disrupting
chromosome alignment leads to defects in the organization of the nucleus during the subsequent
interphase. These data suggest that mitotic chromosome alignment may function to preserve the threedimensional organization of interphase chromosomes from cell cycle to cell cycle.
M135
Microtubule asters grow to span a millimeter-sized egg by nucleation remote from
centrosomes.
K. Ishihara1, P. Nguyen1, A.C. Groen1, C.M. Field1, T.J. Mitchison1;
1
Department of Systems Biology, Harvard Medical School, Boston, MA
The large cells in early vertebrate development face an extreme physical challenge in organizing their
cytoplasm. For example, frog embryos are 1.2 mm in diameter but have to divide every 30 min. In these
cells, radial arrays of microtubules called asters grow, interact and move to precisely position the
cleavage plane. Although the rapid expansion of asters, spanning the entire cytoplasm, is required for
cells to explore its size and shape, it is unknown how microtubules assemble at such large distances
from their presumable nucleation centers or centrosomes. Here, we combine quantitative imaging and
cell-free reconstitution to show that microtubule asters grow by microtubule nucleation away from the
centrosome. Aster growth occurs in the absence of microtubule transport while plus ends undergo both
polymerization and depolymerization. Further, we report that the embryonic interphase cytoplasm
supports spontaneous microtubule assembly independent of centrosomes, consistent with previous
reports in vivo. We propose that aster growth, initiated by the centrosome, is supported by microtubule
nucleation and is stimulated by the presence of pre-existing microtubules.
TUESDAY-ORAL PRESENTATIONS
Minisymposium 15: Cell Motion and Mechanobiology
M136
Nanoscale architecture of tension generation within focal adhesions.
A.H. Mekhdjian1, M. Morimatsu1, A.C. Chang1, S. Tan1, A.R. Dunn1;
1
Chemical Engineering, Stanford University, Stanford, CA
Living cells are exquisitely responsive to mechanical cues from their surroundings. A primary means by
which cells sense and transmit mechanical force is through integrins, a class of heterodimeric,
transmembrane proteins that physically link the cell cytoskeleton to the extracellular matrix (ECM). The
cytoplasmic domains of integrins recruit numerous proteins that collectively comprise focal adhesions
(FAs), micron-sized assemblies responsible for mechanosensing and traction force generation. However,
the underlying mechanisms responsible for the spatial and temporal organization of force generation
within FAs remain poorly understood. We used Förster resonance energy transfer (FRET)-based
molecular tension sensors (MTSs) to directly visualize mechanical forces exerted by individual integrins.
Simultaneous super-resolution imaging of MTSs and GFP-tagged cellular proteins results in maps of the
force-producing structures within FAs with better than 100 nm spatial resolution, allowing us to
correlate local force generation with the presence of specific integrin heterodimers and cytoskeletal
proteins. We find that αvβ3 integrin localizes to high force regions, whereas α5β1 integrin localization is
more diffuse. The canonical FA proteins paxillin, vinculin, talin, and α-actinin colocalize with force
production to varying degrees. Surprisingly, paxillin, which is not generally considered to play a direct
role in force transmission, shows a higher degree of spatial correlation with force than vinculin, talin, or
α-actinin, proteins with hypothesized roles in mechanotransduction. In addition, simultaneous timelapse imaging of either GFP-paxillin or GFP-α-actinin with MTS-measured local traction forces reveals
that paxillin and tension are closely related in both space and time in assembling and disassembling
adhesions, while α-actinin exhibits a more complex relationship with tension. The high degree of spatial
correlation of both paxillin and αvβ3 integrin with mechanical tension suggests that these proteins may
work in concert to regulate both FA dynamics and intracellular, mechanically induced signal
transduction.
TUESDAY-ORAL PRESENTATIONS
M137
Matrix bound BMP2 alters stiffness response through integrin-mediated
mechanotransduction.
L. Fourel1, A. Valat2, E. Faurobert1, R. Guillot2, K. Ren2, I. Bourrin-Reynard1, L. Lafanechere1, E. Planus3, C.
Picart2, C. Albiges-Rizo1;
1
Differentiation and Cell Transformation, Institute Albert Bonniot (IAB) Inserm U823, Grenoble, France,
2
CNRS UMR 5628, LMGP, Grenoble Institute of Technology and CNRS, Grenoble, France, 3INSERM-UJF
U823, Grenoble Cedex 9, France
As extracellular matrix is crucial for binding soluble growth factors and regulation of their distribution,
activation, and presentation to cells, we have designed a biomaterial offering the characteristics of
growth factor presentation with appropriate mechanical properties. BMP-2 presentation in a soft
matrix-bound format alters stiffness response and reveals cooperativity between BMP-2 receptors and
integrin receptors necessary for a BMP-2 coordinated cell response. Specific integrin is required to
mediate BMP-2 induced Smad signaling. Both BMPR and integrin signaling converge to modulate actin
dynamics crucial for Smad activation. Finally, our results indicate that the control of LIMK/cofilin
pathway by specific integrin and BMP receptor is implicated in mechanotransduction and tensional
homeostasis through the modulation of actin dynamics necessary for Smad phosphorylation.
M138
Matrix rigidity sensing drives actin cytoskeleton remodelling and rheology.
M. Gupta1, B.R. Sarangi2, A. Callan-Jones3, R. Mege2, R. Voituriez4, B. Ladoux1,2;
1
Mechanobiology Institute, National University of Singapore, Singapore, Singapore, 2Institut Jacques
Monod, Université Paris Diderot CNRS, Paris, France, 3Matiere et Systemes Complexes, Université Paris
Diderot CNRS, Paris, France, 4UPMC, Paris, France
Rigidity sensing regulates a large variety of cellular processes including cell migration, differentiation and
tumor progression. However, how cells probe substrate stiffness, and hence respond to it, remains
controversial. Here, we show that actin cytoskeleton remodeling governs cell adaptation to matrix
rigidity. Cell adhesion to micropillar substrates of tunable stiffnesses adapts actin rheological properties
and traction forces. Our in vitro experiments and theoretical modeling clearly demonstrate a biphasic
behavior of actin cytoskeleton in fibroblastic cells from viscous fluid on soft substrates towards elastic
solid behaviors on stiffer substrates. Furthermore, we show that the elastic behavior correlates with a
higher polarization of actin stress fibers with increasing substrate stiffness and thus, exhibits an
isotropic-nematic transition similar to elastic nematic gels which is explained in the framework of active
matter theory. We conclude that the actin cytoskeleton itself could act as a force sensor providing an
adaptation mechanism to matrix rigidity that governs cell shape changes and cell polarity.
TUESDAY-ORAL PRESENTATIONS
M139
An Arp2/3 to Formin switch adapts the migratory behavior of dendritic cells to
their function upon maturation.
P. Vargas1, P. Maiuri1, M. Bretou1, P. Pierobon1, E. Terriac2, M. Chabaud1, M. Raab2, A. Alberts3, P.
Suraneni4, R. Li4, R. Voituriez5, M. Piel2, A. Lennon-Dumenil1;
1
Institut Curie, Paris, France, 2UMR 144, Institut Curie, Paris, France, 3Van Andel Res Inst, Grand Rapids,
MI, 4Stowers Institute for Medical Research, Kansas City, MO, 5UPMC, Paris, France
Immature dendritic cells (DCs) patrol the interstitial space of peripheral tissues for the presence of
danger-associated antigens by combining cell migration and extracellular material uptake. Encounter
with such antigen triggers a developmental program referred to as “DC maturation” that ultimately
turns off antigen internalization and up-regulates the surface expression of the chemokine receptor
CCR7. CCR7 allows DC migration along CCL21 gradients that guide them to lymphatic vessels (LVs) and
lymph nodes for activation of T lymphocytes. This migratory event determines the efficiency of the
adaptive immune response. How the core cell locomotion machinery regulates DC migration at their
distinct maturation stages remains unknown. Here we show that upon maturation, DCs increase their
migration persistence and speed through a cell-intrinsic switch in actin-nucleating machineries. We
found that immature DCs are characterized by Arp2/3-dependent actin polymerization at their front,
which, surprisingly, decreases their migration speed and persistence. Arp2/3 is nonetheless required for
antigen internalization in these cells. In contrast, mature DCs exhibit a migration mode characterized by
Formin (mDia1)-dependent actin accumulation at their back. This actin-nucleating protein induces a
transition in DC locomotion from diffusive migration to a persistent random walk, which enables their
efficient response to CCL21 gradients. This transition is further required for DC migration to LVs in
tissues and arrival to lymph nodes in vivo. We conclude that maturation of DCs triggers an Arp2/3 to
Formin molecular switch that optimizes their locomotion mode according to their distinct functional
requirements: antigen capture for immature DCs and fast persistent migration to lymphoid organs for
mature DCs.
M140
Force transduction during adhesion-independent migration.
M. Bergert1,2, A. Erzberger3, R.A. Desai4, A.C. Oates4, G. Charras5, G. Salbreux3, E.K. Paluch1;
1
MRC-LMCB, UCL, London, United Kingdom, 2ETH Zürich, Zurich, Switzerland, 3MPI PKS, Dresden,
Germany, 4MRC NIMR, London, United Kingdom, 5London Centre for Nanotechnology, University College
London, London, United Kingdom
Substrate adhesions have long been considered essential force-transducers during cell migration.
However, recent studies indicate that cells in confinement can migrate without specific substrate
attachment. Adhesion-independent migration is likely to facilitate locomotion during development,
immune response, and cancer metastasis. Yet, force generation mechanisms in the absence of substrate
adhesions remain speculative. We investigate the forces involved in adhesion-free migration. Using a
TUESDAY-ORAL PRESENTATIONS
non-adherent blebbing cell line as a model, we show that actin cortex flows lead to propelling forces via
non-specific substrate friction. A threshold friction is required for migration. Above this friction, we find
that the forces exerted by the cells on the substrate are orders of magnitude lower than, and that force
distribution is opposite compared to, what is observed during adhesion-based motility. This
fundamentally different mode of force transduction may have important consequences for cell-cell and
cell-substrate interactions during migration in vivo.
M141
Nuclear damage in highly constrained migration: from lamina defects to DNA
breaks.
J. Irianto1, A. Athirasala2, R. Diegmiller2, J. Swift3, D.E. Discher1,4;
1
Molecular and Cell Biophysics Lab, University of Pennsylvania, Philadelphia, PA, 2SEAS, University of
Pennsylvania, Philadelphia, PA, 3University of Pennsylvania, Philadelphia, PA, 4Chemical and
Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA
Cells in vivo are sometimes required to migrate through tight spaces that are much smaller than the
largest organelle, their nucleus. Micro-pore migration of lung cancer cells causes nuclear blebs with
segregated lamins as well as DNA tethering and breaks. Nuclear blebs seen in the majority of cells are
enriched in lamin-A and deficient in both lamin-B and DNA, but the cells are viable with a normal rate of
post-migration proliferation. Phosphorylation of lamin-A, which relates to turnover under low stress,
decreases with migration, while phosphomimetic and progeria mutants of lamin-A exhibit distinct
differences. Knockdown of lamin-A induced the frequent formation of DNA tethers that extrude from
the main nuclear body through a gap in lamin-B and to the pore that the cell migrated through. Double
strand breaks also increased and are consistent with subsequent cell death. The findings reveal a crucial
role for the lamins in cell migration and survival, likely through DNA protection.
M142
Somal translocation of Retinal Ganglion Cells: a new mode of microtubule
involvement in neuronal migration.
J. Icha1, C. Norden1;
1
MPI-CBG, Dresden, Germany
Neuronal migration is a key step during brain development and defective neuronal migration can lead to
cognitive impairments. Importantly, neuronal migration modes differ depending on tissue context and
migratory distances covered. Despite this broad variety however, most mechanistic studies have so far
focused on radial glial-guided migration
To get insights into additional neuronal migration modes we investigate the kinetics and molecular
mechanisms responsible for retinal ganglion cell (RGC) migration in zebrafish. RGCs are the earliest born
TUESDAY-ORAL PRESENTATIONS
neurons in the retina and migration from their apical birthplace to the most basal neuronal layer spans
about 50µm. It was proposed that RGCs move via somal translocation but the exact molecular
mechanisms of migration are still elusive.
We image migratory processes in the intact embryos using light sheet as well as confocal microscopy at
high temporal-spatial resolution. This approach revealed that RGC migration occurs unidirectionally and
migratory speeds reach 20 µm/h. To exclude the possibility that somal translocation is a passive process
we inhibited nuclear movements of remaining progenitor nuclei. This treatment did not significantly
impair RGC migration arguing that it is an active process depending on force generation within the cells.
Interestingly, we observe that the centrosomes as well as the Golgi follow the migrating soma during
RGC translocation instead of leading it as seen in glia-guided migration. Nevertheless, like in glial-guided
migration, both, microtubules and dynein play a role in RGC displacement. Microtubules and dynein are
apically enriched during migration, indicating that the force to move cells is generated BEHIND nucleus
and soma. This finding revealed the possibility that microtubules push the nucleus, the bulkiest
organelle of the cell, in the direction of basal migration and/or prevent re-displacements by acting as an
apical barrier. The observation that basal process attachment is crucial for efficient migration underlines
this exciting possibility. We currently test this hypothesis further using laser ablation approaches to
explore whether apical and basal processes feature different mechanical properties. We also test the
possibility that dynein is important to give rigidity to apical microtubules and thereby stabilizes the
apical process.
Overall our study aims to gain a basic understanding of the molecular mechanism involved in somal
translocation that will most likely also apply in brain regions beyond the retina. It furthermore shows
that neurons can achieve successful migration differently in different scenarios and underlines the fact
that neuronal migration has many different flavours several of which still await further exploration.
M143
Myosin II pulls the nucleus forward to increase intracellular pressure and drive 3D
cell movement.
R. Petrie1, H. Koo2, K. Yamada1;
1
NIH/NIDCR, Bethesda, MD, 2Department of Orthodontics, University of Pennsylvania School of Dental
Medicine, Philadelphia, PA
Cells use actomyosin contractility to move through three-dimensional (3D) extracellular matrix.
Contractility also governs the type of protrusions used during integrin-dependent 3D migration by
controlling the switch between lobopodia and lamellipodia. To test the hypothesis that contractility
controls intracellular pressure to dictate the mode of 3D cell migration, we directly measured the
hydraulic pressure exerted by the cytoplasm in primary human fibroblasts migrating using either
lamellipodial or lobopodial protrusions. We found that lobopodia are high-pressure protrusions, and
inhibiting contractility switches cells to low-pressure lamellipodia-based movement. In lobopodial cells,
the nucleus and associated intracellular membranes physically divide the cytoplasm into high and low
TUESDAY-ORAL PRESENTATIONS
pressure forward and rear compartments, respectively. The compartmentalized elevated pressure in
lobopodial cells was associated with a polarized distribution of vimentin intermediate filaments and
myosin IIA in front of the nucleus and the ability of the nucleus to accelerate periodically away from the
trailing edge. Vimentin formed a contractility-dependent complex with actomyosin and the
nucleoskeleton-cytoskeleton linker protein nesprin 3. Knocking-down nesprin 3 abolished the
independent movement of the nucleus and slowed 3D migration in a manner similar to treatment with
blebbistatin. Additionally, nesprin 3 knock-down reduced and equalized intracellular pressure between
the front and back cytoplasmic compartments and switched cells to lamellipodia-based migration.
Inhibition of myosin II in front of the nucleus by the local application of blebbistatin immediately
reduced hydraulic pressure, whereas applying the drug behind the nucleus had no effect. Hence, the
nucleus can act as a piston that is pulled forward by actomyosin contractility, acting on nesprin 3 to
increase the hydrostatic pressure between the nucleus and the leading edge to drive lamellipodiaindependent 3D cell migration. This mechanism is required for the efficient migration of primary human
cells through physiological 3D matrix and highlights the importance of physical factors such as matrix
microenvironment and pressure in mammalian cellular functions.
M144
Integrin-beta3 clusters recruit clathrin-mediated endocytic machinery in the
absence of traction force.
C. Yu1,2, A. Krishnasamy2, M.P. Sheetz3,4;
1
Department of Anatomy, The University of Hong Kong, Hong Kong, Hong Kong, 2Mechanobiology
Institute, National University of Singapore, Singapore, Singapore, 3Mechanobiology Institute, National
University of Singapore, Singapore, NY, 4Biological Sciences, Columbia University, New York, NY
Matrix-integrin activation triggers cell adhesion. Force development at integrins can differentially
regulate adaptor protein recruitment, and micro-partitioned RGD-membrane provides a unique
substrate to study force-dependent signal transduction. Previously, we have used RGD-membrane to
investigate initial activation of integrin clustering and podosome formation. Here we report that
clathrin-associated adaptor protein Dab2 progressively binds to integrin-beta3 after 20-min of cell
adhesion on mobile RGD-membrane. When a single cell adheres on the substrate with both mobile and
immobile ligands, Dab2 is preferentially recruited to integrin-beta3 at mobile RGD regions. Intriguingly,
Dab2 is mutually excluded with other integrin-binding proteins, including talin, kindlin, focal adhesion
kinase, paxillin, and vinculin. Point mutations of tyrosine residues of integrin-beta3 block Dab2 binding.
Increase cellular contractility by RhoA-Q63L and phospho-mimic MRLC mutant impedes Dab2
recruitment on mobile RGD membrane. More importantly, decrease myosin-II activity by blebbistatin
promotes Dab2 binding to integrin-beta3 on immobile matrices. As a result, Dab2 recruits clathrinmediated endocytic machinery and results in RGD-integrin endocytosis. We propose that absence of
traction force causes recruitment of Dab2/clathrin and endocytosis of integrin-beta3.
TUESDAY-ORAL PRESENTATIONS
M145
Osmotic surveillance mediates rapid wound closure through nucleotide release.
W. Gault1, B. Enyedi1, P. Niethammer1;
1
Cell Biology, Sloan-Kettering Institute, New York, NY
While tissue intrinsic wound detection mechanisms exist (e.g. loss of contact inhibition or chemotactic
guidance), little is known about the signals induced by an organism’s extrinsic environment. Injury to
wet epithelia (e.g. gastrointestinal/mucosal surfaces or fish skin) permits entry of environmental liquids
into the tissue and is associated with rapid wound closure. As osmotic cues from the environment
promote wound detection by leukocytes in zebrafish, we tested the hypothesis that environmental
osmolarity is a mediator of wound detection and closure by epithelial cells. Using time-lapse
fluorescence microscopy and intravital luminescence imaging in a zebrafish larval tailfin model of wet
epithelial wound closure, we found that an osmotic difference between the interstitial fluid and the
external environment drives hypotonicity-induced ATP release, leading to long-range activation of basal
epithelial cell migration and rapid wound closure. We determined that the spatiotemporal pattern of
tissue velocity during wound closure is controlled by the extracellular nucleotide degrading
ectonucleoside triphosphate diphosphohydrolase 3 (Entpd3). Our data supports that the larval zebrafish
skin integrates two spatially distinct yet functionally interrelated modes of wound closure: an
environmentally (hypotonicity)-induced lamellipodial sheet migration in the basal cell layer, and an
environmentally independent purse-string contraction at the suprabasal wound margin. Overall, we
define a novel environmental osmotic surveillance circuit, which is integrated with tissue intrinsic
mechanisms to promote rapid, wet epithelial wound closure. These results support that extrinsic
environmental exposure is an important master stimulus of the wound response for both leukocytes
and epithelial cells in vivo.
M146
The Role of Osmotic Engine Model in Cell Migration in Confined Spaces.
K. Konstantopoulos1, K.M. Stroka1, S.X. Sun2;
1
Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, 2Mechanical
Engineering, Johns Hopkins University, Baltimore, MD
Cell migration is a fundamental cellular phenomenon that underlies numerous diverse biological
processes, and is influenced by the complex interplay among intra-/inter-cellular signaling mechanisms
and the physical cues of the microenvironment (i.e., confinement, dimensionality and matrix stiffness).
Much of what we know about the mechanisms of cell migration stems from in vitro studies using twodimensional (2D) surfaces. Cell locomotion in 2D is driven by cycles of actin protrusion, integrinmediated adhesion and myosin-dependent contraction. However, cells in vivo migrate through 3D
extracellular matrices (ECM) of varying pore sizes as well as through 3D pre-existing tracks or channels
created by various anatomic structures. Interestingly, recent intravital microscopy studies reveal that
cells in vivo preferentially migrate along pre-existing 3D longitudinal channels. These channels vary from
TUESDAY-ORAL PRESENTATIONS
highly confining (≤3 µm in width) to larger than the individual tumor cell body (~30 μm), and are 100600 μm in length. Mounting evidence suggests that the physical cues of the microenvironment, such as
confinement and dimensionality, affect the mechanisms of cell locomotion. We recently reported that
actomyosin contractility and β1 integrin-dependent adhesion are dispensable in tumor cell migration
through confined microenvironments. Consistent with these findings, the cell migration speed on a 1Dpatterned surface is not affected by inhibition of myosin contractility, and cell traction forces are
markedly suppressed in 1D relative to 2D migration. Similarly, traction forces are repressed in narrow
(confined) versus wide (unconfined) channels. Importantly, confined migration persists even when Factin is disrupted, and depends largely on microtubule (MTs) dynamics. These findings suggest that a
fundamentally different mechanism of cell locomotion is at play for cells migrating inside narrow
channels. We recently discovered the critical role of water permeation, aquaporins (AQPs) and ion
channels in confined migration, described by the Osmotic Engine Model (OEM). This presentation will
also discuss the roles of transient receptor potential (TRP) channels and microtubules (dynein and
kinesin) in confined migration. We postulate that cells can use different mechanisms depending on the
physical cues of the microenvironment.
Minisymposium 16: Microtubule Assemblies and their Functions
M147
Persistence of microtubule polymerization controls cell migration in 3D matrix.
B. Bouchet1, I. Noordstra1, A. Akhmanova1;
1
Cell Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands
Cooperation between the actin and microtubule cytoskeleton is essential for cell migration, but the
underlying mechanisms differ depending on the cell type and cell culture conditions. In 2D cultures,
actin polymerization typically drives spreading of lamella while actomyosin-based contractility
translocates the cell body towards the leading edge. In this context, microtubules are thought to have
primarily a regulatory function linked to the control of signaling and focal adhesion dynamics. In more
physiologically relevant 3D culture models, cells do not form flat lamella but instead can extend long
pseudopods that penetrate the matrix and form adhesions. Also in these conditions, the actomyosin
network is essential for cell protrusion and translocation, but the role of microtubules is still poorly
understood. Here, we investigated whether microtubule dynamics is an important factor in 3D
migration. We used inactivation of SLAIN2, a recently identified interphase-specific plus-end recruitment
factor for the microtubule polymerase ch-TOG, to disrupt persistent microtubule polymerization. When
SLAIN2 is inactivated, interphase microtubules grow slowly and undergo frequent catastrophes, but the
organization and maintenance of microtubule network is relatively normal and cell divide normally,
because SLAIN2 plays no role in the formation of the mitotic apparatus. Surprisingly, we found that
processive microtubule growth is dispensable for cell spreading and migration in 2D, but is completely
essential for the mesenchymal cell movement in 3D matrix. When persistent microtubule
polymerization is reduced, the formation of long pseudopods that initiate cell movement in 3D is
TUESDAY-ORAL PRESENTATIONS
dramatically inhibited. Short term treatments with very low doses of microtubule-targeting agents that
induced similar perturbation of microtubule growth without destroying microtubule network caused
rapid retraction of the existing pseudopods, indicating that also the maintenance of cell protrusions in
3D depends on persistence of microtubule growth. We further found that rapid microtubule
polymerization antagonizes myosin II-based contractility during pseudopod extension in 3D matrix.
Finally, we showed that persistent microtubule growth is a prerequisite for pseudopod-based invasion
induced by complete epithelial-mesenchymal transition (EMT) in human breast epithelial cells, a process
that is crucial for both normal development and cancer cell metastasis. Our results demonstrate that,
independently from microtubule network maintenance, the microtubule polymerization processivity is a
critical factor required for the elongation of pseudopods that initiate cell motility in 3D matrix.
M148
Moesin is a major regulator of centrosome inactivation in epithelial cells with
extra centrosomes.
R. Basto1, D. Sabino1;
1
Institut Curie, Paris, France
Centrosome amplification has severe consequences during development and is thought to contribute to
a variety of diseases such as microcephaly and cancer. Importantly, however, the longstanding effects of
centrosome amplification in epithelia are not known. In this study we investigated the consequences of
centrosome amplification in the Drosophila wing disc epithelia. Using whole-tissue time-lapse, we find
that epithelia depict mechanisms of clustering and inactivation of extra-centrosomes and the majority of
the mitosis is bipolar. However, unlike in the brain, these mechanisms are not fully efficient and
aneuploidy and consequent cell death are generated. Interestingly, a portion of these aneuploidy cells
are viable, and form tumors when transplanted into WT hosts. In addition, similarly to other aneuploid
conditions, inhibition of cell death results in over-growth and basement membrane degradation that
lead to the formation of abnormal tissue in situ. To identify the reasons why centrosome amplification
results in different phenotypes in the wing disc when compared to the brain, we performed comparative
SILAC to quantitatively compare both tissues and found that Moesin, a FERM domain protein, is
specifically up-regulated in wing discs with extra centrosomes. We show that Moesin localizes to the
spindle and centrosomes during mitosis, and furthermore this Moesin up-regulation correlates with
inefficient centrosome inactivation exclusively in epithelial cells. This work provides a mechanistic
explanation for the increased aneuploidy and transformation potential primed by centrosome
amplification in epithelial tissues.
TUESDAY-ORAL PRESENTATIONS
M149
Observation and regulation of a novel microtubule behavior in vivo: growth
deceleration.
B. Lacroix1, J. Dumont1, P.S. Maddox2, A.S. Maddox2;
1
Institut Jacques Monod, Paris, France, 2Department of Biology, University of North Carolina, Chapel Hill,
NC
Microtubules (MTs) are cytoskeletal polymers that undergo stochastic transitions between growth and
shrinkage phases, behaviors collectively referred to as dynamic instability. MTs are ubiquitous among
eukaryotes but their dynamics and organization vary with cell type, cell function and cell cycle phase.
MT dynamics has been well characterized in diverse contexts including zygotes, cultured cells and in
vitro reconstitution systems. We have developed a high-resolution live imaging approach to visualize MT
behavior during post-embryonic development in intact tissues. Using C. elegans, we recently reported
that MT dynamics change during the differentiation of a single cell lineage from stem-like precursors
(sex myoblasts) into smooth muscle-like cells involved in egg laying (vulval and uterine muscle cells). All
aspects of MT dynamics alter during development, some concurrent with cell-cycle exit and others
differentiation-associated cell morphogenesis. Examining the subcellular specificity of MT dynamics in
the uterine muscle cells, we found that MTs in the thin dorsal extension had distinct dynamics from MTs
deeper in the cell. Careful analysis revealed that MTs in the dorsal extension display a novel behavior,
abruptly decreasing growth rate as they near cell periphery. Interestingly, this MT growth deceleration
coincides with a local reduction of cytoplasmic tubulin concentration. Furthermore, the density of the
actin network is also altered in this region, suggesting a cross-talk between actin and MT organization
and dynamics in this specific cell area. Using RNAi, we identified two novel proteins that are responsible
for this deceleration phenomenon. We initially identified these uncharacterized open reading frames by
homology with the tubulin-sequestering protein stathmin, but they share more similarities with a small,
highly basic, conserved but poorly characterized protein cylicin. In sum, our study revealed a novel
microtubule behavior and began to define its molecular regulation.
M150
In vitro reconstitution of efficient microtubule formation by the combined action
of TPX2 and chTOG.
J. Roostalu1, N.I. Cade1, T. Surrey1;
1
Cancer Research UK London Research Institute, London, United Kingdom
Spindle assembly relies on a number of microtubule-associated proteins that contribute to microtubule
organisation through microtubule crosslinking and transport, regulate microtubule dynamics and/or
facilitate microtubule nucleation. TPX2 is a multi-functional microtubule-binding protein required for
proper spindle formation in all higher eukaryotes. It is one of the key targets of the Ran pathway that is
essential for chromatin-dependent microtubule assembly in Xenopus laevis egg extracts. Total spindle
TUESDAY-ORAL PRESENTATIONS
microtubule mass in Xenopus laevis is additionally controlled by the processive microtubule
polymerases of the XMAP215/Dis1 protein family. The molecular mechanism of how these two proteins
contribute to microtubule formation and whether they directly facilitate the nucleation process is not
understood. Here we use a combination of microscopy-based in vitro reconstitution assays to elucidate
the respective roles of human TPX2 and chTOG, the human ortholog of XMAP215/Dis1, in microtubule
assembly. TPX2 is preferentially recruited to the microtubule lattice at growing microtubule ends by its
central domain, where it prevents microtubule catastrophes without affecting microtubule growth rate.
These properties are markedly different from the microtubule growth-promoting activity of the chTOG
proteins. The microtubule stabilising effect of TPX2 is directly related to its function in microtubule
nucleation: TPX2 also stabilises early sheet-like nucleation intermediates at the initial stages of
microtubule assembly. Efficient tube formation and sheet closure, however, require the additional
presence of the microtubule polymerising activity of chTOG. Together, our results demonstrate that a
combination of very distinct activities of two microtubule binders is both necessary and sufficient for
efficient microtubule formation in solution. Furthermore, these findings provide important insight into
the mechanism of microtubule nucleation stimulated by the Ran pathway during spindle assembly.
M151
Control and function of microtubule overlaps in the bipolar phragmoplast
network.
J. de Keijzer1, J. Verweij1, T. Miki2, G. Goshima2, M. Janson1;
1
Laboratory of Cell Biology, Wageningen University, Wageningen, Netherlands, 2Division of Biological
Science, Nagoya University, Nagoya, Japan
Constriction of the plasma membrane during cell division in animal cells is regulated by short regions of
overlapping microtubules at the centre of the cell division apparatus. Similar regions of limited
antiparallel microtubule overlap are found in the phragmoplast of plants, a bipolar microtubule network
that constructs a cell plate to separate two daughter cells. We used the genetically tractable moss
Physcomitrella patens to gain insight into the function and regulation of these overlaps. We found that
overlaps act as recruitment sites for vesicles containing cell wall material. The short length of overlaps
may thus be a prerequisite to precisely pattern the forming cell plate. To understand the control of
overlap length we investigated the balance between local rates of microtubule growth and microtubule
sliding. The first activity extends overlaps whereas the second decreases overlap length and drives
microtubule flux. By tracking growing microtubule ends and regions of photo-activated microtubule
filaments we show that the bulk microtubule growth velocity is several times faster than the rate of
relative sliding, implying that microtubule growth must be locally down regulated within overlaps. We
therefore functionally analysed kinesin-4 proteins. In agreement with an established role in inhibiting
microtubule growth, deletion of two kinesin-4 genes caused oscillations in overlap length. Moreover,
the initial recruitment of vesicles to overlaps was more diffuse, cell plate construction was delayed, and
completed cell plates were thicker and lacked structural integrity. Our results thus demonstrate the
TUESDAY-ORAL PRESENTATIONS
patterning of the cell plate by the phragmoplast involves kinesin-4 mediated length control of
microtubule overlaps.
M152
Identification of the molecular ruler that defines the 96-nm repeats of cilia/flagella.
T. Oda1, H. Yanagisawa1, R. Kamiya2, M. Kikkawa1;
1
Dept Cell Biology, The University of Tokyo, Tokyo, Japan, 2Dept Life Science, Gakushuin University,
Tokyo, Japan
Primary ciliary dyskinesia (PCD) is an inherited disease caused by abnormal ciliary motility. Majority of
PCD cases associated with axonemal disorganization result from mutations in CCDC39 and CCDC40
genes. However, their localizations and functions in cilia/flagella are not clearly resolved. In this study,
we revealed the molecular mechanism how FAP59 and FAP172, the Chlamydomonas homologues of
CCDC39 and CCDC40, govern the assembly of axonemal ultrastructures. Using biochemical pull-down
assay and cryo-electron tomography, we showed that FAP59 and FAP172 form a complex, and the
absence of the two proteins disrupts 96-nm repeats of axonemes. Structural labeling revealed that
FAP59 and FAP172 take 96-nm long extended conformations along outer doublet microtubules,
suggesting their possible role as a molecular ruler. To test this hypothesis, we elongated the amino acid
sequences of FAP59 and FAP172 by duplicating their coiled-coil domains. Surprisingly, the repeat length
became ~128-nm long, and axonemal components such as radial spokes, inner dynein arms, and dynein
regulatory complexes were duplicated. These results strongly suggest that the FAP59/172 complex is the
molecular ruler that determines the 96-nm repeats in cilia/flagella by regularly recruiting axonemal
components to the correct binding sites.
M153
Regulated assembly of a supramolecular centrosome scaffold in vitro.
J. Woodruff1, O. Wueseke1, V. Viscardi2, J. Mahamid3, K. Oegema2, A. Hyman1;
1
Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany, 2Ludwig Institute for
Cancer Research, La Jolla, CA, 3Max Planck Institute of Biochemistry, Martinsried, Germany
A centrosome comprises a pair of centrioles surrounded by an amorphous protein mass called the
pericentriolar material (PCM). Despite its importance as major microtubule-organizing center, the
mechanism of PCM assembly and its regulation are not understood. In a C. elegans embryo, the coiledcoil protein SPD-5 is essential for PCM assembly. We found that recombinant SPD-5 can polymerize to
form micrometer-sized porous networks in vitro. Network assembly was accelerated by two conserved
regulators that control PCM assembly in vivo, Polo-like kinase-1 and SPD-2/Cep192. Interestingly, only
the assembled SPD-5 networks, and not unassembled SPD-5 protein, functioned as a scaffold for other
PCM proteins. Our results suggest that PCM size and binding capacity emerge from the regulated
formation of a porous network from one coiled-coil protein. Furthermore, these results suggest that
TUESDAY-ORAL PRESENTATIONS
PCM growth is autocatalytic, such that polymerized SPD-5 can recruit additional SPD-5 and its own
positive regulators, leading to rapid network expansion.
M154
A combined cell biological and biophysical strategy to analyze actin/microtubule
crosstalk and its relation to cytoplasmic streaming.
M. Drechsler1, R. Goldstein2, I.M. Palacios1;
1
Department of Zoology, University of Cambridge, Cambridge, United Kingdom, 2DAMTP, University of
Cambridge, Cambridge, United Kingdom
The actin and microtubule (MT) cytoskeletons constitute versatile and highly dynamic protein networks
regulating a broad variety of processes like cargo transport or cellular motility. While there is a good
understanding of the roles of either type of filament, knowledge about how actin and MTs interact
remains rather elusive. Therefore, understanding fundamental concepts of cytoskeletal crosstalk
constitutes an important task in cell biology.
We use the Drosophila oocyte as model of cytoskeletal crosstalk and dynamics. One major function of
these two cytoskeletons in the oocyte is the asymmetric distribution of key developmental
determinants. In addition to cargo transport, MTs and the motor Kinesin-1 (Kin) are essential for the
induction of major bulk movements of the cytoplasm – a process known as cytoplasmic streaming. We
applied particle image velocimetry (PIV) to monitor and quantify cellular fluid dynamics in living oocytes.
With this technique we could detect even subtle changes in Kinesin activity and found that flow features
strongly correlate with the architecture of the MT cytoskeleton[1]. We now aim to understand how flows
feedback on the MT network and how the cell maintains cytoskeletal organization, by simultaneously
monitoring MT dynamics and flows in living oocytes, and subsequent analysis by PIV.
Additionally to Kin and MTs, streaming and cargo localization is regulated by a cytoplasmic F-actin mesh
through a yet unknown mechanism. The formation of this actin mesh is developmentally regulated.
When it is present, oocytes display slow unorganized cytoplasmic flows, while streaming becomes fast
and directed when the mesh dissolves later on. The major task is now to understand how exactly the
actin mesh influences Kin activity and the organization of the MT network, as well as the biophysical
properties of the cytoplasm. In particular we concentrate on the function of actin-MT crosslinking
proteins that might be involved in the interaction of both cytoskeletons. Preliminary data, obtained from
live microscopy, suggest that the absence of flows causes an ectopic correlation between the actin mesh
and MT bundles, indicating an association of both filament types.
The characterization of flows by PIV, in combination with live video microscopy, FRAP and
photoconversion approaches constitutes a novel straight forward experimental setup to gain
mechanistic insights into the crosstalk between F-actin and MTs in relation to cytoplasmic flows.
[1]
Ganguly et al. (2012) “Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and
correlates with the microtubule cytoskeleton architecture” PNAS 18;109(38)13109-14
TUESDAY-ORAL PRESENTATIONS
M155
Cilia autonomous regulation of tubulin transport by IFT.
J.M. Craft1, K.F. Lechtreck1;
1
Cellular Biology, University of Georgia, Athens, GA
Microtubules are the major structural elements of cilia and eukaryotic flagella. During ciliary assembly,
large amounts of tubulin must be transported into the organelle. Intraflagellar transport (IFT) is the
major protein transport system in cilia and IFT has been implicated in tubulin transport. The IFT proteins
IFT81 and IFT72, for example, form a module that binds tubulin dimers in vitro indicating that IFT
particles interact directly with tubulin [1]. Direct in vivo imaging of tubulin during transport, however,
has proved challenging [2]. To analyze ciliary tubulin transport and its regulation, we expressed GFP-αtubulin in wild-type Chlamydomonas reinhardtii. GFP-α-tubulin was incorporated into cytoplasmic and
ciliary microtubules [3]; it was posttranslationally modified and formed heterodimers with β-tubulin
suggesting that GFP-tagged and endogenous α-tubulin behave largely similar. Total internal reflection
fluorescence (TIRF) microscopy revealed that GFP-α-tubulin entered flagella by active transport and by
diffusion. Two-color in vivo imaging showed that GFP-α-tubulin moved on IFT trains inside cilia; thus, IFT
functions as a tubulin transporter. Without of IFT, GFP-α-tubulin continued to enter cilia by diffusion. To
assess how tubulin interacts with IFT, we expressed modified α- and β- GFP-tubulins; this ongoing
project revealed C-terminally truncated GFP-α-tubulin is still transported by IFT and assembled into the
axoneme. During ciliary growth, IFT-based transport of GFP-α-tubulin was increased ~15x over steadystate frequencies and, in growing cilia, the occupancy rate (% of IFT particles carrying GFP-α-tubulin as a
cargo) increased from ~10% to ~80% indicating that tubulin transport by IFT is regulated. Fluorescence
recovery after photobleaching (FRAP) analysis and western blotting revealed a strong increase in the
concentration of soluble GFP-α-tubulin in regenerating over steady-state cilia. In cells possessing both
growing and non-growing cilia, tubulin transport via IFT was elevated only in the growing cilia; likewise,
the concentration of tubulin was increased solely in the matrix of the growing cilia. Thus, cells can direct
tubulin flux specifically into growing cilia and established distinct concentrations of soluble tubulin
within different cilia of a given cell. Our data suggest that tubulin transport via IFT is regulated locally in
a flagella autonomous manner, likely within the basal body-cilium entity. We propose a model in which
IFT functions as a tubulin pump to increase the concentration of soluble tubulin inside cilia, which will
promote the elongation of axonemal microtubules and thereby ciliary growth. 1Bhogaraju et al. 2013.
Science 41:1009. 2Hao et al. 2011. Nat Cell Biol 13:790. 3Rasala et al. 2013. Plant J 74:545.
TUESDAY-ORAL PRESENTATIONS
Minisymposium 17: Organelle Biogenesis and Dynamics
M156
De novo peroxisome formation revisited.
K. Knoops1, A. Krikken1, R.d. Boer1, A. Kram1, I.J. Van Der Klei1;
1
Molecular Cell Biology, Univ Groningen, Groningen, Netherlands
Peroxisomes are ubiquitous cell organelles that play a role in a large range of metabolic pathways. Two
models of peroxisome proliferation have been proposed, namely multiplication by fission of pre-existing
organelles and de novo formation from the endoplasmic reticulum (ER). The latter includes initial sorting
of peroxisomal membrane proteins to the ER, followed by Pex3 and Pex19 dependent formation of two
types of biochemically distinct, ER-derived vesicles. Finally, heterotypical vesicle fusion, mediated by the
AAA-ATPases Pex1 and Pex6, results in the formation of a new peroxisome. The model of the de novo
peroxisome formation pathway predicts that in the absence of Pex3 or Pex19 peroxisomal membrane
proteins accumulate at the ER, whereas in strains lacking Pex1 or Pex6 two types of peroxisomal vesicles
accumulate in the cytoplasm. We re-investigated various yeast PEX deletion strains using advanced
electron and fluorescence microscopy techniques. Our data indicate that pex3 and pex19 mutant cells
harbor peroxisomal membrane vesicles, which contain a subset of peroxisomal membrane proteins and
are the template for de novo peroxisome formation upon reintroduction of Pex3 or Pex19. These data
indicate that Pex3 and Pex19 are not required for exit of peroxisomal membrane proteins from the ER.
Moreover, we observed that pex1 and pex6 mutant cells do not contain different types of preperoxisomal membrane vesicles. Instead, they harbor relatively large peroxisomal membrane ghosts, at
which various peroxisomal membrane proteins co-localize. Our observations shed new light on the
molecular mechanisms of the de novo peroxisome formation process and the role of the ER in
peroxisome biogenesis.
M157
Physical models describing the emergence of organelle biogenesis and
morphology.
M. Rao1,2;
1
Raman Research Institute, Bangalore, India, 2National Centre for Biological Sciences (NCBS), TIFR,
Bangalore, India
A central question in cell biology is how do organelles in the trafficking pathway form de nouvo from
simple rules of organization and how do they achieve morphological and chemical identity. We explore a
whole range of physical models based on nonequilibrium processes of fission-fusion and cisternal
progression and highlight the conditions for morphologically and chemically distinct, stable and robust
compartments. In the second part of my talk, I will discuss how the active processes of fission-fusion
TUESDAY-ORAL PRESENTATIONS
might generically drive the large scale shape of membrane organelles towards tubular, ramified
structures or flattened sacs.
M158
Novel ER shaping proteins Pex30 and Pex31 are involved in pre-peroxisome
vesicle biogenesis.
A.S. Joshi1, W. Prinz2;
1
NIDDK, NIH, Bethesda, MD, 2NIDDK, National Institutes of Health, Bethesda, MD
The peripheral endoplasmic reticulum (ER) forms a network of flat sheet-like cisternae and highly
dynamic interconnected tubules that extend through out the cell. Reticulons and reticulon-like proteins
are required to maintain this structure. S. cerevisiae has three of these proteins: Rtn1p, Rtn2p, and
Yop1p. Interestingly, in cells missing these proteins (rtn1rtn2yop1delta) the peripheral ER is mostly
converted to sheets. However, a few ER tubules are still found in these cells, suggesting that there may
be additional ER tubulating proteins. A novel genetic screen was designed to identify the proteins that
tubulate ER in rtn1rtn2yop1delta. We found that overexpression of Pex30p and Pex31p restores ER
tubules in rtn1rtn2yop1delta suggesting that Pex30 and Pex31 are ER tubulating proteins. Like reticulons
and Yop1p, Pex30p and Pex31p are localized mainly in the peripheral ER and absent from nuclear
membranes. Unlike reticulons and Yop1p, both Pex30p and Pex31p contain dysferlin domain of
unknown function. We show that this domain is required for ER membrane tubulation. Our studies also
establish that dysferlin domain is functionally conserved from humans to yeast. In S. cerevisiae, Pex30p
and Pex31p maintain the number and size of peroxisomes respectively. Mature peroxisomes are derived
from pre-existing vesicles known as pre-peroxisomal vesicles (PPV). While the origin of PPV is not
known, a recent study show that Pex14p containing PPV are present even in pex3atg1delta cells. We
investigated whether ER tubulating proteins play a role in PPV biogenesis. We found a significant
increase in Pex14p containing vesicles in rtn1rtn2yop1pex30pex3atg1delta cells indicating that ER
tubulating proteins negatively regulate PPV formation. Taken together, our results suggest that ER
shaping proteins are essential for tubulating a subdomain of ER involved in PPV biogenesis. The
mechanism of PPV formation remains to be investigated.
M159
Modelling the interplay between maturation and exchange in cellular organelles.
P. Sens1,2;
1
CNRS-ESPCI, Paris Cedex 05, France, 2Institut Curie, Paris, France
Biological cells are compartmentalised into various organelles permanently exchanging material, but
that nevertheless maintain distinct biochemical identities. Heterogeneities of chemical composition are
crucial to the proper maturation and sorting of proteins secreted by the cell, and must be actively
maintained by energy-consuming processes. One of the (many) important questions underlying
TUESDAY-ORAL PRESENTATIONS
intracellular organisation is how biochemical maturation within organelles and transport between (or
through) organelles are coordinated. In the case of the Golgi apparatus, it is still strongly debated
whether intra-Golgi transport occurs via maturation of individual Golgi cisterna or exchange between
cisternae.
In this talk, I will describe a quantitative model of intra-Golgi transport based on a Fokker-Planck
equation, that combines maturation and exchange. Analysis of protein transport data led us to conclude
that even large supramolecular cargo undergo inter-cisternal exchange while traversing the Golgi. I will
then discuss a (very) simple model of secretory pathway, that highlights the importance of the rate of
maturation as a parameter controlling the exchange dynamics between distinct organelles.
M160
PINK1 is a ubiquitin kinase.
L.A. Kane1, M. Lazarou1, A.I. Fogel1, Y. Li1, K. Yamano1, S.A. Sarraf1, S. Banerjee1, R.J. Youle1;
1
NINDS, NIH, Bethesda, MD
PINK1 is a gene mutated in certain autosomal recessive early onset forms of Parkinson’s disease (PD).
During the decade since its identification, it has been shown to be a mitochondrial kinase whose
abundance is controlled by mitochondrial membrane potential. Low mitochondrial membrane
potential, signaling an unhealthy organelle, causes the accumulation of PINK1 on the outer
mitochondrial membrane. This results in the recruitment of Parkin, a cytosolic E3 ubiquitin ligase that is
also mutated in early onset PD. Parkin then induces the recruitment of both the proteasome and
machinery responsible for autophagy, thus facilitating turnover of damaged mitochondria. The
substrates of PINK1 have been a source of debate and many substrates have been suggested in the
literature, including Parkin itself. Using an unbiased proteomic approach, we have now identified that
PINK1 directly phosphorylates ubiquitin (Kane, et al. J. Cell Biol. 205, 143-153 (2014)). This
phosphorylation occurs specifically on Ser65 of ubiquitin and mirrors PINK1’s known phosphorylation of
the ubiquitin-like domain of Parkin. Phospho-Ser65 ubiquitin binds to Parkin and activates its E3 ligase
activity in vitro. Overexpression of a non-phosphorylatable mutant ubiquitin blocks Parkin translocation
to damaged mitochondria in vivo. This is the first identification of a ubiquitin kinase and the first
reported function for phospho-ubiquitin. Phosphorylation of ubiquitin in this clinically-relevant
mitochondrial pathway opens the potential for a broader cellular signaling role for ubiquitin posttranslational modification.
TUESDAY-ORAL PRESENTATIONS
M161
Translocation of cyclin C to the mitochondria mediates stress-induced fission
and programmed cell death in yeast and mammalian cells.
R. Strich1, K. Cooper1, K. Wang1, S. Khakhina2;
1
RowanSOM, Stratford, NJ, 2Molecular Physiology, University of Iowa, Iowa Cite, IA
The decision to undergo programmed cell death (PCD) is controlled by a complex interaction between
nuclear and mitochondrial signals. The mitochondria are highly dynamic organelles that constantly
undergo fission and fusion. Studies have shown that one of the earliest PCD events is a dramatic shift in
mitochondrial morphology toward fission. We have identified the transcription factor cyclin C as the
biochemical trigger for stress-induced hyper-fragmentation in both yeast and mammalian cells. In
response to PCD stimuli such as oxidative stress or anti-cancer drugs, cyclin C is released from the
nucleus then associates with the fission machinery. In both yeast and mammalian cells, loss of cyclin C
function prevents mitochondrial fission while ectopic introduction of cyclin C in the cytoplasm induces
fission in the absence of stress. Recent studies have found that mitochondrial fission occurs at junctions
between the mitochondria and the endoplasmic reticulum (ER). We have found that the number of
these junctions increases upon stress at sites of mitochondrial fission. In addition, the formation of the
additional stress-induced junctions requires cyclin C. The results suggest that cyclin C plays a role in
establishing and/or maintaining these stress-enhanced mitochondrial-ER junctions. Finally, we have
found a direct correlation between with the ability of the cell to undergo extensive mitochondrial fission
and induce PCD. Our analysis revealed that mouse embryonic fibroblasts deleted for cyclin C fail to
undergo mitochondrial outer membrane permeability (MOMP) and do not release cyctochrome c when
subjected to apoptotic stimuli. These results suggest that cyclin C plays two roles in mediating fission
and cell death. The first activity enhances activity of the fission machinery to fragment the
mitochondria. Second, association of cyclin C to the mitochondria stimulates mitochondrial outer
membrane permeability resulting in release of pro-apoptotic proteins sequestered in this organelle. The
importance of correct cyclin C regulation is many fold. In yeast, deleting the anchor protein that
maintains cyclin C in the nucleus in unstressed cells results in constitutive mitochondrial fragmentation
resulting in hypersensitivity to stress and loss of mtDNA integrity and overall organelle function.
Conversely, loss of cyclin C function protects cells from oxidative damage and causes extensive
hyperplasia in mouse tumor models. These findings indicate a new role for cyclin C plays in the stress
response that is independent of its function as a transcription factor.
TUESDAY-ORAL PRESENTATIONS
M162
Nucleolar assembly and growth are governed by a concentration-dependent
phase transition.
S.C. Weber1, J. Berry2, M. Haataja2, C. Brangwynne1;
1
Chemical and Biological Engineering, Princeton University, Princeton, NJ, 2Mechanical and Aerospace
Engineering, Princeton University, Princeton, NJ
Eukaryotic cells exhibit a remarkable degree of spatial organization. In addition to classical membranebound organelles, the cytoplasm and nucleoplasm contain membrane-less, liquid-like droplets of RNA
and protein, many of which function in regulating RNA metabolism. How such structures assemble and
stably persist without a physical boundary holding them together, and how cells might regulate this
process, are still unclear. Here, we use quantitative imaging of live C. elegans embryos combined with
analytical theory and numerical simulations to investigate the assembly and growth dynamics of the
nucleolus, a non-membrane bound organelle important for cell size homeostasis. We find that nucleolar
components condense into nucleoli only above a threshold concentration and that the final size of the
organelle depends linearly on concentration. Furthermore, the growth kinetics of nucleoli are
consistent with a phase separation model in which the thermodynamic attraction between nucleolar
components results in stable, coherent droplets. Our results suggest that phase transitions could play a
general role in functionally organizing the nucleoplasm/cytoplasm and generating organelles of
appropriate size.
M163
A direct interaction between actin filaments and the dynamin-like GTPase Drp1
supports dual roles for the actin cytoskeleton during mitochondrial fission.
A.L. Hatch1, H.N. Higgs2;
1
Dartmouth College, Hanover, NH, 2Dartmouth-Geisel Sch Med, Hanover, NH
As a dynamic network, mitochondria routinely change morphology in response to different cellular
demands and stresses. In this regard, mitochondrial fission and fusion serve several roles: to distribute
mitochondria appropriately in dividing or polarized cells; working as a quality control mechanism to help
eliminate damaged mitochondrial segments; and even as a component of apoptotic pathways.
Defective mitochondrial fission/fusion is found in patients with neurological disorders such as CharcotMarie-Tooth disease (CMTD), Alzheimer’s, Huntington’s, and Parkinson’s disease. The dynamin-like
GTPase Drp1 is a key component of the fission machinery that provides contractile force needed for
mitochondrial fission, but a key unanswered question is how Drp1 gets recruited to the mitochondrial
outer membrane (OMM). While a number of Drp1 “receptors” have been identified on the OMM, there
is also evidence that a mitochondrial pre-constriction step is required prior to Drp1 action. We have
shown that actin filaments contribute to this pre-constriction step through the formin INF2, additionally
requiring myosin II. INF2 is an endoplasmic reticulum (ER)-bound actin polymerization factor, and
TUESDAY-ORAL PRESENTATIONS
mutations in INF2 lead to CMTD. Still, how do actin filaments and pre-constriction lead to Drp1
recruitment? Here, we show that Drp1 binds directly to actin filaments with high affinity. Drp1 binds
evenly along the length of the actin filament (shown by electron microscopy), but the interaction is
highly dynamic (shown by TIRF microscopy). Actin filament binding increases Drp1’s GTP hydrolysis
activity, suggesting that actin binding promotes productive Drp1 oligomer assembly. In mammalian
cells, Drp1 mutants defective in actin binding do not rescue mitochondrial fission defects induced by
endogenous Drp1 suppression. From these results, we propose that actin filaments serve two roles
during mitochondrial fission: 1) to provide the force for OMM pre-constriction; and 2) to recruit Drp1 at
fission sites through direct binding. In the first role, we propose that actin filaments generate force for
pre-constriction with myosin II in a manner similar to cytokinesis. In the second role, actin filaments act
as a “co-incidence detector”, along with Drp1 receptors such as Mff, Fis1, MiD49, and MiD51. Coincidence detection of two signals (in this case actin filaments + a Drp1 receptor) is widely used in cells
to tightly control activation of important processes. We further propose that other molecules, such as
cardiolipin, may serve as independent co-incidence detectors for Drp1 in a similar manner to actin.
M164
Optineurin is an autophagy receptor for damaged mitochondria in parkinmediated mitophagy that is disrupted by an ALS-linked mutation.
Y.C. Wong1, E.L. Holzbaur2;
1
Physiology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA, 2Department of
Physiology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA
Mitophagy is a cellular quality control pathway in which the E3 ubiquitin ligase parkin targets damaged
mitochondria for degradation by autophagosomes. We examined the role of optineurin in mitophagy, as
mutations in optineurin are causative for glaucoma and the neurodegenerative disease Amyotrophic
lateral sclerosis (ALS), diseases in which mitochondrial dysfunction has been implicated. Using live cell
imaging, we demonstrate the parkin-dependent recruitment of optineurin to mitochondria damaged by
depolarization or reactive oxygen species (ROS). Parkin’s E3 ubiquitin ligase activity is required to
ubiquitinate outer mitochondrial membrane proteins, allowing optineurin to stably associate with
ubiquitinated mitochondria via its ubiquitin binding domain (UBD); in the absence of parkin, optineurin
transiently localizes to damaged mitochondrial tips. Following optineurin recruitment, the omegasome
protein DFCP1 (double FYVE-containing protein 1) is transiently recruited to damaged mitochondria to
initialize autophagosome formation. Optineurin then recruits autophagosome formation around
damaged mitochondria via its LC3 interaction region (LIR) domain. Depletion of endogenous optineurin
inhibits LC3 recruitment to mitochondria, leading to increased mtDNA content within cells. This defect in
autophagosome formation and mitochondrial degradation is rescued by wildtype optineurin, but not by
an ALS-associated UBAN mutant (E478G), or by a LIR mutation in optineurin. Optineurin and the
autophagy receptor p62/SQSTM1 are independently recruited to different domains on damaged
mitochondria, and p62/SQSTM1 knockdown does not affect either optineurin or LC3-recruitment to
damaged mitochondria. Thus, our results demonstrates a critical role for optineurin as an autophagy
TUESDAY-ORAL PRESENTATIONS
receptor in parkin-mediated mitophagy. Importantly, as mutations in parkin are linked to Parkinson’s
disease, our study further indicates that defects in a single pathway lead to neurodegenerative diseases
with distinct pathologies.
Minisymposium 18: Small GTPases and Lipids in Membrane
Dynamics
M165
Differential regulation of phosphatidylinositol 4,5-bisphosphate homeostasis by
Nir2 and Nir3 at ER-Plasma membrane junctions.
C. Chang1, J. Liou1;
1
University of Texas Southwestern Medical Center, Dallas, TX
Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) at the inner leaflet of the plasma membrane (PM)
governs many cellular processes, including endocytosis, cytoskeleton dynamics, and store-operated Ca2+
entry (SOCE). Importantly, hydrolysis of PIP2 by receptor-activated phospholipase C (PLC) is pivotal to
initiate signaling events via the production of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG).
Meanwhile, it is necessary to replenish the depleted PIP2 to maintain PIP2-depedent cellular functions
and signaling. It has been shown that PIP2 levels recovered within minutes following receptor-induced
hydrolysis but the mechanisms of PIP2 replenishment remains unclear. In this study, we demonstrate
that non-vesicular delivery of PI originating from the ER is important for rapid PIP2 replenishment at the
PM. Two mammalian PI transfer proteins (PITPs) Nir2 and Nir3 sense phosphatidic acid (PA) production
from PIP2 hydrolysis, and translocate to ER-PM junctions to mediate PIP2 replenishment. The abilities of
Nir2 and Nir3 to mediated PIP2 replenishment are dependent on their translocation to ER-PM junctions
and PI in the ER, suggesting PI transfer at ER-PM junctions. With distinct PITP activities and PA
sensitivities, Nir2 and Nir3 differentially regulate PIP2 homeostasis. Altogether, our findings reveal the
long-sought mechanism that couples PIP2 hydrolysis to its replenishment via Nir2 and Nir3 at ER-PM
junctions to maintain PIP2 homeostasis.
TUESDAY-ORAL PRESENTATIONS
M166
Polyunsaturated phospholipids facilitate membrane deformation and fission by
the endocytic proteins dynamin and endophilin.
M. Pinot1, S. Vanni2, S. Pagnotta3, S. Lacas-Gervais3, L. Payet4, T. Ferreira4, R. Gautier2, B. Goud1, B.
Antonny2, H. Barelli2;
1
Institut Curie, Paris Cedex 05, France, 2Institute of Molecular and Cellular Pharmacology, Valbonne,
France, 3CCMA, Nice, France, 4Inst Physiol Biol Cellu, Poitiers, France
Most cellular membranes contain phospholipids (PLs) with saturated and monounsaturated acyl chains.
However, in a few specialized organelles, such as synaptic vesicles and photoreceptor discs, up to 80% of
PLs contain at least one polyunsaturated acyl chain. Such high levels suggest that polyunsaturated lipids
might endow membranes with specific physicochemical properties, but their effect on membrane
properties is poorly understood. Here, we found that polyunsaturated PLs increased the ability of
dynamin and endophilin to deform and vesiculate synthetic membranes. Furthermore, when cells
incorporated polyunsaturated fatty acids into PLs, the plasma membrane became more amenable to
deformation by a pulling force produced by optical tweezers and the rate of endocytosis was
accelerated, in particular, under conditions in which cholesterol was reduced by the use of methyl-bcyclodextrin. Dyngo 4a, a specific dynamin inhibitor, inhibited this endophilin-driven endocytosis. Thus,
polyunsaturated PLs can facilitate endocytic events that are under the dual control of endophiline and
dynamin. Molecular dynamics simulations and biochemical measurements indicated that
polyunsaturated PLs adapted their conformation to membrane curvature by using their flexible chain to
fill voids in the outer monolayer. Thus, by reducing the energetic cost of membrane bending and fission,
polyunsaturated PLs may help to support rapid endocytosis.
Polyunsaturated PLs are beneficial for health and their abundance in the brain suggests a decisive
advantage for cognitive functions, but the underlying molecular mechanisms are poorly understood. By
showing that polyunsaturated PLs improve the response of model membranes to the mechanical
activities of endocytic proteins, our study offers a potential explanation for extraordinary speed of
endocytosis in the nerve terminal, where polyunsaturated PLs are abundant.
M167
PtdIns(4,5)P2 homeostasis in endosomal trafficking is controled by the Rab35
GTPase and the Lowe syndrome phosphatase OCRL.
A. Echard1;
1
Institut Pasteur, Paris, France
The membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is major regulator of
actin dynamics and activates complexes involved in actin polymerization. Our laboratory has recently
shown that the Rab35 GTPase recruits and directly interacts with the PtdIns(4,5)P2 5-phosphatase OCRL
in late cytokinesis bridges during cytokiensis. This is essential for a normal abscission by preventing local
TUESDAY-ORAL PRESENTATIONS
PtdIns(4,5)P2 and actin accumulation in the intercellular bridge connecting the daughter cells (1,2,3).
Mutations in OCRL are responsible for the Oculo-Cerebro-Renal Syndrome of Lowe, a rare genetic
disease characterized by renal proximal tubular dysfunction, which likely results from defects in the
endosomal pathway (4). How OCRL is localized to endosomes remains a key question. Since the Rab35
GTPase is located on the endosomal system (1) we hypothesized that Rab35 may recruit OCRL on
endosomes to prevent actin accumulation at their surface, by analogy with our results during
cytokinesis. Remarkably, the levels of lysosomal enzymes in the plasma and urine are increased in Lowe
patients, suggesting defects in the intracellular trafficking of the cation-independent mannose 6phosphate receptor (CI-MPR) (4,5,6). Our study suggests that Rab35 critically controls the timing of
OCRL recruitment on clathrin-coated endosomes, and is essential for PtdIns(4,5)P2 hydrolysis and
consequently normal trafficking in the endosomal system.
(1) Kouranti et al., Current Biology 2006 (2) Dambournet et al., Nature Cell Biology 2011 (3) Chesneau et.
al, Current Biology 2012 (4) Vicinanza et al., EMBO J. 2011 (5) Ungewickell et al., PNAS 1999 (6) Norden
et al., Nephrol Dial Transplant 2008
M168
The exchange factor DENND2B activates Rab13 at the leading edge of migrating
cells driving cancer cell metastasis.
M. Ioannou1, E. Bell1, M. Girard1, M. Chaineau1, J. Hamlin1, M. Daubaras1, A. Monast1, M. Park1, L.
Hodgson2, P.S. McPherson3;
1
McGill University, Montreal, PQ, 2Anatomy and Structural Biology, Albert Einstein College of Medicine,
Bronx, NY, 3McGill University, Montreal, QC
The small GTPase Rab13 functions in exocytic vesicle trafficking in epithelial cells. Alterations in Rab13
activity have been observed in human cancers including carcinomas, yet the mechanism of Rab13
activation and its role in carcinoma progression have not been demonstrated. Here we identify the
DENN domain protein DENND2B as the guanine nucleotide exchange factor for Rab13, and develop a
novel FRET-based Rab biosensor to reveal activation of Rab13 by DENND2B selectively at the leading
edge of migrating cells. DENND2B localizes to the cell periphery in a complex with MICAL-L2, a Rab13
effector that binds active Rab13 to induce membrane ruffles. Localized activation of Rab13 also
promotes vesicle fusion with the plasma membrane and together these events stimulate the dynamic
remodeling of the leading edge driving cell migration. Disruption of Rab13-mediated trafficking
dramatically limits the invasive behavior of epithelial cells in vitro and the growth and metastasis of
highly invasive cancer cells in vivo. Thus, blocking Rab13 activation by DENND2B could provide a novel
target to limit the spread of carcinomas.
TUESDAY-ORAL PRESENTATIONS
M169
Divide and Polarize: The Role of Cytokinesis and Endocytic Transport during
Epithelial Cell Polarization.
A. Mangan1, D. Li1,2, R. Prekeris1;
1
Cell and Developmental Biology, Univ Colorado Anschutz Medical Campus, Aurora, CO, 2University of
Colorado Anschutz Medical Campus, Aurora, CO
Epithelial cells are structurally and functionally polarized to transport specific molecules while
maintaining a trans-epithelial barrier. This cellular asymmetry is essential for the proper functioning of
epithelial tissues and depends on polarized endocytic transport routes. Additionally, epithelial cells
coordinate their polarization with neighboring cells to form an apical lumen, a key step in the
establishment of renal and gut architecture, and thereby function. Recent work from several
laboratories including ours identified Rab11 and its binding protein FIP5 as major components that
regulate apical endosome transport during apical lumen formation. We demonstrated that Rab11/FIP5containing endosomes (FIP5-endosomes) mediate the formation of apical lumen by targeted delivery of
apical lumen proteins, and that FIP5 functions by interacting with sorting nexin 18 and Kinesin-2. It was
also shown that targeting of FIP5-endosomes to the apical membrane initiation site (AMIS) is a key step
during the initiation and expansion of the apical lumen during formation of MDCK epithelial cysts in 3D
tissue culture system. Despite recent advances in our understanding of the mechanisms mediating
lumen formation, many questions remains unanswered. How are FIP5-endosomes targeted during apical
lumen formation? How do cells establish the site of single apical lumen? In this study we focused on the
identification of the machinery that mediates AMIS formation and FIP5-endsosmes targeting during
apical lumen formation. We identified a new FIP5-interacting protein, cingulin, which is a tight junction
protein that localizes to the AMIS. Thus, cingulin is perfectly suited to serve as a tether to mediate FIP5endosome targeting during apical lumen formation. Importantly, our data demonstrate that AMIS forms
around the midbody of the mitotic epithelial cells during late telophase. Finally, we demonstrate that
AMIS formation is initiated by Rac1-dependent burst of actin polymerization at the midbody. Based on
all these studies we propose a novel hypothesis that AMIS formation around the midbody and
FIP5/cingulin-dependent endosome targeting to the midbody during late telophase are the first
“symmetry breaking” events leading to the initiation of apical lumen during epithelial morphogenesis.
M170
Control of nutrient signaling and proteostasis by PI3K-C2-mediated PI(3,4)P2
synthesis.
A.L. Marat1, W. Lo1, V. Haucke1;
1
Leibniz Institut Fuer Molekulare Pharmakologie, Berlin, Germany
Eukaryotic cell function depends on a tight balance between biosynthetic growth promoting and
degradative pathways. The mTOR pathway integrates signals elicited by growth and differentiation
TUESDAY-ORAL PRESENTATIONS
factors with the sensing of amino acids to promote protein synthesis and cell growth, while inhibiting
lysosomal protein turnover and autophagy. Activation of mTOR signaling among other factors involves
stimulation of class I phosphatidylinositol 3-kinase to synthesize phosphatidylinositol 3,4,5trisphosphate [PI(3,4,5)P3], which activates Akt to promote S6 kinase-mediated regulation of translation
and shutdown of autophagy by phosphorylation of ULK1, as well as the nutrient stimulated generation
of PI3P via the class III PI 3-kinase mVps34. How mTOR signaling is inactivated following nutrient and
growth factor deprivation and whether and how this process is regulated by phosphoinositides is
unknown. Here we identify class II phosphatidylinositol 3-kinase C2 (PI3K-C2) as a novel negative
regulator of mTORC1 signaling. Depletion of PI3K-C2, an enzyme that specifically synthesizes
phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2], promotes mTORC1 signaling and causes the
dispersion and functional inactivation of LAMP1-positive late endosomes and lysosomes. This phenotype
is rescued by co-depletion of the GTPase Arl8 or its effector SKIP, factors that crucially regulate
lysosomal transport. Furthermore, there is a functional association of PI3K-C2 with the raptor subunit of
mTORC1. Together, these data identify PI3K-C2-mediated PI(3,4)P2 synthesis as a critical switch
between growth promoting nutrient signals and lysosomal proteostasis, by negatively regulating
mTORC1 signaling.
M171
The vacuole/lysosome is required for cell-cycle progression.
Y. Jin1, L.S. Weisman1;
1
Life Sciences Institute, University of Michigan, Ann Arbor, MI
During the cell-cycle, cytoplasmic organelles are distributed to the new daughter cells. For example, in S.
cerevisiae, there is active organelle transport from the mother to the daughter cell. In yeast, the
vacuole/lysosome, which is inherited, has critical functions including maintaining hydrostatic
pressure/pH, as well as recycling, storage and degradation of macromolecules. The daughter cell inherits
the vacuole via the myosin V motor, Myo2, and the vacuole-specific adaptors, Vac17 and Vac8.
When vacuole inheritance is defective, the bud generates a new vacuole that is independent of the
mother vacuole. Moreover, after cytokinesis, the new cell does not form a bud until its vacuole reaches
a specific size. These results suggest that the vacuole is essential for cell viability. If true, then cells
defective in both vacuole inheritance and in the de novo synthesis of a new vacuole, would not be
viable. Indeed, pep12 vac17 and vps45 vac17 double mutants exhibit synthetic growth defects. That the
Pep12, t-SNARE and Vps45, Sec1/Munc18-like molecule are necessary for transport to the pre-vacuolar
compartment, suggests that when a mature vacuole is not inherited, Pep12/Vps45 are required for
maturation of endosomes to a fully functional vacuole. Furthermore, these double mutants show a G1
cell-cycle arrest, indicating that the vacuole is required for cell-cycle progression. A clue to why the
vacuole is required was revealed by the finding that the tor1 vac17 double mutant shows synthetic
growth defects. The Tor1 protein kinase is a member of the TORC1 complex, which signals from the
vacuole. In further studies we found that when a vacuole is not inherited, the newly synthesized vacuole
cannot recruit the major TORC1 target, Sch9. It is tempting to speculate that the newly synthesized
TUESDAY-ORAL PRESENTATIONS
vacuoles lack additional properties essential for normal vacuole function. Taken together, these results
suggest that a functional vacuole is required for cell-cycle progression in part through the TORC1
pathway.
M172
Membrane raft association is a determinant of plasma membrane localization.
B. Diaz-Rohrer1, K. Levental1, I. Levental 1,2;
1
Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston,
Houston, TX, 2Cancer Prevention and Research Institute of Texas, Austin, TX
The organization of metazoan membranes into functional domains is a key feature of their physiology.
The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols,
sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure
and function; however, experimental limitations in defining raft composition and properties have
prevented unequivocal demonstration of their function. In this study we establish a functional
relationship between raft association and subcellular protein sorting. To quantify raft association in
intact plasma membranes without detergent perturbation, we isolated Giant Plasma Membrane Vesicles
(GPMVs) from cultured mast cells. These GPMVs separate into coexisting raft and non-raft phases, as
defined by physical properties and lipid/protein compositions. This system provides an ideal model to
microscopically analyze the structural determinants and functional consequences of protein association
with membrane domains. By systematic mutation of the transmembrane and juxtamembrane domains
of an integral single pass plasma membrane protein, linker for activation of T-cells (LAT), we generated a
panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation,
transmembrane domain length, and transmembrane sequence to be critical and independent
determinants of membrane raft association. Having identified the structural features determining raft
association, we used our panel of mutants to establish a quantitative, functional relationship between
raft association and subcellular protein sorting. We observed that plasma membrane (PM) localization
was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that
raft association is necessary and sufficient for PM sorting of LAT. The specific defect in PM sorting was a
failure to recycle non-raft variants from early endosomes, with abrogation of raft partitioning leading to
mistargeting to late endosomes/lysosomes and subsequent degradation. Finally, we confirmed that
these observations are not specific to LAT, but are general to at least the three other unrelated singlepass transmembrane proteins. These findings identify structural determinants of raft association and
validate lipid-driven domain formation as a mechanism for protein sorting in the endosomal system.
TUESDAY-ORAL PRESENTATIONS
M173
The Intraflagellar Transport Protein IFT27/RABL4 Promotes BBSome Exit from
Cilia through the GTPase ARL6/BBS3.
G.M. Liew1, F. Ye1, A.R. Nager1, J.P. Murphy2, J.S. Lee1, M. Aguiar2, D.K. Breslow1, S.P. Gygi2, M.V.
Nachury1;
1
Dept. of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA,
2
Department of Cell Biology, Harvard Medical School, Boston, MA
The sorting of signaling receptors into and out of cilia relies on the BBSome, a complex of Bardet-Biedl
syndrome (BBS) proteins, and on the intraflagellar transport (IFT) machinery. GTP loading onto the Arflike GTPase ARL6/BBS3 drives assembly of a membrane-apposed BBSome coat that promotes cargo
entry into cilia, yet how and where ARL6 is activated remains elusive. Here, we show that the Rab-like
GTPase IFT27/RABL4, a known component of IFT complex B, promotes the exit of BBSome and
associated cargoes from cilia. Unbiased proteomics and biochemical reconstitution assays show that,
upon disengagement from the rest of IFT-B, IFT27 directly interacts with the nucleotide-free form of
ARL6. Furthermore, IFT27 prevents aggregation of nucleotide-free ARL6 in solution. Thus, we propose
that IFT27 separates from IFT-B inside cilia to promote ARL6 activation, BBSome coat assembly and
subsequent ciliary exit, mirroring the process by which BBSome mediates cargo entry into cilia.
Minisymposium 19: Visualizing and Dissecting How Single Cells Make
Decisions
M174
Two sequential decisions commit human cells to start the cell cycle.
S. Cappell1, S.L. Spencer1, M. Chung1, A. Jaimovich1, T. Meyer1;
1
Department of Chemical and Systems Biology, Stanford University, Stanford, CA
One of the most fundamental decision processes in biology is the commitment of cells to enter the cell
cycle and generate two daughter cells. Cell-cycle commitment in mammalian cells is thought to be
regulated by a single decision point in G1 called the Restriction Point, after which growth factors are no
longer required for the cell cycle to proceed. Using live-cell microscopy of cell-cycle sensors and singlecell analysis, we have identified the moment at which cells cross the Restriction Point and find that more
than three hours pass between the Restriction Point and the actual start of DNA replication.
Furthermore, we find that cell stress after the Restriction Point but before the start of DNA replication
results in cell-cycle exit and a return to a pre-Restriction Point state, providing evidence for another later
decision to commit to the cell cycle. We present evidence for a second bistable switch regulating Sphase entry. The trigger for this second switch is the CDK2/Cyclin E-mediated partial inactivation of
APCCdh1, an E3-ligase that controls the degradation of proteins necessary for S-phase progression. We
show that the switch is driven by a double negative feedback between the regulatory protein Emi1 and
TUESDAY-ORAL PRESENTATIONS
APCCdh1. Markedly, Emi1 functions as both an inhibitor and a substrate of the APC, which generates a
bistable and irreversible switch regulating S-phase entry. Thus, cells commit to the cell cycle by making
two sequential decisions. They first monitor mitogen signals to decide whether to cross the Restriction
Point, and then monitor stress signals to decide whether to irreversibly inactivate APCCdh1, start DNA
replication, and fully commit to the cell cycle.
M175
Intracellular Vibrio parahaemolyticus Escapes Vacuole and Establishes a
Replicative Niche in the Cytosol of Epithelial Cells.
M. de Souza Santos1, K. Orth1;
1
Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX
The marine bacterium Vibrio parahaemolyticus is the leading cause worldwide of seafood-borne acute
gastroenteritis. For decades, the pathogen has been studied exclusively as an extracellular bacterium.
However, recent results have revealed the pathogen’s ability to invade and replicate within host cells.
The present study is the first characterization of V. parahaemolyticus’ intracellular lifestyle. Upon
internalization, V. parahaemolyticus is contained in a vacuole that would in the normal course of events
ultimately fuse with a lysosome, degrading the vacuole’s contents. The bacterium subverts this pathway,
escaping into the cytoplasm prior to lysosomal fusion. Once in the cytoplasm, it replicates prolifically.
Our study provides new insights into the strategies used by this globally disseminated pathogen to
survive and proliferate within its host.
M176
Angiomotins link F-actin architecture to Hippo pathway signaling.
S. Mana-Capelli1, M. Paramasivam1, S. Dutta1, D. McCollum1;
1
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School,
Worcester, MA
The Hippo pathway regulates the transcriptional co-activator YAP to control cell proliferation, organ size,
wound healing, and stem cell maintenance. YAP activity is modulated to act as a molecular switch in
response to stimuli that change the mechanical tension of the cell. When cells are under high
mechanical forces YAP localizes to the nucleus and activates proliferative genetic programs, and when
the mechanical tension drops, YAP is inhibited by relocation to the cytoplasm. Actin stress fibers have
been implicated in YAP regulation. For example, high substrate stiffness or low cell densities promote
mechanical tension and stress fiber formation, which is required for YAP activation. However, the
mechanism that links YAP activity to the actin cytoskeleton is not known. Here we show that the YAP
inhibitory angiomotin proteins (AMOT130, AMOTL1, and AMOTL2) connect F-actin architecture to YAP
regulation. Several lines of evidence support a model whereby angiomotins are sequestered on F-actin
structures, but upon actin perturbation become liberated and free to inhibit YAP. First, we show that
TUESDAY-ORAL PRESENTATIONS
angiomotins are required to efficiently inhibit YAP in the cytoplasm after manipulations that perturb the
actin cytoskeleton. Second, angiomotins associate with F-actin through a conserved F-actin binding
domain, and mutants defective for F-actin binding show enhanced ability to retain YAP in the cytoplasm.
Third, F-actin and YAP compete for binding to AMOT130, explaining how F-actin inhibits AMOT130mediated cytoplasmic retention of YAP. Furthermore, we find that LATS can synergize with F-actin
perturbations by phosphorylating free AMOT130 to keep it from associating with F-actin. Together these
results uncover a mechanism for how F-actin levels modulate YAP localization, allowing cells to make
developmental and proliferative decisions based on diverse inputs that regulate actin architecture.
M177
An endoplasmic reticulum lipid phosphatase regulates plasma membrane lipid
metabolism.
E.J. Dickson1, J.B. Jensen1, O. Vivas1, B. Hille1;
1
Physiology and Biophysics, University of Washington, Seattle, WA
Phosphoinositides are a family of low-abundance phospholipids located on the cytoplasmic leaflet of
cellular membranes that maintain cell structure, cell motility, membrane trafficking, ion channel
functions and also play key roles in signal transduction. Phosphatidylinositol (4,5)-bisphosphate
(PI(4,5)P2) is the signature phosphoinositide of the plasma membrane. How is PM PI(4,5)P2 sourced? We
have reported that plasma membrane PI(4,5)P2 levels are supported by at least two, continuously
supplying, precursor pools of PI(4)P, one in the plasma membrane itself, and the other in the Golgi. How
is plasma membrane PI(4,5)P2 regulated? Many lipid phosphatases and kinases are needed for the
maintenance of each cellular phosphoinositide pool; we focused our attention on an integral membrane
lipid 4-phosphatase enzyme localized to the endoplasmic reticulum (ER), Sac1. It has the ability to
reduce the precursor source of PM PI(4,5)P2, namely PI(4)P. Using a variety of optical (confocal, TIRF,
super-resolution microscopies), protein dimerization, and electrical (ion channel recordings) techniques,
we have discovered that Sac1 aggregates in puncta, intimately apposed to the plasma membrane in
endoplasmic reticulum–plasma membrane (ER-PM) contact sites in tsA-201 cells. Based on FRET
measurements between Sac1 and a plasma membrane marker, Sac1 is within 10 nm of the plasma
membrane. What causes Sac1 to aggregate in ER-PM contact sites? Multiple lines of evidence suggest a
role for the cortical ER protein, extended synptotagmin-2 (E-Syt2). Quantitative super-resolution
imaging analysis reveals strong correlation between Sac1 and E-Syt2 distributions. Overexpressing or
knocking down E-Syt2 increased or decreased the abundance of Sac1 in close proximity to the PM,
respectively, and lead to alterations in plasma membrane PI(4)P and PI(4,5)P2 levels. Activation of Gprotein coupled receptors to deplete plasma membrane PI(4,5)P2 causes a dynamic redistribution of
Sac1 puncta away from the plasma membrane. The reaggregation of Sac1 back into ER-PM contacts is
dependent on the rate of PI(4,5)P2 resynthesis. Similar results were observed in superior cervical ganglia
neurons. Thus, the intimate organization of ER Sac1 next to the plasma membrane gives it the unique
ability to act as a cellular ‘thermostat’ controlling plasma membrane PI(4)P and PI(4,5)P2 levels and with
that many plasma membrane proteins.
TUESDAY-ORAL PRESENTATIONS
Supported by a grant from the NIH National Institute of Neurological Disorders and Stroke R37NS008174
and the Wayne E. Crill Endowed Professorship.
M178
Controlling low rates of cell differentiation through noise and ultra-high feedback.
M.N. Teruel1, R. Ahrends1, K.M. Kovary1, A. Ota1, B. Park1, T. Kudo1;
1
Chemical and Systems Biology, Stanford University, Stanford, CA
Mammalian tissue size is maintained by slow replacement of de-differentiating and dying cells. For
adipocytes, key regulators of glucose and lipid metabolism, the renewal rate is only 10% per year. We
used computational modeling, quantitative mass spectrometry, and single-cell microscopy to show that
cell-to-cell variability, or noise, in protein abundance acts within a network of more than six positive
feedbacks to permit pre-adipocytes to differentiate at very low rates. This reconciles two fundamental
opposing requirements: high cell-to-cell signal variability so that differentiation rates can be kept very
low and low signal variability to prevent differentiated cells from de-differentiating. Higher eukaryotes
can thus control low rates of near irreversible cell fate decisions through a balancing act between noise
and ultra-high feedback connectivity.
M179
Resonator motifs in mechano-chemical signaling pathways revealed by a new
optogenetic method for precise oscillation of signaling circuits.
H. Wang1, M. Vilela2, R. Liu3, G. Danuser2, K.M. Hahn1;
1
Department of Pharmacology, University of North Carolina, Chapel Hill, NC, 2Department of Cell
Biology, University of Texas Southwestern Medical Center, Dallas, TX, 3Eshelman School of Pharmacy,
University of North Carolina, Chapel Hill, NC
We have developed an optogenetic approach to control protein activity that promises to be broadly
applicable and is simple to apply (LOVTRAP, for LOV trapping and release of active proteins). Using
mRNA display we developed Zdk, a small protein (based on the Z domain from protein A) that binds only
to the dark state of the LOV2 domain from Avena sativa phototropin. The LOV2 domain undergoes a
large, reversible conformational change induced by light between 400 and 500 nm. Zdk binds to the dark
state of LOV2 with a Kd of ~27nM, but shows no detectable binding to the lit state. In our new method,
the LOV domain was anchored at the mitochondrian and different proteins of interest were fused to
Zdk. In the dark, the Zdk-protein fusion was sequestered at the mitochondrian, but upon irradiation it
was reversibly released within 1 second. Using different LOV mutants, return to the dark state could be
adjusted for a t1/2 of 2 to 700 seconds. LOVTRAP was used to regulate RhoA, Rac1, Cdc42, Vav2 and a
RhoA inhibitory peptide. By oscillating the intensity of the activating light, LOVTRAP could be used to
generate oscillations in the activity of signaling proteins with precise frequency. We used this approach
to study the cell edge dynamics induce by Vav2, a GEF that activates Rac1, RhoA and Cdc42. Simple
TUESDAY-ORAL PRESENTATIONS
activation of VAV2 or its downstream target Rac1, but not RhoA or Cdc42, increased the frequency of
cell edge oscillations. When Vav2 activity was oscillated at frequencies that were multiples of 3.3 mHz,
strong reinforcement of 3.3 mHz oscillations were observed. Other frequencies, not multiples of 3.3,
produced only an increase across a broad range of frequencies. Frequencies above 3.3 mHz that were
multiples of 3.3 mHz reinforced the Vav2 oscillations at 3.3 mHz, rather than inducing higher frequency
oscillations. This showed that Vav2 was a component of a resonator circuit that controlled cell edge
oscillations. The oscillations induced by VAV2 were dependent on PI3K, suggesting a model for a
resonator based on a positive feedback loop that includes mechanical and biochemical interactions.
Because of the generalizable nature of our approach, we hope that LOVTRAP can open the door to
application of increasingly sophisticated signal processing tools, to study not only cell migration but also
other physiological oscillatory processes.
M180
Endothelial cells use a phosphoinositide-3-kinase-, focal adhesion kinase-, and
Rho-dependent phagocytosis-like process to internalize the bacterium Listeria
monocytogenes.
M. Rengarajan1, J.A. Theriot1,2;
1
Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 2Howard Hughes
Medical Institute, Stanford University School of Medicine, Stanford, CA
To protect organs from toxins, pathogens, and other potentially damaging agents in the bloodstream,
vascular endothelial cells, which line the lumen of blood vessels, act as gatekeepers that selectively
regulate the transport of material from the bloodstream to underlying tissue. Even so, endothelial cells
have been shown to internalize large objects from the bloodstream, including fibrin clots and apoptotic
debris; such internalization suggests a point of vulnerability that can be exploited by invasive bacterial
pathogens. The ubiquitous food-borne bacterium Listeria monocytogenes causes spontaneous abortion
in pregnant women and meningitis in immune-compromised hosts. Bacteria initiate organismal infection
by directly invading intestinal epithelial cells, but systemic dissemination requires bacteria to cross the
endothelial barrier to invade protected tissues such as the placenta and meninges. We have found that
primary endothelial cells (HUVEC) internalize the pathogenic bacterium L. monocytogenes in a
phagocytosis-like process that does not require specific bacterial factors. Molecular regulatory factors
for phagocytosis-like uptake by endothelial cells have not been elucidated, so we used siRNA screening
and subsequent pharmacological and genetic perturbations to determine that uptake of L.
monocytogenes by HUVEC is promoted by a Rho-dependent signaling pathway involving
phosphoinositide 3-kinase and focal adhesions. The actin nucleating formins FHOD1, FMNL3, and INF2
contribute to uptake of free bacteria, while the Arp2/3 complex is not critical. Perturbing this PI3K/focal
adhesions/Rho signaling pathway with inhibitors of Rho kinase, focal adhesion kinase, or formins inhibits
uptake of pathogenic and non-pathogenic bacteria by endothelial cells but does not affect macrophage
phagocytosis of L. monocytogenes. Distinct molecular regulation of endothelial phagocytosis-like uptake
as compared to macrophage phagocytosis should allow for specific perturbation of these processes in
TUESDAY-ORAL PRESENTATIONS
vivo. Furthermore, our results may provide mechanistic insight into how other large objects, such as
stroke-causing clots in small diameter blood vessels, are internalized by endothelial cells. We have also
demonstrated that L. monocytogenes in infected macrophages can be robustly transferred to uninfected
HUVEC via direct heterotypic cell-to-cell spread and that the uptake of a bacterial protrusion by HUVEC
is regulated quite differently from phagocytosis-like uptake of free bacteria; thus, endothelial cells may
utilize distinct mechanisms for internalization of micron-sized objects, depending on the identity of the
cargo.
M181
Switch-like precision of cholesterol-sensing proteins arises due to limited
accessibility of membrane cholesterol.
A. Gay1, D. Rye1, D. Frias1, A. Radhakrishnan1;
1
Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX
Cholesterol levels in mammalian cells are maintained within narrow limits by a network of proteins that
measure cholesterol concentrations and then regulate its synthesis and uptake. This regulatory protein
network resides in the ER membrane where the cholesterol concentration fluctuates around a set-point
of 5 mole% of total lipids. The cholesterol sensor in this system is Scap, a transmembrane protein that
regulates the transport and subsequent proteolytic activation of SREBP. The sensitivity of Scap as a
sensor is sharp and switch-like; a small increase in ER cholesterol halts both synthesis and uptake. The
molecular mechanism by which Scap can measure ER cholesterol with such precision is largely unknown.
The sigmoidal response of Scap could arise from several mechanisms, including positive cooperativity
(protein effects) and limited accessibility of cholesterol (membrane effects). A convenient model for
eukaryotic membrane-bound Scap is a family of soluble bacterial toxins that show identical switch-like
specificity for endoplasmic reticulum membrane cholesterol. Using this model, we show that sigmoidal
responses can arise primarily due to membrane effects. Truncated versions of these toxins fail to form
oligomers but still show sigmoidal binding to cholesterol-containing membranes. The non-linear
response emerges because interactions between bilayer lipids control cholesterol accessibility to toxins
in a threshold-like fashion. Around these thresholds, affinity of toxins for membrane cholesterol varies
by >100-fold, generating highly cooperative lipid-dependent responses independent of protein-protein
interactions. Such lipid-driven cooperativity may control the sensitivity of many cholesterol-dependent
processes.
TUESDAY-ORAL PRESENTATIONS
M182
From intracellular signaling to collective behavior: Understanding the dynamical
origins of collective cAMP oscillations in Dictyostelium discoideum.
A. Sgro1, D. Schwab1, J. Noorbakhsh2, P. Mehta2, T. Gregor1;
1
Physics and Lewis-Sigler Institute, Princeton University, Princeton, NJ, 2Physics, Boston University,
Boston, MA
Collective behaviors are a common feature of a diverse range of biological systems, from flocking birds
and schooling fish to swarming bacterial colonies and embryonic morphogenesis. Such population-level
behaviors in cellular collectives are controlled by complex biochemical signaling networks that reside
within individual cells to coordinate cell-cell communication. One of the most striking examples of these
behaviors is the cAMP-coordinated transition from an independent, single-celled state to a multicellular
aggregate in the eukaryotic social amoebae Dictyostelium discoideum. However, describing how these
population-level collective behaviors arise from intracellular signaling network dynamics is challenging.
Even in well-studied model systems many of the dynamics of underlying signaling networks remain
uncharacterized, complicating efforts to build a predictive model that takes into account each network
component and interaction. Physical systems have taught us that collective behaviors do not depend on
all the details of the system and that in regimes with behavioral changes only a few types of behaviors
exist. Using these principles, we have condensed the amoeba’s complex signaling network dynamics
into a simple two-variable phenomenological model. We have confirmed our model’s success at
capturing these dynamics through quantitative experimental measurements, using microfluidics to
control the extracellular environment and a FRET reporter for intracellular cAMP to monitor cellular
responses. By using the single-cell model as a building block for a multicellular model, we are able to
predict and experimentally verify novel population-level behaviors. Together, our model and
experiments demonstrate that stochasticity is a key player both in the initiation and the ongoing
coordination of collective behaviors, allowing cells to communicate in noisy extracellular environments.
Our results lay the groundwork for using these types of models to identify common principles of how
cellular collective cellular behaviors arise in nature.
E.B. Wilson Medal Presentation and Address
G4
Romancing the Mitotic apparatus (MA) and cytoplasmic microtubule complex
(CMTC) in eukaryotic cells.
B. Brinkley1;
1
Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX
After over 50 years of investigations in our laboratory and elsewhere, the cytoskeleton and mitotic
apparatus has attracted considerable creative research interests, worldwide. My research began in
TUESDAY-ORAL PRESENTATIONS
1964 as a postdoctoral fellowship in the laboratory of Professor T.C. Hsu, at the M.D Anderson Cancer in
Houston, Texas. Newly trained in transmission electron microscopy (TEM), I was most anxious to
explore the ultrastructure of chromosomes and the mitotic apparatus of mammalian cells using
Professor Hsu's novel sources of cell lines with low chromosome numbers, i.e., the rat kangaroo (2n=11),
and Indian muntjacs deer (2n=6). Relatively few EM studies had been done on mammalian mitotic cells
at that time, and explorations of ultrathin sections of these remarkable cells resulted in many novel
“discoveries of the week.” Using various mitotic inhibitors such as, colcemid and nocodazole to arrest
cells in mitosis and subsequently reversing the blockage, we analyzed and characterized microtubule
assembly sites (centrioles, centrosomes, kinetochores) throughout mitosis. Of special importance was
the discovery of the trilaminar plate-like kinetochores at the centromeres found to be widely conserved
on mitotic chromosomes of most all eukaryotic species. Subsequently, we extended our studies of
microtubule assembly to include lysed cell models to characterize microtubule assembly sites in vitro. In
collaboration with investigators at the M.D. Anderson Cancer Center in Houston, TX, we investigated the
Aurora kinases and their role in chromosome instability and aneuploidy in normal and neoplastic cells.
Through a valuable collaboration with Professor G.M. Fuller at the University of Alabama at Birmingham,
we produced the first monospecific antibody against 6s tubulin in rabbits, thus obtaining remarkable
immunofluorescent images of the intact mitotic spindles and the microtuble cytoskeleton in mammalian
cell monolayers. Although novel at the time, today, such immunofluorescence probes are widely used
to illuminate and characterize the eukaryotic cytoskeleton and many other cellular structures and
activities.
G5
A personal history of EM-visualization of the cytoskeleton.
J.E. Heuser1,2;
1
Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO,
2
Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan
This will be a simple recounting of the history of the quest for understanding the cytoskeleton by
electron microscopy. It all started with Keith Porter, who considered it his most important goal and
pursued it his whole career, and occupied countless other researchers, as EM evolved and molecular
biology developed. The history will focus on the images themselves, so that no one will leave without a
better view of the cytoskeleton in their mind's eye. It will obviously be biased toward the approach to
viewing the cytoskeleton I hit upon: namely, replication of freeze-dried cells. It will also be totally
anecdotal, since I will show my 'bests' from a series of collaborations with outstanding figures in the
ASCB, and will dote briefly on how great it was to work with each one of them.
TUESDAY-ORAL PRESENTATIONS
G6
Studies on Cilia: Onwards from the cradle.
P. Satir1;
1
Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY
This presentation records a voyage of discovery from the “cradle of cell biology” to the present, focused
on the biology of the oldest known cell organelle, the cilium. Initially ciliary beat stages were captured
by quick fixation to examine the mechanism of motility. In the “romper room of cilia and microtubule
biology”, the sliding microtubule hypothesis of ciliary motility was born. From the “summer of love”,
students and colleagues joined the journey to test switch point mechanisms of motility. In the new
century, interest in non-motile (primary) cilia, never lost from the cradle, was rekindled, leading to
discoveries relating to PDGFRα signaling, a recriprocal relationship of ciliogenesis and autophagy and
hypotheses of how molecules cross ciliary necklace barriers for cell signaling.
WEDNESDAY-ORAL PRESENTATIONS
ORAL PRESENTATIONS- Wednesday, December 10
Minisymposium 20: Cytoskeletal Filaments and Motors
M183
Tuning myosin driven sorting on cellular actin networks.
R.F. Hariadi1, R. Sommese1, S. Sivaramakrishnan1;
1
Cell and Developmental Biology, University of Michigan, Ann Arbor, MI
Unconventional myosin function in cellular processes emerges from the interaction of multiple myosins
on an organelle scaffold with the complex actin cytoskeleton. Currently, the role of myosins in
membrane sorting has been extensively studied in reconstituted systems, but these have focused
almost exclusively on understanding single actin-myosin interactions. To bridge the gap between single
molecule and cellular function, we pair DNA origami scaffolds, containing a defined number of
antagonistic myosins, with a model cellular actin network. Scaffolds with antagonistic myosins (V and VI)
exhibit unidirectional motion, unlike the bi-directional movement previously reported for groups of
kinesin and dynein. For scaffolds with equal numbers of myosin V and VI, minus and plus-end directed
movement is equally favored. This two-dimensional flux is significantly lower than that of single
filament, emphasizing the importance of actin architecture on myosin behavior. In ensembles with
asymmetric motor composition, the net flux of trajectories can be finely tuned by the relative number of
myosin V and myosin VI. Thus, modulating the relative number of motor engagement sites represents
an elegant sorting mechanism in cells. Finally, the net flux is dependent on the structural properties of
the myosin lever arm. Our study demonstrates a novel regulatory function for the myosin lever arm
beyond its canonical function as a structural amplifier in the chemomechanical cycle.
M184
Removal of the fission yeast myosin-II lever arm slows motility and accelerates
contractile ring constriction during cytokinesis.
Q. Tang1, G. Murray1, M.J. Lord1;
1
Molecular Physiology and Biophysics, University of Vermont, Burlington, VT
The myosin-II heavy chain contains a light chain-binding region that associates with the essential (ELC)
and regulatory (RLC) light chains. This region forms a rigid ‘lever arm’ that amplifies a conformational
change in the motor domain that drives the displacement and motility of associated actin filaments.
Previous studies in fission yeast indicated that RLC phosphorylation ensures optimal rates of Myo2p
motility and actomyosin ring constriction during cytokinesis. We find that removal of RLC-mediated
regulation (via deletion of the entire Myo2p lever arm) significantly slows down Myo2p motility while
WEDNESDAY-ORAL PRESENTATIONS
promoting significantly faster rates of ring constriction in vivo. Our findings indicate that maximal rates
ability of the RLC to control Myo2p lever arm rigidity. We performed a high-copy suppression screen to
search for proteins capable of rescuing growth in the absence of the RLC. We identified the highly
conserved Rad23 homolog (Rhp23p), a factor involved in mediating protein degradation by the ubiquitin
proteasome system (UPS). Further characterization revealed a role for Rad23 in myosin-II and
actomyosin ring function, suggesting that alternative pathways of cellular regulation contribute to the
complex process of cytokinesis.
M185
Shs1 is required for an organized septin higher order structure in vivo.
M. McQuilken1, S. Abrahamsson2, S. Mehta3, G. Harris3, A. Verma3, R. Oldenbourg3, A.S. Gladfelter1;
1
Biological Sciences, Dartmouth College, Hanover, NH, 2Rockefeller University, New York, NY, 3Cellular
Dynamics, Marine Biological Laboratory, Woods Hole, MA
Septins are conserved filament-forming proteins that act in cytokinesis, membrane remodeling, cell
polarization, and migration. They closely associate with membranes and can act as diffusion barriers to
restrict the diffusion of proteins within membranes. Although septin function is critical for diverse cell
events, it is not well understood how they assemble in vivo or how they are remodeled throughout the
cell cycle. GFP can be excited by and emit linear polarized light and linking GFP in a constrained manner
to an endogenous septin enables analysis of septin organization in vivo by polarization microscopy.
Polarized fluorescence analysis has previously suggested that septins filaments are paired in higher
order structures, and that organized septin filaments undergo a coordinated 90° reorientation during
cytokinesis in vivo. We developed a Multifocus Polarization Microscope (MF-PolScope) to evaluate
septin organization in 3D through time to capture the assembly and rearrangement of higher-order
septin structures, and analyze mutant yeast strains with abnormally organized septins. This method
allows simultaneous detection of 9 focal planes at a single instant so as to analyze the degree of
coordination of events in different areas of the cell and assess if filaments in mutant strains are likely
paired. MF-PolScope imaging has led to the identification of septin interactors important for distinct
aspects of the assembly, stability, and rearrangement of septins. One interactor necessary for ordered
assembly of septin higher order structures is the septin, Shs1. The septin structure is disordered in shs1
mutant cells in a manner consistent with the absence of paired septin filaments. Our work demonstrates
the power of this new imaging approach to assess dynamic rearrangements of the cytoskeleton in
whole, live cells.
WEDNESDAY-ORAL PRESENTATIONS
M186
Microtubule-dependent transport and dynamics of vimentin intermediate
filaments.
C. Hookway1, L. Ding2, M.W. Davidson3, J.Z. Rappoport1,4, G. Danuser2, V.I. Gelfand1;
1
Department of Cell and Molecular Biology, Northwestern University, Feinberg School of Medicine,
Chicago, IL, 2Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX,
3
National High Magnetic Field Laboratory and Department of Biological Science, Florida State University,
Tallahassee, FL, 4Center for Advanced Microscopy, Northwestern University, Chicago, IL
We studied two aspects of vimentin intermediate filaments dynamics, transport of filaments and
subunit exchange. Using Structured Illumination Microscopy (SIM) of live cells, we directly observed
transport of long intermediate filaments at the cell periphery. To study filament transport elsewhere in
the cell, we used the photoconvertible protein mEos3.2 fused to vimentin. This technique enabled us to
selectively label a subset of filaments within the dense vimentin network by photoconversion of green
fluorescence to red in a small area of the cytoplasm. We then followed red filaments by time-lapse Total
Internal Reflection Fluorescence Microscopy (TIRFM), which demonstrated that photoconverted
filaments are rapidly transported along linear tracks. Filament transport was microtubule-dependent,
likely due to transport by microtubule-based motors. Transport was independent of microtubule
dynamics and/or interaction of vimentin with the microtubule plus-tip binding protein APC. We also
used mEos3.2-vimentin and photoconversion to study subunit exchange in vimentin filaments over the
course of several hours. We found that even 17 hours after photoconversion, photoconverted (red)
vimentin formed distinct patches and was not completely intermixed with green vimentin filaments.
This pattern was also observed in cells that divided after conversion. These data show that vimentin
filaments are stable and do not disassemble into individual subunits even during cell division. Instead,
dynamics of intermediate filaments include severing and re-annealing. Together, these results
contribute to the understanding of intermediate filament organization, which is important for
maintaining mechanical integrity during cell migration. This work was supported by the Intermediate
Filament Program Project Grant, NIGMS # P01GM09697.
M187
Katanin regulates microtubule length.
M. Bailey1, J.L. Ross2;
1
Molecular and Cellular Biology, University of Massachusetts, Amherst, MA, 2Physics, Univ
Massachusetts-Amherst, Amherst, MA
Microtubules require regulation to maintain organization, which entails constant remodeling in order to
properly form networks and perform the necessary functions of the cell. This remodeling is performed
by microtubule associated proteins (MAPs), which control microtubule length and dynamics by
stabilizing or destabilizing microtubules. While stabilizing MAPs are relatively well understood, little is
known about destabilizing MAPs, such as microtubule severing enzymes. Katanin, the first-discovered
WEDNESDAY-ORAL PRESENTATIONS
microtubule severing enzyme, is a AAA enzyme that oligomerizes into a hexamer and uses ATP
hydrolysis as an energy source to sever microtubules. In vivo these enzymes must be tightly regulated to
ensure the microtubule network remains in tact and the cells can continue to function properly. We
observe, using total internal reflection fluorescence (TIRF) microscopy, purified katanin p60 perform
complete rapid severing on taxol-stabilized microtubules in a matter of seconds. We investigate how
katanin and its severing activity can be regulated by other MAPs, such as the neuronal protein, tau, the
type of microtubule substrate, the free tubulin concentration, and the ATP concentration. Interestingly,
free tubulin disrupts katanin activity, and severing activity is rescued by the presence of MAPs,
counterintuitive to previously hypothesized regulation mechanisms.
M188
Genetically engineered kinesin motors amenable to selective small molecule
inhibition.
M.F. Engelke1, Y. Yue1, F. Teloni1, P. Soppina1, S. Reddy1, T.L. Blasius1, S. Shastry2, W.O. Hancock2,3, K.J.
Verhey1;
1
Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI,
2
Department of Biomedical Engineering, Pennsylvania State University, University Park, PA,
3
Interdisciplinary Graduate Degree Program in Bioengineering, Pennsylvania State University, University
Park, PA
The human genome codes for 45 kinesins that contain a highly conserved kinesin motor domain and
divergent tail domains for their specific functions. Genetic methods such as RNAi and overexpression of
dominant negative species have been used to explore the cellular roles of different kinesins. These
methods have yielded valuable information, but are slow acting and prone to off-target effects and
hence not suitable to address complex and dynamic biological questions. The identification of small
molecule inhibitors specific to different kinesins is highly desirable, yet only a handful of such inhibitors
have been identified due at least in part to the high conservation of the kinesin motor domain. Here we
demonstrate that the kinesin motor domain can be engineered to maintain motility yet be inhibited by
small, cell permeable molecules. Using kinesin-1 as a prototype, we pursued two strategies to obtain
inhibitable motors: I) We inserted the six amino acid tetracysteine tag into surface loops of the motor
domain such that binding of biarsenic dyes conformationally distorts and thereby inhibits motility. II) We
fused DmrB dimerization domains to the motor heads such that addition of B/B homodimerizer crosslinks the motor domains and inhibits motor stepping. We show using cellular assays that the engineered
kinesin-1 motors are able to transport artificial cargoes similarly to the wild type motor, but are
efficiently inhibited by the addition of inhibitor. In vitro assays revealed that inhibitor addition reduces
the number of active motors on the microtubule, with minor effects on motor run length and velocity.
Future studies will enable us to deploy the inhibitable kinesin-1 motors in cells and animals to study
cellular kinesin function with high specificity and temporal resolution. It is likely that these inhibition
strategies can be successfully applied to other members of the kinesin superfamily, owing to the high
WEDNESDAY-ORAL PRESENTATIONS
conservation of the kinesin motor domain. MFE was supported by an early postdoc mobility fellowship
(PBZHP3_141433) from the Swiss National Science Foundation.
M189
MYO19 ensures symmetric partitioning of mitochondria and coupling of
mitochondrial segregation to cell division.
J.L. Rohn1, J.V. Patel2, O.A. Quintero3, B. Baum2;
1
Centre for Clinical Science and Technology, University College London, London, United Kingdom,
2
Laboratory for Molecular Cell Biology, University College London, London, United Kingdom,
3
Department of Biology, University of Richmond, Richmond, VA
During animal cell division, an actin-based ring cleaves the entire cell into two. Defects in this process
can lead to chromosome mis-segregation, a hallmark of cancer, as well as to defects in cytoplasmic
inheritance and the partitioning of organelles. Although a great deal is known about how chromosome
segregation is coupled to the process of cell division, the way organelles coordinate their inheritance
during partitioning to daughter cells is less well understood. Here, using a high-content live-imaging
siRNA screen, we identify myosin-XIX (MYO19) as a novel regulator of cell division. Previously this actinbased motor was shown to control the interphase movement of mitochondria. Using live-cell imaging,
our analysis shows that MYO19 is indeed localised to mitochondria, and that its silencing leads to
asymmetric segregation of mitochondria during anaphase and telophase, as well as to defects in
mitochondrial partitioning at cytokinesis. Moreover, MYO19 RNAi cells frequently fail to complete
division. This cell division failure phenotype persists in cell lines stably expressing shRNA against
MYO19, as these cell lines display a higher percentage of multinucleate cells, compared to controls. This
phenotype appears to be due to the physical interference of cytokinesis machinery by mitochondria, as
the phenotype can be mimicked using a treatment that blocks mitochondrial fission and rescued by
decreasing mitochondrial fusion. The phenotype can also be rescued by exogenous expression of GFPMYO19 refractory to RNAi treatment. Taken together, these data support a model whereby the actinbased myosin motor, MYO19, is responsible for the faithful segregation of mitochondria during cell
division, and highlights the importance of coupling organelle inheritance to cytokinesis.
WEDNESDAY-ORAL PRESENTATIONS
M190
Direct observation by cryo-EM of cytoplasmic dynein motors stepping along
microtubules.
H. Imai1, T. Shima2, K. Sutoh3, M.L. Walker4, P.J. Knight1, T. Kon5,6, S.A. Burgess1;
1
School of Molecular and Cellular Biology and Astbury Centre for Structural Molecular Biology, Faculty of
Biological Sciences, Univ Leeds, Leeds, United Kingdom, 2QBiC, RIKEN, Osaka, Japan, 3Waseda University,
Tokyo, Japan, 4MLW Consulting, Cornwall, United Kingdom, 5Department of Frontier Bioscience, Hosei
University, Tokyo, Japan, 6JST PRESTO, Saitama, Japan
Cytoplasmic dynein motor proteins perform critical roles in eukaryotic cells, transporting subcellular
cargoes towards the minus ends of microtubule (MT) tracks and maintaining the positions of organelles.
Tracking of fluorescently-tagged dimeric dynein suggested an uncoordinated stepping by the two heads,
but there is also evidence of communication between them. We have now imaged directly by cryoelectron microscopy individual Dictyostelium discoideum dynein dimers flash-frozen while stepping
along MT at near-physiological Mg-ATP concentration. About half of the molecules show diverse
configurations of the two motor heads. The other half of the molecules has the two heads closely
superposed with both their linker domains in post-powerstroke conformations. This unpredicted
superposed configuration reveals more clearly the coiled-coil stalk domains and their terminal
stalkheads that bind the dimer to the MT. The stalks are angled at ~42±11° to the MT, and are flexible
mainly about the stalkhead-stalk junction, resulting in axial and radial variation of the heads. The
superposed configuration is different from any configuration of the two other cytoskeletal motor
families, kinesin and myosin, and thus direct motor-motor interactions may be functionally important in
stepping by dynein.
M191
Tubulation of Lysosomal Membranes Containing Niemann Pick Type C1 Requires
StARD9 – the First Transmembrane Kinesin.
A.M. Brumfield1, K. Huegel1, P.S. Vaughan1, M. Joyce1, W. Boggess1, E.H. Hinchcliffe2, K.T. Vaughan1;
1
University of Notre Dame, Notre Dame, IN, 2Univ Minnesota-Hormel Inst, Austin, MN
Late Endosomes and Lysosomes (LE/Ls) are microtubule-dependent organelles that play an important
role in cholesterol transport and acquire pathological accumulations of cholesterol in lysosomal storage
diseases such as Niemann Pick Type C (NPC) disease. 95% of NPC cases are caused by mutations in NPC1,
a 13-pass lysosomal transmembrane protein. LE/Ls containing NPC1 undergo alternating, bidirectional
excursions along microtubules (MTs), and project dynamic membrane tubules that are also MTdependent. Because the projection of dynamic membrane tubules is lost in cells expressing mutant but
not wild-type NPC1, we used a proteomic survey to identify differences between these two membrane
populations. StARD9 emerged as a novel membrane protein present in wild-type but absent in
membranes containing I1061T mutant NPC1. This 4700 amino acid protein contains a conserved kinesin
WEDNESDAY-ORAL PRESENTATIONS
domain at the N-terminus, a dileucine, lysosomal targeting signal and a C-terminal StAR domain that
overlaps with predicted transmembrane segments. StARD9 expression reveals MT-binding by N-terminal
constructs and lysosomal accumulation by constructs also containing the StAR domain. Full-length
StARD9 targets to lysosomes and incorporates into membrane tubules. ShRNA-driven depletion of
StARD9 reduces centrifugal excursions of LE/Ls and projection of membrane tubules. These activities
were rescued by shRNA-resistant wild-type but not a P-loop mutant StARD9 construct. Whereas coexpression studies reveal that wild-type NPC1 incorporates into lysosomes containing StARD9, mutant
NPC1 fails to incorporate into StARD9-containing lysosomes. These findings identify StARD9 as the first
transmembrane kinesin and suggest that loss of StARD9 in mutant NPC1 membranes could explain
defects in cholesterol transport.
Minisymposium 21: The Mechanics of Cell Division
M192
Kinetochore architecture encodes a mechanical toggle-switch to control the
spindle assembly checkpoint.
P. Aravamudhan1, A.A. Goldfarb2, A.P. Joglekar1,2;
1
Biophysics, University of Michigan, Ann Arbor, MI, 2Cell and Developmental Biology, University of
Michigan, Ann Arbor, MI
How microtubule attachment to the kinetochore silences the Spindle Assembly Checkpoint (SAC) has
puzzled cell biologists for over two decades. It is clear that the architecture of kinetochore changes
following microtubule attachment, and that this altered architecture disrupts SAC signaling. However,
the network of protein complexes that forms the kinetochore can change in innumerable ways following
microtubule attachment, and one or more of these changes can disrupt any of the steps in SAC
activation. Therefore, defining the molecular mechanism that enables this attachment sensitive
signaling has been challenging. To overcome this challenge, we asked how the well-defined architecture
of attached kinetochore disrupts SAC signaling. We show that microtubule attachment to the
kinetochore changes the separation between two conserved kinetochore proteins, Ndc80 and Spc105 to
disrupt phosphorylation of Spc105, an essential step in SAC activation.
We used a comprehensive analysis of the activity of SAC proteins artificially localized at specific
kinetochore positions to find that the architecture of attached kinetochore prevents Mps1 kinase from
phosphorylating Spc105. Furthermore, attachment could control the phosphorylation of Spc105 only
when Mps1 was localized in the outer kinetochore, proximal to the microtubule-binding domain of
Ndc80. Mps1 localized in the inner kinetochore constitutively activated the SAC, irrespective of the
attachment state. This suggested that Mps1 must bind to the outer kinetochore to enable attachmentdependent SAC signaling. An engineered sensor for Mps1 activity revealed that this was the case. These
observations, when combined with structural and biochemical properties of Ndc80 and Spc105
suggested a simple toggle-switch like mechanism that controls the SAC: In unattached kinetochores, the
WEDNESDAY-ORAL PRESENTATIONS
microtubule-binding domain of Ndc80 and the phosphodomain of Spc105 are in close proximity. This
allows Mps1 bound to Ndc80 to phosphorylate Spc105 and turn the SAC on. Microtubule attachment to
the kinetochore separates the two domains, and prevents phosphorylation of Spc105 to turn the SAC
off. FRET between the microtubule-binding domain of Ndc80 and the phosphodomain of Spc105
confirmed that the two domains are in close proximity (less than 10 nm) in unattached kinetochores,
and are separated by microtubule attachment. We further demonstrated the working of the toggleswitch by artificially reducing the separation between its terminals: moving the phosphodomain of
Spc105 close to the microtubule binding domain of Ndc80 activated the SAC irrespective of the
attachment state of the kinetochore. Our results demonstrate how a specific change in the architecture
of the kinetochore disrupts SAC signaling.
M193
Force on spindle microtubule minus ends moves chromosomes.
M.W. Elting*1, C. Hueschen*1, D. Udy1, S. Dumont1;
1
University of California, San Francisco, San Francisco, CA
*These authors contributed equally to this work.
The spindle is a dynamic self-assembling machine that coordinates mitosis. The spindle’s function
depends on its ability to organize microtubules into poles and maintain pole structure despite
mechanical challenges and component turnover. Although we know that dynein and NuMA mediate
pole formation, our understanding of the forces dynamically maintaining poles is limited: we do not
know where and how quickly they act or their strength and structural impact. Using laser ablation to cut
spindle microtubules, we identify a force that rapidly and robustly pulls severed microtubules and
attached chromosomes poleward, overpowering opposing forces and repairing spindle architecture.
Molecular imaging and biophysical analysis suggest that transport is powered by dynein pulling on
minus ends of severed microtubules. NuMA and dynein/dynactin are specifically enriched at new minus
ends within seconds, re-anchoring minus ends to the spindle and delivering them to poles. This force on
minus ends represents a newly-uncovered chromosome transport mechanism that is independent of
plus end forces at kinetochores and is well-suited to robustly maintain spindle mechanical integrity.
WEDNESDAY-ORAL PRESENTATIONS
M194
Adaptive changes in the kinetochore architecture suppress erroneous
attachments and accelerate spindle assembly.
R. Paul1, V. Magidson2, N. Yang2, J. Ault2, C. O’Connell2, B. McEwen2, A. Khodjakov2, A. Mogilner3;
1
Indian Association for Promotion of Science, Kolkata, India, 2Wadsworth Center, Albany, NY, 3Courant
Inst and Dept of Biology, New York University, New York, NY
Assembly of the mitotic spindle is driven by the stochastic capture of microtubules at the kinetochores.
Both the efficiency and fidelity of spindle assembly are greatly affected by spatial constraints and
specifically by the size and shape of the kinetochore. Large kinetochores are expected to accelerate
spindle assembly but they would also increase the number of erroneous captures connecting a single
kinetochore to both spindle poles (merotelic) or both sister kinetochores to the same spindle pole
(syntelic). Conversely, smaller kinetochores would suppress erroneous attachments but make
microtubule capture inefficient. Minimalistic computational models in fact predict an inversed relation
between the conditions that promote efficiency vs. fidelity of microtubule capture. Noteworthy is that
thus far no successful simulation of fast yet low-error spindle assembly has been produced. Seeking to
gain additional insights into the role of geometric constraints in kinetochore/microtubule interactions
we characterized the architecture of centromere in human cells by 3-D light microscopy as well as by
correlative light/electron microscopy. Our analyses reveal that the kinetochore size and shape change
dramatically during spindle assembly. Upon mitotic entry, kinetochores expand into large crescents that
subsequently compact into discrete objects on opposite sides of the centromere only upon the
formation of end-on attachments. Kinetochores that laterally interact with microtubules but have not
formed end-on attachments remain enlarged. From the standpoint of classic search-and-capture these
changes are counterproductive, as they would promote erroneous attachments. However, a newlydeveloped computational model that accounts for partial rotation of centromeres during the stage of
lateral interactions makes a surprising prediction that enlargement of kinetochores at the onset of
spindle assembly actually reduces both the time of spindle assembly and attachment errors. The
simultaneous improvement of both efficiency and fidelity of microtubule attachment is a result of
restricted angular orientation of centromeres that is achieved during lateral interactions. Importantly,
such a ‘pre-orientation’ of centromeres by lateral interactions with microtubules has been observed
during normal spindle assembly in human cells (Magidson et al., 2011, 146:555-567). Our simulations,
for the first time, explain how the spindle assembles in
WEDNESDAY-ORAL PRESENTATIONS
M195
Mechanics of the acto-myosin cortex during cytokinesis.
R. Khaliullin1, L. Shi2, M. Berns2, A. Desai3, K. Oegema3;
1
Department of Cellular and Molecular Medicine, Ludwig Institute for Cancer Research, La Jolla, CA,
2
University of California, San Diego, La Jolla, CA, 3Ludwig Institute for Cancer Research, La Jolla, CA
Cytokinesis is the process that completes mitosis, physically partitioning the contents of a single cell into
the two daughter cells. Cytokinesis is driven by the mechanical constriction of a cortical contractile ring
that assembles around the cell equator. The contractile ring is a part of the larger cell cortex, an elastic
actomyosin network that underlies the cell membrane. An important question in cytokinesis is where
cortical surface is added, how the contractile ring and cortex interact, and whether the rate of ring
ingression is affected by cortical remodeling. Here, we investigate these questions using the C. elegans
embryo as a model system. During the first embryonic division, cortical surface area increases by
roughly 30% as the contractile ring constricts asymmetrically within the division plane (ring center
moves along an axis from one side of the division plane to the other as the ring constricts). To determine
whether this surface area increase is uniform versus spatially restricted (such as behind the furrow or at
the poles), we generated a map of cortical flow by imaging a strain expressing a GFP fusion with the
cortical component myosin II. Data collected in ~100 embryos were computationally combined to
generate a 360° map of cortical flow. This analysis revealed that the cortex in the central 60% of the
embryo flows at a constant velocity towards the division plane like a carpet being dragged across a floor.
Rather than occurring uniformly, cortical surface increase occurs primarily at the cell poles. Laser
ablation experiments revealed that cutting the cortex does not accelerate the rate of ring closure,
indicating that cortical surface increase follows ring closure rather than limiting it. In addition, inhibition
of ARP2/3, which is predicted to weaken the cortex by preventing the assembly of branched actin
filaments, did not alter ring closure dynamics. An analysis of cortical flux revealed that the total amount
of surface area flowing across the boundary into the division plane was greater than that required,
suggesting that there is significant cortical compression within the division plane. To predict whether
the ring constricts asymmetrically or symmetrically and the pattern of cortical compression, we
developed a finite element model. Based on the experimentally measured flow pattern, this model
predicts uniform shrinkage of the contractile ring around its circumference and asymmetric cortical
compression near the ring that reflects the distribution of myosin II.
M196
Profiling of the mammalian mitotic spindle proteome reveals a transmembrane
ER protein, OSTD-1, as being necessary for cell division and ER morphology.
M. Bonner1, B. Han1, A.R. Skop1;
1
Genetics, University Wisconsin-Madison, Madison, WI
Cell division is important for many cellular processes including cell growth, reproduction, wound healing
and stem cell renewal. Failures in cell division can often lead to tumors and birth defects. To identify
WEDNESDAY-ORAL PRESENTATIONS
factors necessary for this process, we implemented a comparative profiling strategy of the published
mitotic spindle proteome from our laboratory. Of the candidate mammalian proteins, we determined
that 77% had orthologs in C. elegans and 18% were associated with human disease. Of the C. elegans
candidates, we determined that 34 genes functioned in embryonic development and 56% of these were
predicted to be membrane trafficking proteins. A secondary, visual screen to detect distinct defects in
cell division revealed 21 genes that were necessary for cell division. One of these candidates, OSTD-1, an
transmembrane ER resident protein, was further characterized. We determined that OSTD-1 plays a role
in maintaining the dynamic morphology of the ER during the cell cycle. In addition, 65% of all ostd-1
RNAi-treated embryos failed to correctly position cleavage furrows, suggesting that proper ER
morphology plays a necessary function during animal cell division.
M197
How the kinetochore harnesses microtubule force and centromere stretch to
move chromosomes revealed by a FRET tension sensor within Ndc80 protein.
A. Suzuki1, B.L. Badger1, J. Haase1, T. Ohashi2, H.P. Erickson2, E.D. Salmon1, K.S. Bloom1;
1
Biology, University of North Carolina, Chapel Hill, NC, 2Department of Cell Biology, Duke University
Medical Center, Durham, NC
The Ndc80 complex (Ndc80, Nuf2, Spc24, Spc25) is a highly conserved kinetochore protein essential for
end-on anchorage of kinetochore microtubule (kMT) plus-ends and for force generation coupled to
attached plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex
binds the kinetochore. The N-terminal tail and CH domains of Ndc80 at the other end bind microtubules
(MTs). An internal loop domain of Ndc80 may be linked to microtubule-associated proteins (MAPs) such
as the Dam1 complex. To determine how the MT and MAP binding domains of Ndc80 contribute to
force production at kinetochores in budding yeast, we have inserted a characterized FRET tension sensor
into Ndc80 protein about halfway between its CH and loop domains. During the cell cycle, the FRET
sensor reported low tension from late anaphase though interphase and high tension at metaphase,
when pericentromeric chromatin is maximally stretched between sister kinetochores. In addition, we
found that both Stu2, which concentrates at MT plus ends during polymerization, and tension reported
by the FRET sensor fluctuated over time indicating that tension at the MT binding domains of Ndc80 is
different for the polymerization and depolymerization phases of kMT dynamic instability. Surprisingly,
FRET sensor measurements showed that reducing MT dynamicity with low dose benomyl at metaphase
caused a major drop in tension at the MT binding domains of Ndc80 without loss of normal centromere
stretch. In addition we found that tension at the MT binding domains of Ndc80 is abnormally low with a
Dam1 mutation (DAM1-765, Shimogawa et al., 2006) that enhances Dam1 complex affinity for MTs and
produces at metaphase hyper-centromere stretch. The above data suggest that Dam1 complex bound to
Ndc80 complex at a site inside the FRET tension sensor has a dominant role, compared to the MT
binding domains of Ndc80, for attachment to kMT plus ends at metaphase. Based on the above in vivo
studies and in silico simulations, we propose a mechanical model for the Ndc80 force coupler at budding
yeast kinetochores. During depolymerization, pushing forces from peeling protofilaments against the
WEDNESDAY-ORAL PRESENTATIONS
Dam1 complex pulls on the Ndc80 complex to stretch the centromere and drags the MT binding
domains of Dam1 and Ndc80 poleward along their kMT at the velocity of depolymerization until a switch
occurs to polymerization. Then, during polymerization, pulling force from centromere stretch on the
Ndc80 complex drags the MT binding domains of Dam1 and Ndc80 complexes away from the pole along
their kMTs until a switch occurs to depolymerization.
M198
Remodeling of cell-cell junctions during cytokinesis.
T. Higashi1, T.R. Arnold1, R.E. Stephenson1, A.L. Miller1;
1
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI
Cell-cell junctions are required for maintenance of mechanical integrity and barrier function in epithelial
tissues. During cytokinesis, the dividing cell and its neighbors undergo major changes in shape and
tension as the contractile ring pinches the cell in two. Junctional integrity and barrier function are
thought to be preserved even during cell division. However, our understanding of how junctions are
maintained and remodeled during cytokinesis in epithelial cells is lacking. In this study, we investigated
the behaviors of three types of cell-cell junctions, tight junctions (TJs), adherens junctions (AJs), and
tricellular tight junctions (tTJs), during cytokinesis in the Xenopus gastrula by immunofluorescence
microscopy and live-imaging of fluorescently-tagged junction proteins. Using a small fluorescent tracer
molecule, we demonstrated that the barrier function of TJs remained intact throughout cell division. We
found that both TJs and AJs invaginated together with the cleavage furrow; this is in contrast to reports
in the Drosophila epithelium indicating that AJs are locally disengaged at the division site. Notably, the
invagination of AJs preceded that of TJs. Furthermore, we observed that two nascent tTJs were formed
at the end of cytokinesis, one on each side of the midbody. LSR/angulin-1, a protein unique to tTJs, was
recruited to the newly formed tTJ first and was quickly followed by a second tTJ component, tricellulin.
Our data provide the first in-depth characterization of dynamic cell-cell junction remodeling during
vertebrate cytokinesis and show for the first time how nascent tTJs are formed following cell division.
M199
Coordinated chromosome movement in the absence of physical connection in
spider spermatocytes.
A.O. Nedo1, C.M. Andreychik1, R.N. Doan1, L. Jamrog1, L.V. Paliulis1;
1
Biology Department, Bucknell University, Lewisburg, PA
In cell division, physical connection guarantees that movement of partner chromosomes is coordinated;
i.e. connected sister chromatids move together toward their associated spindle pole in anaphase I of
meiosis. Interestingly, there are some cell types in which unconnected chromosomes always move
together, allowing study of how chromosomes communicate their position in the cell in the absence of
physical connection. We use spiders with the X1X1X2X2 (female)/X1X20 (male) sex determination system
WEDNESDAY-ORAL PRESENTATIONS
to study coordinated chromosome movements. In males the nonhomologous X1 and X2 chromosomes
always associate with the same spindle pole from prometaphase I through anaphase I. After seeing a
visible gap between X1 and X2, we hypothesized that the gap was reflective of a lack of connection
between X1 and X2. We found that X1 and X2 are easily separable using a micromanipulation needle in
metaphase I, showing that they move together in the absence of physical connection. When we used a
micromanipulation needle to separate X1 and X2 such that they associated with opposite poles, we
found that cells could progress into anaphase. This showed that cosegregation of X1 and X2 is not
monitored by the cell following initial chromosome attachment in prometaphase I, which would
seemingly lead to error-prone segregation of sex chromosomes in male meiosis. We found that
consistently correct segregation of X1 and X2 occurs because X1 and X2 appear to associate with a spindle
pole while in the nuclear envelope and, under our observation, never reorient during meiosis I,
suggesting that continuous monitoring of the position of X1 and X2 is not necessary in normal meiosis I.
Spindle breakdown and reassembly during prometaphase I could, however, lead to sex chromosome
aneuploidy.
M200
The equatorial position of the metaphase plate ensures symmetric cell divisions.
P. Meraldi1, C.H. Tan1, S. Huber Reggi2, I. Gasic1, M. Barisic3, H. Maiato3,4;
1
Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland, 2University of Zürich,
Zurich, Switzerland, 3Chromosome Instability Dynamics Laboratory, Instituto de Biologia Molecular e
Celular, Universidade do Porto, Porto, Portugal, 4Cell Division Unit, Department of Experimental Biology,
Faculdade de Medicina, Universidade do Porto, Porto, Portugal
Chromosome alignment in the middle of the bipolar spindle is a hallmark of metazoan cell division.
When we force human cells to build asymmetric spindles by creating an asymmetric distribution of
centrioles numbers at each spindle pole, we find that they always re-center the position of the
metaphase plate before anaphase onset. This plate centering mechanism is made possible by delaying
satisfaction of the spindle assembly checkpoint, to provide cells enough time to correct metaphase plate
position before anaphase onset. Cells with only one centriole at each pole do not elicit a checkpoint
response, indicating that the spindle assembly checkpoint response depends on the asymmetry of
centriole distribution, and not on the change in centriole numbers per se. The checkpoint response in
cells with an asymmetric centriole distribution is not elicited by unattached or tension free kinetochores,
but by minor defects in the maturation of kinetochore-microtubule attachments, which arise as a
consequence of an imbalance in microtubule stability between the two half-spindles. Stabilizing these
attachments by depleting the microtubule depolymerases KIF2a and MCAK satisfies the checkpoint, and
results in anaphase entry with asymmetric spindles that lead to asymmetric cell divisions. We thus
postulate that the symmetric metaphase plate position plays in essential role for the control of cell
division symmetry.
WEDNESDAY-ORAL PRESENTATIONS
Minisymposium 22: Mechanotransduction of Disease
M201
A Drosophila model of cerebral cavernous malformations.
Y. Song1, A. Ghabrial1;
1
Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA
As many as 1 in 200 individuals in the general population will suffer from cerebral cavernous
malformations – gross dilations of capillary tubes in the brain. These vascular lesions may be
asymptomatic, cause headaches, or result in stroke. Familial forms of the disease arise from mutation in
one of three genes (CCM1-3) [1]. Of these, only CCM3 is conserved from flies to human. Although CCM3
is known to bind the Germinal Center Kinase III family (3 in human) of STE20Ks, the consequences of this
interaction are unclear, and the cell biological processes regulated by CCM3/GCKIII remain controversial
[2-4]. We have generated dCCM3 knockout flies (unpublished), and find that they confer a tube dilation
defect in the larval tracheal system. Likewise, we showed that mutations in the sole Drosophila GCKIII
result in an identical tracheal tube dilation [5]. As true for human disease, only the smallest vessels of
the organ system are susceptible to dilation – in flies, the intracellular tubes produced by terminal cells.
We have identified another gene required to prevent tube dilation (MO25, unpublished), and have also
identified a number of mutations that suppress tube dilation, including mutations in Crumbs, Varicose
[5]and Rab11 (unpublished). We present a model in which MO25 activates GCKIII, and CCM3 stabilizes
it. Together, these proteins regulate Rab11-dependent recycling to the apical membrane domain, such
that Rab11 levels and subcellular distribution maintain an appropriately sized and shaped apical
membrane domain.
1. Revencu, N., and Vikkula, M. (2006). Cerebral cavernous malformation: new molecular and clinical
insights. J Med Genet 43, 716-721.
2. Louvi, A., Chen, L., Two, A.M., Zhang, H., Min, W., and Gunel, M. (2011). Loss of cerebral cavernous
malformation 3 (Ccm3) in neuroglia leads to CCM and vascular pathology. Proc Natl Acad Sci U S A 108,
3737-3742.
3. Zheng, X., Xu, C., Di Lorenzo, A., Kleaveland, B., Zou, Z., Seiler, C., Chen, M., Cheng, L., Xiao, J., He, J., et
al. (2010). CCM3 signaling through sterile 20-like kinases plays an essential role during zebrafish
cardiovascular development and cerebral cavernous malformations. J Clin Invest 120, 2795-2804.
4. He, Y., Zhang, H., Yu, L., Gunel, M., Boggon, T.J., Chen, H., and Min, W. (2010). Stabilization of VEGFR2
signaling by cerebral cavernous malformation 3 is critical for vascular development. Sci Signal 3, ra26.
5. Song, Y., Eng, M., and Ghabrial, A.S. (2013). Focal defects in single-celled tubes mutant for Cerebral
cavernous malformation 3, GCKIII, or NSF2. Dev Cell 25, 507-519.
WEDNESDAY-ORAL PRESENTATIONS
M202
Joined forces of cancer cells and fibroblasts against the basement membrane.
A. Glentis1, V. Gurchenkov1, M. Schoumacher1, F. Zaccarini2, P. Mariani3, D. Vignjevic1;
1
Institut Curie, Paris, France, 2AP-HP Hopitaux de Paris, Paris, France, 3Hopital Curie, Paris, France
In carcinoma in situ, the basement membrane represents a physical barrier that prevents spreading of
primary tumor to adjacent tissues. It is believed that cancer cells perforate basement membranes.
However, stromal cells such as carcinoma-associated fibroblasts also secrete matrix proteinases.
Therefore, the question is who is invading whom – do cancer cells invade stroma or possibly stroma is
invading tumor cells? Using human colon cancer cells and primary human fibroblasts isolated from
tumors and adjacent normal tissues, we addressed if cancer cells and fibroblasts are invading the
basement membrane simultaneously or they work together but have distinct functions. Analyzing
human colon cancer samples, we observed that invasive tumors contain higher amount of fibroblasts in
general and higher proportion of CAFs (αSMA+) compared to non-invasive tumors or healthy tissues. We
isolated fibroblasts from fresh human colon tumors of different stages (CAFs) and from the adjacent
normal tissues (NAFs). A combination of markers was used to discriminate fibroblasts from other cell
types to validate the purity of isolated cells and to discriminate NAFs from CAFs. In co-culture
experiments on Matrigel-coated transfilters, both NAFs and CAFs induced migration and invasion of
HT29, intrinsically non-invasive colon cancer cells. On contrary, in an assay containing native, mesenteric
basement membrane that separates cancer cells on one side and fibroblasts embedded in collagen I on
the other, we found that only CAFs are able to stimulate invasion of cancer cells. CAFs stimulated
invasion of cancer cells when physically present in the assay and, in a lesser amount, via paracrine ways.
Proteomic study using SILAC, showed that CAFs secrete more proteases, extracellular matrix proteins,
and proteins that modify the matrix compared to NAFs, pointing to a matrix-remodeling role in invasion.
Live cell imaging showed that fibroblasts and cancer cells are communicating through the basement
membrane via cellular protrusions long time before the actual translocation of cancer cells is detected.
We are currently testing a role of CAF-derived molecules in basement membrane remodeling in order to
dissect the interplay between cancer cells and CAFs in basement membrane invasion.
M203
Tumor genotype dictates the mechanophenotype and fibrotic behavior of
pancreatic ductal carcinoma.
H. Laklai1, Y.A. Miroshnikova1, M. Pickup1, J. Lakins2, S. Novitskiy3, R. Kalluri4, H.L. Moses3, V.M. Weaver2;
1
University of California, San Francisco, San Francisco, CA, 2Center for Tissue Engineering and
Regenerative Medicine, University of California, San Francisco, San Francisco, CA, 3Vanderbilt University
School of Medicine, Nashville, TN, 4MD Anderson Cancer Center, Houston, United States
Pancreatic ductal adenocarcinoma (PDAC) is amongst the most lethal malignancies due largely to an
inability to detect these tumors early and a lack of effective therapies. PDAC is characterized by a strong
desmoplastic response including elevated extracellular matrix (ECM) deposition and remodeling that
WEDNESDAY-ORAL PRESENTATIONS
severely compromises treatment and surgical resection. Nevertheless, the significance of tissue fibrosis
to pancreatic transformation and aggression remain unclear. Tissue fibrosis increases tissue tension and
we showed that ECM stiffness and epithelial cell tension drive squamous carcinoma and mammary
transformation. We therefore asked whether pancreatic fibrosis could promote PDAC progression and
aggression by stiffening the ECM and increasing tissue tension and how. Using two standard PDAC
mouse models which incorporate the KrasG12D mutation along with either loss of the TGFb receptor 2
(PKT) or expression of a P53 mutation (KPC), we showed that PDAC progression is accompanied by
significant fibrosis and inflammation that correlate strongly with collagen deposition and LOXdependent cross-linking and ECM stiffening. Interestingly, PDAC progression in the PKT mice associated
significantly with activated myosin, FAK and YAP and enhanced contractility in the pancreatic
epithelium, whereas KPC mechanosignaling and epithelial contractility was much less pronounced and in
fact was markedly similar to that observed in mice expressing the KrasG12D transgene alone in vivo or in
isolated cells from the KrasG12D mouse. Intriguingly, we showed that PKT contractility was functionally
linked to tissue inflammation through a reciprocal Cxcr2 receptor - GPCR- JAK-STAT3 - ROCK - FAK
signaling circuit and our data suggested that this circuit contributes substantially to PDAC aggression.
Indeed, we observed that the pancreas of mice expressing a targeted β1 integrin V737lox/lox clustering
mutant, which drives integrin-dependent mechanosignaling in their epithelium developed a profound
fibrosis and chronic inflammation and when combined with the KrasG12D oncogene the mice developed
a profound pancreatitis within 3 months. These data identify a vicious positive feedback mechanism,
mediated through tissue tension, whereby tissue fibrosis promotes pancreatic tumor progression by
enhancing Stat3-dependent chemokine expression and tissue inflammation, at least in the PKT mouse
model. Ongoing studies now aim to explore the efficacy of anti JAK-Stat inhibitors as viable treatment
for TGFb mutant pancreatic cancer subtypes. Whether the molecular mechanisms regulating tissue
pancreatic tumor fibrosis and mechanophenotype are in fact specified by the specific tumor genotype
and if this in turn dictates the choice of therapeutic strategy is now being investigated.
M204
Pharmacological activation of myosin II to correct pancreatic cancer cell
mechanics.
A. Surcel1, Q. Zhu2, E.S. Schiffhauer1, H. West-Foyle1, R.A. Anders2, D. Robinson1,3;
1
Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, 2Department
of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, 3Department of
Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD
Pancreatic ductal adenocarcinoma (PDAC) annually affects 44,000 people in the U.S. and has an abysmal
five-year survival rate of around 6%, which is nearly unchanged over the past 40 years. Pharmacological
strategies for treating cancer have primarily focused on inhibiting cell growth through specific genetic
pathways, which typically either fail to abolish the disease or lead to compensatory regulatory changes
and subsequently, to drug resistance. Importantly, alterations in mechanical properties are a common
WEDNESDAY-ORAL PRESENTATIONS
feature of cancer cells, yet targeting cell mechanics remains an under-utilized approach for drug
development.
Here we develop a system for targeting cell mechanics for the discovery of novel therapeutics. We
designed a live-cell, high-throughput chemical screen to identify mechanical modulators in Dictyostelium
discoideum. We characterized 4-hydroxyacetophenone (4-HAP), which increases the cellular cortical
tension by enhancing the cortical localization of the mechanoenzyme myosin II, independent of myosin
heavy-chain phosphorylation regulation. To shift cell mechanics, 4-HAP requires myosin II, including its
full power stroke. We further establish that changes in key cytoskeletal protein distributions correlate
with the changes in the biomechanical profile of PDAC progression. In addition to actin-crosslinkers, we
detect that non-muscle myosin II distributions vary across PDAC states: specifically myosin IIA increases,
myosin IIB decreases, and myosin IIC increases in metastatic cells. We further demonstrate that invasive
pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical
profile that was partially corrected with 4-HAP. Tests of 4-HAP in mouse models of metastatic
pancreatic disease are underway. Overall, 4-HAP modifies non-muscle myosin II-based cell mechanics
across phyla and disease states and provides proof-of-concept that cell mechanics offer a rich drug
target space, allowing for possible corrective modulation of tumor cell behavior.
M205
Mechanical induction of the oncogenic Beta-catenin pathway by tumour growth
pressure.
S. Barbier1, M. Fernández Sánchez1, J. Whitehead1, G. Bealle2, A. Michel2, H. Latorre-Ossa3, C. Rey4, L.
Fouassier4, A. Clapéron4, L. Brullé1, E. Girard1, N. Servant1, T. Rio-Frio1, H. Marie5, S. Lesieur5, C. Housset4,
J. Genisson3, M. Tanter3, C. Menager2, S. Fre1, S. Robine1, E. Farge1;
1
Institut Curie, Paris, France, 2Laboratoire PHENIX Physico-chimie des Electrolytes et Nanosystèmes
InterfaciauX, UPMC Univ Paris 06, Sorbonne Universités, Paris, France, 3ESPCI ParisTech, CNRS
UMR7587, Inserm U979, Paris, France, 4INSERM UMRS 938, Paris, France, 5CNRS UMR 861, Paris, France
The tumour microenvironment, in terms of fibrotic stiffness or mechanical pressure developed by
mitotic cells confinement, is known to influence tumour progression. Conversely, the oncogenic role of
the mechanical pressure developed by tumour growth applied to the non-tumorous environing
epithelial cells has been unexplored. Previously, we have found ex-vivo that pressure, potentially
associated to intestinal transit or tumour growth, triggers the activation of the primary oncogen
program in genetically predisposed pretumoral APC+/- mice colon tissues. Here we show that in the
early stage of mouse colon tumour development, tumour growth pressure activates the oncogenic
mechanosensitive Beta-catenin pathway in the mechanically stressed non-tumorous epithelial cells in
vivo. We have developed an innovative method allowing stable magnetization of deep tissues
(colorectal tissues) on the weeks to months time scale, by intra-venous injection of ultra-magnetic
vesicles, in the presence of a strong magnetic field gradient due to a small intense magnet positioned
dorsally under-skin close to the colon. Magnetic pressure quantitatively mimicked in vivo the
endogenous early tumour growth stress on the order of 1200 Pa with no stiffness modification, as
WEDNESDAY-ORAL PRESENTATIONS
monitored by acoustic imaging in situ. Mimicking tumour growth pressure in vivo led to the
phosphorylation of Ret kinase in the healthy epithelium followed by phosphorylation of -catenin at
tyrosine 654, known to impair its interaction with the E-cadherin in adherens junctions, increased
nuclear translocation of Beta-catenin, and expression of the myc, axin-2 and zeb-1 target genes. This
oncogenic mechanosensitive Beta-catenin pathway was revealed to be induced both in predisposed
APC+/- genetic background (after one month) and in wild type (after 2 months). Mechanical activation
of the oncogenic Beta-catenin pathway suggests new modes of tumour propagation based on
mechanical induction of oncogenic pathways in healthy epithelial cells by tumour growth pressure.
M206
Preclinical intravital microscopy of cancer cell invasion, metastasis and therapy
response.
P. Friedl1,2;
1
Cell Biology, Radboud Univ Nijmegen-Nijmegen Ctr Molec Life Scis, Nijmegen, Netherlands, 2GU
Medical Oncology, The University of Texas, MD Anderson Cancer Center, Houston, TX
The tumor microenvironment supports both cancer cell invasion and growth/survival programs, with
impact on response to therapy and prognosis. Using intravital near-infrared/infrared multiphoton
microscopy, we have established sarcoma and melanoma models for spontaneous collective cancer cell
invasion and distant metastasis to lymph nodes and lungs. Interstitial dissemination away from the
primary lesion occurred along blood vessels, myofibers, nerves, adipocytes and collagen bundles, as
guided migration along preformed multi-interface conduits of 1D, 2D or confined 3D geometry.
Molecular targeting of β1 and β3 integrin-mediated mechanotransduction resulted in anoikis induction
in the primary tumor, but intact local invasion, collective-to-amoeboid transition and enhanced distant
metastasis. Thus lowering integrin-dependent mechanocoupling induces plasticity of migration
programs and release of cancer cells from the primary site. Once populated by cancer cells, this invasion
niche enabled tumor cell survival and relapse of disease even after high-dose hypofractionated
radiotherapy, but was sensitive to combined anti-β1/β3 integrin and radiation therapy. This establishes
tumor-stroma interaction niches of overlapping invasion and resistance programs which can be
exploited by combining conventional with with integrin-targeted therapy.
WEDNESDAY-ORAL PRESENTATIONS
M207
Septins enhance the mesenchymal-like motility of renal epithelia by promoting
stress fiber connectivity and focal adhesion maturation.
L. Dolat1, J.L. Hunyara1, J.R. Bowen1, E.P. Karasmanis1, V. Galkin2, E.T. Spiliotis1;
1
Biology, Drexel University, Philadelphia, PA, 2Physiological Sciences, Eastern Virginia Medical School,
Norfolk, VA
Epithelial-mesenchymal transition (EMT) underlies renal fibrosis and the metastasis of renal cell
carcinomas (RCCs). EMT is characterized by disruption of cell-cell adhesions, cell scattering and the
enhancement of cell motility with a front-rear polarity. While the disruption of cell-cell adhesion during
EMT is extensively studied, little is known about the molecules and mechanisms that enhance the
mesenchymal-like motility of epithelial cells. Septins are filamentous G proteins that are over-expressed
in metastatic RCCs, but their functions in the mesenchymal-like motility of epithelial cells is unknown.
Using the Madin-Darby canine kidney (MDCK) model of partial EMT that allows the study of epithelial
motility in 2D and 3D matrices after stimulation with hepatocyte growth factor (HGF), we discovered a
novel network of septin filaments that underlies the organization of the transverse arc and radial
(dorsal) stress fibers in the leading lamella of migrating epithelia. We show that this lamellar network of
septin filaments is required for the maturation of nascent focal adhesions as septin depletion results in
smaller and more transient and peripheral focal adhesions with increased levels of phosphorylated
paxillin-Y118. In addition, septin-depleted cells consisted of a disorganized transverse arc and shorter
and fewer radial stress fibers. These phenotypes were independently rescued by alpha-actinin and the
actin-binding motor-dead myosin II N93K, but not by alpha-actinin-deltaABD, which cannot bind or
bundle actin, or the constitutively active myosin regulatory light chain RLC-DD. These data suggested
that septins function as actin-binding and cross-linking proteins. Using low speed sedimentation assays
and negative stain EM, we show that pre-assembled actin filaments are bundled by septin 9 (SEPT9),
whose expression is increased after induction of renal epithelial motility with the hepatocyte growth
factor. We show that SEPT9 over-expression enhances renal cell migration in 2D and 3D matrices, while
SEPT9 knock-down decreases the velocity and alters the mesenchymal-like shape of epithelial cells
migrating in 3D matrices. Taken together, these results suggest that septins promote epithelial motility
by reinforcing the connectivity of lamellar stress fibers and thereby, stabilizing nascent focal adhesions
in the leading edge of motile epithelia.
WEDNESDAY-ORAL PRESENTATIONS
M208
N-WASP/WIP mediated matrix adhesion site maturation drives 3D cancer cell
migration through direct force coupling to the nucleus.
T. Zech1, O. Chatzidoukaki1,2, A. Charles-Orszag2, S. Sender2, L. Machesky2;
1
Institute of Translational Biology, Dept of Cellular and Molecular Physiology, University of Liverpool,
Liverpool, England, 2CRUK Beatson Inst Cancer Res, Glasgow, Scotland
The actin nucleation promotion factor N-WASP is up-regulated in breast cancer and is coupling
pseudopod extension and matrix degradation to facilitate invasive cancer cell migration. Cells migrating
through 3D matrices form hybrid adhesion structures termed “actin-hotspots”, which contain N-WASP
and display hallmarks of both focal adhesions and invadopodia. We have identified a novel interaction
between the N-WASP Interacting Protein, WIP, and the guanine nucleotide exchange factor
ARHGEF7/bPIX. ARHGEF7 localises to actin-hotspots in 3D matrices. Loss of ARHGEF7 abolished cancer
cell invasion, but increased matrix degradation and pseudopod extension in collagen matrices. These
seemingly contradictory results can be explained by our finding that ARHGEF7 is part of a NWASP/WIP↔ARHGEF7↔PAK2 signalling cascade that is required for adhesion site turnover and force
coupling. Loss of ARHGEF7 leads to less tension being applied by actin on adhesion sites as well as the
nuclear envelope, resulting in a loss of nuclear movement and subsequently cell motility. The nucleus
can act as limiting factor in 3D cell migration. Migrating cells need to actively squeeze the nucleus
through matrix pores. Knockdown of nuclear envelope actin binding proteins called Nesprin-1 and -2
severely affect 3D cell migration. We have found a reciprocal regulation of Nesprin-2 and ARHGEF7
function. We here show, using a novel nuclear membrane FRET/FLIM force biosensor, that direct force
coupling from actin-hotspots to the nuclear membrane is required for 3D cell migration and we propose
that this could be the mechanism to establish polarity during 3D cell migration.
M209
Macrophage-dependent activation of a non-canonical NOTCH-RhoA signaling
pathway regulates tumor cell intravasation.
J.J. Bravo-Cordero1, M. Roh-Johnson1, J. Pignatelli1, L. Hodgson1, J.S. Condeelis1;
1
Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY
The tumor microenvironment plays an important role during tumor progression and metastasis.
Immune cells have been shown to play a pro-tumorigenic role during several steps of the metastatic
cascade. In particular, tumor-associated macrophages facilitate tumor cell invasion and intravasation in
vitro and in vivo. Recently, our group has shown that heterotypic cell contact between tumor cells and
macrophages induce the formation of invadopodia in tumor cells, invasive structures necessary for
matrix degradation and tumor cell intravasation. Using high resolution FRET imaging, we further found
that macrophage-induced invadopodium formation is dependent on RhoA activation. However, what
remained to be determined was the signaling pathway that regulated this heterotypic cell contact-
WEDNESDAY-ORAL PRESENTATIONS
mediated phenomenon. NOTCH signaling is known to be involved in homotypic cell-cell communication,
and has recently been shown to be involved in invadopodium formation (Diaz et al., 2013). Thus, we
explored the role of NOTCH in mediating macrophage-dependent tumor cell invadopodium formation.
We found that upon NOTCH depletion the number of invadopodia in tumor cells remains unchanged.
But surprisingly, in the absence of Notch signaling, macrophage-induced invadopodium formation and
tumor cell intravasation are abolished. Moreover, RhoA is no longer activated in tumor cells upon
macrophage contact when NOTCH signaling is perturbed. These results suggest that NOTCH signaling
regulates heterotypic cell contact mediated invadopodium formation through RhoA activation, and
reveals a novel mechanism for both invadopodium formation and the NOTCH signaling pathway.
Minisymposium 23: Nuclear Architecture and Dynamics
M210
Regulated targeting of genes to the nuclear pore complex promotes
interchromosomal clustering and regulates transcription and chromatin
structure.
D.G. Brickner1, C. Randise-Hinchliff1, D. Egecioglu1, J. Brickner1;
1
Molecular Biosciences, Northwestern University, Evanston, IL
The interaction of chromatin with nuclear pore proteins (Nups) is a conserved phenomenon that has
important effects on transcriptional regulation. In metazoa, Nups interact with genes both at the
nuclear pore complex (NPC) and in the nucleoplasm. In yeast, these interactions occur exclusively at the
NPC. We have identified a number of transcription factors that bind to "DNA zip codes" in the
promoters of these genes that are necessary and sufficient to promote interaction with the NPC. This
function is, in some cases, separable from their more canonical roles is regulating transcription. In
addition to promoting interaction with the NPC, these transcription factors also cause interchromosomal
clustering of genes to which they bind. Most genes interact with the NPC in a regulated fashion that
correlates with transcriptional activation. We find that there are (at least) three different mechanisms
by which recruitment to the NPC is regulated: 1) repression of nearby zip codes by transcriptional
repressors, 2) conditional production of transcription factors that bind to zip codes and 3) signaldependent DNA binding by transcription factors. Targeting to the NPC can also occur after repression, a
phenomenon called transcriptional memory. Memory is regulated by distinct mechanisms and has
distinct outputs: altered chromatin structure and poised RNA polymerase II.
M211
Functional role of chromosome refolding during mating type switching in yeast.
B. Avsaroglu1, K. Li1, G. Bronk1, J.E. Haber2, J. Kondev1;
1
Department of Physics, Brandeis University, Waltham, MA, 2Department of Biology, Rosenstiel Basic
WEDNESDAY-ORAL PRESENTATIONS
Medical Sciences Research Center, Brandeis University, Waltham, MA
Haploid yeast, Saccharomyces cerevisiae, cells can switch their mating type by replacing one MAT allele
with the DNA sequence of the opposite mating type, copied from a distant locus. Mating type switching
in yeast is a highly choreographed repair event in which a DNA double strand break (DSB) created at the
MAT locus is repaired by homologous recombination using one of the two silent donors, HMLα or
HMRa, located near the two ends on the same chromosome. MATa cells recombine with HML 90% of
the time, and this preference to use HML is dependent on a nearby cis-acting locus, the recombination
enhancer (RE). The RE sequence physically interacts with the MAT locus after a DSB, thereby increasing
the frequency of collisions between HML and MAT.
To observe the kinetics of switching and changes in chromosome conformation, we imaged two-color
fluorescent reporters inserted near MAT and HML, and measured the three-dimensional distances
between the two loci in live cells or in fixed cells that were collected at different time points before and
after a DSB is induced. In addition we measured the percent usage of HML during MAT switching.
We have compared these experimental observations to a polymer model of yeast chromosome III to
compute the distribution of distances between MAT and HML loci before and after the break. We find
that the polymer model predictions are in quantitative agreement with the experimental distances
before and after the break, thus establishing a biophysical basis of this gene conversion event. Moreover
our calculations show that by refolding chromosome III after a DSB, via tethering RE at MAT, polymer
entropy drives donor preference in yeast mating type switching.
M212
Anti-SON protein “TSA-omics” Reveals a Gradient in Gene Density and Elevated
Gene Expression as a Function of Proximity to Nuclear Speckles.
Y. Chen1, Y. Zhang2, J. Ma2, A.S. Belmont1;
1
Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, IL,
2
Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL
Here we report development of a new genomic mapping method that measures genome proximity to
nuclear speckles. Unexpectedly, this method also revealed Mbp-scale looping between the nuclear
periphery and nuclear speckles from a single genomic plot. Genome-wide computational analysis
demonstrates that a gradient in nuclear speckle proximity is mirrored by a gradient in both gene density
and the distribution of highly expressed genes, with genomic regions closest to nuclear speckles showing
peaks in gene density and the highest enrichment of highly expressed genes.
Our approach uses Tyramide Signal Amplification (TSA) immunostaining to create a gradient of biotin
labeling from the target throughout the nucleus. Importantly, labeling can be directly visualized by
immunofluorescence microscopy prior to DNA isolation and genomic analysis. Using nuclear speckle
marker protein SON as the labeling target, we mapped the whole genome position relative to nuclear
WEDNESDAY-ORAL PRESENTATIONS
speckles in human erythroleukemia K562 cells, revealing distinct genome domains: speckle-proximal,
speckle-distal, and connecting transition zones. Speckle-proximal domains correlate with gene-rich
chromosome “R” bands, consistent with earlier cytological observations. Speckle-distal domains,
appearing as valleys in the genomic proximity plot, correlate strongly with Lamina-Associated Domains
(LADs). Transition zones extending over several Mbp frequently appear as linear ramps in the proximity
plot, connecting valleys with peaks of speckle association. 3D immuno-FISH confirms that speckleproximal domains associate at high frequency with nuclear speckles, while speckle-distal domains locate
away from speckles, frequently near the nuclear periphery. FISH visualization of a 5Mbp transition zone
revealed a linear signal typically stretching between the nuclear periphery and an interiorly located
nuclear speckle, suggesting the capability of revealing Mbp-scale looping of interphase chromosomes
between the nuclear periphery and nuclear speckles directly from the TSA genomic plot. Besides a
gradient in gene density and highly expressed genes relative to nuclear speckle proximity,
computational analysis also revealed a striking correlation of the density of specific histone
modifications and chromatin-related marks with distance to nuclear speckles.
We anticipate this simple proximity-mapping method will provide a valuable tool to measure the spatial
proximity of the genome to a variety of nuclear compartments. Simultaneous mapping of genome
proximity to different nuclear compartments may allow deconvolution of 3D nuclear organization. The
simplicity of this mapping approach makes it ideal for investigating the dynamics of nuclear organization
and its correlation with changes in gene expression.
M213
Dynamics of Replication Clusters of Single Chromosome Territories in Nuclei of
Living Cells.
W. Xiang1, M.J. Roberti1, S. Huet2, J. Ellenberg1;
1
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany,
2
Faculté de Médecine, Université de Rennes 1, Rennes Cedex, France
Chromatin architecture is of fundamental importance to understand genome regulation. Chromosome
conformation capture based approaches have revealed signatures of topological domains below a
megabase pair in size based on biochemical averaging over large cell populations. However, how such
domains are manifested in single cells and how they affect the dynamic properties of chromatin as a
physiological polymer in living cells remains unknown.
To address this, we have systematically studied the dynamic organization of fluorescently labeled
replication clusters of single chromosome territories in nuclei of living cells. After co-replicative labeling
of the DNA backbone with a pulse of fluorescent nucleotides, labeled chromosomes were diluted to
single territories by several rounds of cell division. In these cells, we first investigated replication cluster
size and structure with super-resolution microscopy. We next mapped the dynamic properties of
replication domains by tracking ~1700 domains in single chromosome territories of 145 cells over
several minutes. The resulting trajectories allowed us to obtain a global view of interphase chromatin
WEDNESDAY-ORAL PRESENTATIONS
dynamics and systematically examine effects of subnuclear and subterritorial position, as well as cell
cycle stage. Our data reveals that during interphase, late replicating heterochromatin positioned in
direct contact with the nuclear envelope or nucleoli is essentially immobile. Early and mid replicating
nucleoplasmic euchromatin is able to move, however highly constrained, characterized by slow
anomalous diffusion with median short range diffusion coefficient Ds=0.0065 µm2/s and anomality
α=0.57±0.31. More dynamic behavior is extremely rare and only observed for very few labeled foci.
Short-range diffusion of euchromatin was strictly energy dependent, but independent of subnuclear or
subterritory position. Interestingly, euchromatin dynamics changed with cell cycle progression, with a
higher diffusion coefficient in G1 and G2 and a progressive reduction during from early to late S phase.
Using chase labeling with two colors of fluorescent nucleotides, we could correlate the motion of
neighboring replication domains which revealed a short typical persistence length of chromatin. Taken
together our quantitative data on the structural dynamics of native chromatin allow us to propose a
comprehensive model for higher order organization of chromatin in nuclei of single living cells.
M214
Investigating live chromosome dynamics in Xenopus using the CRISPR/Cas
system.
A.B. Lane1, M. Strzelecka1, A.W. Ettinger2, A.W. Grenfell1, T. Wittmann2, R. Heald1;
1
Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, 2Department of Cell and
Tissue Biology, University of California, San Francisco, San Francisco, CA
Proper chromosome architecture, organization and dynamics are essential for a multitude of biological
processes, from transcription and cell division to development and genome evolution. A major
roadblock to progress in understanding chromosome behaviors has been the inability to specifically
visualize individual chromosomes in living systems. We are modifying recently described CRISPR gene
editing technology to paint chromosomes and follow them live in Xenopus egg extracts and embryos. By
fusing nuclease-deficient Cas9 to a fluorescent protein, and loading it with guide RNAs that recognize
sequences on Xenopus laevis chromosomes, we targeted distinct nucleotide repeats near the
centromere, at telomeres, and at a locus previously reported to be single-copy in single- and
simultaneous dual-color experiments. We have termed this technique CLISP for CRISPR Live In Situ
Probe. By fluorescence time-lapse microscopy, probes bound to Xenopus chromosomes throughout the
cell cycle and revealed co-localization of a subset of pericentromeric and telomeric loci. Chromosomes
observed by two-color CLISP maintained the patterns expected when using either probe alone,
suggesting that exchange between loaded dCas9-guide RNA complexes is slow once complexes are
formed. To label large regions of chromosomes, we have developed a novel strategy that employs
enzymatic processing of target sequences to generate CRISPR libraries. We have successfully generated
libraries targeting loci using this approach and validated them by sequencing and CLISP. This method is
an inexpensive alternative to utilizing array-synthesized oligonucleotides to generate guide RNAs, and
can be applied to any DNA source. Thus, CLISP provides a powerful tool to investigate how genomic loci
and chromosomes move and remodel during cell growth, division and differentiation.
WEDNESDAY-ORAL PRESENTATIONS
M215
Investigating repair of DNA double-strand breaks in live S. pombe cells using a
lacO/lacI-GFP system to monitor DSB resection, position, and dynamics.
B.A. Leland1, M.C. King1;
1
Department of Cell Biology, Yale University, New Haven, CT
A cell must accurately correct DNA damage to maintain its genome. One pathway used to repair DNA
double-strand breaks (DSBs) is homologous recombination (HR). In HR, the broken strand of DNA is
paired with a homologous donor strand (usually the sister chromatid in G2) that is then used as a
template for repair. A key, early step in HR is nucleolytic degradation of the 5’ ends flanking the DSB, a
process termed resection. Resection commits a DSB to repair by the HR pathway and facilitates pairing
with the homologous donor. Much is known about the molecular details of HR, including its regulation
throughout the cell cycle and many of the enzymes that mediate the various steps of repair. However,
less is understood about how HR (and more broadly, all types of DNA repair) is influenced by its cellular
context: the densely packed and dynamically organized nucleus.
To investigate the role of sub-nuclear compartmentalization in DSB repair by HR, we developed a
microscopy-based assay to observe the initial steps of repair in live S. pombe cells. Our assay uses a
rapid induction system to generate a single, site-specific DSB that is directly adjacent to a lac operator
(lacO) array. Binding of lacI-GFP to the lacO array allows us to track the position of the DSB during the
course of repair. After DSB induction, loss of dsDNA sequence at the lacO array flanking the DSB due to
resection provides a means to monitor various properties that influence the resection rate.
Unexpectedly, our data have revealed that the rate of resection is quite variable within a population of
live cells in a manner independent from the cell cycle. We are currently addressing the molecular basis
for this heterogeneity by visualizing repair proteins (such as Rad52-mCherry) and assaying strains
deficient in various repair proteins. We are also investigating the role of DSB position, especially
tethering at the nuclear periphery, in regulating resection.
M216
Dynamic and structural properties of interphase chromatin mapped in vivo with
fluorescence correlation spectroscopy and quantitative modelling.
M. Wachsmuth1, T.A. Knoch2;
1
European Molecular Biology Laboratory (EMBL), Heidelberg, Germany, 2Biophysical Genomics, Dept.
Cell Biology and Genetics, Erasmus MC, Rotterdam, Netherlands
The three-dimensional organization of chromosomes of eukaryotic interphase cells is emerging as an
important parameter for the regulation of storage, replication and expression of the genome. While a
range of techniques like electron microscopy (EM), fluorescence in situ hybridization (FISH), or
chromosome conformation capture techniques (3C, 4C, 5C, HiC, or the novel targeted chromatin
capture, T2C) have been very successfully used to study chromosomal architecture, many details of
WEDNESDAY-ORAL PRESENTATIONS
structural and especially of dynamic properties remain unresolved. Here, we present a novel approach
to dissect intramolecular polymer dynamics from fluorescence intensity fluctuations measured with
fluorescence correlation spectroscopy (FCS) to investigate the higher order chromatin dynamics in living
cells. Using fluorescently tagged linker histone H1 and core histones H2A and H2B as tracer molecules,
we find distinct chromatin relaxation times of ~160 ms for open and ~90 ms for dense chromatin areas,
corresponding to radii of gyration of 240 and 300 nm for the topologically independent chromatin units.
According to their genomic content of ~1 Mb, these units correspond to the distinct topologically
associating domains (TADs) found recently by 3C-derived techniques and to subchromosomal domains
seen earlier by FISH and in vivo chromatin labelling. We have also obtained light-sheet microscopybased FCS maps of chromatin domain dynamics. Based on these results, we have developed a
quantitative analytical and numerical model of chromatin dynamics that provides access to mass
density, persistence length and topological information of chromatin. It allows to extract these
parameters from dynamics and 5C/HiC results, to predict chromatin conformation and distance data,
and to identify complex looping as crucial for domain formation. Data and model suggest the existence
of several connected loops of ~100 kb each per domain. Especially in combination with the recently
developed highly selective T2C method (see abstract T.A. Knoch & M. Wachsmuth, “Determination of
the three-dimensional organization of chromatin by modelling-supported targeted chromosomal
interaction capture (T2C)”), which provides very good signal-to-noise ratio at high genomic resolution,
we present a comprehensive systematic approach for the understanding of chromatin dynamics and its
relation to structure as well as for insight into the impact on gene regulation.
M217
Cytoskeletal tension induces the polarized architecture of the nucleus.
D. Kim1, D. Wirtz1,2;
1
Physical Sciences-Oncology Center, Johns Hopkins University, Baltimore, MD, 2Department of Pathology
and Oncology and Sydney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of
Medicine, Baltimore, MD
Accumulating evidence suggests that the three-dimensional organization of the nucleus regulates gene
expression through lamina-chromosome interactions. The nuclear lamina, which is composed of
filamentous lamin proteins and lamin-associated proteins, provides mechanical support to the nucleus
and regulates essential cellular processes, including DNA replication, chromatin organization, cell
division and differentiation. Conventional fluorescence and electron microscopies have long suggested
that the nuclear lamina forms a spatially isotropic thin shell confined to a narrow region underneath the
nuclear envelope with a few filamentous structures extending to the intranuclear space. Inside the
nuclear lamina, chromosomes are condensed differently along the radial direction from the nuclear
center to the nuclear periphery, which is associated with the radially differentiated chromatin
accessibility for gene expression. However, 3D confocal reconstruction reveals instead that major lamin
protein lamin A/C forms an apically polarized Frisbee-like dome structure in the nucleus of adherent
cells. Here we show that this lamin dome is mediated by cytoskeletal tension provided by the
WEDNESDAY-ORAL PRESENTATIONS
perinuclear actin cap, a subset of contractile actin fibers organized on the apical surface of the nucleus
and dynamically connected to the nuclear lamina through LINC protein complexes. Mechanical coupling
between actin cap and lamina in turn induces an apical distribution of transcription-active subnucleolar
compartments and epigenetic markers of transcription-active genes. Cells that do not feature an actin
cap (e.g., epithelial cells, cancer cells) form evenly distributed lamins that line the inner nuclear
membrane and specific disruption of the actin cap turns the apically polarized lamins into an isotropic
thin shell. These results demonstrate that intra-nuclear structures are apically polarized through the
extra-nuclear actin cap in a wide range of somatic adherent cells. We anticipate this study broadens our
understanding of 3D nuclear architecture and opens new prospects in the field of laminopathy models
and cellular mechanotransduction.
M218
Association of chromatin with the nuclear envelope supports stable nuclear
mechanics.
S.M. Schreiner1, P. Koo2, Y. Zhao3, S. Mochrie3, M.C. King1;
1
Department of Cell Biology, Yale University, New Haven, CT, 2Physics, Yale University, New Haven, CT,
3
Applied Physics, Yale University, New Haven, CT
Cells must constantly withstand mechanical stresses derived from both extracellular and intracellular
forces. These forces can be transmitted to the cell’s nucleus through connections with the cytoskeleton,
raising the possibility that defects in nuclear mechanics can impact the cell’s ability to withstand
mechanical stress. The nuclear lamina, in particular lamin A, is often considered to be the primary
mechanical defense of the mammalian nucleus. However, lamins are part of an integrated network of
proteins, lipids, and chromatin, all of which contribute to the ensemble mechanical properties of the
nucleus. Yeast, which lack a nuclear lamina, provide a model system in which to study the laminindependent contributions of chromatin and proteins residing in the nuclear membrane to nuclear
mechanics. Here, we have combined a quantitative imaging platform capable of measuring 3D nuclear
contours in live cells with an in vitro optical tweezers assay to probe the mechanical properties of S.
pombe nuclei. In live cells, we find that association of chromatin with the inner nuclear membrane (INM)
through integral membrane proteins is required for a normal mechanical response to microtubule (MT)
forces. Increasing loss of integral INM proteins results in highly deformable nuclei that are subject to
catastrophic failures in nuclear envelope integrity, specifically in response to exogenous forces from
MTs. Loss of integral INM proteins that associate with the centromeres results in the most highly
deformed nuclei in vivo. This observation supports a model in which the association of heterochromatin
with the nuclear envelope adjacent to the spindle pole body, the yeast centrosome equivalent, helps
buffer MT-driven forces. Further, our findings suggest that organisms without a nuclear lamina and with
similar heterochromatin-centrosome interfaces have adopted a chromosome organization that
contributes to the mechanical stability of nuclei. Using optical tweezers, we find that nuclei lacking
integral INM proteins are less stiff than wild type nuclei, particularly when force is applied at rates that
recapitulate the kinetics of MT dynamics in vivo. Wild type mitotic nuclei, in which chromatin is globally
WEDNESDAY-ORAL PRESENTATIONS
released from the INM, are extremely soft, raising the possibility that a transition in nuclear mechanics
may support segregation of daughter nuclei during a period when forces are instead balanced between
spindle poles. Together our data support a model in which the physical association of chromatin with
the INM provides the stiffness necessary to buffer cytoskeletal forces in interphase while the
deformability of mitotic nuclei supports nuclear division.
M219
Directed reorganization of chromatin to the nuclear lamina is mediated by
chromatin state, YY1 and A-type lamins.
J.C. Harr1, T.R. Luperchio2, X. Wong2, E. Cohen2, S. Wheelan3, K.L. Reddy4;
1
Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, 2Biological
Chemistry, Johns Hopkins University, Baltimore, MD, 3Oncology Biostatistics and Bioinformatics, Johns
Hopkins University, Baltimore, MD, 4Biological Chemistry and Center for Epigenetics, Johns Hopkins
University School of Medicine, Baltimore, MD
Nuclear organization has been implicated in regulation of gene activity. Recently, large developmentally
regulated regions of the genome that dynamically associate with the nuclear lamina, the so called
Lamina Associated Domains (LADs), have been identified. Intriguingly the dynamic re-organization of
these regions has been implicated in cell-type specific gene regulation. We and others have identified
developmentally regulated genes that reside in these dynamically associating regions, which we call
variable LAD (vLADs). However, little is known about the mechanisms underlying LAD organization,
establishment and scaffolding to the nuclear lamina. In order to identify DNA sequences able to
establish a de novo LAD, we utilized our tagged chromosomal insertion site system (TCIS). Using TCIS we
identified small sequences from borders of fibroblast specific vLADs that are sufficient to target these
ectopic sites to the nuclear periphery. These relocating sequences are enriched in motifs for Ying-Yang1
(YY1), BTB/POZ domain transcription factors (i.e. Zbtb7b) and CTCF. Knockdown of these proteins or
lamin A/C, but not lamin A, led to a loss of lamina association. In addition, targeted recruitment of YY1
proteins facilitated ectopic LAD formation dependent on histone H3 lysine 27 trimethylation
(H3K27me3) and histone H3 lysine di- and tri-methylation (H3K9me2/3). Additionally, we show that
endogenous loci are dependent upon lamin A/C, YY1, H3K27me3 and H3K9me2/3 for maintenance of
lamina proximal positioning. Taken together, these results reveal a mechanism for LAD recruitment
implicating the involvement of tissue specific factors and epigenetic modifications.
WEDNESDAY-ORAL PRESENTATIONS
M220
Structural Analysis of the Nuclear Lamina by Nanoscale Resolution Microscopy.
T. Shimi1, Y.G. Turgay2, M. Kittisopikul3, A.E. Goldman1, S.A. Adam1, Y. Guo4, K. Jaqaman3, Y. Zheng4,5, O.
Medalia2, R.D. Goldman1;
1
Department of Cell and Molecular Biology, Northwestern University, Feinberg School of Medicine,
Chicago, IL, 2Department of Biochemistry, University of Zürich, Zurich, Switzerland, 3Department of
Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, 4Department of Embryology,
Carnegie Institution for Science, Baltimore, MD, 5Department of Biology, Johns Hopkins University,
Baltimore, MD
In mammalian cells, nuclear lamins are the major components of the nuclear lamina which underlies the
nuclear envelope (NE). They are subdivided into the A-type lamins (LA and LC) and the B-type lamins
(LB1 and LB2). However, the structural organization of each of these lamin isoforms within the lamina
remains unclear. The elucidation of the 3D structural organization of each type of lamin is essential for
understanding their structure and functional interactions with peripheral heterochromatic regions of
interphase chromosomes. In order to study this, 3D Structured Illumination Microscopy (3D-SIM)
combined with computational image analysis was performed on WI-38 human diploid fibroblasts and
mouse embryonic fibroblasts (MEFs). Interestingly, LA, LB1, LB2 and LC form separate fibrillar structures
which appear to overlap at their ends to form complex meshworks. Using LB1 null (lmnb1-/-) MEFs, we
also show that the lamina meshwork formed by LA/C and LB2 fibrils becomes abnormally enlarged
compared to WT MEFs. The enlarged complexes of these fibrils coalign with peripheral elements of
heterochromatin as determined by staining with anti-H3K27me3. These results suggest that LB1
regulates both the normal structure of the lamina and the normal structural links to peripheral
heterochromatin. To go one step further in determining nanoscale structures of the lamina, we have
used vimentin null MEFs (Vim-/- MEFs), the nucleus of which lies in close proximity to the plasma
membrane due to the loss of the juxtanuclear intermediate filament cage. This allows us to perform
Stochastic optical reconstruction microscopy (STORM) with total internal reflection fluorescence
microscopy (TIRF) to achieve even higher resolution images of the lamina in situ. These nuclei can also
be imaged in situ by advanced cryo-electron tomography which reveals that the major filamentous
structures in the lamina are not 10nm in diameter but rather are about 5 nm in diameter. We propose
that these lamin nanofilaments probably associate laterally to form the lamin fibers resolved by 3D-SIM
and STORM. These results are being confirmed by the observation that lamin null MEFs in which
vimentin expression is silenced and Vim-/- MEFs stably overexpressing lamins exhibit significant changes
in number of lamin nanofilaments. Our results demonstrate that the lamin fibers assemble into a
complex hierarchical network which changes its structure and function regionally within the nuclear
lamina. This work is supported by National Institutes of Health grants (GM106023) and (CA03176).
WEDNESDAY-ORAL PRESENTATIONS
Minisymposium 24: Protein Sorting to Intracellular Compartments
M221
ER export of yeast plasma membrane proteins requires dual interaction with the
COPII coat.
S. Pagant1, E.A. Miller1;
1
Biological Sciences, Columbia University, New York, NY
Many polytopic membrane proteins that reside at the yeast plasma membrane require the cargo
receptor, Erv14, for efficient ER export. We recently found that the yeast ABC transporter, Yor1, similarly
requires Erv14 for traffic despite having its own diacidic ER export signal. We sought to ask why a
membrane protein that directly interacts with the Sec24 subunit of the COPII coat to facilitate ER export
also requires a dedicated cargo receptor. Here we present a detailed structure-function analysis of Erv14
in the context of both Yor1 and a comprehensive array of additional polytopic membrane clients. We
show that Erv14 indeed functions as a receptor, with a cargo-binding site contained within its second
transmembrane domain that recognizes a subset of clients. The COPII binding signal of Erv14 is essential
for traffic of all of its clients, further supporting a role as a classical cargo receptor. However, Sec24 itself
seems to be the primary driver of ER export since overexpression of Sec24 could rescue Erv14 defects, at
least with respect to Yor1 traffic. We propose that for a large class of polytopic membrane proteins,
Sec24 binds to a dual signal that forms from two independent COPII binding signals: one on the cargo
protein and one on Erv14, which in turn binds to cargo via a TMD-TMD interaction. This added affinity
for the coat may improve packaging efficiency of large, bulky proteins that could be difficult to capture
into a spatially constrained vesicle.
M222
Quality control of GPI-anchored proteins in the secretory pathway.
P. Satpute-Krishnan1, J. Lippincott-Schwartz2,3;
1
Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and
Human Development, NIH, Bethesda, MD, 2Physiology Course at Marine Biological Laboratory, Woods
Hole, MA, 3Cell Biology and Metabolism Program, NICHD, NIH, Bethesda, MD
Accumulation and aggregation of misfolded secretory and membrane proteins can lead to various
degenerative diseases. To avoid this, cells employ quality control systems that route misfolded proteins
for degradation, including the well studied ER associated degradation (ERAD) pathways. ERAD involves
retrotranslocation of the protein from the ER to the cytosol for proteasomal degradation. However, not
all misfolded secretory pathway proteins are efficient ERAD substrates, requiring alternate degradation
pathways. In particular, we found that misfolded GPI-anchored proteins (GPI-APs) are poorly recognized
by ERAD systems. GPI-APs encompass a major class of proteins that play essential roles in critical life
processes including embryogenesis, fertilization, neurogenesis, and immunity (1). The GPI-anchor is a
WEDNESDAY-ORAL PRESENTATIONS
post-translational modification conserved in all eukaryotes (1). Despite the prevalence and ubiquity of
GPI-APs, little is known about how their quality control is regulated.
To elucidate how cells handle misfolded GPI-APs, we created a diverse panel of fluorescent proteintagged misfolded variants of GPI-APs and monitored them by live cell microscopy combined with
biochemical analysis. We identified a novel degradation pathway that operates constitutively but is
markedly enhanced during ER stress (2). At the onset of stress, misfolded GPI-APs dissociate from ER
chaperones and through requisite interactions with p24 ER-export factors leave the ER to the secretory
pathway. Inhibiting this pathway through depletion of its key ER-export factor results in their
aggregation in the ER. We named this stress-inducible mechanism RESET (for rapid ER stress-induced
export) as it helps to reset ER homeostasis during the critical period before induction of the unfolded
protein response.
Intriguingly, after release from the ER into the secretory pathway, misfolded GPI-APs transiently access
the cell-surface before destruction in lysosomes, implicating plasma membrane-level quality control.
Although misfolded transmembrane proteins have been shown to be downregulated from the plasma
membrane after ubiquitination on their cytosolic regions, GPI-APs lack a cytosolic domain, implicating a
distinct pathway for rapid downregulation of misfolded GPI-APs. We are now using the tractable
experimental systems that we have developed to reveal novel mechanisms in plasma membrane quality
control.
(1) Fujita, M., and Kinoshita, T. (2012) GPI-anchor remodeling: potential functions of GPI-anchors in
intracellular trafficking and membrane dynamics. Biochim Biophys Acta 1821, p1050-1058 (2) SatputeKrishnan et al. (2014) ER stress-induced clearance of misfolded GPI-anchored proteins via the secretory
pathway, Cell, In Press
M223
The golgin coiled-coil proteins tether vesicles.
M. Wong1, S. Munro1;
1
Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
A key step in membrane traffic is tethering, which provides initial association between transport vesicles
and their target membranes prior to SNARE-mediated membrane fusion. The golgins are one class of
putative vesicle tethering factors found on the Golgi apparatus. Their precise functions have been
unclear, partially due to redundancy suggested by mild phenotypes of mutant models. In this work, we
adopted a relocation strategy to test for sufficiency rather than necessity. Ten golgin proteins were
systematically relocated to the mitochondria through substituting their C-termini, which have been
shown to confer Golgi localisation, by a mitochondrial transmembrane domain. We found that individual
golgins are capable of capturing ER-derived cargos, Golgi residents, or endosome-to-TGN cargos to the
mitochondria. Furthermore, ultrastructural studies show striking accumulation of vesicles around
mitochondria decorated with particular golgins. Our results demonstrate for the first time in vivo that
WEDNESDAY-ORAL PRESENTATIONS
golgins are indeed vesicle tethers, and that they exhibit specificity towards different classes of transport
vesicles that travel to or through the Golgi.
M224
The HOPS/Class C Vps complex tethers membranes by binding to a Rab GTPase
in one membrane and directly via a curvature-sensing motif to a second, highlycurved membrane.
C. Stroupe1, R. Ho1;
1
Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA
Membrane tethering factors are thought to function by simultaneously binding to two apposed
membranes. However, the sites on tethered membranes that are bound by tethering factors in order to
form membrane-bridging physical linkages remain unknown. Here, we use liposomes and purified
proteins to identify the physical interactions needed for membrane tethering by the HOPS/Class C Vps
complex, an
effector for the yeast vacuolar Rab GTPase Ypt7p. We find that HOPS can tether two
low-curvature membranes using its two Ypt7p binding sites – one in its Vps41p subunit, the other in its
Vps39p subunit – to bind a molecule of Ypt7p in each of the apposed membranes. HOPS also can tether
a low-curvature membrane to a high-curvature membrane by binding to the low-curvature membrane
via Ypt7p and to the highly-curved membrane using a curvature-sensing ALPS motif in Vps41p. A protein
construct containing only the Ypt7p-binding site and ALPS motif from Vps41p was unable to tether
membranes, while a HOPS complex lacking Vps39p was similarly unable to perform tethering. We
therefore conclude that HOPS tethers low-curvature membranes to high-curvature membranes by
binding via Vps39p to Ypt7p in the low-curvature membrane, and via its ALPS motif to the highcurvature membrane. These results suggest a model for how HOPS tethers both high-curvature
membranes (e.g. small transport vesicles) and low-curvature membranes (e.g. late
endosomes/multivesicular bodies) at the large, low-curvature vacuole/lysosome.
M225
“Structure of a lipid-bound extended synaptotagmin indicates a role in lipid
transfer”.
C.M. Schauder1, X. Wu2, Y. Saheki3, P. Narayanaswamy4, F. Torta4, M. Wenk5, P. De Camilli3, K.M.
Reinisch6;
1
Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 2Harvard Medical School,
Boston, MA, 3Department of Cell Biology, Howard Hughes Medical Institute, Program in Cellular
Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, CT,
4
Department of Biological Sciences, National University of Singapore, Singapore, Singapore,
5
Department of Biochemistry, National University of Singapore, Yong Loo Lin School of Medicine,
Singapore, Singapore, 6Department of Cell Biology, Yale University School of Medicine, New Haven, CT
WEDNESDAY-ORAL PRESENTATIONS
It has become increasingly apparent that membrane contact sites, regions of close apposition between
two distinct membrane components, facilitate lipid exchange. The mechanisms underlying this type of
lipid transfer are largely unknown. SMP domains are proposed lipid binding modules that are found
only in proteins localized to membrane contact sites. The extended synaptotagmins (E-Syts) are a
family of proteins that tether the endoplasmic reticulum (ER) to the plasma membrane at contact sites
and contain an N-terminal ER anchor sequence, an SMP domain and three or more C2 domains. Here
we report at 2.44 Å resolution the crystal structure of an N-terminal fragment of human E-Syt2, which
includes the SMP domain and two adjacent C2 domains (A,B). The C2A and C2B domains interact to
form an arch, which likely is not rigidly positioned with respect to the SMP domain in solution. The SMP
beta-barrel fold identifies these domains as members of the tubular-lipid-binding (TULIP) superfamily.
In E-Syt2, the SMP domain dimerizes to form a 90-Å-long cylinder. A cavity lined almost exclusively with
hydrophobic residues runs the length of the cylinder and is connected to solvent via a “seam”. The
electron density within the SMP domain cavity revealed the presence of diacylglycerol lipids, which were
subsequently identified as glycerophospholipids via mass spectrometry. These results strongly support a
role for E-Syt2 in transferring glycerophospholipids between the ER and the PM and with the
identification of the SMP domain as a lipid binding domain, have much farther reaching implications.
M226
Targeting of Peripheral Proteins to Lipid Droplets and other Organelles.
A. Copic1, S. Bouvet1,2, C. La Torre-Garay1, C. Jackson1;
1
Institut Jacques Monod - CNRS, Université Paris Diderot, Sorbonne Paris Cité, Paris, France, 2Present
Address: Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland
Lipid droplets (LDs) are dynamic organelles that interface with membrane trafficking pathways, and are
involved in lipid homeostasis and signaling. LDs differ from other cellular organelles in having a core of
neutral lipids (triglycerides and cholesterol esters) surrounded by a phospholipid monolayer rather than
an aqueous interior surrounded by a phospholipid bilayer. How these unique features of LDs influence
targeting of peripherally associated proteins to their surface is poorly understood. Our previous work
has shown that two classes of amphipathic helices (AHs) can target membranes of different composition
through direct protein-lipid interactions (Pranke et al. 2011). We showed that the physical properties of
membranes, notably their curvature, lipid packing and electrostatics, can allow the selective adsorption
of AHs that possess a complementary chemistry. Because AHs enter the interfacial region of only the
cytosolic leaflet of a bilayer, they are also ideal localization devices for targeting of proteins to the
monolayer surface of an LD. We are addressing the question of how the AHs of LD-associated proteins
are targeted to the LD surface, using a combination of cell biology, biochemistry and in silico
approaches.
The Arf1 exchange factor GBF1, along with its substrate Arf1 and effector COPI, function both at the
Golgi and on lipid droplets, and localize to both organelles. We have shown that localization of GBF1 to
the Golgi and LDs requires the HDS1 domain, immediately downstream of the catalytic Sec7 domain
(Bouvet et al. 2013). The HDS1 domain of GBF1 contains an AH that mediates localization to LDs in cells,
WEDNESDAY-ORAL PRESENTATIONS
and to both liposomes and artificial droplets in vitro (Bouvet et al. 2013). In contrast to HDS1 alone, a
Sec7-HDS1 domain tandem was cytosolic in cells and failed to bind lipids in vitro, suggesting that the
Sec7 domain inhibits the targeting of HDS1 to organelles. Hence the GBF1 AH represents a class of
motifs that targets both lipid droplets and Golgi membranes in a manner regulated by other domains of
the protein. We are studying the LD localization of other AHs present in LD-associated proteins. The
chemical properties of AHs that target both LDs and other organelles vary widely, suggesting that the LD
surface can be quite permissive for AH binding. However, one class of AH appears to specifically target
LDs in cells, using a mechanism that we are defining through bioinformatics and biochemical analysis.
Pranke IM, Morello V, Bigay J, Gibson K, Verbavatz JM, Antonny B, Jackson CL. (2011) J Cell Biol. 194:89103.
Bouvet S, Golinelli MP, Contremoulins V, Jackson CL. (2013) J Cell Sci. 126:4794-805.
M227
Structure and function of the N-terminal domains of Sec7/BIG1/2 regulating Arf1mediated trafficking from the trans-Golgi network.
B.C. Richardson1, J.C. Fromme2;
1
Cornell University, Ithaca, NY, 2MBG/WICMB, Cornell University, Ithaca, NY
Export of trafficked proteins from the trans-Golgi network (TGN) is an essential function in all
eukaryotes, controlled by the small GTPase Arf1. The proximal upstream regulator of Arf1 is the
conserved Sec7 (yeast) / BIG1/2 (mammal) / Sec71 (fly) guanine nucleotide exchange factor (GEF), which
activates Arf1 specifically at the TGN. This action initiaties the terminal cascade of events at the Golgi
leading to cargo sorting and vesicle formation with multiple different cargos and cargo adaptors.
Sec7 consists of a single well-characterized catalytic domain responsible for its GEF activity on Arf1, and
six similarly sized domains, two N-terminal and four C-terminal to the GEF domain. We recently
demonstrated that these latter six domains serve varying and essential functions both to localize Sec7 to
its point of action and to regulate its activity negatively when unlocalized and positively when properly
localized. Together, the regulatory domains of Sec7 tightly restrict its activity to the trans-Golgi
network, ensuring proper maturation and targeting of all transported proteins. However, the
mechanistic details of how each domain mediates its autoregulatory functions and its interactions with
other proteins remain obscure.
Here, we present the 2.7 Å crystal structure of the two N-terminal regulatory domains of Sec7,
representing the first structural model available for regions of Sec7 outside the catalytic domain. The
structure identifies a pair of conserved surface regions, one in each domain, mutation of which affects
the activity of Sec7 on Arf1 both in vitro and in vivo. Paired with small-angle x-ray scattering and multiangle light scattering data of a suite of Sec7 fragments, this structure provides insight into the nature of
dimerization mediated by the N-terminal domains and into the relationship between the catalytic
domain and the N-terminal domains.
WEDNESDAY-ORAL PRESENTATIONS
M228
Defining the assembly determinants and architecture of the yeast exocyst
complex.
M.R. Heider1, C.M. Duffy1, Z. Hakhverdyan2, R. Kalia3, M. Gu3, M. Rout2, A. Frost3, M. Munson1;
1
Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester,
MA, 2Laboratory of Cellular and Structural Biology, The Rockefeller University, New York, NY,
3
Biochemistry, University of Utah, Salt Lake City, UT
The exocyst is a highly conserved, hetero-octameric protein complex, which is proposed to function at
the tethering step of exocytosis in all eukaryotes. The classification of the exocyst as a multisubunit
tethering complex (MTC) stems from its known interacting partners, polarized localization at the plasma
membrane, and structural homology to other putative MTCs. The presence of 8 subunits begs the
questions: why are so many subunits required for vesicle tethering and what are the contributions of
each of these subunits to the overall structure of the complex? Additionally, are subunit or subcomplex
dynamics a required feature of exocyst function? In order to answer these questions, it was critical that
we accurately define the subunit connectivity within the endogenous exocyst complex. Using
Saccharomyces cerevisiae as our model system, we purified intact exocyst complexes under
physiological conditions. Regardless of the exocyst subunit used as purification handle, all 8 subunits are
stoichiometric. In contrast to early studies of the exocyst, these complexes remained assembled over a
wide range of pH and ionic strength conditions, suggesting greater intra-complex stability than
previously expected. However, at higher extremes of pH and salt concentration, biochemically stable
subcomplexes including Sec10-Sec15 and Sec3-Sec5-Sec6-Sec8 emerged. To further dissect these
connections and because most of the exocyst subunits are encoded by essential genes, we used the
auxin-inducible degradation (AID) system to selectively and rapidly degrade individual exocyst subunits
and monitor the effects on complex assembly. Using the AID system, we identified critical subunits that
are required for the overall assembly and for connecting modular subcomplexes. However, given the
highly stable assembly of the complex, we hypothesize that any subunit dynamics or complex
disassembly require additional factors. To address this hypothesis, we are using the AID system to
identify which components within the post-golgi secretory pathway are required for the assembly and
disassembly of the exocyst. By selectively depleting GTPases, myosin, and SNAREs and then purifying
endogenous exocyst complexes, we will pinpoint the steps in the pathway when these complex
rearrangements may occur. Finally, piecing together these subunit connections into the overall structure
of the exocyst will provide clues as to how the subunits are poised to capture secretory vesicles. To this
end, we visualized its structure for the first time using negative stain electron microscopy. 2D class
averaging reveals an elongated conical structure, and we are currently working to position the individual
exocyst subunits within this overall structure.
WEDNESDAY-ORAL PRESENTATIONS
M229
EHD proteins coordinate membrane reorganization and fusion to initiate early
steps of ciliogenesis.
C. Insinna (Kettenhofen)1, Q. Lu1, C.M. Ott2, U. Baxa3, S. Lopes4, J. Lippincott-Schwartz5,6, S. Caplan7, P.K.
Jackson8, C.J. Westlake9;
1
LCDS, National Cancer Institute-Frederick, Frederick, MD, 2NIH/NICHD, Bethesda, MD, 3NIH/NCI/Leidos,
Frederick, MD, 4Faculdade de Ciências Médicas, CEDOC, Lisboa, Portugal, 5Physiology Course at Marine
Biological Laboratory, Woods Hole, MA, 6Cell Biology and Metabolism Program, NICHD, NIH, Bethesda,
MD, 7Univ Nebraska Med Ctr, Omaha, NE, 8Department Microbiology and Immunology, Stanford
University School of Medicine, Stanford, CA, 9National Cancer Institute-Frederick, Frederick, MD
The primary cilium is a membrane-bound, microtubule based sensory organelle that plays essential roles
in development and disease pathways. Cilia biogenesis requires coordination of a series of processes
including, mother centriole to basal body transformation, recruitment of intraflagellar transport (IFT)
and transition zone (TZ) proteins, and axoneme formation and association with a developing ciliary
membrane. Membrane association with the distal appendages of the mother centriole is a critical step
in ciliogenesis initiation. The membrane trafficking Rab GTPase Rab11-Rab8 cascade plays key roles in
early ciliary membrane assembly, but the molecular details remain unclear. Here, we show that the
membrane shaping proteins EHD1 and EHD3, in association with the Rab11-Rab8 ciliogenesis cascade,
function in cilia assembly in zebrafish and mammalian cells. We discovered that EHD proteins localize to
Rab11 pre-ciliary vesicles that dock to the distal appendages of the mother centriole, membranes we
refer to as distal appendage vesicles (DAV). Using live and super-resolution imaging, as well as electron
microscopy approaches, we established that EHD proteins are essential for the formation of the larger
pre-axonemal ciliary vesicle (CV) from DAVs. Furthermore, we show that EHD1-dependent CV formation
is critical for initiating mother centriole to basal body transformation and recruitment of IFT20 and
transition zone proteins. Surprisingly, we found that Rab8 is recruited for ciliary membrane growth only
after these steps, and in coordination with axonemal assembly. Investigations into the molecular
mechanism of these early ciliogenesis initiation steps suggested that EHD proteins tubulate DAVs,
bringing them in close proximity to allow fusion into the CV. This step is required for CP110 removal
from the distal end of the mother centriole prior to recruitment of IFT20 and TZ proteins. Based on
these findings we predicted that SNAREs, regulators of membrane fusion, would be important for CV
assembly and ciliogenesis progression. We show that the SNARE SNAP29, an EHD1 interacting protein, is
required for ciliogenesis and localizes with EHD1/3 on early ciliary membrane structures. Together, our
studies provide new molecular mechanisms informing the classically described intracellular ciliogenesis
pathway and uncover a previously uncharacterized step in ciliary assembly.
WEDNESDAY-ORAL PRESENTATIONS
Minisymposium 25: Stem Cell Proliferation in Organogenesis
M230
The Hippo Pathway Effector Yap Controls Patterning and Differentiation of Airway
Epithelial Progenitors.
W.V. Cardoso1;
1
Columbia Center for Human Development, Department of Medicine, Columbia University Medical
Center, New York, NY
The mechanisms by which epithelial progenitor cells integrate local signals to balance expansion with
differentiation during organogenesis are still little understood. Here we provide evidence that the Hippo
pathway effector Yap is a key regulator of this process in the developing lung. We demonstrate that
when epithelial tubules are forming and branching, a nuclear-cytoplasmic shift in the localization of Yap
marks the boundary between the progenitors of the distal lung and the airway compartment. At this
transition zone, Yap specifies a transcriptional program that controls Sox2 expression and is ultimately
required to generate the airway epithelium and its branched tubular structures. Without Yap, epithelial
progenitors are unable to properly respond to local TGFβ-induced cues and control levels and
distribution of Sox2 to form airways. Moreover, Yap levels and subcellular localization markedly
influence Sox2 expression and differentiation in adult airways. Our data reveal a crucial role for the
Hippo-Yap pathway in integrating growth factor-induced cues in the developing and adult progenitors,
potentially key for lung homeostasis and regeneration-repair.
M231
RAS-ERK1/2 signaling controls mitotic spindle rotation in airway tube
morphogenesis.
Z. Tang1, F. Li1, X. Wang2, W. Marshall3, N. Tang1;
1
National Institute of Biological Sciences, Beijing, China, 2Institute of Biophysics, Chinese Academy of
Sciences, Beijing, China, 3Biochemistry and Biophysics Dept., University of California, San Francisco, San
Francisco, CA
The sizes and shapes of lung epithelial tubes are critical for lung function. Our previous study
demonstrated that the RAS-ERK1/2 signaling plays a key role in regulating airway shape by influencing
mitotic spindle orientation in embryonic airway epithelial cells. Our data suggest that RAS-ERK1/2
signaling functions as a switch to change the cell division orientation with respect to the longitudinal axis
from parallel to random. How ERK1/2 signaling influences spindle orientation remains an interesting
question. By employing several approaches including mouse genetics, quantitative cell biology with
three-dimensional time-lapse imaging, we investigated the cellular mechanisms by which ERK1/2
signaling controls mitotic spindle orientation. We found that mitotic spindles rotate in airway epithelium
during cell divisions. Furthermore, we revealed there is a link between spindle rotation behavior and
WEDNESDAY-ORAL PRESENTATIONS
mitotic spindle orientation. We demonstrated that the spindle rotation behavior is controlled by ERK1/2
activity. We therefore propose a novel cellular mechanism by which ERK1/2 signaling controls mitotic
spindle orientation to ensure proper development of the mammalian lung.
M232
Molecular and Cellular Mechanisms of C. elegans Neuroblast Development.
G. Ou1;
1
School of Life Sciences, Tsinghua University, Beijing, China
C. elegans Q neuroblasts undergo asymmetric cell division, migration and apoptosis to generate three
distinct types of neurons at the L1 larval stage. We have developed fluorescence live cell imaging
technique to document these events using spinning disk confocal microscopy. In additional to the classic
genetics, we have also devised efficient conditional knockout strategies with the use of somatically
expressed TALENs or CRISPR-Cas9 system. We combined live imaging and genetic approaches to
understand mechanisms underlying Q neuroblast development. We will report our recent progresses on
Q neuroblast asymmetric division. We previously showed that the polarized distribution of myosin
during cytokinesis may produce unequal contractile force that contributes to the asymmetry of daughter
cell size and fate. We performed large-scale forward genetic screens to isolate essential molecules
governing this process; and we have also performed small-scale candidate screen to study the function
of embryonically essential genes in neuroblast asymmetric division using somatic CRISPR-Cas9. Equally
intriguing is the asymmetric segregation of cell fate determinants, and we will report our work of the
fate of midbody in Q neuroblast lineages. Taken together, these results will advance our understanding
of neuroblast development.
M233
Prolonged mitosis of neural stem cells alters cell fate in the developing brain.
L. Pilaz1, J. McMahon1, E. Miller1, A. Lennox1, A. Suzuki2, E.D. Salmon2, D.L. Silver1;
1
Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, 2Biology,
University of North Carolina, Chapel Hill, NC
Mitosis defects in neural progenitors are posited to induce cell fate changes associated with reduced
brain size, termed microcephaly. Yet the underlying mechanism by which aberrant mitosis impacts
production and survival of neurons and progenitors is unclear. Here we demonstrate that prolonged
prometaphase of neural progenitors drives specification of neural cell fates. We assess mitosis using live
imaging of embryonic brain slices from a mouse microcephaly model haploinsufficient for Magoh. We
find a significant proportion of mutant progenitors are delayed in prometaphase for >1 hour, with most
eventually completing mitosis. Magoh mediates mitotic progression by regulating centrosome
separation, but is not essential for intact kinetochore structure or centrosome maturation. Another
subset of mutant progenitors exhibits randomized spindle orientation but normal prometaphase
WEDNESDAY-ORAL PRESENTATIONS
duration, revealing these two processes can be uncoupled and may independently influence cell fate.
Consistent with this, live imaging reveals mutant progenitors with prolonged prometaphase directly
generate more neurons and apoptotic progeny than those with shorter prometaphase. We next
independently established a causal link between prometaphase duration and cell fate using
pharmacological inhibitors in brain slices to reversibly and selectively lengthen prometaphase. This assay
was coupled with EdU pulse chase to indelibly mark mitotically delayed progenitors and their direct
progeny. Strikingly, prometaphase-delayed progenitors generate ectopic neurons at the expense of
progenitors, and apoptotic progeny. We further show that neuronal specification and apoptosis are
mutually exclusive outcomes as the former is P53-independent whereas the latter is P53-dependent.
Together our results expose an essential role for prometaphase length in neural cell fate specification
and define a new paradigm to understand why mitosis perturbations cause microcephaly.
M234
Dissecting cell fate potential and cytoarchitectural dynamics in developing neural
stem cells in vitro reveals functional correlates to human corticogenesis.
Y. Yaffe1, R. Edri1, O. Ziv1, A. Zaritsky2, M.J. Ziller3, E. David4, I. Gat-Vics4, A. Meissner3, Y. Elkabetz1;
1
Department of Cell and Developmental Biology, Tel Aviv University, Tel Aviv, Israel, 2Department of Cell
Biology, University of Texas Southwestern Medical Center, Dallas, TX, 3Broad Institute of MIT and
Harvard, Cambridge, MA, 4Deaprtment of Cell Research and Immunology, Tel Aviv University, Tel Aviv,
Israel
Early neuroepithelial progenitors derived from pluripotent stem cells (PSCs) serve as a primary in vitro
neural stem cell (NSC) source for generating neural cell type diversity. However, the heterogeneity of
neuroepithelial cell cultures and their rapid transition through distinct neural stem and progenitor cell
types impedes development of in vitro strategies for understanding lineage transition and neural cell
type specification. This is further challenged by the lack of in depth cellular and molecular
characterization of progenitor cells, together introducing a major challenge for harnessing their full
potential in vitro. To tackle these limitations, here we dissected the transition of neuroepithelial cells
through distinct potencies by prospective isolation of primary progenitor cells derived from human ES
cells during long-term neural differentiation based on their Notch activation. We demonstrate that
Notch activation in neuroectodermal cells is required for the establishment of neuroepithelial cells with
broad developmental potential and strong proliferation capacity. Notch active neuroepithelial cells
rapidly progress into early and mid neurogenic cerebral radial glial (RG) cells followed by gliogenic RG in
a Notch dependent manner. Transition through these cell types correlates with forebrain specification,
cortical lamination and glial transformation at both functional and molecular levels. Furthermore, we
found a functional correlation between Notch activation in early RG cells and their ability to form neural
rosette structures - the in vitro counterpart of cortical RG cells. Mechanistically, Notch activation in RG
cells confers a strong apicobasal cell polarity, which in turn enables their organization into rosette
structures demarcating ventricular zone- and subventricular zone-like equivalents. We further
demonstrate that rosette organization and Notch activation enable interkinetic nuclear migration and
WEDNESDAY-ORAL PRESENTATIONS
cell division at rosette apical sites, strongly suggesting that these features interplay to ensure the
maintenance of neurogenic RG cell pools in vitro. Transcriptional analysis and subsequent shRNA screen
validation reveal essential key factors involved in relaying Notch activation through neuroepithelial cell
induction, radial glial cell transition and glial transformation. Our observations assign Notch activation
and rosette formation as essential components orchestrating NSC ontogeny in vitro, by establishing
identity of neuroepithelial cells, maintaining their numbers, and dictating their transition through
distinct potencies. Our cellular and molecular observations provide a first insight into human NSC
ontogeny and propose a well-controlled platform to dissect the development of normal and pathogenic
NSCs and their progeny.
M235
Abscission delay in Germline Stem Cells is controlled through intrinsic
modifications to cytokinesis coupled with extrinsic regulation by adjacent
somatic cells.
K. Lenhart1, S. DiNardo2;
1
University of Pennsylvania, Philadelphia, PA, 2Department of Cell Developmental Biology, University of
Pennsylvania , Philadelphia, PA
Incomplete cytokinesis is a deeply conserved feature of germ cells required for their differentiation. In
Drosophila, germline stem cells (GSCs) significantly delayed completion of cytokinesis but the eventual
abscission of daughter cells is necessary to maintain a functioning tissue. Very little is known about how
delayed abscission in GSCs is regulated, how it differs from incomplete cytokinesis of differentiating
germ cells and why such a delay in membrane scission might exist. Through extended live imaging, we
have identified three distinct regulations of cytokinesis specific to GSCs in the testis. Specifically, an actin
ring-based block to cytokinesis mediated by Cofilin, a novel role for Aurora B Kinase in controlling the
rate of cytokinesis progression and a non-autonomous requirement for somatic cell encystment in
triggering GSC abscission. We speculate that these regulations exist to promote the coordination
between stem cell lineages required for robust production of sperm and maintenance of the tissue.
Together, these findings shed significant insight into the mechanisms by which cytokinesis is inhibited
and reinitiated in GSCs and why such complex regulation might exist within the stem cell niche.
M236
The Wnt signaling and cytoskeletal regulator APC2 controls stem cell niche size
and architecture.
S.L. Oliver1, B.M. McCartney1;
1
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA
Stem cell behavior is controlled by the cellular neighborhood, or niche, in which stem cells reside. Niche
function relies on specific tissue architecture and cell-cell signaling to maintain tissue homeostasis.
WEDNESDAY-ORAL PRESENTATIONS
Signaling within the niche is well studied. However, regulation of niche architecture is not well
understood. Here we report that the Wnt signaling and cytoskeletal regulator, Adenomatous polyposis
coli (APC2) is required for proper niche size and architecture in the germarium. 5-7 Cap cells (Cpc), a
central component of the female germline stem cell niche, reside at the anterior tip of the germarium
and are essential for maintaining stem cells in the niche. Loss of APC2 results in an increased number of
Cpc, and their displacement away from the anterior tip. Interestingly, there does not appear to be a
direct relationship between the increase in Cpc number and the defect in Cpc position. In addition, APC2
null mutants exhibit an increased number of germline stem cells (GSCs) that may result from increased
Cpc number. Preliminary data using a separation of function allele of APC2 suggest that Cpc number is
regulated by Wnt signaling, while Cpc position requires the actin regulation function of APC2. In the
syncytial embryo, APC2 regulates cortical actin through a collaboration with the formin Diaphanous. We
are currently testing the hypothesis that this collaboration regulates Cpc position. Consistent with our
hypothesis, selective reduction of the actin organizers Spectrin and Kelch in Cpcs results in Cpc
displacement, and Spectrin is mislocalized in APC2 mutants. These results suggest that the assembly and
organization of cortical actin is required for proper Cpc position. Taken together, our data suggest that
APC2 plays a dual role in the stem cell niche. APC2 regulates niche size by restricting Cpc number
through negative regulation of Wnt signaling, and controls Cpc position within the germarium through
the regulation of cortical actin.
M237
Zebrafish intestinal stem cells are located at the base of the inter-villus pocket
between villi ridges and populate both sides of the flanking ridges.
S. Tavakoli1, P.T. Matsudaira1;
1
Mechanobiology Institute, National University of Singapore, Singapore, Singapore
In contrast to the stereotypic villus-crypt organization of the bird and mammalian intestine, the
zebrafish intestinal epithelium is folded into villar ridges and crypts are absent. Previous studies have
shown the dividing cells are located at the base of the villus ridge while cell death occurs at or near the
villus tip. This simple organization suggests that intestinal stem cells (ISCs) maintain the villus epithelium
along a base-to-tip axis. We tested the dynamics of zebrafish intestinal epithelium renewal by label
retention assay, creation of mosaic intestinal tissues and finally lineage tracing. We examined the
intestinal stem mammals’ cell markers candidate in zebarfish. The promoters of prmt1 and lrig1 genes,
as the most reliable intestinal stem cell markers in zebrafish, were used to express CreERT2 in the
generated transgenic lines. Activating the CreERT2 at desired time helps to trace the Cre mediated
recombination in tissue. Our result suggests that renewal of the zebrafish epithelium is similar to birds
and mice with the newly divided cells at the base of the inter-villus pocket, which complete their
translocation to the ridge. Furthermore, similar recombination pattern of the inter-villus pocket flanking
sides of the villi ridges, demonstrates the bilateral migration of newly reproduced cells toward the
flanking ridges. In another words, intestinal stem cells are located at the inter-villus pocket between villi
ridges and populate facing ridges. Therefore, the facing ridges share the stem cells at the base and show
WEDNESDAY-ORAL PRESENTATIONS
similar expression pattern. In contrast, two sides of a ridge may show different expression pattern as
originate from different stem cells.
M238
Molecular ties between the cell cycle and differentiation in embryonic stem cells.
V. Li1, M.W. Kirschner1,2;
1
Department of Systems Biology, Harvard Medical School, Boston, MA, 2Systems Biology, Harvard
Medical School, Boston, MA
Attainment of the differentiated state during the final stages of somatic cell differentiation is closely tied
to cell cycle progression. Much less is known about the role of the cell cycle at very early stages of
embryonic development. Here we show that molecular pathways involving the cell cycle can be
engineered to strongly affect embryonic stem cell differentiation at early stages in vitro. Strategies
based on perturbing these pathways can shorten the rate and simplify the lineage path of ES
differentiation. These results make it likely that pathways involving cell proliferation intersect at various
points with pathways that regulate cell lineages in embryos and demonstrate that this knowledge can be
used profitably to guide the path and effectiveness of cell differentiation of pluripotent cells.
Symposium 7: New Perspectives on the Nucleus
S16
The role of the LINC complex in the 53BP1-driven mobility and NHEJ of
dysfunctional telomeres.
F. Lottersberger1, R.A. Karssemeijer1, N. Dimitrova2, T. de Lange1;
1
Laboratory of Cell Biology and Genetics, The Rockefeller University, New York, NY, 2David H. Koch
Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA
The removal of the shelterin component TRF2 from the telomeres elicits an ATM-dependent DNA
damage response that leads to NHEJ-mediated telomere fusions. A key role in this repair pathway is
played by the DNA damage factor 53BP1, which is recruited at deprotected telomeres in an ATMdependent manner, promotes NHEJ, increase telomere mobility and inhibits 5’ end resection (Dimitrova
et a., 2008; Lottersberger et al, 2013; Zimmerman et al., 2013).
Here we address the mechanism by which 53BP1 enhances the mobility of dysfunctional telomeres.
Treatment with nocodazole and low concentrations on vincristine, which depolymerize microtubules,
inhibited the 53BP1-driven mobility of dysfunctional telomeres, whereas the microtubule stabilizing
drug taxol had no effect. The effect of nocodazole was readily reversible upon wash-out of the drug. This
indicated that telomere mobility dependent on a microtubule mediated movement. We therefore
examined the role of the LINC (LInker of the Nucleoskeleton and Cytoskeleton) complex, which was
WEDNESDAY-ORAL PRESENTATIONS
previously implicated in telomere movement movement in meiosis in S. pombe and mammals
(Chikashige et al., 2006; Ding et al., 2007; Schober et al., 2009). Using SV40 immortalized TRF2
conditional knockout MEFs we tested shRNAs to various LINC components for their effect on telomere
fusions, making the assumption that the mobility of dysfunctional telomeres is required for efficient
fusion. These experiments implicated the inner nuclear envelope proteins SUN1 and SUN2, the KASH
domain protein Nesprin-4, as well as kinesin-1 and kinesin-2 in the 53BP1-driven mobility of
dysfunctional telomeres. Live-cell imaging of dysfunctional, TRF2-depleted telomeres in conditional
TRF2/SUN1/2 double knockout cells indicates that SUN1 and SUN2 are indeed required for the
movement of dysfunctional telomeres. Thus, 53BP1 mediates the movement of dysfunctional telomeres
by promoting their interaction with the LINC complex and allowing their kinesin-mediated movement
along microtubules.
S17
Cracking the Nucleus: Visualizing the Higher Order Coding Structures of DNA.
H.D. Ou1, S. Phan2, T.J. Deerinck2, M.H. Ellisman2, C.C. O'Shea1;
1
Molecular And Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, 2University
of California, San Diego, La Jolla, CA
In 1953 Watson and Crick discovered that DNA forms a double helix, which revealed not only DNA’s
long-sought atomic structure but how genetic information is stored and transferred. However, the
double helix captures only the first-order structure of DNA. It is now clear that the biological functions
and activity of DNA in our genomes cannot be predicted by linear sequence information alone. To fit
within the nucleus DNA assembles into chromatin and coils into spatially defined territories that
determine if genes are active or silent through still poorly understood mechanisms. The higher order
coding structures of the human genome have yet to be visualized throughout an intact cell.
Fluorescent labels can mark where a macromolecule is located in the cell but do not reveal its
underlying ultrastructure. The latter requires electron microscopy (EM). Unfortunately, conventional EM
stains do not enable DNA to be distinguished from the complex mix of proteins and other nucleic acids
within the cell nucleus. Our textbook pictures of chromatin forming different compaction states within
the nucleus remain cartoon drawings, extrapolated from in vitro reconstitution studies or extracted
nuclei, with no consensus on a model for its actual forms within the cell. This represents a fundamental
gap in our understanding of DNA and the structural code that determines if a gene is in an ‘active’ versus
‘repressed’ state.
To overcome this, we have developed ChromEM, which exploits a cell permeable fluorescent small
molecule that binds specifically to DNA and upon excitation can be used to paint its surface with an
electron dense polymer that enables the 3D ultrastructure of chromatin to be visualized at nearnucleosome resolutions. The first 3D movies of DNA in the interphase nucleus indicate that the
predominant structural unit of chromatin is a 10nm fiber that coalesces into domains of different
densities. We are using ChromEM together with additional labels to visualize how viral and cellular
WEDNESDAY-ORAL PRESENTATIONS
protein complexes remodel chromatin ultrastructure in the nucleus to modulate gene activity in health
and disease. These technological advances are revealing new insights into the structure and organization
of DNA in the nucleus that have broad and exciting applications for cell biology and genomic medicine.