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Late Abstracts 1
2764/L001
Understanding the Spindle Checkpoint and the Error Correction within the Kinetochore
Framework.
S. Santaguida, S. Maffini, C. Vernieri, A. Ciliberto, A. Musacchio; European Institute of Oncology, Milano,
Italy
The Spindle Assembly Checkpoint (SAC) is a safety device that monitors the microtubule-kinetochore
attachment process and delays anaphase onset until all sister chromatids have achieved bipolar
attachment to the mitotic spindle. Protein phosphorylation by mitotic kinases is a crucial event in
regulating SAC functions. Among the core components of the SAC, several proteins have a kinase
domain, such as Aurora B, Bub1, BubR1, Mps1. Here, we report the characterization of two checkpoint
kinases, Aurora B and Mps1, using cell-permeable small molecules selectively targeting their kinase
activities. Firstly, we describe the characterization of a new Mps1 inhibitor named Reversine as a novel
inhibitor of the Spindle Assembly Checkpoint. Importantly, Reversine treatment led to undetectable levels
of Zwilch and Mad1 at unattached kinetochores which is consistent with the powerful SAC override
observed in the presence of this drug. Intriguingly, we found that after a recovery from a STLC block (that
leads to a monopolar spindle whit several synthelic KT-MT attachments), Reversine-treated cells show a
high degree of mis-aligned chromosomes suggesting that Mps1 can play a role in the error correction
mechanism. Aurora B was also implicated in the error correction mechanism whereas its role in the SAC
is controversial. We found that commonly used concentrations of Aurora inhibitors did not lead to a
complete inactivation of Aurora B kinase activity and that the residual activity can still trigger the SAC.
Using the Aurora inhibitor Hesperadin, we noticed that Aurora B is strictly required for the SAC since
complete ablation of its kinase activity leads to severe impairment of localization of SAC proteins at
unattached kinetochores. Finally, our findings suggest that the function of a core component of the SAC
in the response to unattached as well as tensionless kinetochores is intimately intertwined with its role in
the error correction mechanism establishing a new paradigm in the SAC.
2765/L002
comet
and Protein Synthesis.
Regulation of the Anaphase Promoting Complex/Cyclosome by p31
G. Varetti, C. Guida, A. Musacchio; European Institute of Oncology, Milan, Italy
The fidelity of chromosome segregation in eukaryotic cells is ensured by the Spindle Assembly
Checkpoint (SAC). The SAC is a feedback control mechanism that prevents the anaphase onset until all
the chromosomes are correctly attached to the mitotic spindle. The ultimate target of the SAC is to inhibit
the Anaphase Promoting Complex/Cyclosome (APC/C), an E3 ubiquitin ligase whose activity is required
for mitotic exit and chromosome separation. The inactivation of the APC/C by the SAC relies on the
formation of the inhibitory Mitotic Checkpoint Complex (MCC), a complex made up of the SAC proteins
Mad2, BubR1 and Bub3, and of the APC/C coactivator Cdc20. Upon SAC satisfaction, the MCC is
disassembled and anaphase ensues. The mechanisms leading to MCC disassembly are currently under
debate. We combined cell imaging and biochemistry approaches to show that the checkpoint response is
comet
and proteolysis are required for MCC
the result of different opposing forces. The protein p31
comet
continuously
dissociation from the APC/C in cells. As shown by in vitro ubiquitination assays, p31
favors APC/C activity in mitosis, as well as Cdc20 turnover and mitotic slippage. On the other hand, we
find a critical role for protein synthesis in MCC formation and SAC maintenance. Together, our results
indicate that the regulation of MCC concentration in mitosis is controlled dynamically to ensure optimal
APC/C inhibitory capacity as well as rapid mitotic exit when the checkpoint is satisfied.
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2766/L003
Cytoprotective Effect of Trolox against Oxidative Damage in NRK-52e Cells Induced by Melamine.
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H. Yuan , Z. He , L. Wen , C. Guo , J. Yi , L. Yuan , S. Deng , Y. Lu , J. Wu , L. Xue ; College of
2
Veterinary Medicine, Hunan Agricultural University, Changsha, China, Biochemistry and Molecular &
Cellular Biology, Georgetown University Medical Center, Washington, DC
This study was designed to explore oxidative damage of renal NRK-52e cell line induced by melamine
and the protective effect of trolox. Half inhibition concentration (IC50) of melamine was test by MTT
assay. Several groups with various doses of melamine without or with trolox were set up. The cells were
lysated and total SOD activity was determined by NBT staining method. The GSH-Px activity was
detected by UV colorimetric assay and MDA content was determined by thiobarbituric acid assay. LDH
activity in culture medium and reactive oxygen species (ROS) levels of cells were measured. The IC50 of
melamine on NRK-52e cells was 28.22mM. The activity of antioxidant enzymes of SOD and GSH-Px
decreased with the increasing concentration of melamine. Compared to the control group, the activity of
SOD and GSH-Px in 8 mM group decreased significantly, and the other two groups decreased very
significantly. MDA content of 8 mM group was significantly higher, and other two groups of 16 mM and 24
mM was very significantly difference, compared to control group. In protection groups of trolox, SOD
activity of 16 mM group was significantly difference than its toxin group. GSH-Px activity of 24 mM group
was significantly higher than that of melamine-treated group whereas its MDA content was significantly
lower. LDH activity of 8 mM group in culture medium was increased with the rising concentration of
melamine with trolox. ROS levels in 24 mM and 16 mM groups were significantly lower than the control
group. These results suggest that melamine decreases the activity of antioxidant enzymes to accelerate
cell lipid peroxidation and destroys the cell membrane integrity. Melamine promotes the cells to produce
excessive reactive oxygen species, which may cause cell damage and induce cell apoptosis. The
damage induced by melamine decreases by adding membrane protective chemical trolox.
2767/L004
Dehydroepiandrosterone Inhibits Proliferation and Migration and Induces Death of Breast Cancer
Cells.
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2 1
R. López-Marure , J. S. Dillon ; Biología Celular, Instituto Nacional de Cardiología "Ignacio Chávez",
2
México D. F., Mexico, Veterans Affairs Medical Center, University of lowa, Iowa, IA
Cancer invasion and metastasis are the leading causes of mortality in patients with breast cancer.
Dehydroepiandrosterone (DHEA) has a protective role against cancer, however, the mechanism by which
DHEA has this effect remains poorly understood. The present study was aimed at investigating the
actions of DHEA on the proliferation, cell cycle, death and migration of breast cancer cell lines. We used
estrogen receptor (ER) positive MCF-7 cells and ER negative MDA-MB-231 and Hs578T cells for these
studies. Cell proliferation was evaluated by crystal violet staining, cell cycle by flow cytometry, and cell
death by the carboxyfluorescein FLICA analysis of caspase activation. Migration was evaluated by
transwell cell migration and wound assay. We also determined the expression of ECM-1 protein by
western blot and PCR in real time. DHEA inhibited the proliferation of all breast cancer cell lines. This was
associated with an arrest in the G1 phase of the cell cycle and death in MCF-7 cells. There was no
alteration in phase of the cell cycle or death in DHEA treated MDA-MB-231 or Hs578T cells. DHEA also
suppressed the migration of all breast cancer cell lines, independently of the presence of ERs and
decreased the expression of ECM-1 protein in Hs578T cells. These results suggest that the mechanism
of DHEA actions against breast cancer involves the inhibition of cell proliferation and the suppression of
migration, indicating that DHEA could be useful in the treatment of breast cancer.
2768/L005
XLF in the Repair of Replication-Dependent DNA Double-Strand-Breaks and Senescence.
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D. Liu , L. Chen , X. Deng , Y. Cong , X. Xu ; College of Life Science, Capital Normal University,
2
Beijing, China, College of Life Science, Beijing Normal University, Beijing, China
DNA double-strand-breaks (DSBs) are the most serious DNA damage, which can be repaired mainly by
two pathways, namely homologous recombination repair (HR) and non-homologous-end-joining (NHEJ).
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Failure to repair DSBs can lead to genome instability which is fundamentally important in senescence in
all eukaryotes. XLF (XRCC4-1ike factor) is a newly identified NHEJ protein, whose loss-of-function
mutations result in human immunodeficiency with microcephaly. It has been reported that XLF facilitates
repair of replication-independent DSBs induced by ironing radiations. In this report, we found that
depletion of XLF expression by siRNA impaired the repair capacity of DSBs induced by CPT (a
topoisomerase I inhibitor), sensitized cells to DNA replication inhibitors, and increased senescence. We
also found that XLF is a positive regulator of its transcription. Taken together, these results indicate that
XLF may play a role in the repair of replication-dependent DSBs and senescence.
2769/L006
Biochemical Characterization of the Immunoisolated Bi-Lobe of Trypanosoma brucei.
L. Gheiratmand, C. He; NUS, SINGAPORE, Singapore
Trypanosoma brucei is an infectious pathogen causing African sleeping sickness in humans and Nagana
in cattle. Understanding how this single-celled parasite duplicates and divides its single-copy organelles
during the cell cycle is important for the development of effective parasiticidal strategies. A bi-lobed
structure was previously shown to be important for the tight regulation of the organelle duplication and cell
division. Several conserved proteins with functional implications in cell cycle such as TbCentrin2,
TbCentrin4 and a homologue of the Polo-like kinase TbPLK1 have been identified on the bi-lobe,
supporting a critical role of this structure. More recently, a new bi-lobe component TbLRRP1 was
identified in our lab by comparative proteomic methods.It exclusively labelled the bi-lobe and was
therefore used to immunoisolate the structure for further biochemical and morphological studies.
Preliminary studies showed that the flagellar pocket collar (FPC) protein TbBILBO1 cofractionated with
TbLRRP1 through the purification steps, suggesting a tight association between the bi-lobe and the FPC.
2770/L007
Protective Effects of Thymoquinone on the Neuronal Injury in Frontal Cortex after Chronic
Toluene Exposure.
M. Kanter; Histology and Embryology, Faculty of Medicine, Trakya University, Edirne, Turkey
Objective: The aim of this study was designed to evaluate the possible protective effects of thymoquinone
(TQ) on the neuronal injury in the frontal cortex after chronic toluene exposure in rats. Methods: The rats
were randomly allotted into one of three experimental groups: A (control), B (toluene treated) and C
(toluene treated with TQ); each group contain 10 animals. Control group received 1ml serum physiologic
and toluene treatment was performed by inhalation of 3000 ppm toluene, in a 8 hr/day and 6 day/week
order for 12 weeks. The rats in TQ treated group was given TQ (50 mg/kg body weight) once a day orally
for 12 weeks starting just after toluene exposure. Tissue samples were obtained for histopathological
investigation. Results: To date, no histopathological changes of neurodegeneration in the frontal cortex
after chronic toluene exposure in rats by TQ treatment have been reported. In this study, the morphology
of neurons in the TQ treatment group was well protected. The caspase 3 immunopositivity was increased
in degenerating neurons of the frontal cortex tissues following toluene exposure. Treatment of TQ
markedly reduced the immunoreactivity of degenerating neurons after chronic toluene exposure. The
amount of neuronal cells apoptosis was quantitatively higher in the toluene treated group than the control
group. Treatment of TQ markedly reduced the reactivity and the number of apoptotic cells (TUNEL
positive cells). Chronic toluene exposure caused severe degenerative changes, shrunken cytoplasma,
severely dilated cisternae of endoplasmic reticulum, markedly swollen mitochondria with degenerated
cristae and nuclear membrane breakdown with chromatin disorganization in neurons of the frontal cortex.
Conclusions: We conclude that TQ therapy causes morphologic improvement on neurodegeneration in
frontal cortex after chronic toluene exposure in rats. We believe that further preclinical research into the
utility of TQ may indicate its usefulness as a potential treatment on neurodegeneration after chronic
toluene exposure in rats.
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2771/L008
SYTOX® Blue for Dead Cell Discrimination with RFP Expressing Cells.
S. Jaron, P. Melquist; Life Technologies, Eugene, OR
There is a growing awareness of the need for a dead cell discriminator when assessing cells expressing
Red Fluorescent Protein (RFP) using flow cytometry. However, for the user without a UV or Violet laser,
there is no good solution for determining dead cells with RFP. The gold standard for dead cell
identification by flow is Propidim Iodide (PI), but its emission is broad and often uses multiple channels,
including the optimal channel for RFP emission. SYTOX® Green is another common option for 488 nm
excitation, but it too has a large overlap into the RFP channel that is difficult to compensate for when
combined with low cellular expression of RFP. However, it turns out that SYTOX® Blue, a dead cell
discriminator developed for use with a 405 nm excitation, has little overlap in the RFP channel and is
adequately excitable using 488 nm light. Herein, we demonstrate that SYTOX® Blue gives accurate and
reproducible results with a 488 nm excitation, and that the minimal bleed-through into the RFP channel
makes it an excellent choice for dead discrimination for RFP expressing cells. Mammalian cells were first
transduced with virus containing an RFP vector at varying concentrations and then measured on the BD
FACSCalibur™ with and without the addition of SYTOX® Blue, PI, or SYTOX® Green. In all cases, gating
was varied to include all events, or a subset of the total events, to prove that: (1) SYTOX® Blue provides
accurate and reproducible dead cell statistics when excited by the 488 nm laser; (2) unlike SYTOX®
Green, minimal compensation of the SYTOX® Blue out of the RFP channel is necessary; and (3)
discrimination of dead cells using SYTOX® Blue leads to more accurate information about expression
levels in the cell population over simply gating by Forward vs Side Scatter (FSC vs SSC). In conclusion,
we demonstrate the importance of using a fluorescent method to identify dead cells when determining the
population statistics of cells expressing RFP as well as the usefulness of SYTOX® Blue as that dead cell
discriminator.
2772/L009
Lats2 Links Chk1-Mediated DNA Damage Signaling Pathway to the P-Body Formation through
Phosphorylation of 14-3-3.
H. Suzuki, N. Okada, S. Mukai, N. Yabuta, H. Nojima; Osaka University, Osaka, Japan
Proper response to DNA damage is essential to maintain the integrity of the genome and to avoid cancer
malignancy. Lats2 (Large tumor suppressor 2), a member of the conserved Lats kinase family, is a
human tumor suppressor gene. Previous studies elucidate that Chk1 and Chk2 is phosphorylated by
ultraviolet (UV) irradiation. Here we show that in response to ultraviolet (UV) radiation, Lats2 is
phosphorylated by Chk1 and Chk2 at S408. Additionally, we have found Lats2 which is phosphorylated at
S408 induced phosphorylation of 14-3-3 proteins at S59 (p14-3-3), namely, I identified a novel Chk1Lats2-14-3-3 signaling pathway. Immunocytochemical studies to investigate the role of p14-3-3 have
revealed that p14-3-3 translocates to the P-body where mRNA degradation, translational repression, and
mRNA surveillance occur, and these fluorescence signals were enhanced in response to UV damage.
Furthermore, the depletion of Lats2 or 14-3-3 reduced the P-body localization of a putative P-body
scaffold protein, GW182. This suggests that p14-3-3 has functions as a novel important scaffold protein
for P-bodies. Moreover, U2OS/Lats2 S408A mutants increased cell death as compared with S408D
mutants and WT. Based on these findings, we propose that the Chk1/2-Lats2-14-3-3 axis is a novel DNA
damage signaling pathway that is critical for regulation of P-body assembly. In addition, this signaling
pathway may suppress cell death and promote the repair of DNA damage by UV radiation.
2773/L010
Inhibition of Plk1 Induces Mitotic Infidelity and Embryonic Growth Defects in Developing Zebrafish
Embryos.
K. Jeong; Seoul National University, Seoul, Republic of Korea
Polo-like kinase 1 (Plk1) has crucial functions in mitotic progression. Most of Plk1’s function in vertebrates
has been done in cultured cell lines and some in knockout mice. Mice deficient in Plk1 allele are
embryonic lethal, making it difficult to define the actual function of Plk1 in vertebrate development.
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Because it is well conserved from fish to human, we attempted to reveal the in vivo functions of Plk1 in
vertebrate development in zebrafish embryos. The embryos of the zebrafish are transparent and the eggs
are laid ex utero without limitation in numbers. Therefore, we attempted to visualize mitosis in developing
embryos at the whole organism level. Taking the advantages of zebrafish embryos, we here show the
essential functions Plk1 in vivo. First, we generated transgenic fish that expresses GFP-H2B. Then timelapse microscopy was performed in GFP-H2B transgenic zebrafish embryos with morpholino injections.
Combining all the cell biological tools, including imaging technique, we show that Plk1 is essential for the
mitotic fidelity in growing embryos. Plk1 morphant embryos display mitotic infidelities such as abnormal
centrosomes and irregular spindle assembly. Furthermore, we observed severe mitotic infidelity when the
embryos were soaked in BI 2536, a recently developed Plk1-specific small molecule inhibitor. These
results reveal that zebrafish embryos are valuable tools in validating mitotic kinase inhibitors. Collectively,
we propose that zebrafish will be an efficient and economical in vivo system for studying mitosis and in
validating anti-mitotic drugs.
2774/L011
The N-Terminally Truncated Lats1 Kinase Causes Chromosomal Instability and Tumorigenesis in
Nude Mice.
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S. Mukai , N. Yabuta , H. Suzuki , N. Okada , D. Miura , H. Nojima ; Dep.of Mol. Genet., RIMD, Osaka
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Univ., Suita, Japan, Dep. of Pharm., Hyogo University. of Health Sci., Kobe, Japan
Lats1 and Lats2 kinases are the tumor suppressors that regulate cell cycle progression and apoptosis
through “Hippo pathway”. Lats1and Lats2 share 85% sequence identity in the kinase domain, but their
non-kinase regions at the N-terminus are distinct except for Lats conserved domain 1 (LCD1) and LCD2.
Thus, their N-terminal regions may be important for Lats1/2-specific functions. Here, we report on
ΔN/ΔN
knockout (KO) mice that express N-terminally truncated Lats1 protein. Our previous study
Lats1
-/-/revealed that Lats2 KO (Lats2 ) mice were embryonic lethal and the Lats2 mouse embryonic fibroblasts
(MEFs) were deficient in mitosis showing centrosome fragmentation and cytokinesis defects that are
-/ΔN/ΔN
associated with nuclear enlargement and multinucleation. Unlike Lats2 KO mice, however, Lats1
ΔN/ΔN
KO mice were born safely and grew normally with slightly smaller body size. Nonetheless, Lats1
MEFs harbored drastic defects in mitosis, showing enhanced centrosome overduplication, chromosomal
misalignment, multipolar spindle formation, chromosome bridging, and cytokinesis failure. Moreover, they
displayed accelerated cell cycle, formed foci in tissue culture plate like transformed cells, and exhibit
anchorage independent growth in nano culture plate (NCP) that allows three-dementional cell culture, and
in soft agar. Moreover, these MEFs produced tumors in nude mice after subcutaneous injection, although
the tumor growth rate was slower than ordinary cancer cells. Interestingly, the abnormal centrosome
overduplication and multipolar spindle formation became less apparent in proportion to the progression of
population doubling level (PDL). Furthermore, the tumor size was suppressed in proportion to the
accumulated cell passage, suggesting that abnormal centrosome amplification is closely associated with
the tumor size in nude mice. Taken together, these results indicate that the N-terminal region of Lats1 is
required for chromosomal stability and tumor suppression.
2775/L012
Egr1 Mediates P53-Independent C-Myc-Induced Apoptosis via a Noncanonical ARF-Dependent
Transcriptional Mechanism.
D. Boone, Y. Qi, Z. Li, S. Hann; Vanderbilt, Nashville, TN
c-Myc is frequently deregulated in human cancers. While deregulated c-Myc leads to tumor growth, it also
triggers apoptosis in partnership with tumor suppressors such as ARF and p53. Apoptosis induced by cMyc is a critical fail-safe mechanism for the cell to protect against unrestrained proliferation. Despite the
plethora of information on c-Myc, the molecular mechanism of how c-Myc induces both transformation
and apoptosis is unclear. Oncogenic c-Myc can indirectly induce the expression of the tumor suppressor
ARF, which leads to apoptosis through the stabilization of p53, but both c-Myc and ARF have proapoptotic activities that are independent of p53. In cells without p53, ARF directly binds to c-Myc protein
and inhibits c-Myc-induced hyperproliferation and transformation with a concomitant inhibition of
canonical c-Myc target gene induction. However, ARF is an essential cofactor for p53-independent c-
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Myc-induced apoptosis. Here we show that ARF is actually necessary for c-Myc to drive transcription of a
novel noncanonical target gene, Egr1. In contrast, c-Myc induces another family member, Egr2, through a
canonical mechanism that is inhibited by ARF. We further demonstrate that Egr1 is essential for p53independent c-Myc-induced apoptosis, but not ARF-independent c-Myc-induced apoptosis. Therefore,
ARF binding switches the inherent activity of c-Myc from a proliferative to apoptotic protein without p53
through a novel noncanonical transcriptional mechanism. These findings also provide evidence that
cofactors can differentially regulate specific transcriptional programs of c-Myc leading to different
biological outcomes.
2776/L013
NECC2 As a Novel Component of Caveolae in PC12 Cells. Role in NGF-Mediated PC12 Cell
Differentiation.
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J. A. Diaz-Ruiz , Y. Rabanal , Y. Jimenez-Gomez , D. Cruz-Garcia , J. R. Peinado , R.
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Vazquez-Martinez , M. M. Malagon ; Dept. Cell Biology, Physiology and Immunology, University of
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Cordoba, Cordoba, Spain, IMIBIC, Cordoba, Spain, CIBERobn, ISCIII, Spain
Objective: We report the molecular characterization of Neuroendocrine long-coiled coil protein 2
(NECC2), which shows preferential expression in endocrine and neuronal tissues and displays structural
characteristics of a scaffolding factor. We analyzed NECC2 in relation to TrkA receptor signalling and
actin dynamics in the neuroendocrine cell line PC12. In these cells, TrkA receptor is compartmentalized in
specialized microdomains of the plasma membrane, the caveolae, which are organized by cortical actin
and scaffold proteins such as caveolin-1. Methods: Colocalization studies were performed by
immunocytochemistry against NECC2 combined with transfection of a GFP-TrkA construct. The
importance of actin organization in NECC2 localization was assessed by disrupting actin cytoskeleton
with 5 μM latrunculin B for 30 min. To analyze the effect of NECC2 overexpression in NGF-induced
phosphorylation of ERK1/2 and Akt, cells were transfected with c-myc-NECC2 or c-myc constructs and
protein extracts were subjected to western blotting for the downstream TrkA-effectors ERK1/2/phosphoERK1/2 (pERK1/2) and Akt/phospho-Akt (pAkt). Results: Confocal microscopy revealed that NECC2
distributes within the cytosol in most PC12 cells under basal conditions, yet some cells exhibited NECC2
immunofluorescence in close apposition to the cell surface, wherein it colocalizes with caveolin-1.
Treatment of PC12 cells with latrunculin-B led to a loss of NECC2 and caveolin-1 colocalization,
suggesting that actin cytoskeleton is necessary for NECC2 membrane localization. Interestingly,
treatment with NGF (100 ng/ml) for 0-120 min revealed a time-dependent translocation of NECC2 to the
cell surface, wherein it overlapped with TrkA. Moreover, NECC2 overexpression decreased by 50% the
NGF-induced phosphorylation of ERK1/2 after 120 min NGF treatment, but did not affect Akt
phosphorylation. Conclusion: Taken together, our data indicate that NECC2 participates as a molecular
scaffold in caveolae organization and support the involvement of this protein in the regulation of
NGF/TrkA signalling. Support: MICINN/FEDER (BFU2007-60180/BFI), FIS/ISCIII (CIBEROBN CB03/06),
J. Andalucía (CVI-0139, P07-CTS-03039), Spain.
2777/L014
Hmd Activity Is Compromised by a Knockout of hcgd in M. maripaludis.
D. M. Pak; Department of Biology, Swarthmore College, Swarthmore, PA
Methanococcus maripaludis is a hydrogenotrophic organism in the methanogenic Archaea group, also
known as methanogens, which are strictly anaerobic organisms. This methanogen has a complex energyproducing metabolic pathway in which it fully reduces CO2 into methane with the oxidation of H2 or
formate. A key intermediate step of this pathway is a redox reaction involving the reduction of methenylH4MPT+ to metheylene-H4MPT and the oxidation of H2 to H+. This step is catalyzed by [Fe]hydrogenase, encoded by hmd, which exists as a homodimer with two intertwined units and two active
sites. It is only enzymatically active when bound to an essential iron guanylylpyridine cofactor (FeGP).
The genes involved in the biosynthesis of this cofactor are hypothesized to be the seven hcgs (hmd cooccurring genes) found in almost all methanogens with hmd. Knockouts of hmd and hcgA in a ΔfrcΔfru
background have shown that a ΔhcgA mutant follows a very similar growth pattern to a Δhmd mutant.
This similarity indirectly indicates that a ΔhcgA mutant compromises hmd activity. Both mutants also
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display a slow growth phenotype compared to the background ΔfrcΔfru strain. These results suggest that
the seven hcgs are somehow involved in some unknown pathway that synthesizes the FeGP cofactor
essential for the activity of the [Fe]-hydrogenase encoded by hmd. We constructed a ΔhcgD mutant in the
ΔfrcΔfru background to further support the theory that the seven hcgs are involved in the biosynthesis of
the essential FeGP cofactor. The ΔhcgD mutant displayed a slow growth phenotype similar to the
phenotypes of the Δhmd and ΔhcgA mutants, indirectly indicating that: 1) A ΔhcgD mutant compromises
hmd activity and 2) HcgD is involved in the biosynthesis of the FeGP cofactor, further supporting the
theory that all seven hcgs are involved in an unknown pathway that synthesizes the cofactor.
2778/L015
Quantification of CENH3/CENP-A and NDC80 Complexes in Individual Arabidopsis Kinetochores.
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J. S. Ramahi , R. Maruthachalam , P. N. Kwong , S. Chan ; Plant Biology, UC Davis, Davis, CA,
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Chemistry and Biochemistry, UCLA, Los Angeles, CA
Kinetochores mediate chromosome segregation in both mitosis and meiosis. Saccharomyces cerevisiae
kinetochores assemble on a single nucleosome containing the centromere-specific histone CENH3,
allowing their protein stoichiometry to be measured using quantitative fluorescence microscopy. The
number of CENH3 nucleosomes in large animal and plant kinetochores based on simple tandem repeats
is unknown. We replaced Arabidopsis thaliana CENH3 and NUF2 with GFP-tagged proteins, and
quantified their stoichiometry using yeast GFP-tagged lines as fluorescence standards. During mitosis,
individual Arabidopsis kinetochores contained 350 GFP-CENH3 molecules, and 150 NUF2-GFP
molecules. Therefore, kinetochores assemble on a small subset of centromere DNA, and the ratio of
CENH3 nucleosomes to microtubule binding proteins differs from that in budding yeast. Interphase
kinetochores have similar size to metaphase kinetochores, but some are substantially smaller and may
have been remodeled. Meiosis I kinetochores are paired, and contain fewer CENH3 molecules. Our
results show kinetochore structure is fundamentally different between yeast point kinetochores and large
kinetochores of animals and plants.
2779/L016
The Cdh1 Inhibitor Acm1 Is Required for Anaphase Spindle Integrity.
J. S. Martinez, M. C. Hall; Biochemistry, Purdue University, West Lafayette, IN
The anaphase-promoting complex (APC) is a cell cycle-regulated ubiquitin ligase with important functions
in promoting passage through mitosis and establishment of a stable G1 phase. APC activity during mitotic
cell division is controlled by the availability of the activator proteins Cdc20 and Cdh1, which function in
part by helping to recruit specific substrates to the APC for ubiquitination. Activator proteins are the
primary targets for regulating APC activity during the cell cycle and a common mechanism for regulation
of these proteins is inhibition by pseudosubstrates. We have been characterizing a yeast
pseudosubstrate inhibitor of Cdh1 called Acm1. Although much of the biochemical mechanism and cell
cycle regulation of Acm1 is known, its biological importance has been unclear because no phenotype has
been reported for yeast cells lacking Acm1. Here were demonstrate that Acm1 is important for
maintaining spindle integrity during anaphase, particularly under conditions that delay anaphase
completion due to mis-positioning of the nucleus. Cells lacking Acm1 exhibit extensive spindle breakage
and abnormal morphologies when arrested in late anaphase due to inactivation of the mitotic exit
network. We present evidence suggesting that spindle instability in cells lacking Acm1 impairs proper realignment of mis-positioned spindles. The spindle integrity defects are dependent on Cdh1, but
surprisingly, we have not yet found any known APC substrates that are destabilized in the absence of
Acm1. Since Acm1 competitively inhibits substrate binding to Cdh1, our results suggest either that
destabilization of an unknown Cdh1 target is responsible for the observed phenotypes, or that Cdh1
inappropriately binds and interferes with substrate function independent of APC in the absence of Acm1.
We are currently testing these two possibilities. The spindle integrity defect is the first reported phenotype
of cells lacking Acm1 and explains why this protein has been widely conserved in budding yeast species.
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2780/L017
The Cell Cycle-Dependent Expression and Localization of LAMMER Kinases Dsk1p and Kic1p.
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L. Taggart-Murphy , J. Portillio , M. Luca , B. Ngo , C. Chang , H. Palomino , E. Sachdev , T. Wen , J.
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Murray , A. Carr , Z. I. Tang ; W.M. Keck Science Center, The Claremont Colleges, Claremont, CA,
2
Genome Damage and Stability Center, University of Sussex, Falmer, United Kingdom
LAMMER kinase family members, Dsk1p and Kic1p, are the orthologues of human SRPK1 and
mammalian CLK1, respectively in fission yeast Schizosaccharomyces pombe. Previous studies
demonstrated their overlapping functions in cell growth, mitosis, and cytokinesis. We hypothesize that if
the Dsk1p and Kic1p play roles in nuclear division and cell separation, their expression and cellular
localization may alter to perform the temporal tasks during the cell cycle. The goal of our investigation is
to determine the expression and localization patterns of the two protein kinases during the cell cycle as to
gain more understanding of their functions. To accomplish this goal, we TAP (tandem affinity purification)and GFP (green fluorescent protein)-tagged dsk1+ and kic1+ genes at their genomic loci. We also used
ectopic expression systems by transforming plasmids containing the genes that encode the N-terminal
TAP- and GFP-tagged kinases into fission yeast wild-type (wt) and some temperature-sensitive (ts) cdc
strains. In addition to observing the cellular localization of GFP-tagged Dsk1p and Kic1p by fluorescence
microscopy, we generated their expression profiles during the cell cycle by detecting the TAP-tagged
kinases. Our studies have shown that both proteins are mainly concentrated in the nucleus. Dsk1p can
shuttle between nucleus and cytoplasm, and it is enriched in the cytoplasm during G2 and S phases.
Furthermore, using centrifugal elutriation for cell synchronization, we have shown that the
phosphorylation pattern and expression level of Dsk1p and Kic1p change during the cell cycle.
Interestingly, both proteins are expressed at high level during late mitosis and cytokinesis, consistent with
their roles in the cell cycle of fission yeast. Although the cellular localization of Kic1p during cell cycle
remains to be further determined, these data allow us to conclude that the two kinases play important
roles in both nucleus and cytoplasm, and their functions are regulated in a cell cycle-dependent manner.
2781/L018
Importazole, a Small Molecule Inhibitor of the Ran-Importin-β Pathway in Interphase and Mitosis.
1
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J. F. Soderholm , S. Bird , Y. Sampathkumar , P. Kalab , M. Uehara-Bingen , K. Weis , R. Heald ;
1
2
Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, The Small Molecule
Discovery Center, University of California, San Francisco, San Francisco, CA
During interphase, the transport receptor importin-β carries cargoes into the nucleus. In the nucleus, it
binds to RanGTP and releases the cargo. A similar mechanism operates in mitosis to generate a gradient
of active spindle assembly factors around mitotic chromosomes. Though the Ran pathway is crucial
throughout the cell cycle, the precise roles of this pathway during different stages of the cell cycle are
poorly understood. Small molecules present a promising new method of studying this pathway in mitosis.
We implemented a Fluorescence Resonance Energy Transfer (FRET)-based, high-throughput small
molecule screen to find compounds that interfered with the interaction between RanGTP and importin-β.
We identified importazole, a 2,4-diaminoquinazoline, a reversible small molecule inhibitor specific to the
RanGTP/importin-β pathway. Importazole blocks importin-β-mediated nuclear import both in Xenopus egg
extracts and cultured cells, without disrupting transportin-mediated nuclear import or Crm1-mediated
nuclear export. During mitosis, importazole causes both predicted and novel defects in spindle assembly.
Biochemical assays indicate that importazole binds importin-β and causes a conformational change that
alters its interaction with Ran. Together, these experiments point towards importazole as a valuable tool
for evaluating the function of the Ran pathway at specific stages during the cell cycle.
2782/L019
Aurora B Kinase Interactions and Functions Prior to Mitosis.
D. Nash, S. D. Slattery, R. M. Hall, B. R. Brinkley; Molecular and Cell Biology, Baylor College of
Medicine, Houston, TX
Aurora B kinase is a key factor regulating multiple events in mitosis including the mitotic spindle assembly
checkpoint and cytokinesis. In our investigations, we have identified novel signaling pathways, interacting
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partners, and substrates for Aurora B kinase and have determined that it may have additional functions in
stages of the cell cycle preceding mitosis. Utilizing standard affinity purification and coimmunoprecipitation we identified a DNA/RNA helicase, DHX9, as a novel interacting partner for Aurora B
kinase. DHX9 domains were expressed as recombinant protein and utilized as substrate for Aurora B
kinase in in vitro kinase assays and the C-terminal domain was found to be robustly phosphorylated.
Immunofluorescent localization revealed a partial colocalization of DHX9 and Aurora B kinase in
interphase. Further investigation revealed Aurora B kinase accumulation at sites of replication. Inhibition
of Aurora B kinase during S phase results in a delay during mitosis. These data suggest that Aurora B
kinase has a functional role during S phase.
2783/L020
Mitotic Slippage in Non-Transformed Human RPE1 Cells Does Not Require Caspase Activity.
K. Lee, C. L. Rieder; Wadsworth Center, Albany, NY
Based on indirect studies of HeLa and other cancer cell lines it has been suggested that various
caspases are required during mitosis for proper spindle assembly, mitotic checkpoint (MC) function and/or
chromatid distribution. To directly test this idea we have followed non-transformed human RPE1 cells as
they entered and completed mitosis in the presence of small molecule broad-spectrum (QVD-OPh or ZVAD (OMe)-FMK) or individual caspase inhibitors, as well as after knocking down selected caspases by
RNAi. These live cell studies revealed that individual or broad-spectrum caspase inhibitors neither
prolonged mitosis nor lead to defects in chromosome segregation, and neither did knocking down
caspases individually or in combination. From these data we conclude that caspase activity is not
required for a normal mitosis. When RPE1 were treated with nocodazole or dimethylenastron (an Eg5
inhibitor) concentrations that prevented satisfaction of the MC, 20-25% died in mitosis while the
remainder slipped into the next G1 after approx. 21 hrs. Under these conditions broad spectrum caspase
inhibitors completely prevented cell death in mitosis, and death was also largely prevented by
simultaneously knocking down both caspase 3 and 9. Thus, when MC cannot be satisfied death of RPE1
in mitosis is mediated entirely by apoptosis. When the MC cannot be satisfied the duration of mitosis in
the presence of broad-spectrum caspase inhibitors, or after simultaneously knocking down various
caspases, was never longer than controls (approx. 21 hrs), although it was sometimes 25% shorter (15
versus 21 hrs) as after simultaneously knocking down C3 and C9. Thus caspase activity is not required
for a functional MC. These novel findings combined with previous data that BubR1 and MAD2 are not
destroyed in RPE1 during mitotic slippage (Brito and Rieder, Curr Biol., 16:1194-2000, 2006), imply that
slippage in RPE1 cells is not precipitated by the caspase-mediated destruction of MC proteins as
suggested by others for HeLa.
2784/L021
Differential Activation of Neuregulin1-ErbB Downstream Effectors by Regulation of Receptor
Endocytosis in Schwann Cells.
K. Reddy, H. A. Kim; Biology, Rutgers University, Newark, NJ
Neuregulin1-ErbB interaction is one of the major regulators of Schwann cell (SC, the myelinating glia of
the peripheral nervous system) development and function. Neuregulins expressed by the neurons bind to
the ErbB2 and ErbB3 receptors on the SCs to elicit diverse functions. Neuregulin1 is classified into 3
types of which, type I and II (GGF) are soluble, while type III (CRD-Nrg1) is membrane-tethered. CRDNrg1 is involved in promoting myelination via the PI3kinase pathway, while, high-doses of soluble GGF
are known to inhibit myelination via the Erk1/2 pathway. Both neuregulins bind the same receptor
complex. The mechanism by which activation of the ErbB receptor complex elicits multiple biological
functions in SCs is unclear. Differential activation of receptor downstream signaling pathways is often
mediated by regulation of ligand-dependent receptor trafficking. Studies in other cell types have shown
that receptor endocytosis is required for Erk1/2 signaling, while PI3kinase signaling is predominantly
occurring at the cell surface. However, the fate of activated ErbB2 is not known in SCs. We hypothesized
that membrane-bound CRD-Nrg1 sustains membrane-associated PI3kinase signaling whereas soluble
GGF causes internalization of receptors thereby mediating Erk1/2 activation. To test this, we first studied
GGF-induced receptor degradation and show that both ErbB2 and ErbB3 show dose-dependent
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degradation. ErbB receptor internalization was confirmed by biotinylation assays, immunocytochemistry
and confocal imaging on live cells. We then analysed the kinetics of Akt and Erk1/2 activation in the
presence of both endocytosis inhibitor drugs (Monodansylcadaverine (MDC)& Dynasore) and
temperature-sensitive dynamin-1 mutants. Inhibition of endocytosis by the use of drugs and temperaturesensitive dynamin-1 mutants showed an attenuation in kinetics of Akt and not Erk1/2 signaling. This
suggests a role for ErbB receptor endocytosis in regulating Akt activation in Schwann cells.
2785/L022
Response of Proliferating and Quiescent Fibroblasts to Proteasome Inhibition.
A. Legesse-Miller, E. Haley, I. Raitman, D. Wang, B. Lund, D. Wolle, A. Liao, H. Coller; Molecular
Biology, Princeton University, Princeton, NJ
Quiescence, the reversible arrest of cell proliferation, is critical for normal cellular development and tissue
maintenance. Quiescence is also a mechanism for cellular survival. For example, certain subsets of cells
within a tumor, through quiescence, avoid apoptosis normally induced by anticancer agents. We
hypothesized that proliferating and quiescent cells may have distinct functional requirements for protein
catabolism pathways. We have found that quiescent fibroblasts are resistant to proteasome inhibition
mediated cell death. This result suggests that, in quiescent fibroblasts, other pathways may be activated
to protect quiescent cells from proteasome inhibition mediated cell death. This finding is of particular
interest because proteasome inhibitors have been used for treatment of specific tumor types. Therefore,
understanding the mechanisms by which cells are resistant to proteasome inhibition is of basic and
clinical importance. We discovered that in primary fibroblasts proteasome inhibition and quiescence
greatly increase autophagy and MnSOD, a reactive oxygen species (ROS) detoxifying enzyme.
Proteasome inhibition in proliferating fibroblasts leads to induction of ROS, DNA damage and apoptotic
cell death. However, in quiescent fibroblasts, proteasome inhibition leads to reduced ROS levels,
selective up-regulation of MnSOD, and less DNA damage. Inhibition of autophagy leads to induction of
DNA damage both in proliferating and quiescent cells. These results suggest that in quiescent cells, ROS
detoxifying pathways and autophagy may serve as protective mechanisms to maintain quiescent cell
viability and reversibility in adverse conditions.
2786/L023
Construction of a Cell-Based Systems Model of a Triple Negative Breast Cancer Cell Line.
M. Y. Fink, M. Sapre; Biomedical Sciences, LIU-CW Post, Brookville, NY
Triple-negative breast cancer currently has no targeted therapeutic options. The current study was
undertaken in order to comprehensively map an individual triple-negative cell model in order to elucidate
regulatory properties/interactions amenable to targeting. All literature references for BT20 cells were
curated into a network using CellDesigner software. Initial construction of the network from the literature
provided a picture of “what we know”. This was further expanded by including proteins that are
transcriptionally upregulated in these cells as assessed by gene expression assays from public
databases. This network was used to identify prominent signaling features of BT20 cells. (1) Several
receptor-ligand pairs were both identified in the literature and found to be overexpressed. These include
IGF1-R, EGFR, and MET. (2) We identified multiple interactions between VDR and receptor activity. (3)
This approach identified several tumor/metastasis suppressor pathways such as TGFβ and Kiss-1. (4)
This approach approximates at least 25 receptor systems operating in the BT20 cell line. Ongoing work is
investigating the connections between the Oxytocin receptor, which we show as upregulated in these
cells, and intracellular signaling pathways. This study presents the first systems-level analysis of a Triplenegative cancer cell and provides a basis for further focused investigation and preclinical discovery.
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2787/L024
Curcumin Inhibits Fibroblast Proliferation and Migration and Induces Apoptosis in a CathepsinDependent Manner.
1,2
1,2 1
2
D. Zhang , D. Bromme ; The University of British Columbia, Vancouver, BC, Canada, OBMS, UBC,
Vancouver, BC, Canada
Aim: Fibroblast proliferation and migration play a key role in pulmonary fibrosis. Preliminary studies
revealed that curcumin, an antioxidant from the Indian spice tumeric can protect mice from bleomycin
induced pulmonary fibrosis. However, little is known how curcumin relates to apoptosis in fibroblasts.
Methods: Bleomycin stimulated fibroblasts were exposed to different concentrations of curcumin. MTT
was used to study fibroblast proliferation. A cell wound healing assay was employed to study fibroblast
proliferation and migration. Western blotting was used to evaluate the expression of cathepsins K and L,
TGF-b1, bcl-2, bax, and caspase-3. FACS analysis was used to analysis cell cycle distribution. Results:
At various concentrations, curcumin can inhibit fibroblast proliferation, slow down the wound healing,
especially at 30µm (p<0.01). Cell cycle distribution was changed with the treatment of curcumin.
Curcumin stimulates a three-fold increase in cathepsins K and L expression, and a two-fold decrease in
the expression of TGF-b1. The expression of caspase-3 and the ratio of bax/bcl-2 were increased twofold in a curcumin dose-dependent manner. A potent cathepsin inhibitor was able to rescue cells from
death. Conclusion: Curcumin can inhibit bleomycin induced fibroblast proliferation and migration, and
affects the cell cycle. These effects are associated with an increasing expression of cathepsins L and K, a
decreasing expression of TGF-b1and an induction of apoptosis in fibroblasts. Key Words: Curcumin,
Cathepsins, TGF-b1, Caspase-3
2788/L025
Cripto-1 Enhances the Canonical Wnt/β-Catenin Signaling Pathway in Mouse Embryonal
Carcinoma F9 Cells.
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2
T. Nagaoka , K. Watanabe , M. Gonzales , N. Castro , M. Rangel , H. Karasawa , N. Held , J. S. Rubin ,
1
1 1
D. S. Salomon , C. Bianco ; Mammary Biology and Tumorigenesis Laboratory, National Cancer Institute,
2
Bethesda, MD, Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD
Cripto-1 is a membrane bound protein that belongs to the epidermal growth factor (EGF)- Cripto/FRL1/cryptic protein family. Cripto-1 is a co-receptor for the transforming growth factor-β related protein Nodal
and together with Nodal induces Smad-2/3 phosphorylation through the serine-threonine kinase receptor
ALK-4. Furthermore, Cripto-1 can also bind to Glypican-1, Caveolin-1, GRP78, TGFβ1, Activin, Notch and
Wnt11 proteins. Cripto-1 is important for the self-renewal and maintenance of embryonic stem cells and is
associated with the initiation and progression of carcinomas in a number of tissues including the colon
and breast. These properties are also associated with an activated canonical Wnt/β-catenin signaling
pathway which can regulate Cripto-1 expression. We therefore investigated the stabilization of β-catenin
that is a central effector of the canonical Wnt signaling pathway, using mouse embryonal carcinoma F9
-/cells that express high levels of endogenous Cripto-1 protein and Cripto-1 null F9 (F9 Cr-1 ) cells.
Interestingly, we found that F9 cells contained a greater amount of stabilized cytoplasmic β-catenin than
-/F9 Cr-1 cells after the cells were treated with Wnt3a. Moreover, transfection of the β-catenin-TCF/LEF
reporter gene super TOP-FLASH also revealed a significant increase in luciferase activity in F9 cells
-/compared to F9 Cr-1 cells following Wnt3a treatment. We treated F9 cells with Wnt3a and the ALK-4, 5
and 7 inhibitor SB-431542 which had no significant effect on activation of TOP-FLASH activity in
response to Wnt3a treatment. This result suggests that the canonical Wnt signaling pathway is
independent from Smad-2/3 pathway in F9 cells. We transfected Cripto-1 and the Wnt co-receptors Lrp5
or Lrp6 into 293T cells and performed co-immunoprecipitation assays. We found that both Lrp5 and Lrp6
are precipitated with Cripto-1. In conclusion, our results demonstrate that Cripto-1 can enhance the
canonical Wnt signaling pathway in mouse embryonal carcinoma cells by modulating Lrp5 and Lrp6
activity. Therefore, modulation of the canonical Wnt pathway by Cripto-1 might perform an important role
in embryonic stem cells and embryonal carcinoma cells that are expressing high levels of Cripto-1.
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2789/L026
Weighing the Effects of mTOR Inhibition on the Growth Cycle of Single-Cells.
1,2
2
2
1,2
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1,2 1
Y. Weng , K. Wood , S. Son , A. Bryan , D. Sabatini , S. R. Manalis ; Biological Engineering, MIT,
2
3
Cambridge, MA, David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, Biology,
MIT, Cambridge, MA
The mTOR pathway regulates a myriad of cellular functions among which are cell cycle progression, cell
growth, and cell survival. While conventional methods used to study mTOR have revealed an extensive
amount of information regarding the biochemistry that regulates the pathway, the effects of mTOR on how
an individual cell grows or progresses through the cell cycle is not well understood. The suspended
microchannel resonator (SMR) is a mass-based sensor which enables a single-cell’s weight to be
precisely tracked for the duration of entire cell cycles. We have developed a method whereby mTOR
inhibitors (torin and rapamycin) are delivered to the cell being monitored by the SMR so that the response
as seen in changes in growth rate can be elucidated. Combining this technology with microscopy we can
deduce very specific information regarding a cell’s current state in the cell cycle. As proof of concept, we
have demonstrated that the effects of 250nM Torin on a cell’s growth are not immediate; the cell will
continue to maintain its growth rate post treatment for two to three hours prior to slowing down. Cells that
have undergone DNA replication and are in G2 will go through mitosis and its daughters cells will either
grow very slowly or stop growing altogether. Cells treated with Torin in G1 phase will extend its
interdivisionary period and will divide at a much smaller size than that of an average cell in culture. These
results serve to explain why post Torin treatment proliferation of the culture will take a toll and why the
average distribution of the population will shift to a smaller size.
2790/L027
The Human Centromere and Kinetochore Proteins: 183 and Counting.
S. Liu, A. R. Tipton, K. Wang, P. Oladimeji; Biol Sci, University of Toledo, Toledo, OH
Extensive literature review has led to a compilation of 183 human centromere/kinetochore proteins, all
with experimental evidence supporting the subcellular localization. By interrogating online resources, we
have mined for genes/proteins that display transcriptional co-expression or protein-protein interaction
(PPI) with 64 core centromere/kinetochore components that have better-characterized mitosis-specific
expression patterns and functions. Top-ranked hubs in either co-expression or PPI network not only are
highly enriched with known mitosis regulators including some that have been identified only recently, but
also contain candidates previously unknown to have mitotic functions. Characterization of two candidate
genes, KIAA1377 and TRIP13, confirmed that both interacted with the mitotic checkpoint silencing protein
p31comet. In addition, GFP-KIAA1377 and GFP-TRIP13 are localized at centrosomes and kinetochores,
respectively, strongly implicating their functions during mitosis. We conclude that transcriptional coexpression and PPI network analyses with known human centromere/kinetochore proteins as queries will
help identify novel mitosis regulators.
2791/L028
A Cell Cycle-Regulated Proteolytic Mechanism Independent of Ubiquitin Conjugation to the
Substrate.
E. Choi, H. Hall, M. C. Hall; Biochemistry, Purdue University, WEST LAFAYETTE, IN
Regulated proteolysis via the ubiquitin proteasome system is a hallmark of the eukaryotic cell cycle.
Generally, substrates are recognized by ubiquitin ligases in a cell cycle-dependent manner and modified
with polyubiquitin chains conjugated to substrate lysine residues. The polyubiquitin chains target the
substrates to the 26S proteasome for destruction. We have been characterizing the proteolytic
mechanism of the yeast Acm1 protein, which is a pseudosubstrate inhibitor of the anaphase-promoting
complex. Acm1 expression is strictly cell cycle dependent. Phosphorylation of Acm1 by cyclin-dependent
kinase (Cdk) stabilizes Acm1 during S phase and mitosis. An Acm1 mutant lacking Cdk phosphorylation
sites is constitutively unstable. At the end of mitosis, dephosphorylation by Cdc14 phosphatase triggers
the rapid destruction of Acm1. Here we report several lines of evidence suggesting that Acm1 is subject
to a unique mode of recognition by the 26S proteasome. We tested all known yeast ubiquitin ligases for
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effects on the extreme instability of Acm1 in G1 cells without a positive result. Overexpression of a
ubiquitin mutant lacking lysine residues failed to stabilize Acm1, in contrast to typical substrates of the
ubiquitin proteasome system, suggesting that polyubiquitin chain assembly is not required for Acm1
proteolysis. Importantly, an Acm1 mutant lacking lysines and containing an epitope tag to block the native
N-terminus exhibited the same half-life as wild-type Acm1 in both G1 and S phases. This demonstrates
that conventional ubiquitin conjugation to Acm1 is not required for its recognition and destruction by the
proteasome. Surprisingly, Acm1 was completely stabilized in a conditional uba1 mutant strain,
demonstrating that its proteolysis is still dependent on the ubiquitin conjugation system in some way.
Possible models for this novel mode of cell cycle-regulated proteolysis will be presented.
2792/L029
Proper Mitotic Function of HURP Is Governed by Importin β.
F. Ye, L. Tan, C. Li, Y. Liou; Department of Biological Sciences, National University. of Singapore,
Singapore, Singapore
The accurate segregation of chromosomes during mitosis requires the proper connection between
spindle microtubules and sister kinetochores (bi-orientation), defects of which often leads to chromosome
instability and aneuploidy. The classical “search-and-capture” model suggests that RanGTP gradient
centered around the chromosomes is the key signal to guide the probing microtubules to kinetochores.
Recently, Hepatoma Up-Regulated Protein (HURP), a novel member of MAPs, was characterized as an
important factor for microtubule-kinetochore capture in a RanGTP-Importin β dependent manner. As a
result of Importin β binding, HURP locates on the plus end of the kinetochore microtubules at the vicinity
of chromosomes (K-fiber). Here we report a novel regulatory motif of HURP (278-368 aa) that modulates
its binding to Importin β, and thereby affects the spindle localization of HURP. Deletion of this regulatory
domain results in mis-localization of HURP and leads to chromosome congression defect as well as
mitotic arrest. Fluorescence Recovery After Photobleaching (FRAP) study and microtubule cold-stability
assay show that HURP traction mutants that lack of this importing β binding regulatory domain would
hyperstabilize microtubules and reduce the microtubule dynamics during interphase and mitosis. Our
study suggests a novel mechanism of how HURP mitotic function is mediated by importin β.
2793/L030
Nucleoporin Nup88 Associates with Cdc20 during Mitosis.
C. Hashizume, R. Wong; Kanazawa University, Kanazawa, Japan
Nuclear pore complexes (NPC) are massive multiprotein channels responsible for traffic between the
nucleus and cytoplasm, and are composed of approximately 30 proteins, termed nucleoporins (Nup).
Nup88 has been proposed as a tumor marker. Recently, we identified Nup88 was localized on the
spindles during mitosis. In this study, we further show that a fraction of Nup88 is located at kinetochores
through its N-terminal domain, which is responsible for interacting with Cdc20 as a bona fide binding
partner, suggesting that Nup88 might target to kinetochores through binding cdc20. Overexpression of
Nup88 enhanced multinucleated cell formation. RNA interference-mediated knockdown of Nup88 caused
a severe chromosome segregation defects and altered Cdc20 expression and localization. Next, we
performed a series of rescue and dominant negative experiments to confirm that Nup88 orchestrates
proper chromosome segregation through interaction with Cdc20. Our data indicate that Nup88 functions
as a spatial and temporal regulator of Cdc20, ensuring the efficient recruitment of securin degradation to
promote proper anaphase formation.
2794/L031
The Novel Function of Nuclear ErbB-2.
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1
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L. Li , H. Chen , Y. Hsieh , Y. Wang , H. Chu , M. Hung ; Center for Molecular Medicine and Graduate
2
Institute of Cancer Biology, China Medical University and Hospital, Taichung, Taiwan, Department of
Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, TX
We have demonstrated that ErbB-2 translocates into the nucleus through importin β1-mediated nuclear
import by endosomal sorting, and transactivates transcription of cyclooxygenase-2 which has been known
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to be involved in tumor progression and metastasis, suggesting nuclear ErbB-2 may also contribute to the
tumor malignancy (Oncogene 2010, 29: 3997-4006; Mol. Cell. Biol. 2005, 25: 11005-11018; Cancer Cell
2004, 6: 251-261). However, the biological functions of the nuclear ErbB-2 are not yet completely
understood. Here, we have identified novel ErbB-2-interacting nuclear proteins. Nuclear ErbB-2 physically
interacts with actin and RNA Pol I, and coincides with active RNA Pol I transcription sites in nucleoli.
Knocking down ErbB-2 reduces pre-rRNA and protein synthesis, whereas pre-rRNA level and protein
production are augmented by wild-type ErbB-2 but not by ErbB-2 mutant, which is defective in nuclear
translocation. Chromatin immunoprecipitation assays reveal that ErbB-2 binds to rDNA together with RNA
Pol I and actin. Our results indicate that nuclear ErbB-2 co-occupies rDNA with actin and RNA Pol I,
stimulates rRNA production and protein synthesis/cell growth, thus links the nuclear ErbB-2 to a novel
function in regulating cellular translation, which may contribute to tumorigenesis. [This work was
supported by National Health Research Institutes NHRI-EX99-9603BC, National Science Council NSC952311-B-039-002 and NSC 98-3111-B-039-004 and Department of Health DOH98-TD-I-111-TM002 (to
LYL), Taiwan].
2795/L032
Mitochondrial ClpXP Is Essential for Survival of Human Cells in Culture.
Y. Zhang, M. Maurizi; NCI, Bethesda, MD
ClpXP is a chaperone/protease machine consisting of the ATP-dependent protein unfoldase, ClpX, and
the compartmentalized protease, ClpP. ClpXP plays a key role in a numerous regulatory pathways in
bacteria and has been implicated in the unfolded protein response in eukaryotes. We investigated the
effects of knockdown and over expression of human ClpP (hClpP) in cultured human cells. Addition of
HCLPP siRNA to HeLa cells causes a rapid loss of HCLPP mRNA and leads to a complete loss of hClpP
protein within 24 h. In cells depleted of hClpP, the pro-apoptotic protein Bax translocates to mitochondria,
the ratio of Bax/Bcl2 is increased, and the cells undergo apoptotic cell death. After partial knockdown of
hClpP cells become more sensitive to anticancer drugs, such as the DNA-damaging agent cisplatin or the
cAMP-dependent kinase inhibitor staurosporin. Stable transfectants of the human hepatocarcinoma cell
line, Huh-4, over expressing hClpP displayed resistance to killing by cisplatin. Corresponding delays in
the appearance of activated PARP indicated that the cells were impaired in apoptosis. In Huh4 cells over
expressing hClpP, AIF release into the cytosol following cisplatin treatment was lower, and activation of
caspases 3, 7, and 9 were delayed, indicating that excess hClpP exerts an anti-apoptotic effect upstream
of both caspase-dependent and -independent pathways. Our data indicate that hClpP is required for
mitochondrial integrity and plays a role in homeostatic mechanisms that influence mitochondrial signaling
in cell death pathways. Future work will focus on trapping mitochondrial substrates of hClpXP, pulling
down their co-factors, and the role of hClpXP in maintenance of mitochondrial membrane potential.
2796/L033
Regulation of Mammalian Sterile20-Like Kinase 1 (Mst1) by Peroxiredoxin 1.
2,1
1,2 1
2
S. Jalan , J. Chernoff ; fox chase cancer center, Philadelphia, PA, drexel university, philadelphia, PA
The Mammalian Ste20-like kinase 1 (Mst1) is a serine/threonine protein kinase, that belongs to the class
II germinal center kinase (GCK-II) sub-family of protein kinases. Mst1 becomes activated in response to
various apoptotic or stress stimuli (i.e. Hydrogen peroxide, UV radiation, serum starvation), which results
in the autophosphorylation of Mst1 at multiple sites. Mst1 has been found to play an important role in
various biological processes, such as, cell proliferation, apoptosis and cell motility. Recently, Mst1 has
become a subject of intense investigation, as it has been shown to have tumor suppressor activity.
Despite this interest, little is known about the regulation of Mst1 activity and its cellular targets. Using
tandem affinity chromatography, we identified Peroxiredoxin 1 (Prdx1) as a Mst1 interacting protein under
conditions of oxidative stress. Prdx1 is a member of the Peroxiredoxin family of non-seleno peroxidases,
which reduces H in cells and thereby maintain normal Hydrogen peroxide levels. We confirmed the
association between Mst1 and Prdx1 by co-immunoprecipitation and found that the interaction between
these two proteins is induced by oxidative stress. However, catalytic inactive mutant of Prdx1,
C52A/173A, did not show oxidative stress induced interaction, indicating that catalytic activity of Prdx1 is
required for interaction with Mst1. We also demonstrated that Prdx1 does not act as Mst1 substrate.
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Further, we found that Prdx1 inhibits Mst1 kinase activity in the presence of Hydrogen peroxide, in vitro.
Our results suggest that the interaction between Mst1 and Prdx1 might be important for regulating stress
signals in the cell and therefore, further studies will be carried out to establish the functional
consequences of this interaction.
2797/L034
Two Step Release of Cdc14 with Two Step Re-Sequestration.
1
1,2 1
2
S. Ramachandran , R. Li ; Rong Li Lab, Stowers Institute, Kansas, MO, Department of Molecular and
Integrative Physiology, University of Kansas Medical Center, Kansas, KS
Cdc14 play a major role in mitotic exit through two signaling networks, the Cdc14 early anaphase release
(FEAR) and the mitotic exit network(MEN), which ensures the proper chromosome segregation between
the mother and daughter cell.Cdc14 phophatase antagonize Cdk activity by dephosphorylating of Cdk
substrates and down regulation of Cdk by dephosphosphorylating anaphase-promoting complex (APC)
activatory subunit Cdh1 and Cdk inhibitor Sic1. Cdc14 phosphatase is sequestered and kept inactive in
the nucleolus by binding with Net1. As Cdc14 release from the nucleolus is central to mitotic exit control,
we have developed tools and methods to determine the precise release kinetics of Cdc14 in single cells.
Most of the published studies employed immunoflourescence staining to visualize Cdc14 in fixed cells
and this method does not permit kinetic measurement with high temporal resolution at the single cell
level. So we done a new approach to quantitative imaging to determine the in vivo dynamics of protein
involved in the mitotic exit control. An unresolved question is whether Cdc14 is released in two distinct
steps that are controlled by FEAR and MEN, or in a single step that is triggered abruptly by FEAR
network with the MEN playing a role in sustaining the released state. Our novel technique confirming that
there are two different phases of Cdc14 release. MEN dependent release of Cdc14 is totally independent
from FEAR dependent. More interestingly there is a re-sequestration of Cdc14 between FEAR and MEN,
in addition to the final re-sequestration after the mitotic exit. Through this approach we are elucidating the
role of SLK19, SPO12, LTE1, BUB2, and DYN1 in mitotic exit and also placing the gene in the correct
phase of the releases. In dyn1 mutant with binucleate mother cell shows 1st phase of release (FEAR
dependent) as normal, then Cdc14 is re-sequestered back until the 2nd phase (MEN) trigger, which can
delay up to 35 minutes, where as in wt type this gap phase( 1st re-sequestration phase) is only for five
minutes.
2798/L035
Cell Surface of Aging Yeast Saccharomyces cerevisiae.
1
2
3
1 1
A. Pichova , Z. Burdikova , J. Plasek , K. Sigler ; Institute of Microbiology, Academy of Sciences of the
2
Czech Republic, Prague, Czech Republic, Institute of Physiology, Academy of Sciences of the Czech
3
Republic, Prague, Czech Republic, Faculty of Mathematics and Physics, Charles University, Prague,
Czech Republic
During aging the cells are undergoing many changes on cellular and molecular level. We compared the
most remarkable change, i.e. the overall appearance of the whole cell (cell shape, features of cell wall
etc.) in replicatively old and young cells of Saccharomyces cerevisiae. Beside the known enlargement of
mother cells and growing number of bud scars, significant remodeling of cell surface was revealed using
wide-field fluorescence and confocal microscopy, electron scanning microscopy and also atomic force
microscopy. Replicatively young cells possess smooth cell surface and bud scars with rounded rim, while
old cells display rough cell surface with cell wall wrinkling and exfoliation, and bud scars with sharp rim.
Additionally, staining of old cells with Calcofluor, a fluorochrome that binds to glucan microfibrils in cell
wall and thus makes it possible to visualize aging-related changes in its texture, showed fading of bud
scars and cell wall during aging. Further, we described four categories of the terminal phenotype or the
pre-mortal cells; the most remarkable of them is the formation, by some very old mother cells, of
extremely elongated buds giving rise to normal progeny in the next generations. These data show that the
cell shape and cell surface, i.e. the external cell skeleton, are suitable markers of aging yeast.
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2799/L036
A Specific MAPK Cascade Downstream RalA and Exocyst Complex Regulates Focal Adhesion
Organization for Cell Division Accomplishment.
1
1
1
2
1,2 1
I. Cascone , R. Selimoglu , A. Santos , E. Del Nery , J. Camonis ; Inserm U830, Institut Curie, Paris,
2
France, Translational Departement, Institut Curie, Paris, France
The Ras family G-proteins RalA and RalB make critical contributions to the oncogene Ras-dependent
tumourigenesis; and we showed that they regulate the Exocyst complex during cytokinesis, the final step
of cell division. Now, we have identified and characterized a specific cascade of stress activated MAP
kinases (MAPK) downstream RalA and Exocyst, and we show that this pathway regulates de novo focal
adhesion(FA)organization during cytokinesis. We demonstrate that the MAP4K, MAP4K4, triggers a
MAP3K8-MAP2K7 cascade that activates the MAPK JNK and phosphorylates paxillin at the intracellular
bridge. RalA and Sec5 regulate this cascade via MAP4K4. During mitosis cells round up and focal
adhesions FAs disassemble, than during cytokinesis FA structures re-organize. We demonstrate that
paxillin phosphorylation is a signal for post-furrowing spreading necessary for cytokinesis progression and
cell division accomplishment. This pathway could act as a check point of cell division timely coordinating
cell spreading and telophase progression.
2800/L037
The Drosophila Gene Doublefault Is Required for Chromosome Segregation and Cytokinesis
during Male Meiosis.
1
2
1 1
S. Sechi , R. Piergentili , M. Giansanti ; Istituto di Biologia e Patologia molecolari, CNR, Roma, Italy,
2
Dipartimento di Biologia e Biotecnologie, Università La Sapienza di Roma, Roma, Italy
Drosophila male meiosis provides an excellent cell system for the molecular dissection of cytokinesis.
These cells are considerably larger than most somatic cells, making them amenable to cytological
analysis. In addition, the spindle assembly checkpoint is not stringent in spermatocytes, allowing the
characterization of genes whose products are required for multiple stages of cell division. We have
isolated a novel male sterile mutation affecting both chromosome segregation and cytokinesis that failed
to complement the P-induced mutant doublefault (dbf), previously mapped in 32A2. DNA sequencing
revealed that our (dbf) allele caused a premature stop codon in the annotated CG17098 Drosophila gene.
Based on these results dbf encodes a predicted 73kDa polypeptide, containing a C2H2-like zinc finger
domain involved in nucleic acid binding. During meiotic division dbf affected spindle morphology,
chromosome alignment and segregation. Moreover dbf mutants were unable to assemble both the central
spindle and the contractile ring during telophase. Imaging of spermatocytes expressing a GFP-tagged
protein revealed that Dbf was enriched on microtubules during prophase and accumulated around the
spindle envelope and the spindle poles during meiosis. Interestingly dbf mutants disrupted the localization
of RanGAP a protein involved in the establishment of a RanGTP gradient across the nuclear envelope
and in the nucleocytoplasmic transport. dbf mutations also abolished the localization of the Chromosomal
passenger (CPC) proteins at both the kinetochores and the spindle midzone. Our results suggest that Dbf
might be involved in Ran-driven RNA transport and RNA-mediated recruitment of the CPC at the
kinetochores required for spindle dynamics and cytokinesis.
2801/L038
RalBP1, a Centrosomal Protein Required for Golgi Morphology and Cell Cycle Progression.
J. Fillatre, D. Delacour, J. Moreau; Institut Jacques Monod CNRS, Paris, France
The Ras/Ral pathway is actively involved in oncogenesis and its effects are partly mediated by the protein
RalBP1 (Ral Binding Protein 1). RalBP1 is overexpressed in many cancers where it is supposed to
protect the tumoral cells from apoptosis. It has been reported that RalBP1 is involved in endoctyosis and
cell detoxification. Recently we have identified a role of RalBP1 in actin cytoskeleton remodelling during
the early development. Here we aimed at extending the knowledge of RalBP1 function during the
development. For that purpose, we overexpressed a truncated form of the protein in the animal cap of two
cell stage xenopus embryos. The injected cells exhibited severe defects in cell division, with incomplete
cleavage furrow ingression and abnormal nuclear segregation. These results showed a new function of
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RalBP1 in mitosis. Furthermore, a two hybrid screen and microinjection experiments allowed the
identification of a new partner of RalBP1, Golgin-160. Co-expression with our RalBP1 mutant form
together with Golgin-160 rescued the cell division arrest, demonstrating a functional interaction. In HeLa
cells, colocalization between these two proteins is observed during late telophase as the Golgi apparatus
starts to reform around centrosomal RalBP1. Besides, the overexpression or the silencing of RalBP1 led
to Golgi apparatus defects, including fragmentation of the pericentriolar Golgi stacks into Golgi blobs
scattered throughout the cytoplasm. Altogether, these data highlight a new function for RalBP1 in the
regulation of the Golgi structure and precise its function in Ral induced oncogenesis.
2802/L039
Int6, a New Regulator of Metaphase-Anaphase Transition in Drosophila S2 Cells.
1
2
2
1
1
1
2 1
M. Somma , F. Renda , V. Naim , E. Bucciarelli , C. Pellacani , A. Palena , M. Gatti ; Ist Biol e Pat Mol,
2
CNR, Rome, Italy, Dip. Biologia e Biotecnologie, Università La Sapienza, Rome, Italy
We identified int6 in recent RNAi-based screen aimed at the identification of new Drosophila mitotic
genes. int6 encodes a protein product that is a component of the translation initiation complex and is
probably also part of both the 26S proteasome and the COP9 signalosome. RNAi-mediated depletion of
Int6 in Drosophila S2 cells results in a metaphase arrest phenotype, with short spindles and
elongated/distorted centromere/kinetochore regions. To elucidate the role of Int6 we performed double
RNAi experiments to simultaneously deplete both Int6 and another protein required for spindle formation.
We found that co-depletion of the MT-interacting kinetochore component Ndc80 or the kinesin-type MT
depolymerases Klp10A and Klp67A, or the spindle assembly checkpoint (SAC) proteins Mad2 and BubR1
rescues the phenotypes elicited by Int6 single depletion. These results indicate that the int6 mutant
phenotype requires a functional SAC and is partially suppressed when the kinetochore-MT interaction or
the MT flux are inhibited. We thus propose that the int6 phenotype results MT-mediated tension at the
kinetochore in the presence of a SAC mechanism that persistently blocks metaphase-anaphase
transition.
2803/L040
Harnessing Functional Genomics to Dissect the Final Stages of Cell Division in Human Cells.
1
1
2
1 1
S. Sundaramoorthy , S. Lekomtsev , M. Howell , M. Petronczki ; Cell Division and Aneuploidy, Cancer
2
Research UK London Research Institute, Potters bar, United Kingdom, High Throughput Screening,
Cancer Research UK London Research Institute, London, United Kingdom
Cytokinesis is the final step of cell division and forms the basis for cell multiplication on our planet. The
primary goal of our project is to use available functional genomic data to identify novel regulators of
cytokinesis in human cells. Using a list of candidate genes emerging from the published screens as a
starting point, we try to decipher the role of individual proteins in initiating, conducting and exiting the
process of cell division. Preliminary data emerging from the screen suggests that existing genome wide
screens have been successful to varying degrees in pinpointing cytokinetic regulators. Secondary
screens and further characterization are being done to confirm the hits from the primary screen. This will
help to narrow down the list of potential genes involved in cytokinesis, thereby facilitating focussed
studies to decipher the roles of a select group of genes in regulating cytokinesis.
2804/L041
Delayed Release of Chromosome Cohesion in Chromosomes That Are Not Attached to the
Spindle at Anaphase Onset.
A. Martens, L. V. Paliulis; Biology, Bucknell University, Lewisburg, PA
In mitosis, release of sister chromatid cohesion allows sister chromatids to separate from one another. In
meiosis I, release of sister chromatid arm cohesion allows homologues to separate from one another.
Sister chromatids remain connected via cohesion between centromeres, which is released in anaphase II.
In this study, we examine the timing and progression of cohesion release in anaphase. We have found
that complete release of cohesion takes approximately 5 minutes in both mitosis and meiosis in the
grasshopper Melanoplus sanguinipes and in the cricket Acheta domesticus. Cohesion release initiates
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near centromeres and progresses toward chromosome ends in both mitosis and meiosis. In previous
studies (Paliulis and Nicklas, 2004. Current Biology. 14:2124-2129), we showed that release of
chromosome cohesion did not require spindle attachment or tension on the chromosome at anaphase
onset. We now show that, while chromosomes detached from the spindle with a micromanipulation
needle separate in anaphase, the separation is delayed. We propose that spindle attachment speeds
separation by allowing transport of molecules required for cohesion release to the chromosome at
anaphase onset.
2805/L042
Holocentric Chromosome Separation in Mitosis and Meiosis in the Milkweed Bug, Oncopeltus
fasciatus.
S. Thibault, L. V. Paliulis; Biology Department, Bucknell University, Lewisburg, PA
Milkweed bugs (Oncopeltus fasciatus) have holocentric chromosomes. In mitosis, the holocentric
chromosomes have kinetochore activity all along their length. In meiosis, milkweed bug chromosomes
behave like monocentric chromosomes, designating a small region on each chromosome to have
kinetochore activity. The aim of this study was to determine whether there were differences in
chromosome separation between monocentric and holocentric chromosomes. We have determined that
holocentric and monocentric chromosomes have very different chromosome separation in anaphase;
mitotic cells underwent complete chromosome segregation in less than 30 seconds with sister chromatids
separating almost simultaneously, while meiotic cells with monocentric-like chromosomes underwent
complete chromosome segregation in approximately 7 minutes. We used micromanipulation to determine
the impact of chromosome attachment to the spindle on the timing and progression of chromosome
separation.
2806/L043
Characterization of a Novel Raptor Phosphorylation Site Targeted by Intestinal Cell Kinase.
Z. Fu, D. Wu; Medicine, University of Virginia, Charlottesville, VA
Intestinal Cell Kinase (ICK), originally cloned from the intestine, is a highly conserved and ubiquitously
expressed serine/threonine protein kinase that requires the dual phosphorylation of a MAPK-like TDY
motif for full activation. Recently we have shown that knocking down the ICK expression in cultured
intestinal epithelial cells induced growth retardation and G1 cell cycle delay. We also reported that the
expression and/or activity of several key regulatory components of G1 cell cycle progression (c-Myc and
Cyclin D1) and of major regulators of protein translation and cell growth in the mTORC1 pathway (mTOR,
Raptor, S6K1) were significantly altered. Furthermore, we provided biochemical evidence suggesting that
ICK may directly interact with the mTOR/Raptor complex and Raptor is a putative substrate of ICK. In this
report, we investigated whether ICK targets Raptor to regulate the mTOC1-mediated signaling events.
Using the ICK consensus sequence [RPX-S/T-P/A/T/S], we identified a putative phosphorylation site
908
RPGT T for ICK in human Raptor that is conserved across vertebrate orthologs of Raptor. Raptor-T908
is directly phosphorylated by ICK as determined by in vitro kinase assay and site-directed mutagenesis.
However, Raptor-T908 phosphorylation by ICK is blocked when Raptor is associated with mTOR.
Although Raptor phosphorylation at T908 does not affect mTOC1 integrity or in vitro kinase activity as
assessed by S6K1 phosphorylation in response to insulin, expression of a phosphorylation site mutant of
Raptor (T908A) enhances insulin-stimulated ERK1/2 phosphorylation and activation. Our results suggest
Raptor-T908 phosphorylation by ICK may act in mTOR-independent mechanisms to regulate specific
signaling events such as the response of ERK1/2 to insulin.
2807/L044
Dynamic Localization of the Proline-Rich Akt Substrate of 40kda (PRAS40) throughout the Cell
Cycle.
J. J. Havel, Z. Li, Y. Du, H. Fu; Pharmacology, Emory University, Atlanta, GA
PRAS40 has recently been identified as a component and substrate of the mammalian Target of
Rapamycin Complex 1 (mTORC1), a growth factor- and nutrient-sensitive kinase that acts as a master
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regulator of protein synthesis and cell growth. When in it’s non-phosphorylated form, PRAS40 binds
mTORC1 and inhibits its kinase activity. In the presence of growth factors, Akt phosphorylates PRAS40,
causing it to bind 14-3-3 proteins and dissociate from mTORC1, thereby neutralizing PRAS40’s inhibitory
function and contributing to growth factor-induced activation of mTORC1. Somewhat surprisingly,
PRAS40 has been found to exhibit pro-survival activity in a rat model of traumatic spinal cord injury as
well as in a murine tumor xenograft model. Because PRAS40’s only currently known function is the
inhibition of the pro-growth, pro-survival kinase mTORC1, we reason that PRAS40 may possess a
separate molecular function that mediates its pro-survival effects. To explore this possibility we began by
studying the subcellular localization of PRAS40. We demonstrate that while PRAS40 is a primarily
cytoplasmic protein, a subpopulation of PRAS40 is located within the nucleus and that nuclear
localization is controlled at least in part by a functional nuclear export signal sequence. Furthermore,
studies in synchronized HeLa cells show that nuclear PRAS40 levels are highest at the G1/S phase
transition, indicating a potential role in regulation of DNA replication. Because the putative pro-survival
function of PRAS40 is likely to be executed when PRAS40 is in its Akt-phosphorylated, non-mTORC1associated state, we also explored the specific subcellular localization of phosphorylated PRAS40.
Interestingly, immunofluorescence imaging reveals a striking dynamic localization of p-PRAS40
throughout mitosis. p-PRAS40 immunoreactivity is found to localize to the spindle poles and along spindle
fibers during metaphase, to the midzone during anaphase and telophase, and at the midbody during
cytokinesis. Taken together, our results demonstrate that PRAS40 sub-cellular localization is dynamically
controlled throughout the cell cycle and suggest roles for PRAS40 in the regulation of DNA synthesis and
cell division.
2808/L045
Investigating Meiotic Spindle Scaling between Xenopus laevis and Xenopus tropicalis.
1
2
1
1 1
K. Helmke , R. Loughlin , J. Wilbur , R. Heald ; Molecular and Cell Biology, University of California
2
Berkeley, Berkeley, CA, Biophysics Graduate Group, University of California Berkeley, Berkeley, CA
Bipolar spindles must separate chromosomes by the appropriate distance during cell division, but how
spindle and cell size are coordinated is not understood. We are using cytoplasmic egg extracts to identify
factors that scale meiotic spindles smaller in the species X. tropicalis compared to X. laevis. We have
characterized the role of microtubule depolymerization, and found that the microtubule severing factor
katanin is more active in X. tropicalis and contributes to spindle length regulation, but does not account
completely for spindle size differences. We have also identified differences in the chromatin-mediated
pathway governed by the small GTPase Ran. The guanine exchange factor (GEF) that activates Ran is
bound to mitotic chromatin and creates a diffusible gradient of RanGTP that locally releases spindle
assembly factors (SAFs) from inhibitory interactions with importins, promoting microtubule polymerization
and bipolar spindle organization. Using Ran mutants, we have found that the smaller spindles in X.
tropicalis extracts assemble properly in the absence of a RanGTP gradient and still localize Ranregulated SAFs to the microtubules. X. laevis extracts, however, require this pathway to form and
maintain a larger spindle. We hypothesize that compensatory centrosome-driven spindle assembly
pathways may be upregulated in the small X. tropicalis spindles, and we are currently characterizing the
downstream effectors that underlie the altered requirement for RanGTP in meiotic spindle assembly that
could contribute to spindle size regulation between the two species.
2809/L046
Dynein/Dynactin-Mediated Transport of Kinetochore Components off Kinetochores and onto
Spindle Poles Induced by Nordihydroguaiaretic Acid.
1
1
2
1,3 1
L. Vos , J. Famulski , J. B. Rattner , G. Chan ; Oncology, University of Alberta, Edmonton, AB,
2
3
Canada, Anatomy and Cell Biology, University of Calgary, Calgary, AB, Canada, School of Cancer,
Engineering and Imaging Sciences, University of Alberta, Edmonton, AB, Canada
The mitotic checkpoint functions to ensure accurate chromosome segregation by regulating the
progression from metaphase to anaphase. Once the checkpoint has been satisfied, it is inactivated in
order to allow the cell to proceed into anaphase and complete the cell cycle. The minus end-directed
microtubule motor dynein/dynactin has been implicated in the silencing of the mitotic checkpoint by
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“stripping” checkpoint proteins off kinetochores. A recent study suggested that Nordihydroguaiaretic acid
(NDGA) stimulates dynein/dynactin-mediated transport of its cargo including ZW10 (Zeste White 10). We
analyzed the effects of NDGA on dynein/dynactin dependent transport of the RZZ (Zeste White 10,
Roughdeal, Zwilch) complex as well as other kinetochore components from kinetochores to spindle poles.
Through this approach we have catalogued several kinetochore and centromere components as
dynein/dynactin cargo. These include hZW10, hZwilch, hROD, hSpindly, hMad1, hMad2, hCENP-E,
hCdc27, Cyclin-B and hMps1. Furthermore, we found that treatment with NDGA induced a robust
accumulation and complete stabilization of hZW10 at spindle poles. This finding suggests that NDGA may
not induce dynein/dynactin transport but rather interfere with cargo release. Lastly, we determined that
NDGA induced accumulation of checkpoint proteins at the poles requires dynein/dynactin-mediated
transport, hZW10 kinetochore localization and kinetochore-microtubule attachments but not tension or
Aurora B kinase activity.
2810/L047
Molecular Mechanism of Anillin’s Role in Contractile Ring Asymmetry.
C. A. Patino-Descovich, L. Zhang, T. Nguyen, J. F. Dorn, A. S. Maddox; IRIC, Université de Montréal,
Montreal, QC, Canada
A frequently observed, but poorly understood feature of cytokinesis is asymmetric furrowing, such that the
center of the contractile ring is not coincident with that of the division plane. Asymmetric furrowing occurs
in marine invertebrate and Xenopus eggs and epithelial cells in a monolayer. However, coverslip-axis
images indicate that furrowing is symmetric in the popular cultured cell models HeLa and Drosophila S2
cells. Using four-dimensional time-lapse imaging, we found that if the division plane is viewed end-on,
asymmetric furrowing towards the coverslip invariably occurs. We previously reported asymmetric
furrowing in the C. elegans zygote. Because of the efficiency of protein depletion and ease fluorescent
protein expression, we used this system to examine the molecular mechanism of this phenomenon. In C.
elegans early embryos depleted of Anillin, a conserved scaffold protein, cytokinesis still occurs, but is
abnormally symmetric. To study how Anillin impacts ring asymmetry, we tested the ability of Anillin
truncations to replace function of the endogenous Anillin in furrow asymmetry. To quantify contractile ring
kinetics and asymmetry, we created custom image-processing software. Anillin-depleted cells expressing
a truncation lacking the myosin-binding domain had abnormal furrow symmetry. However, the transgene
is expressed at lower levels than endogenous Anillin. To determine the expected phenotypes over a
range of levels of functional Anillin, we performed a depletion time-course, measuring Anillin levels by
western blotting and monitoring asymmetry by live cell imaging of embryos expressing GFP-tagged
myosin. By consulting our standard curve, we concluded that the truncated protein is fully functional.
Thus, Anillin’s myosin-binding domain is not required for asymmetry. In contrast, using the same
approach we found that the Anillin homology and PH domains predicted to bind Rho, RhoGAP, and
septins are required for furrow asymmetry. In sum, we are working to understand the molecular
mechanism of asymmetric furrowing, a universal feature of animal cell cytokinesis. We will also present
evidence that asymmetric furrowing is energetically favourable.
2811/L048
Intracellular Localization and Secretion of Two Glial Cell Line-Derived Neurotrophic Factor Splice
Isoforms.
S. Leppänen, L. Lonka-Nevalaita, E. Jokitalo, M. Saarma; Institute of Biotechnology, University of
Helsinki, Helsinki, Finland
Glial cell line-derived neurotrophic factor (GDNF), a member of GDNF-family ligands (GFLs), regulates
the development and maintenance of neuronal populations in brain and periphery. Outside the nervous
system GDNF regulates kidney development and spermatogeneses. Because of its neurotrophic effects,
GDNF has been widely studied as a therapeutic agent for neurodegenerative diseases, particularly
Parkinson’s disease. Although the function of GDNF has been extensively studied, very little is known
about the basic cell biology of GDNF and its precursor forms pre-(α)pro-GDNF and pre-(β)pro-GDNF. Our
aim was to characterize the intracellular localization and secretion of two GDNF splice isoforms. All
commercially available anti-GDNF antibodies detect the mature region of GDNF only. In oder to detect
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both pro-GDNF and GDNF proteins spesifically, we produced a new polyclonal peptide antibody anti-proGDNF against the GDNF pro-region. We used immunofluorescence (IF) analysis and immune electron
microscopy to study the intracellular localization of overexpressed proteins. Our results show that proteins
encoded by pre-(α)pro-GDNF and pre-(β)pro-GDNF cDNAs localize to Golgi compartment and secretory
vesicles. Our western blotting analyses revealed the secretion of both pro-GDNF/GDNF isoforms. The
secretion of (α)pro-GDNF and (β)pro-GDNF was confirmed in IF analysis and surface labeling, which was
used as an additional method to detect extracellular pro-GDNF proteins. The results from IF analysis
show that noncleaved (α)pro-GDNF and (β)pro-GDNF or cleaved pro-regions together with mature GDNF
are localized to the cell surface in the presence of GDNF family ligand receptor α1. The functional
importance of pro-neurotrophins has been studied recently. Our study indicates that the proteolytic
cleavage of pro-GDNF into mature GDNF may also occur outside the cells, most likely in the extracellular
matrix. In addition, pro-GDNF proteins may remain uncleaved and have specific roles outside the cells.
2812/L049
G1-S Cell Cycle Progression Is Dependent on Presence of Normal Levels of a Mitochondrial
Protein of the Ribonuclease III Family in Drosophila.
S. Mukherjee, G. C. Sen, J. McDonald; Molecular Genetics, LRI, Cleveland Clinic, Cleveland, OH
Ribonucleases (RNases) play an important role in maturation of precursor RNAs and are central to
cellular defense against RNA viruses. The Type III RNases have an additional RNA-protein recognition
domain called the double-stranded RNA binding domain (DRBD). Mouse knock-out models of DRBD
containing proteins (DRBPs) are severely compromised in organism development. This prompted us to
study an uncharacterized member of the RNase III family called MRPL44 (mitochondrial ribosomal protein
L44), which contains a single RNase domain and a DRBD. RNase III proteins are evolutionarily
conserved from archaebacteria to mammals. We sought to characterize MRPL44 in Drosophila. Since
MRPL44 is annotated as mitochondrial, we examined its sub-cellular localization. Intriguingly, transfection
of a C-terminal GFP-tagged protein in Drosophila S2 cells shows that the protein can localize to the
mitochondria or nucleus in different cells. Similar to other members of the DRBP family of proteins, we
find that MRPL44 is essential for viability in Drosophila. Gene disruption by transposon insertion causes
lethality at first instar larval stage. Ubiquitous knock-down of this gene by RNAi, which presumably has
some remnant functional protein, delays the onset of lethality to pupal stage. This allows us to examine
the developmental problems underlying lethality in late larval adult precursor tissues called imaginal
discs. From our studies in Drosophila S2 cells, we know that the amount of MRPL44 expressed in cells is
critical for normal cell proliferation. Both, down-regulation and over-expression of MRPL44 in these cells
results in G1 arrest (FACS). To examine if knocking down MRPL44 in vivo also had the same effect, we
immunostained imaginal discs with cell cycle markers BrDU and Phospho-histone 3 (PH3). We find
significantly less BrDU and PH3 positive cells in MRPL44 knock-down imaginal discs compared to wildtype, suggesting a G1 arrest. Strikingly, tissue-specific knock down of MRPL44 results in malformations in
the adult that phenocopy knock-down of the G1/S cell cycle regulator, Cyclin E, in the same tissues.
Thus, we propose a novel role for a mitochondrial protein of the RNase III family in G1-S cell cycle
progression.
2813/L050
Aurora B Is Required for Chromosome Segregation but Not for Bivalent Resolution during
Xenopus oocyte Meiosis I.
H. Shao, C. Ma, J. Liu; Ottawa Hospital Research Institute, Ottawa, ON, Canada
Aurora B, as a key component of the chromosome passenger complex, is best known to function in
correcting merotelic chromosome attachment in mitosis. This function appears to be conserved in yeast
meiosis. One key feature of mitosis in general, and meiosis in yeast, is the presence of dominant
kinetochore microtubule(s) emanating from the centrosomes (or spindle pole bodies in yeasts) to capture
each pair of kinetochores in a bipolar manner. This feature is essential to explain how Aurora B, which is
localized to the inner centromeres and therefore at the interface between the opposing kinetochores,
detects and destabilizes kinetochore microtubules that have strayed over to the wrong kinetochore.
However, it is not clear whether this function of Aurora B is also conserved in animal oocytes undergoing
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meiosis, as animal oocytes lack centrosomes or dominant kinetochore microtubules. Previous studies
have demonstrated that in C elegans oocytes, inhibition of Aurora B results in failure of chromosome
segregation, suggesting a defect in timely removal/degradation of cohesin. We have developed a novel
method of karyotyping the giant Xenopus oocytes. Combining karyotype analysis with live cell imaging,
we demonstrated that inhibiting Aurora B, by either inhibitory antibodies or by dominant negative Aurora
B, did not prevent the timely resolution of bivalents to dyads at anaphase I. However, the resolved
homologous dyads failed to segregate. In Aurora B-deficient oocytes, polar body protrusion initiated but
eventually retracted, with all chromosome dyads returning inside the oocyte. These results suggest that
Aurora B is required for chromosome segregation in Xenopus oocyte meiosis I but not for bivalent
resolution.
2814/L051
A Previously Uncharacterized Protein Nsk1 Interacts with Cdc14 Phosphatase and Dynein to
Promote Chromosome Segregation.
1,2
1,2
1
1,2 1
J. Chen , D. M. Clifford Hart , R. Ohi , K. L. Gould ; Department of Cell and Developmental Biology,
2
Vanderbilt University School of Medicine, Nashville, TN, Howard Hughes Medical Institute, Nashville, TN
The conserved Cdc14 phosphatase is a proline directed dual specificity protein phosphatase. In yeast,
Cdc14 phosphatases antagonize cyclin-dependent kinases (CDKs) during mitosis and are key regulators
of late mitotic events. In this study, the previously uncharacterized protein Nsk1 was identified as a Clp1
(Cdc14 homolog in fission yeast S. pombe)-interacting protein through tandem affinity purification (TAP)
followed by liquid chromatography mass spectrometric (LC-MS) analysis. The interaction was validated
by Co-IP. Fluorescence microscopic studies showed that Nsk1 localized to the nucleolus during
interphase, and was released into nucleoplasm at the beginning of mitosis, becoming enriched on
kinetochores. During anaphase Nsk1 was also detected on spindles. Nsk1 was highly phosphorylated
during mitosis and became de-phosphorylated upon exit of mitosis. Cdk and Clp1 contribute to the
phosphorylation and de-phosphorylation, respectively. Mutational analysis showed that the phosphomimic mutant Nsk1-18D localized preferentially to kinetochores whereas the non-phosphorylatable
mutant Nsk1-18A localized preferentially to the spindle, indicating that phosphorylation status dictates
Nsk1 localization. Deletion of Nsk1 caused a spindle checkpoint-dependent delay of anaphase onset
suggesting that Nsk1 is important for spindle function. Consistent with this notion, nsk1Δ cells
missegregate chromosomes at elevated levels and exhibit abnormal kinetochore dynamics. The interkinetochore distance and the frequency of kinetochore collisions with spindle pole bodies are increased in
nsk1Δ cells, suggesting that Nsk1 is required for coordinated movement of sister chromatids during
chromosome congression at prometaphase. In addition, nsk1 deletion suppressed the temperature
sensitivity of an ark1 (Aurora B homolog in fission yeast) mutant, suggesting that Nsk1 acts in opposition
to Ark1 to stabilize kinetochore-microtubule attachments. We also find that dynein light chain (Dlc1)
interacts directly with Nsk1 and is required for Nsk1 to target kinetochores. We propose that Nsk1
interacts with Cdc14 phosphatase and dynein to stabilize kinetochore-microtubule attachments and
thereby promote proper chromosome segregation.
2815/L052
B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding
Yeast.
M. K. Chee, S. B. Haase; Biology, Duke University, Durham, NC
Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the
mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified.
It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5
Cdc4
ubiquitin ligase are required for the separation of the spindle poles and assembly
motors, and the SCF
of a bipolar spindle. It has been suggested that in budding yeast, B-type cyclin/CDK (Clb/Cdc28)
complexes promote spindle pole body (SPB) separation by inhibiting the degradation of the kinesins-5,
Cdh1
). We have determined, however, that the
Kip1 and Cin8, by the anaphase-promoting complex (APC
Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here,
we show that Kip1 and Cin8 are in vitro substrates of Clb2/Cdc28 kinase, and that the mutation of
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conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting either the
protein’s in vivo localization or its abundance. Mass spectrometry analysis confirms that two CDK sites in
the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a
Cdc4
target that inhibits spindle pole separation in cells lacking
Clb/Cdc28-specific inhibitor, is the SCF
functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by
direct phosphorylation of kinesin-5 motors. We are currently working to characterize the motor activity of
Kip1 and Cin8 in the presence and the absence of Clb/Cdc28 kinase in order to investigate the manner in
which the budding yeast kinesins-5 are regulated by phosphorylation.
2816/L053
The Nup98-Rae1 Nucleoporin Complex Protects Securin from Unscheduled APC/CCdh1-Mediated
Degradation in G2 Phase.
1
1
1,2 1
L. A. Malureanu , K. B. Jeganathan , J. M. van Deursen ; Pediatric and Adolescent Medicine, Mayo
2
Clinic, Rochester, MN, Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN
Anaphase onset is initiated by APC/C-mediated proteosomal degradation of Securin and Cyclin B1 after
sister chromatid bi-orientation has been accomplished. The established view is that Cdc20-activated
APC/C (APC/CCdc20) mediates the destruction of both Securin and Cyclin B1, although earlier gene
knockout studies in mice have suggested a prominent role for Cdh1-activated APC/C (APC/C Cdh1) in
the degradation of Securin. These studies further revealed that the Nup98-Rae1 nuclear pore
subcomplex binds to and inhibits APC/CCdh1 in early mitosis to couple Securin degradation to
chromosome bi-orientation and mitotic checkpoint silencing. To better understand the role of the Nup98Rae1 subcomplex in Securin stabilization and destruction during cell cycle progression, we monitored
Securin-YFP levels in mutant MEFs with low amounts of Nup98 and Rae1 (Nup98+/-/Rae1+/-) by live-cell
imaging. Strikingly, we observed that the kinetics of Securin degradation after nuclear envelope break
down (NEBD) was similar in Nup98+/-/Rae1+/- and wild-type MEFs. However, Nup98+/-/Rae1+/- MEFs
consistently entered mitosis with much lower Securin levels than their wild-type counterparts. This was
due to proteosomal degradation rather than impaired synthesis, as Securin-YFP levels increased to
normal after MG132 treatment. Furthermore, expression of non-degradable Securin-YFP(ΔD-box/ΔKenbox) was high in both Nup98+/-/Rae1+/- and wild-type G2 MEFs. Co-immunoprecipitation experiments
indicate that Nup98 and Rae1 form a complex with APC/C and Cdh1 not only in mitosis, as reported
earlier, but also in G2 phase. In contrast, Nup98 and Rae1 failed to precipitate Cdc20 throughout the cell
cycle. Together, the data suggest that Nup98-Rae1 complexes act to inhibit APC/CCdh1 degradation of
newly synthesized Securin in G2 phase to allow for proper timing of Separase activation and sister
chromatid separation. Although Nup98-Rae1 complexes prevent APC/CCdh1 mediated degradation of
Securin prior to mitosis entry, analyses of the kinetics of Securin degradation Cdc20- and Cdh1-deficient
MEFs imply that APC/CCdc20 is the main ubiquitin E3 ligase complex driving Securin degradation after
NEBD.
2817/L054
Expression of 14-3-3 Sigma in Primary Human Mammary Epithelial Cells and Cell Lines.
S. D. Dimitriejvich, J. Shankardas; Orthopedic Surgery, UNT Health Science Center, Fort Worth, TX
Purpose: 14-3-3 is a highly conserved and ubiquitously expressed family of proteins consisting of seven
known mammalian isoforms (beta, epsilon, gamma, eta, theta, sigma, and zeta). Several isoforms have
been implicated in cancer and the epithelial cell specific 14-3-3 σ is believed to be involved in beast
cancer. The purpose of our study is to determine the cellular localization and expression levels of the 143-3 proteins in primary human mammary epithelial cells. We previously reported that σ, γ and ζ isoforms
are secreted and we are also interested in the extracellular role of these isoforms. Methods: Using
immunofluorescence, we determined the expression of all the 14-3-3 isoforms in primary human
mammary epithelial cells (HMEC). Western blot analysis was used to quantitate and compare these
expression levels with those in MCF-7 and MCF10A cells. We used TIRF microscopy to determine the
interaction of 14-3-3 σ with HMEC cell membranes. The conditioned medium from HMEC cells was also
examined for the presence of 14-3-3 σ using western blot analysis. Results: Indirect immunofluorescence
and western blot analysis show that the HMEC cells express all the seven 14-3-3 isoforms. These
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proteins are also shown to be predominantly cytosolic. We also show that the expression level of 14-3-3 σ
isoform is significantly higher in HMEC than in MCF7 and MCF10A cell lines. TIRF microscopy shows
membrane association of 14-3-3 σ protein in HMEC cells and that HMEC cells secrete 14-3-3 σ into the
conditioned medium. Conclusions: All the seven 14-3-3 family members are expressed by HMEC cells.
The higher expression of 14-3-3 σ in the primary HMEC cells then that in the MCF7 and MCF10A cell
lines correlates with the cell cycle alterations these cells. We also show for the first time that in HMEC
cells 14-3-3 σ is both cytosolic and membrane associated and that some of the 14-3-3 σ is secreted into
the conditioned medium.
2818/L055
Ciap1 (Cellular Inhibitor of Apoptosis Protein 1) Mediates the Regulation of Eif4e (Eukaryotic
Translation Initiation Factor 4E) by CHIP (Carboxyl-Terminus of Hsc70 Interacting Protein).
S. Lee, S. Yoo; KyungHee University, Seoul, Republic of Korea
In eukaryotes, most mRNAs are translated in a cap-dependent manner. A key player in translation is
5’cap-binding protein eIF4E (Eukaryotic translation initiation factor 4E), which is the rate-limiting
component for initiation of translation. Up-regulation of eIF4E level was observed in many of human
malignancies especially in breast cancer and its over-expression triggered oncogenic transformation,
indicating that dysregulation of eIF4E is closely linked to oncogenic transformation and to understand
molecular mechanism how eIF4E is regulated is important to propose more potential therapeutic targets.
Although CHIP (Carboxy-terminus of Hsc70 interacting protein), a U-box type E3 ligase recognizing
Hsp70 client proteins as a cochaperone, was reported to ubiquitinate eIF4E in heat shock condition, but
exact regulatory mechanism of eIF4E is largely not known. The inhibitor of apoptosis protein(IAPs) known
as a caspase inhibitor physically interacts with caspases through BIR (Baculoviral IAP Repeat) domains.
Also IAP contains another domain called RING, which can function as an E3 ubiquitin ligase. Several
recent findings have suggested that IAPs are involved in various intracellular events other than apoptosis
especially through their RING domain. Previously, we have shown that Diap1 (Drosophila Inhibitor of
Apoptosis Protein) which is a cell death inhibitor in fly directly binds to and regulate deIF4E (Drosophila
translation initiation factor 4E). In this study, we questioned whether the interaction between Diap1 and
deIF4E in fly is conserved in human, whether IAP molecules, if any, regulate eIF4E and how two different
E3s, IAP and CHIP regulate eIF4E in similar or distinct way, eventually regulating cap-dependent
translation. We identified that cIAP1 (Cellular Inhibitor of Apoptosis Protein 1) among five IAPs which
contain RING domain in human interacts with eIF4E. We also found that cIAP1 interacts with CHIP
specifically and cIAP1 actually mediates the interaction and regulation between CHIP and eIF4E. Taken
together, our results suggest that cell might have a sophisticated regulatory strategy which involves two
different E3s cooperatively to prevent malignant transformation.
2819/L056
Regulation of Endonuclease G by Hsp70 and Cochaperone CHIP.
J. Lee, S. Yoo; Kyung Hee University, Seoul, Republic of Korea
Programmed cell death (PCD) pathways are subdivided into three major types. These include apoptosis,
autophagic cell death and programmed necrosis. Apoptosis is mediated by caspases, while the other cell
deaths are not dependent on caspase activities. Neuronal cells by various stimulus undergo programmed
necrosis in which Endonuclease G(EndoG) is suspected as an important mediator. EndoG is a
mitochondrial nuclease that was originally identified as an enzyme involved in mitochondrial DNA
replication process in C. elegans. But when cell death was triggered by certain types of stimulus, EndoG
carried out “death process” in caspase-independent manner by cleaving chromosomal DNA following
translocation to nucleus. EndoG is reported to be a critical mediator of caspase-independent cell death
occurred in brain trauma, ischemia and neurodegenerative disorders caused by oxidative damage. Thus
understanding how EndoG is regulated is an important step to propose potential therapeutic target for
those neurological disorders. To understand how EndoG is regulated, we sought to identify regulator to
interact with EndoG. Since Hsp70 is reported as an important modulator of EndoG, we tested a wellknown Hsp70 interacting E3 ubiquitin ligase, CHIP(Carboxyl terminus of Hsp70-Interacting Protein) for its
interaction with EndoG. In our study, CHIP interacts with EndoG and is able to regulate endoG protein
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level through ubiquitin-proteasome system. CHIP can also regulate mtEndoG which is a mutant form
mitochondrial targeting sequence is deleted and localized in cytoplasm rather than in mitochondrial
intermembrane space where wtEndoG is localized. CHIPK30A mutant which cannot interact with Hsp70
do not interact with EndoG and depletion of Hsp70 results in no EndoG ubiquitination, indicating that
EndoG interaction with CHIP is dependent on Hsp70. Depletion of CHIP lead to accelerated cell death
induced by oxidative insults. Altogether, our results suggest that CHIP and Hsp70 may play an important
role in regulating EndoG and EndoG-dependent cell death and might be a candidate of therapeutic target
for neurological disorders.
2820/L057
Deciliation of Kidney Epithelial Cells: A Potential Marker of Carcinogenic Effects.
R. Radford, C. Slattery, M. P. Ryan, T. McMorrow; S.B.B.S., U.C.D., Dublin, Ireland
Primary cilia are organelles which are found on the majority of differentiated mammalian cell types. In
addition to their role in maintenance of cellular polarity, growth factor signalling, flow sensing, and in some
cases phototransduction, primary cilia are now believed to act as a physical barrier which prevent
quiescent cells from re-entering the cell cycle. It has been suggested that exposure to potential
carcinogens elicit early effects on primary cilia. Therefore in this study we have examined the effects of
carcinogenic compounds on the primary cilia of human kidney epithelial cells. Preliminary experiments
have shown that exposure of human kidney epithelial cells (RPTEC/TERT1) to relatively low doses of
carcinogenic compounds including ochratoxin-A and KBrO3 result in significant deciliation of the epithelial
monolayer. Analysis of the effects of cell cycle confirms that the deciliation observed following exposure
to these compounds correlates with an increase in the percentage of the cell population in S-phase,
G2/M-phase and also multi-nucleate cells. Effects have also been observed on tight junction maintenance
downstream of deciliation, including deregulation of expression of the pore forming tight junction protein
claudin-2. In addition to this, microarray analysis has also shown perturbations in the expression of
multiple genes which are involved in mitosis, ciliary maintenance, and cellular polarity. Stimuli which
cause deciliation remove a physical checkpoint preventing differentiated cells from re-entering mitosis.
We have shown that multiple carcinogens induce human kidney epithelial cells to deciliate and re-enter
the cell cycle demonstrating that the primary cilium is a sensitive marker of early carcinogenic effects. As
such, prevention of deciliation may impede cell cycle re-entry and uncontrolled cellular proliferation.
2821/L058
Role of Conserved Orb6 Kinase in the Control of Cell Growth.
F. Verde; University of Miami, Miami, FL
David Wiley, Maitreyi Das and Fulvia Verde Department of Molecular and Cellular Pharmacology,
University of Miami Miller School of Medicine, Miami, FL, 33101-1015, USA. [email protected] The
conserved NDR kinase Orb6 controls cell morphogenesis in different organisms, ranging from yeast to
neuronal cells.. Experimentally reducing cellular Orb6 protein levels leads to shortened cell size and cell
length at mitosis. Thus, we investigated the effects of decreased Orb6 protein levels on cell growth and
protein translation. We found that cell expressing reduced levels of Orb6 kinase or cells over-expressing
kinase-inactive Orb6-K122A display an increased 80S/polysomes ratio and a reduced rate of radiolabeled amino-acid incorporation into proteins. Further, we found that Orb6 kinase has a role in the
control of conserved RNA binding protein Sts5/Orb4. Our observations suggest that Orb6 kinase, in
addition to its role in the control of cell polarity, has a function in the control of cell growth. In addition, our
data indicate that Orb6 protein levels are modulated in response to the nutritional status of the cell.
2822/L059
Poly-Unsaturated Fatty Acids Inhibit Depolymerisation of Cortical Actin Fibers and Cell Motility in
E. histolytica.
D. Manna; Biochemistry, Bose Institute, Kolkata, India
Entamoeba histolytica is the causative agent for dysentery and the formation of liver-abscess in human.
The motility of E. histolytica trophozoites is due to its actin cytoskeletal network. Analysis of a novel
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galacto-glycerolipid (GGL) that killed E. histolytica trophozoites showed that the anti-amoebic activity was
due to the poly-unsaturated alpha-linolenic acid (ALA) side chain in GGL. We discovered that like ALA,
several other unsaturated fatty acids were extremely effective in killing amoeba and the potency of killing
increased with the degree of unsaturation in the fatty acid. Real time microscopy showed that addition of
poly-unsaturated fatty acids to E. histolytica trophozoites led to the polarization of F-actin at one end of
the cells with prominent filopodial extensions. Accumulation of surface receptors and signaling molecules
(Eh Gal/GalNac lectin, glycoproteins and Eh Rho1) was observed adjacent to the polarized actin and
filopodia. Con- induced cap formation also showed polarization of F-actin with surface receptors. In
contrast to ALA, filopodial extensions were absent or much shorter in the Con-A treated caps.
Furthermore, ALA treatment led to inhibition of cell motility and lysis of cells while Con-A treated cells
remained actively motile and periodically shed the cap without loss of membrane integrity. Unlike known
actin polymerizing (Jasplakinolide) and depolymerizing agents (Cytochalasin-D or Latrunculin-A), polyunsaturated fatty acids cause cortical actin induced capping, lengthening of filopodial extensions and
membrane lysis. Our results suggest that poly-unsaturated fatty acids kill E. histolytica cells by preventing
depolymerisation of cortical actin fibers and consequent inhibition of cell motility and finally leading to cell
lysis.
2823/L060
Light-Mediated Activation Reveals a Key Role for Rac in Collective Guidance of Cell Movement In
Vivo.
1
1
2
2
1 1
X. Wang , L. He , Y. I. Wu , K. M. Hahn , D. Montell ; Johns Hopkins School of Medicine, Baltimore,
2
MD, University of North Carolina, Chapel Hill, Chapel Hill, NC
The small GTPase Rac induces actin polymerization, membrane ruffling and focal contact formation in
cultured single cells but can either repress or stimulate motility in epithelial cells depending on the
conditions. The role of Rac in collective epithelial cell movements in vivo, which are important for both
morphogenesis and metastasis, is therefore difficult to predict. Recently, photoactivatable analogues of
Rac (PA-Rac) have been developed, allowing rapid and reversible activation or inactivation of Rac using
light. In cultured single cells, light-activated Rac leads to focal membrane ruffling, protrusion and
migration. Here we show that focal activation of Rac is also sufficient to polarize an entire group of cells in
vivo, specifically the border cells of the Drosophila ovary. Moreover, activation or inactivation of Rac in
one cell of the cluster caused a dramatic response in the other cells, suggesting that the cells sense
direction as a group according to relative levels of Rac activity. Communication between cells of the
cluster required Jun amino-terminal kinase (JNK) but not guidance receptor signalling. These studies
further show that photoactivatable proteins are effective tools in vivo.
2824/L061
Dynamic Microtubules Are Involved in Human Endothelial Cells Barrier Function.
1,2
2
1
2
2
2
2 1
I. Alieva , E. Zemskov , I. Kireev , B. Gorshkov , D. Wiseman , S. Black , A. Verin ; 1Electron
2
Microscopy Department, Moscow State University, Moscow, Russian Federation, Vascular Biology
Center, Medical College of Georgia, Augusta, GA
The primary function of the endothelial cells (EC) lining the inner surface of all vessels is to regulate
permeability of vascular walls and control the exchange between circulating blood and tissue fluids. EC
cytoskeleton plays a crucial role in maintaining endothelial barrier function. Cytoskeleton reorganization
changes the cell shape and provides structural basis for an enhancement as well as a loss of barrier
function. EC adherens junctions (AJs), largely composed of vascular endothelial cadherin (VE-cadherin),
are the basic structure of endothelial permeability regulation because of their dynamic ability to open and
close. The role of actin cytoskeleton in the formation, maintenance, and functionality of AJs in EC is well
characterized. However, the potential link between VE-cadherin AJs and microtubules (MTs) in barrier
function has not been well investigated. Our previous data that the MT disassembly in EC barrier
dysfunction allowed us to assume that MT dynamics is an early event in the changes of pulmonary EC
barrier permeability. To understand how MTs contribute to the dynamic reorganization of the EC
cytoskeleton, we established an EC model expressing EB3-GFP, a protein that marks MT plus-ends.
Using this model, we were able to measure MT growth rate at the centrosome region and near the cell
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periphery of a single human EC and in the EC monolayer. We demonstrate that the majority of MTs in EC
are dynamic, the growth rate of their plus-ends is highest in the internal cytoplasm, in the region of the
centrosome. Growth rate of MT plus-ends decreases from the cell center toward the periphery. Our data
suggest the existing mechanism(s) of local regulation of MT plus-ends growth in EC. MT growth rate in
the internal cytoplasm of EC in the monolayer is lower than that of single EC suggesting the regulatory
effect of VE-cadherin AJs. Centrosomal MT growth rate distribution in single EC indicated the presence of
two subpopulations of MTs with “normal” (similar to those in monolayer EC) and “fast” (three times as
much) growth rates. Our results indicate functional interactions between VE-cadherin AJs and MTs,
dynamic MTs are capable of adjusting existing contacts and can adjust endothelial permeability.
2825/L062
Transport and Recycling Dynamics of the Budding Yeast Class V Myosin Myo2p.
K. W. Donovan, A. Bretscher; Molecular Biology and Genetics, Cornell University, Ithaca, NY
The polarization of proteins, lipids, and organelles within a eukaryotic cell allows for the spatial regulation
of numerous biological processes. Saccharomyces cerevisiae displays exaggerated polarized growth of
its plasma membrane during budding through the directed transport of secretory vesicles. In addition,
several organelles are actively transported into the growing bud. These processes are accomplished
through an interplay between formin-nucleated actin cables extending from the bud tip and neck and the
class V myosin motor Myo2p. While many of the components linking Myo2p to its various cargoes are
known, the dynamic behavior of the motor and how its dynamics is regulated at the molecular level
remains unclear. We have used molecular standards to define the number of motors bound to secretory
vesicles in transit to sites of growth. Further, photobleaching experiments indicate that the residency time
in the bud for an individual motor is on the order of seconds and that motor release from vesicles is a
regulated process. Taken together, these results support a model where Myo2p efficiently transports
secretory cargo to sites of growth and is rapidly released from cargo in a regulated manner.
2826/L063
Investigating Function of Nck Adaptor in Tyrosine Kinase Signaling to the Actin Cytoskeleton.
1,2
2,1
1,2
2,1 1
S. Borinskaya , J. A. Ditlev , L. M. Loew , B. J. Mayer ; R. D. Berlin Center for Cell Analysis and
2
Modeling, University of Connecticut Health Center, Farmington, CT, Raymond and Beverly Sackler
Laboratory of Genetics and Molecular Medicine, University of Connecticut Health Center, Farmington, CT
Many extracellular signals are transduced through Receptor Tyrosine Kinases. We investigate RTK
signaling to the actin cytoskeleton through the Nck adaptor protein. The SH2 domain of Nck binds
phosphorylated tyrosine residues of RTK and then SH3 domains of Nck bind and activate N-WASP
(Neural Wiskott-Aldrich Syndrome Protein). Activation of N-WASP involves release of the VCA domain
(verprolin-homology, central and acidic domains). Simultaneous binding of G-actin monomer and actin
nucleation factor Arp2/3 to the VCA domain initiates nucleation of a new actin branch. Aggregation of the
Nck SH3 domains on plasma membrane in vivo results in the formation of long, dynamic, moderately
dense actin comet tails. We utilize a variety of techniques to study this artificially induced localized actin
polymerization: cell culture and quantitative western blotting, confocal fluorescent microscopy and
quantitative image analysis, biochemical modeling in the Virtual Cell environment. Our model
recapitulates important features of Nck actin comets: length of the comet tails, F-actin concentration
within the tails, distance from the head of the aggregate to the maximum F-actin concentration location,
and dependence of these parameters on comet velocity and the number of Nck molecules in the
membrane cluster. However the model does not account for the fact that Nck binding proteins (WIP, PAK,
Abl) can have an effect on actin polymerization. The VCA domain of N-WASP is directly responsible for
Arp2/3 activation and can be used in our experimental system to bypass the need for adaptor proteins
such as Nck to initiate actin polymerization. Aggregation of VCA domains on the membrane in vivo
produced actin “blobs” - short thick structures. Understanding the differences between the Nck and VCA
induced localized actin assemblies will advance our knowledge of RTK signaling to mobilize and direct
actin cytoskeleton.
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2827/L064
Mechanisms for Cell-Width Control in Gram-Negative Bacteria.
C. Tropini, D. Swem, M. Hidestrand, S. Sinha, K. Huang; Stanford, Stanford, CA
A longstanding question in bacterial physiology is the mechanism by which cells select and maintain a
particular shape. Despite extensive biochemical investigation of the cell wall, even the question of how E.
coli robustly selects a precise diameter remains unanswered. Interestingly, many closely related rodshaped Gram-negative bacteria adopt radii ranging from 400 nm to 2 microns, and in each case width is
robustly maintained. Recent in vitro experiments have shown that the enzyme PBP2, responsible for
glycan strand synthesis during cell elongation, acts with a species-dependent processivity. Our
quantitative modeling predicts that, in response to an overall decrease in distribution of glycan strand
lengths, the cell increases in its radius as the glycan strands straighten and elongate with increased
tension. To test the relevance of this variation experimentally, we explore a systematic transplantation of
penicillin binding protein 2 (PBP2) between related rod-shaped Gram-negative bacteria with different
radii. Preliminary results find that substitution of Escherichia coli PBP2 with the other homologs gives rise
to E. coli cells showing altered diameter but normal morphology.
2828/L065
MTBindingSim: A Computer Program Designed to Help Students and Researchers Understand
Microtubule Binding Proteins.
1
2
3
1
1 1
J. T. Philip , C. H. Pence , E. Bunker , A. Slabbekoorn , H. Goodson ; Chemistry and Biochemistry,
2
University of Notre Dame, Notre Dame, IN, Program in History and Philosophy of Science, University of
3
Notre Dame, Notre Dame, IN, Department of Chemistry, Evergreen State College, Olympia, WA
In order to understand the function and mechanism of microtubule binding proteins (MTBPs), it is
important for experimentalists to establish how these proteins bind to MTs. Many MTBPs exhibit binding
behavior that cannot be explained by simple first order binding, and several models have been proposed
to account for the experimentally observed binding data. We have developed MTBindingSim, a MATLAB
program designed to simulate binding curves for MAPs binding to MTs using various binding models. The
purpose of this program is to help researchers develop intuition regarding protein binding behavior in
different binding models as well as to assist researchers in designing binding experiments that will be
able to distinguish between these models. The program can simulate three different experimental modes,
corresponding to experiments commonly found in the literature. The first is a "classic" binding experiment
where the MAP concentration is held constant and the MT concentration is varied. In the second
experimental mode the MT concentration is held constant while the MAP concentration is varied. The
third mode simulates a competition experiment involving two different MAPs, A and B. In these
competition experiments, the concentrations of A and MTs are held constant and the concentration of B is
varied. All experimental modes can either simulate single curves or multiple curves for easy comparison.
MTBindingSim is freely distributed using Google Code under the GNU General Public License.
2829/L066
Centrosome Migration to the Cortex during Symmetry Breaking in C. elegans Zygotes.
D. Bienkowska, C. Cowan; IMP, Vienna, Austria
Establishment of cell polarity is a fundamental process required for cell differentiation, providing a
vectorial axis for the distribution of cellular compartments and regulatory molecules. A spatial cue, either
from the environment or from within the cell, breaks symmetry and allows polarization. In one-cell C.
elegans embryos, functional symmetry breaking occurs in the acto-myosin cortex, where a local change
in contractility leads to the establishment of global cortical polarization. Symmetry breaking depends on
the sperm-provided centrosomes that move from the cell interior to the cortex sometime during the
approximately twenty minutes between fertilization and polarity establishment. How do centrosomes find
the cortex? We investigated the mechanism of centrosome migration using fluorescent timelapse
microscopy and quantitative analysis of centrosomal movement. The majority of centrosome motion - until
approximately one minute before symmetry breaking - resembled a random walk with a bias toward the
cortex. The bias appeared to become stronger with decreasing centrosome-cortex distance. Cortical bias
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was prevented by actin depolymerization or by depletion of the centrosome component SPD-5. Bulk
cytoplasmic flow did not provide directionality, suggesting cortex-directed migration was a unique property
of centrosomes. However, centrosomes did not always reach the cortex during this time. Close
approximation of centrosomes to the cortex occurred during a short period of highly processive migration
at increased velocity about one minute before symmetry breaking, implying a change in the centrosome
migration mechanism. Thus centrosomes appear to use a random strategy to come into proximity of the
cortex, which then may facilitate a switch to directed motility of centrosomes to the cortex during
symmetry breaking.
2830/L067
Control of Metastasis by Selective Activation of Akt1/Pkbα.
E. Kim, S. Yun, J. Ha, Y. Kim, I. Jin, D. Woo, C. Kim, S. Bae; Pharmacology, Pusan National University,
Yangsan, Republic of Korea
The mammalian target of rapamycin (mTOR) which is a highly conserved serine/threonine kinase
activated in response to growth, proliferation, and according to recent findings, is also critical for cell
migration. mTOR exists in two complexes, mTORC1 and mTORC2 which mediates growth and
proliferation, respectively. It has been reported that P-Rex1 forms molecular complex with mTORC2 and
mediates Rac activation. However, it is still not clear whether P-Rex1/mTORC2 complex-mediated
regulation of cell migration is also regulated by Akt. Here, we showed that molecular complex formation of
Rictor/P-Rex1/Akt1 is necessary for insulin-like growth factor-1 (IGF-1)-induced migration of human ovary
carcinoma (SKOV3) cells. To dissect the functional roles of Rictor/P-Rex1/Akt1 complex during IGF-1induced cell migration, we have established knock-down cell lines by lentiviral-mediated shRNA. As
results, silencing of mTOR and P-Rex1 completely abolished IGF-1-induced SKOV3 cell migration and
invasion. Interestingly, silencing of Akt1 showed impairment of IGF-induced cell migration and invasion
whereas silencing of Akt2 did not affect IGF-1-induced SKOV3 cell migration and invasion. In correlation
with this result, Akt1 preferentially associated with Rictor, on the other hand, association of Akt2 with
Rictor was relatively weak. In addition, IGF-1 induced Akt activation was reduced in cells silencing PRex1. GTP-Rac1 was translocated form cytosol to the leading edge of membrane upon IGF-1 stimulation.
However, P-Rex1, Rictor knock-down cell abolished IGF-1-induced Rac1 translocation and Rac1
activation. Finally, in order to confirm our previous data, we used nude mice to check the metastasis of
cancer cells. Interestingly, injection of Akt1 knock-down cells impaired tumor metastasis. Given these
results, we suggest that SKOV3 cell metastasis is specifically regulated by Rictor/P-Rex1/Akt1 complex
thereby selective activation of Rac small G protein.
2831/L068
The Small GTPase Rab18 Participates in Microtubule-Based Secretory Granule Transport.
1,2,3
1,2,3
1,2,3
1,2,3
R. Vazquez-Martinez , M. R. Pulido , F. Almabouada , F. Gracia-Navarro , S. Garcia1,2,3
1,2,3 1
Navarro , M. M. Malagon ; Dept. Cell Biology, Physiology and Immunology, University of Cordoba,
2
3
Cordoba, Spain, IMIBIC, Cordoba, Spain, CIBERobn, ISCIII, Spain
Several Rab family members control vesicle trafficking by regulating the activity of different components
of the vesicle transport machinery. Particularly, Rab18 inhibits secretory granule movement, but how the
GTPase accomplishes this role and the identity of Rab18 effectors remain unknown. Objective: We
compare the motility of Rab18 granules with that exhibited by granules carrying the secretory peptide
NPY, investigate the relationship of Rab18 granules with microtubules, and identify certain Rab18
effectors related to cargo movement. Methods: Rab18 granule dynamics were studied by time-lapse
microscopy followed by vesicle tracking analysis. In addition, Rab18 interacting proteins were identified
by FRET. Results: Video-microscopy revealed that most NPY and Rab18 granules exhibited saltatory
movements. The percentage of NPY and Rab18 granules displaying anterograde and retrograde
movements were similar (47%), as were their diffusion coefficients. However, after K+ stimulation,
percentage of NPY granules moving anterogradely increased (64%), as did NPY granule DC, whereas
the number of anterograde Rab18 granules (39%), and their DC decreased. We also analyzed
distribution of Rab18 granules in relation to microtubules by confocal microscopy. We found a close
spatial relationship between both structures that was compromised by microtubule depolymerization with
SUNDAY
nocodazole. This treatment also blocked Rab18 recruitment to granules. Given that Rab18 modifies
granule transport dynamics, we investigated whether its function involves modulation of components of
the microtubule-based transport machinery by protein-protein interactions. Thus, FRET experiments
showed that Rab18 interacts with kinesin-1 light chain in its active form (after K+ treatment or using the
Rab18 constitutive active mutant) and with huntingtin in its inactive conformation. Conclusion: Altogether,
these data suggest that Rab18 impairs transport of secretory granules by regulating the activity of certain
components of the microtubule-based transport apparatus. Support: MICINN/FEDER (BFU200760180/BFI, HF2007-0002), FIS/ISCIII (CIBEROBN CB03/06), J. Andalucía (CVI-0139, P07-CTS-03039),
Spain.
2832/L069
Mechanical Regulation of Epithelial Cell Collective Migration.
M. R. Ng, J. Brugge; Cell Biology, Harvard Medical School, Boston, MA
Collective migration is a mode of cell migration characterized by the movement of a cell group, strand or
sheet that maintains cell- cell junctions. Although it is an important process in morphogenesis, tissue
repair, and tumor invasion, the mechanisms governing collective migration are not well understood.
Orchestration of collective migration involves maintenance and coordinated regulation of both cell-cell
and cell- matrix adhesions. To understand how crosstalks between these two types of adhesions enable
collective migration, we first investigated the contribution of cell-matrix adhesions by modulating substrate
stiffness. Using a modified wound-healing assay, we found that the average rate of wound closure
increases with substrate stiffness. This is correlated with an increase in individual cell migration speed,
persistence and directionality on stiffer substrates, as well as differential organization of actin
cytoskeleton and focal adhesions. To address the importance of cell-cell adhesion in this effect, we
disrupted cell-cell adhesions using a dominant inhibitory cadherin mutant, and found that loss of cell-cell
contacts increases wound closure rate on soft substrates but not on stiff substrates. This suggests that
the role of cell-cell adhesions in mediating collective migration is dependent on the nature of cell-matrix
interactions. Ongoing studies include the use of fluorescence imaging and traction force microscopy to
characterize the nature of cell-matrix adhesions and cell-cell mechanical coupling on different substrate
stiffnesses.
2833/L070
Investigating the Roles of Centrioles in Mouse Development.
H. Bazzi, K. V. Anderson; Developmental Biology, Sloan-Kettering Institute, New York, NY
Our lab has discovered that a tiny organelle, the primary cilium, is required for Sonic Hedgehog (Shh)
signaling in mice. Primary cilia, like other microtubule based cilia and flagella, use a centriolar template
which is at the core of the centrosome. The centrosome has historically been regarded as the microtubule
organizing center of animal cells especially due to its localization at the spindle poles during mitosis,
although recent studies suggest that its most important role may be in nucleation of the primary cilium.
Because the centrosome also houses many signaling molecules like kinases (protein kinase A, Aurora
kinase, etc.), cyclins (E and A) and proteasomes, we hypothesize that the centrosome also functions as a
signaling center. To investigate this hypothesis we obtained a mouse line carrying a gene trap mutation in
Sas4, which encodes a protein essential for centriole duplication. We found that Sas4 gene trap insertion
causes only a partial loss of gene function. The resulting mutants live to adulthood, are fertile, but are
smaller than their normal siblings perhaps due to skeletal defects. Using cre recombinase we further
disrupted the engineered locus to generate a null allele. The null mutant embryos die at mid-gestation
(e8-e9). These embryos have no detectable interphase centrosomes, but retain mitotic centrosomes, and
possess no cilia. Sas4 mutant embryos have widespread cell death but are still capable of forming
relatively normal embryonic organizers like the midline. Gene trap/null embryos die before birth with extra
digits on all limbs, a phenotype consistent with partial disruption of the Hedgehog pathway. Our work
supports a role for centrioles in both Hedgehog signaling and other aspects of mouse development.
SUNDAY
2834/L071
A Tgfβ1-Generated ‘EMT-Like’ Response Is Coupled with Non-Muscle Myosin II Activation in
Epidermal Keratinocyte Migration.
1
1
1
2
2
1 1
J. Monslow , J. Beach , J. Crish , J. Mack , E. Maytin , T. T. Egelhoff ; Cell Biology, Cleveland Clinic,
2
Cleveland, OH, Biomedical Engineering, Cleveland Clinic, Cleveland, OH
Cell migration is an essential feature of re-epithelialization during wound healing. Non-muscle myosin II is
required for the mechanics of cell movement, but surprisingly little attention has been devoted to this
machinery in the context of epidermal wound closure. TGFβ signaling is one of the major pathways
regulating wound healing. Early studies suggested TGFβ signaling promoted the healing process by
facilitating epithelial cell migration, fibroblast differentiation and matrix remodeling. However, more recent
studies have found that it can have detrimental effects. Our collaborators (Mack and Maytin) have
observed enhanced TGFβ signaling in epidermal wound margins by increased nuclear staining of
phosphorylated SMAD2. We therefore sought to determine the specific role of TGFβ1 on epidermal reepithelialization alone, along with the related function of non-muscle myosin II in this process. TGFβ1
enhanced the expression of non-muscle myosin IIA and IIB (but not IIC) in both primary human
keratinocytes and a rat epidermal keratinocyte (REK) cell line. This was coupled by an increase in
markers of epithelial to mesenchymal transition (EMT), including vimentin, fibronectin and S100-A4, as
well as a decrease in E-cadherin. These changes correlated with accelerated cell migration as observed
in REK scratch-wound assays and a marked decrease in cell proliferation at the wound margin.
Additionally, the increased cell motility by TGFβ1 was prevented by inhibition of myosin activity
(blebbistatin) and EGF receptor signaling (AG1478). These results show that TGFβ1 signaling appears to
promote a pro-migratory phenotype in keratinocytes, and that re-epithelialization is partially driven by an
EMT-like transformation. We are now reproducing this phenomenon in a 3D model of epidermal wound
closure, and determining the specific roles of myosin IIA and IIB (by selective gene knockdown) in this
process.
2835/L072
Control of Vascular Tube Formation by Microtubule Tip Complex Proteins in 3D Collagen
Matrices.
D. Kim, G. E. Davis; Medical Pharmacology and Physiology, University of Missouri-Columbia, Columbia,
MO
Microtubules are major building blocks for cell structure and play a crucial role in intracellular transport,
cell polarity, cell division, cell motility and cell morphogenesis. The functional role of microtubules and
microtubule plus-end regulatory proteins during vascular tube morphogenesis has not been previously
addressed. To begin to address the molecular basis for how microtubule dynamics affect EC lumen and
tube formation, we have examined the role of microtubule plus-end regulatory proteins, as well as
microtubule post-translational modifications during these events. An si-RNA based approach was used to
screen for microtubule associated proteins that affect vascular lumen and tube formation in 3D collagen
matrices. Suppression of EB1, p150Glued and Clasp1 (microtubule tip complex proteins) expression
using at least two independent si-RNAs leads to marked blockade of EC lumen and tube formation. EB1
siRNA suppression also blocks kinase activation cascades during EC lumenogenesis including Src, Pak2,
Pak4 and Erk1/2 and shows reduced vascular guidance tunnels which form secondary to MT1-MMPmediated proteolysis. We also evaluated the post-translational modification of microtubules during
vascular tube formation. Microtubule acetylation and polyglutamylation are increased during early stage
of lumen formation while interestingly, detyrosinated tubulin and delta-2 tubulin are detected following
establishment of EC tube networks. ECs depleted of EB1 leads to blockade of microtubule
posttranslational modifications (i.e. acetylation, polyglutamylation and detyrosination). Re-expression of
adenovirus EB1 in EB1 knockdown cells restores microtubule acetylation as well as EC tube formation,
whereas overexpression of control-GFP in EB1 knockdown ECs fails to restore microtubule acetylation
and tube formation. Taken together, EB1 can regulate microtubule dynamics including post-translational
tubulin modifications and is necessary for vascular lumen formation by regulating various microtubule
functions. This work describes the novel involvement of microtubule tip complex proteins during EC lumen
and tube formation in 3D collagen matrices.
SUNDAY
2836/L073
Regeneration of Giant Ciliary Comb Plates in Ctenophores.
S. L. Tamm; Biology, Boston University, Boston, MA
A long section of a comb row including 20-30 locomotory comb plates was surgically removed from
Mnemiopsis leidyi. The wound rapidly closed and healed within 3-4 hours, increasing the distance
between comb plates near the former cut edges by variable amounts (up to 10x the normal interplate
spacing). Video microscopy followed the regeneration of new comb plates in the gaps between the
widely-separated ones. A localized widening of the interplate ciliated groove (ICG) appears first, followed
by outgrowth of two opposing groups of comb plate cilia on either side of the ICG widening. The split
halves of a new comb plate elongate as their bases extend laterally outward and continue ciliogenesis.
Ciliary lengths and bases of split halves are often asymmetrical at this early stage. The growing halves
move closer together into the ICG widening until they merge into a single comb plate of uniform length
with a common base that interrupts the ICG. Video DIC snapshots of dissected gap rows 1.5-3 days after
the operation show that ICG widenings and/or new plates do not all appear at the same time or with
uniform spacing within a gap. As a result the lengths of young plates in a gap are quite variable. Stereo
microscopy of intact animals 3-4 days after the operation show that all of the new plates that will form in a
gap are present, fairly evenly spaced and similar in length, but smaller and closer together than normal.
Later the regenerated comb plates attain the characteristic length and regular spacing of the other plates
in the comb rows. In conclusion, the puzzling spatial and temporal variability in onset of new comb plate
formation is somehow transformed into the precise morphological pattern of mature comb rows.
2837/L074
Microtubule Motility in Crowded Conditions.
1,2
2 1
L. Liu , J. L. Ross ; Biochemistry and Molecular Biology, University of Massachusetts Amherst,
2
Amherst, MA, Physics, University of Massachusetts Amherst, Amherst, MA
Microtubules and their associated proteins form a complex network in the cell to perform many functions
such as cellular trafficking. Motor proteins such as kinesin-1 travel on microtubules to transport various
cargo across the crowded cell. Many in vitro assays only study the biophysical properties of microtubules
and motors at low density. In the gliding assay low concentrations of fluorescently labeled microtubules
are visualized gliding as motors immobilized on a cover slip push them. By adapting this assay to high
density, gliding assays can study the physical properties of these biomolecules in a more physiological
setting. We find that gliding velocity is dependent on both motor and microtubule concentrations. Until a
critical concentration is reached, the gliding velocity increases with increasing motor concentration after
which the motors become less efficient. Conversely, increasing the microtubule concentration causes an
increase in the gliding velocity. At the maximum microtubule concentration, self organized patterns such
as vortices appear. These different behaviors give insight into how crowded conditions, such as those in
the cell, might affect motor behavior and cytoskeleton organization.
2838/L075
Interactions between Tau and Microtubules.
A. Slabbekoorn, H. Goodson; University of Notre Dame, Notre Dame, IN
Tau is a neuronal microtubule (MT) associated protein found abnormally aggregated into plaques in the
brains of Alzheimer’s patients. Tau’s normal cellular function is to stabilize MTs, which are cylindrical,
intracellular tubes composed of tubulin subunits. MTs can dynamically grow (polymerize into long
filaments) and shrink (depolymerize/ fall apart) in order to perform many critical cellular functions, such as
chromosomal separation during mitosis and intracellular cargo transportation. MT dynamicity is regulated
by MT-associated proteins including tau. To better understand the role of tau in cellular function and
disease, we are studying the interaction of tau with MTs. Surprisingly, previous analyses have reported a
wide range for tau-MT binding ratios and affinities. The origins of this variability are not known. We
hypothesize that the tau-tau interactions which occur in Alzheimer’s disease might also occur under
normal conditions, resulting in different binding behaviors at different tau concentrations. To test this
hypothesis, we are measuring the affinity of tau for MTs under different tau concentrations. Our
SUNDAY
preliminary data indicate that the apparent affinity of tau depends on the concentration of tau, suggesting
that tau-tau interactions may contribute to normal tau function. To better understand the MT stabilizing
mechanism of tau, we are also testing the sensitivity of tau for different tubulin conformations. Our
analyses thus far show that tau binds to GMPCPP-MTs more weakly than it binds to Taxol-stabilized
GDP-MTs. GMPCPP-MTs are believed to mimic the conformation of the GTP cap at the dynamic MT end,
while Taxol-stabilized GDP-MTs represent the conformation of the MT lattice. The observation that tau
binds weakly to GMPCPP-MTs suggests that it may direct its stabilizing activity towards the MT lattice, in
part by actively avoiding the dynamic MT end.
2839/L076
Golgi Localization Is Impaired in Dcx-/Y Or Dclk-/- Deficient Neurons.
1
2
1 1
X. Fu , C. A. Walsh , J. S. Liu ; neuroscience, Children's national medical center, Washington, DC,
2
Neurology and Program in Neuroscience, Harvard Medical School, Boston, MA
In neurons, the Golgi apparatus forms outposts in dendrites and trafficking from dendritic Golgi has been
implicated in growth during neuronal development. In primary cultures of Dclk-/- or Dcx-/y deficient
neurons, Golgi outpost formation in dendrites is significantly decreased compared to those in wildtype
neurons. The outpost formation of Golgi in dendrites is further impaired in neurons which are doubly
deficient in both Dcx and Dclk. This result is obtained by measuring the distance of Golgi outposts in
dendrites to the nearest edge of nucleus. To understand which portion of Dcx is important for the Golgi
outpost formation in dendrites, we co-transfected Dcx siRNA with Dcx C-terminus or N-terminus in
cultured neurons. The expression of Dcx N-terminus in Dcx deficient neurons can rescue the phenotype
of impaired Golgi outpost formation in dendrites, but not the C-terminus of DCX. The N-terminus of Dcx is
the microtubule binding domain. Our data show Dcx and Dclk1 are involved in Golgi outpost formation
and they support the idea that these proteins are involved in protein sorting and trafficking.
2840/L077
Dynein Facilitates Cell Migration in 3-Dimensional Environment.
D. Kim, S. Rhee; Department of Life Science, Chung-Ang University, Seoul, Republic of Korea
The biomechanical features of the extracellular matrix (ECM) surrounding cells are critical for cell
morphogenesis and migration. However, the molecular mechanisms underlying these features remain
poorly understood. We have previously found that microtubule function is necessary for cell spreading in
3-dimensional (3D) soft collagen matrices. In this study, we tested the effects of microtubule motor
proteins on cell migration in different mechanical environments. Interference with activities of myosin II or
its upstream activator, Rho kinase, caused low tensional status in cells but still promoted cell migration in
response to serum stimulation in a 2-dimensional (2D) condition. However, the combination of a dynein
inhibitor (EHNA) with myosin II or Rho kinase inhibitors markedly decreased cell migration on a 2D
surface and in 3D soft collagen matrices. Silencing of the dynein intermediated chain and KIF3A
expression revealed that dynein was specifically involved in cell migration in low tensional environments.
Finally, ablation of dynein expression did not induce cell migration on a soft polyacrylamide matrix,
supporting the notion that microtubule function through dynein may contribute to cell migration in a soft
matrix such as a 3D environment.
2841/L078
Calcium Dynamics Regulate the Persistence of Dendritic Cell Motility.
1
1
2
2
1 1
P. Vargas , P. Solanes , G. Gautier , P. Launay , A. Lennon-Duménil ; Inserm U932, Institut Curie,
2
Paris, France, Inserm U699, Hôpital Bichât, Paris, France
The success of the adaptive immune response relies on the ability of leukocytes to coordinate their
individual immune function with cell migration. This particularly applies to dendritic cells (DCs), which
sample peripheral tissues for the presence of potential infectious agents by coupling efficient antigen (Ag)
uptake and processing to high cell motility. We have shown that migration and Ag processing are coregulated in DCs by a protein complex that contains the MHC class II-associated Invariant Chain (Ii) and
the actin-based motor protein Myosin II. While the association between Ii and Myosin II promotes Ag
SUNDAY
processing for presentation onto MHC class II molecules, it impairs DC motility, suggesting that the use of
common regulators enables DCs to coordinate Ag processing and cell migration in time and space. In this
study, we investigated whether calcium signaling is involved in the regulation of Myosin II activity by Ii. We
found that, while migrating into confined space, Ii-deficient DCs display important calcium flashes that are
not observed in their wild-type counterpart. Accordingly, the influx of calcium induced upon liberation of
ER calcium stores is increased in Ii-/- DCs, suggesting that Ii may regulate Myosin II activity and DC
motility by altering calcium dynamics. In agreement with this hypothesis, the increased in the persistence
of migration observed in Ii-deficient cells is lost when preventing calcium influx. Altogether these data
indicate (1) that calcium dynamics are regulated by Ii in migrating DCs and (2) that such control allows the
tight coupling between their motility and their antigen presentation function. Our results further suggest a
key role for calcium signaling in the regulation of migration persistence and maintenance of cell polarity.
2842/L079
Protein Interactions of Gamma-Tubulin Reveal Its Multiple Roles in Acentrosomal Plant Cells.
P. Binarova, A. Doskocilova, H. Kourova, O. Plihal, J. Volc, P. Halada, L. Kohoutova; Inst. Microbiology
AS CR, v.v.i., Prague 4, Czech Republic
Broad cellular distribution of plant gamma-tubulin in cytoplasm, with microtubules, membranes, nuclear
envelope, and with kinetochore region of chromosomes is consistent with acentrosomal nature of the
plant cells where several microtubular arrays are organized from undefined dispersed sites. We found,
that compared to well-defined cytoplasmic gamma-tubulin ring complexes in animal cells, complexes of
plant gamma-tubulin with homologues of GCP2, GCP3, and GCP4 proteins were more heterogeneous in
size and localized in the cytoplasm, with membranes and microtubules. Beside the GCP proteins that
present conserved components of gamma-tubulin complexes we identified several novel protein
interactions for plant gamma-tubulin. One of them designated as NodGS is a fusion protein composed of
an amidohydrolase domain and a GSI domain. The NodGS that belongs to yet uncharacterized proteins
was found to be widespread across plant species. The oligomeric assembly of the NodGS was the
dominant protein form in the cytoplasm and in microsomal fraction, a minor portion of the protein pool was
associated with a subpopulation of heterogeneous gamma-tubulin complexes. The phenotype of RNAi
Arabidopsis seedlings showed retardation of primary root growth that was accompanied by increased
level of lateral and adventitious root formation and cell division defects. Expression of the NodGS protein
was upregulated after Flg22 elicitor treatment. Our data suggest that the NodGS is a morphogenetic
factor responding to biotic stress. Supported by MŠMT LC545, IAA500200719, GACR 204/09/H084, and
GACR204/07/1169.
2843/L080
Multiplex Biosensors for Quantifying Activation of Parallel MAPK Pathways in a Single Living Cell.
1
1,2,3 1
C. Hsu , K. M. Hahn ; Division of Medicinal Chemistry and Natural Products, University of North
2
Carolina at Chapel Hill, Chapel Hill, NC, Department of Pharmacology, University of North Carolina at
3
Chapel Hill, Chapel Hill, NC, Lineberger Comprehensive Cancer Center, University of North Carolina at
Chapel Hill, Chapel Hill, NC
The mitogen-activated protein kinase (MAPK) signaling cascades govern numerous cellular processes
and are regulated mainly through MAPK kinases (MAPKK). Subtle differences in the relative timing and
location of activation for different MAPKK can lead to highly divergent cell response (i.e. apoptosis versus
cell motility). No biosensors for MAPKK are currently available. To visualize the spatiotemporal dynamics
of multiple MAPKK in a single living cell, we are developing sensitive biosensors that use complementary
wavelengths for simultaneous imaging of two or more MAPKK. MAPK, the substrates of MAPKK are
mutated and covalently labeled with environment-sensitive fluorescent dyes to act as substrate-based
biosensors of MAPKK activity. Recently, we synthesized a library of cysteine-reactive versions of red,
near infrared and ratiometric merocyanine dyes for use with MAPKK biosensors. Labeling MAPK with
these new dyes, our preliminary results indicate that dye-labeled ERK2 and p38α are substrate-based
biosensors for MEK1/2 and MKK3/6, respectively. These biosensors respond only to the active
conformation of the corresponding MAPKK.
SUNDAY
2844/L081
Syntaxin 6 Plays a Role in Α5β1 Integrin Recycling and Endothelial-Cell Motility.
1
1
2
2
1 1
A. Tiwari , J. Jung , C. Brown , A. Goel , A. Choudhury ; Anatomy and cell biology, University of Iowa,
2
Iowa City, IA, Department of Radiation Oncology, University of Iowa, Iowa City, IA
The α5β1 integrin mediates endothelial-cell adhesion and plays an important role during angiogenesis.
Cell-surface levels of α5β1 integrin are regulated by endocytic recycling as well as by secretory transport
of newly synthesized receptors to the plasma membrane. In an ongoing study, we have demonstrated
that the cell-surface level of α5β1 integrin is regulated by syntaxin 6, a t-SNARE protein that localizes to
both the trans-Golgi network and endosomes. Loss of syntaxin 6 function results in decreased α5β1
integrin recycling and enhanced degradation. Our data suggest that syntaxin 6 regulates turnover of α5β1
integrin primarily from the endosome to the cell surface, we will discuss the implications of these findings
in the regulation of integrin-mediated endothelial-cell dynamics.
2845/L082
2+
Lipid Rafts Is Involved in Ca /Calpain-Dependent Degradation of Ci-Sp17 for Activation of Sperm
Motility in the Ascidian Ciona Intestinalis.
L. Zhu, A. Hozumi, K. Inaba; Shimoda Marine Research Center, University of Tsukuba, Shimoda City,
Japan
Lipid rafts are specialized microdomains, which function as a platform for cell signaling across plasma
membrane in many animals and plants. Disruption of lipid raft by methyl-β-cyclodextrin (MCD) showed
inhibition of the activation of sperm motility by an egg-derived sperm activating- and attracting-factor
(SAAF) in the ascidian Ciona intestinalis. Theophylline-induced motility activation, however, was not
affected by MCD, suggesting that lipid rafts are needed in the pathway between SAAF reception and the
2+
activation of adenylyl cyclase in SAAF-induced activation of sperm motility. On the other hand, Ca
ionophore A23187 induces the activation of Ciona sperm motility. The activation was inhibited in MCD2+
treated sperm, suggesting that lipid rafts is involved the pathway between SAAF reception and Ca
2+
influx. Transient changes in intracellular Ca are required for sperm chemotaxis. We showed that MCD
inhibits the formation of asymmetric flagellar waveform and sperm chemotactic behavior. Thus, these
2+
results suggest that SAAF-dependent Ca influx is lipid raft-dependent and is necessary for both motility
activation and chemotaxis of Ciona sperm. Biochemical analysis indicated that several proteins related to
SAAF-dependent sperm activation, such as 14-3-3 proteins, p97, F0-d and Ci-Sp17, are present in lipid
raft fraction. We found that a cancer-testis antigen Ci-Sp17 is degraded at activation of sperm motility in a
2+
Ca /calpain-dependent pathway. MCD-treated sperm showed inhibition of Ci-Sp17 degradation. Taken
2+
together, lipid raft may be necessary to induce Ca influx in response to SAAF reception, leading to
activate calpain for degradation of Sp17 and ultimately resulting in the activation of adenylyl cyclase.
2846/L083
Differential Mechanoregulation of FilGAP and Integrin Binding to Filamin A.
1
2
1
1
1 1
A. Ehrlicher , D. A. Weitz , J. H. Hartwig , T. P. Stossel , F. Nakamura ; Medicine, Brigham & Women's
2
Hospital, Boston, MA, Physics, Harvard University, Cambridge, MA
Cells sense and respond to mechanical stimuli by altering their shapes, growth, and differentiation.
Filamin A (FLNa) is a flexible F-actin crosslinker and scaffolding protein, whose structure can be altered
by F-actin-transduced mechanical force. We have focused on two FLNa binding partners, FilGAP and
integrin. FilGAP-, a GTPase-activating protein specific for Rac, dependent cell spreading and protection
of cells from force-induced apoptosis requires FLNa. We hypothesized that strain alters FLNa’s
conformation to inhibit FilGAP binding, while facilitating integrin binding, and determined the binding
kinetics of FilGAP or integrin tagged with the photoconvertible fluorophore, PA-GFP, in FLNa F-actin
networks under shear forces by Fluorescence Loss After photoConversion (FLAC), a novel high-speed
alternative to FRAP. Binding of activated PA-GFP-FilGAP to FLNa decreased after application of shear
stress while strain increased the affinity of the integrin to FLNa. As a more physiological alternative to the
shear cell device, we used myosin II in the presence of photolabile ATP to generate controlled internal
mechanical stresses. Activation of myosin II by uncapping ATP modulated the binding kinetics for FilGAP
SUNDAY
and integrin within FLNa-F-actin networks in similar fashion to the application of external strain. These
findings demonstrate that mechanical stress regulates the FLNa-FilGAP and FLNa-integrin interactions
and suggest that mechanical regulation of FLNa-binding partner interaction occurs under physiological
conditions.
2847/L084
Expression Pattern and Localization of LINC Complex Components during Mouse Retinal
Development.
G. Peebles, D. Razafsky, S. Zang, D. Hodzic; Ophthalmology and Visual Sciences, Washington
University School of Medicine, St Louis, MO
Transmembrane nuclear envelope Sun proteins (Sun1 and Sun2) and Nesprins (Nesprin 1, 2, 3 and 4)
interact within the perinuclear space to provide macromolecular scaffolds (called LINC complexes) that
span the NE and connect the nuclear lamina to cytoskeletal networks. Even though the functional role of
LINC complex components in nuclear migration and anchorage is now well established, little is known
about their temporal expression and localization during mammalian tissues development. Mammalian
retinal development provides an excellent model to analyze the expression pattern of LINC complexes in
the CNS. Indeed, the accessibility of the mammalian retina allows for qualitative, quantitative and
localization analyses of LINC complexes components at different developmental stages. Furthermore,
retinal development is accompanied by different types of nuclear movement such as interkinetic nuclear
migration within retinal precursor cells, nuclear translocation during laminar position specification and
nuclear anchorage in the mature laminated retina. Here, using RT-PCR and immunofluorescence
microscopy, we characterized the expression pattern of A- and B-type lamins, Sun proteins and Nesprins
during mouse retina development and show that the expression of some of its components is
developmentally regulated and/or preferentially associated to a restricted set of neurons. These results
indicate that the composition of LINC complexes is developmentally regulated and varies with cellular
identity.
2848/L085
1
2
Do Gradients Really Matter in Directing Stem Cell Migration from a Cell Source? E. D. Miller , K. Li ,
3
3,5
4
1,5 1
T. Kanade , L. Weiss , L. M. Walker , P. Campbell ; Biomedical Engineering, Carnegie Mellon
2
University, Pittsburgh, PA, Electrical and Computer Engineering, Carnegie Mellon University, Pittsburgh,
3
4
PA, Robotics Institute, Carnegie Mellon University, Pittsburgh, PA, Chemical Engineering, Carnegie
5
Mellon University, Pittsburgh, PA, Institute for Complex Engineered Systems, Carnegie Mellon
University, Pittsburgh, PA
The purpose of this study was to investigate how engineered gradients of immobilized exogenous growth
factors influence stem cell population collective guidance over extended time (>1 day) and length (>1
mm) scales. Patterns of low-to-high, high-to-low, and uniform concentrations of heparin-binding epidermal
growth factor-like growth factor were inkjet printed at precise locations on fibrin substrates. Manual and
automated computer vision-based cell tracking were used to image and analyze the response of
mesenchymal stem cells seeded at the pattern origins. Cells on high-to-low patterns exhibited the highest
collective cell population movement immediately out from the cell sources. However, over time, as cell
populations moved further away from the cell source, collective cell guidance on all printed patterns
behaved more similarly. The overall trend was for all printed patterns to preferentially direct collective cell
guidance away from the cell source compared to unprinted controls. Although there were differences in
the forces driving collective cell guidance on each pattern the most predominant driving force was “cell
diffusion” resulting from high-to-low cell-to-cell confinement pressure gradients originating at the cell
starting lines. Spatial regions of higher cell proliferation, in register to localized higher growth factor
surface concentrations at the origins of printed high-to-low and uniform patterns, maintained the
confinement gradient in comparison to unprinted control and printed low-to-high growth factor gradient.
These results suggest that collective cell diffusion down a confinement gradient, but not the net sum of
directed migration of individual cells up a growth factor concentration gradient, is the principal driving
force for directing mesenchymal stem cell population outgrowth from a cell source.
SUNDAY
2849/L086
Identification, Localization and Interacting Networks of New Sun1 Isoforms.
D. Razafsky, S. Zang, G. Peebles, D. Hodzic; Ophthalmology and Visual Sciences, Washington
University School of Medicine, St Louis, MO
Transmembrane nuclear envelope Sun proteins (Sun1 and Sun2) and Nesprins (Nesprin 1, 2, 3 and 4)
interact within the perinuclear space to provide macromolecular scaffolds (called LINC complexes) that
span the NE and connect the nuclear lamina to cytoskeletal networks. Sun1 has been associated to a
surprisingly wide range of physiological functions such as centrosome anchoring at the NE, the
determination of nuclear pore spacing, chromosomes focal convergence at the nuclear envelope during
meiotic prophase and the decondensation of mitotic chromosomes. Here, we report that mouse and
human Sun1 are encoded by at least six variants originating from the alternative splicing of the central
domain of their nucleoplasmic region. The expression of these variants is regulated in a developmentaland tissue-specific manner. We also provide compelling evidence for the targeting of Sun1 to
mitochondria. Our results imply that the functional diversity of SUN/KASH-based molecular assemblies at
the nuclear envelope is underlined by the modularity of the nucleoplasmic region of Sun1 in addition to
the known modularity of Nesprins’ cytoplasmic region.
2850/L087
AZX100 Reduces Myofibroblast Contraction and Expression of Myofibroblast Markers in Attached
Collagen Gels.
E. Rousseau, B. Pullins, M. Sheller; Capstone Therapeutics Corp., Tempe, AZ
The myofibroblasts found in healing wounds and persistent scar tissue are characterized by an increase
in number of stress fibers and expression of α-smooth muscle actin (α-SMA) and connective tissue
growth factor (CTGF). Functionally, myofibroblasts increase contraction of the surrounding extracellular
matrix composed of collagen. Previous research has shown that AZX100, a 24-amino acid
phosphopeptide analogue of Heat Shock Protein Beta 6 (HSPB6 or HSP20) containing a protein
transduction domain, results in depolymerization of stress fibers in TGFβ1-induced myofibroblasts. This
study was designed to determine the effect of AZX100 treatment on myofibroblast contractile behavior
and differentiation in a fixed 3D collagen matrix simulating late stage wound healing. Normal human
dermal fibroblasts (NHDF) were seeded into attached collagen gels and incubated for 4 days in 2% FBS.
Myofibroblast differentiation was then induced with TGFβ1 (2.5ng/ml), with or without AZX100 (12.5µM to
500µM). RNA was extracted from cells in the attached gels to determine the effect of AZX100 on
myofibroblast markers via real-time PCR. Floating collagen gel assays, simulating early stage wound
healing, were also performed with NHDF to determine if AZX100 (100µM-300µM) had the potential to
hinder normal fibroblast reorganization of collagen. AZX100 significantly reduced myofibroblast
contraction in attached gels by up to 13%. In addition, AZX100 reduced the expression of the
myofibroblast markers α-SMA and CTGF by 14% and 16%, respectively. The peptide did not affect
normal fibroblasts reorganization of collagen in the floating gels. These data suggest that in closed
wounds AZX100 disruption of stress fibers may lead to decreased contraction of ECM, a decreased
myofibroblast population and potentially decreased fibrosis.
2851/L088
Syndecan 4 Regulates the Endocytosis of Fibroblast Growth Factor Receptor 1 via a Rac1Dependent Macropinocytic Pathway.
1,2,3
2
3
1
2 1
A. Elfenbein , A. Lanahan , A. Yamasaki , M. Matsuda , M. Simons ; Graduate School of Biostudies,
2
Kyoto University, Kyoto, Japan, Section of Cardiovascular Medicine, Yale University, New Haven, CT,
3
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH
Fibroblast growth factor 2 (FGF2) is an angiogenic protein that induces endothelial migration and
proliferation via two classes of cell surface receptors: tyrosine kinase receptors, such as fibroblast growth
factor receptor 1 (FGFR1), and heparan sulfate proteoglycans, such as syndecan 4 (S4). Originally
characterized as an FGFR co-receptor because of its ability to stabilize the ligand-receptor interaction, S4
is now understood to also signal independently of the FGFRs. In particular, S4 is required for the
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regulation of Rac1, a GTPase that orchestrates actin polymerization. While the downstream signaling
events of both FGFR1 and S4 have been extensively characterized, the mechanisms underlying their
downregulation from the cell membrane remain incompletely understood. In the current study, we
investigated the endocytic pathway by which the FGFR1/S4 signaling complex becomes internalized
following stimulation with FGF2. Using live cell microscopy and quantitative assays of endocytosis by flow
cytometry, we hereby demonstrate that FGFR1 endocytosis proceeds via a clathrin-independent,
dynamin-independent macropinocytic pathway. S4 is critical to this process due to its control over Rac1.
Shortly after internalization, the GTPase Rab5 becomes activated at macropinosomes containing FGFR1,
mediating vesicular transport toward fates of either membrante recycling or lysosomal degradation. These
findings set the FGFR1 endocytic pathway apart from those of other tyrosine kinase receptors, such as
vascular endothelial growth factor and epidermal growth factor receptors. They also establish a novel role
for S4 in FGFR1 signaling, as a mediator of receptor complex internalization.
2852/L089
AZX100 Reduces α-SMA Expression through Altered MRTF-A Localization.
D. Derkach, T. N. Trevino, K. Perkins, M. Sheller; R&D, Capstone Theraputics, Tempe, AZ
Keloid and hypertrophic scars are characterized by the persistent presence of myofibroblasts, which are
distinguished from fibroblasts by the expression of α-Smooth Muscle Actin (α-SMA) and Connective
Tissue Growth Factor (CTGF). Previous studies have demonstrated that AZX100, a 24-amino acid cellpermeable phosphopeptide analog of Heat Shock Protein 20 (HSP20), binds to 14-3-3 protein, displacing
phospho-cofilin and increasing cofilin-dependent depolymerization of actin. Moreover, increasing the Gactin pool results in a subsequent decrease in α-SMA and CTGF expression. α-SMA gene activation is
regulated by Serum Response Factor (SRF) and its unique co-activator, Myocardin-related Transcription
Factor A (MRTF-A). MRTF-A is found in the cytoplasm in unstimulated cells where it is bound to G-actin.
Upon stimulation, for example, by lysophosphatidic acid (LPA), the amount of G-actin decreases,
resulting in translocation of MRTF-A into the nucleus. MRTF-A then binds with SRF, initiating α-SMA
transcription. In this study, the expression of α-SMA and translocation of MRTF-A during AZX100
treatment were investigated. NIH 3T3 cells were stably transfected with a vector-expressing luciferase
under control of the α-SMA promoter. Cells stimulated with 20 μM LPA demonstrated a two-fold induction
of luciferase after 24 hours. AZX100 treatment reduced luciferase expression by ~30% during the 24 hour
treatment. In addition, immunofluorescence imaging demonstrated an abundance of MRTF-A in the
nucleus and cytoplasm of unstimulated cells and a shift of MRTF-A into the cytoplasm during serum
starvation. Upon stimulation with 20 μM LPA, MRTF-A translocated to the nucleus as expected. In
contrast, pretreatment of NIH 3T3 cells with AZX100 for 10 minutes reduced translocation of MRTF-A into
the nucleus upon the same LPA stimulation. In summary, AZX100-induced reduction of α-SMA is
mediated through increasing the pool of G-actin, which results in sequestering MRTF-A in the cytoplasm
and subsequently reducing SRF/MRTF-A-dependent transcription of α-SMA.
2853/L090
Mouse Myosinviia Is a Slow, Monomeric, High Duty Ratio Motor.
1
2
1
4
2
3
1
1
J. Haithcock , N. Billington , K. Choi , J. Fordham , J. Sellers , W. F. Stafford , H. White , E. Forgacs ;
1
2
Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA, Laboratory of Molecular
3
Physiology, NIH/NHLBI, Bethesda, MD, Integrative Protein Biology Program, Boston Biomedical
4
Research Institute, Watertown, MA, Randall Division of Cell and Molecular Biophysics, King’s College,
London, United Kingdom
MyosinVIIa is crucial in hearing and visual processes. We examined the kinetic and association
properties of the baculovirus expressed, truncated mouse myosinVIIa construct containing the head, all
5IQ motifs and the putative coiled coil domain (myosinVIIa-5IQ). The construct appears to be monomeric
as determined by analytical ultracentrifugation experiments, and only single headed molecules were
-1
detected by negative stain electron microscopy. The relatively high basal steady-state rate of 0.18 s is
-1
activated by actin only by ~3.5-fold resulting in a Vmax of 0.7 s and a KATPase of 11.5 µM. There is no
single rate-limiting step of the ATP hydrolysis cycle. The ATP hydrolysis step (M-T ↔ M-D-P) is slow (12
-1
s ) and the equilibrium constant (KH) of 1 suggests significant reversal of hydrolysis. In the presence of
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-1
-1
actin ADP dissociates with a rate constant of 1.2 s . Phosphate dissociation is relatively fast (>12 s ), but
the maximal rate could not be experimentally obtained at actin concentrations
≤ 50 µM because of the
weak binding of the myosinVIIa-ADP-Pi complex to actin. At higher actin concentrations the rate of
-1
attached hydrolysis (0.4 s ) becomes significant and partially rate limiting. Our findings suggest that the
myosinVIIa is a “slow”, monomeric molecular motor with a duty ratio of 0.7.
2854/L091
Microtubule Plus-End Accumulation of Dynactin p150 in Aspergillus nidulans Requires Its Basic
Domain Rather Than the CAP-Gly Domain.
X. Yao, J. Zhang, H. Zhou, E. Wang, X. Xiang; Biochemistry & Molecular Biology, USUHS, Bethesda,
MD
The p150 subunit of the dynactin complex interacts with cytoplasmic dynein and enhances dynein
processivity in vitro. The N-terminus of p150 contains a microtubule (MT)-binding region that includes a
CAP-Gly domain and a basic domain, and the functional significance of this MT-binding region is under
debate. Here we investigated the function of this region in the filamentous fungus Aspergillus nidulans in
which cytoplasmic dynein is required for nuclear distribution and the retrograde movement of early
endosomes. We first made an in-frame deletion of the MT-binding region of A. nidulans p150 by
homologous recombination. The p150 mutant protein is able to interact with dynein normally as
evidenced by a pull-down assay result. Colonies formed by the mutant appear much healthier than those
formed by the dynein heavy chain deletion mutant, but a partial nuclear-distribution defect is detected in
the mutant. In A. nidulans, GFP-labeled dynein and dynactin both accumulate at the plus-ends of
microtubules, and our previous studies indicate that dynein and dynactin depend on each other and on
kinesin-1 for this localization. Here our live cell imaging studies indicate that the microtubule plus-end
accumulation of GFP-dynein heavy chain and p150-GFP is diminished in the mutant missing the p150
MT-binding region. To test the in vivo functions of the CAP-Gly domain and the basic domain within the
MT-binding region, we made deletion mutants specifically missing either one of these domains.
Interestingly, the mutant missing the basic domain exhibits a clear defect in the microtubule plus-end
accumulation of p150. While plus-end p150-GFP comets can be observed in the mutant missing the
CAP-Gly domain, they can no longer be detected in the mutant missing the basic domain. In addition, the
mutant missing the basic domain also exhibits a partial defect in nuclear distribution, similar to what was
observed in the mutant missing the entire MT-binding region. Our results suggest that p150 is able to use
mechanisms other than those involving direct interactions between CAP-Gly with other protein motifs to
accumulate at the microtubule plus ends. Currently we are examining the effects of these mutations on
early endosome transport.
2855/L092
HER2 Signaling Regulates Fn14 Receptor Expression in Breast Cancer Cells.
K. Asrani, J. Winkles; UMB, Baltimore, MD
Human epidermal growth factor receptor (HER)-2 overexpression occurs in ~25% of all breast cancers
and these cancers have a very poor prognosis. HER2 requires HER3 to mediate cellular transformation
and the HER2-HER3 heterodimer functions as a powerful oncogenic unit. Heregulin (HRG)-β1 is a natural
ligand for HER3 and elicits the formation of potent HER2-HER3 heterodimers. HRG is expressed in ~30%
of breast tumors and is also a risk factor for invasive breast cancer. Fibroblast growth factor-inducible 14
(Fn14), a member of the TNF receptor superfamily, is overexpressed in breast tumors, where high
expression levels strongly correlate with both the invasive HER2+/ER- intrinsic subtype and indicators of
poor prognosis. Hence, we investigated whether HER2 activation in breast cancer cells could directly
induce Fn14 gene expression. We found that transient or stable transfection of MCF-7 cells with HER2
increased Fn14 protein expression. Breast tumor tissue from HER2-transgenic mice showed increased
expression of Fn14, over normal mammary gland. Also, HRG-β1 induced Fn14 protein expression in a
dose- and time-dependent manner in MCF-7 cells. Both the HER2 and HRG-β1-induced increase in Fn14
expression in MCF-7 cells could be blocked by treatment with the HER1/2 tyrosine kinase inhibitor
lapatinib. Also, siRNA knockdown of HER2 and/or HER3 decreased Fn14 expression in HER2overexpressing cells, as did treatment with the MEK inhibitor U0126 and the PI3K inhibitor Wortmannin.
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Preliminary results indicate that Fn14 may play a role in HRG-induced migration in SKBR3 cells. Studies
are in progress to elucidate the mechanisms involved in the increase in Fn14 expression and whether
Fn14 contributes to HER2-triggered proliferation, migration and invasion. These studies should indicate
whether Fn14 could be a new therapeutic target for HER2+ tumors with either intrinsic or acquired
resistance to HER2-targeted drugs.
2856/L093
The Effect of Alpha Tubulin (TUBA1A) Mutations on Neuronal Migration and Morphology.
N. Lamba, J. Liu; Center for Neuroscience Research, Children's National Medical Center, Washington
DC, DC
Microtubules are apart of the cytoskeletal system of neurons and play a role in the migration of neurons
during the development of the brain. Previous studies have confirmed the roles of the microtubuleassociated genes Lissencephaly 1 (LIS1) and doublecortin (DCX) in lissencephaly, a class of neuronal
migration disorders. Lissencephalic patients lack the normal sulci and gyri of the cerebral cortex due to
defects in neuronal migration. Recent studies have proven that causative mutations in TUBA1A, a tubulin
isoform gene, result in lissencephaly. In this study, we sought to introduce mutations in the TUBA1A gene
that have been previously identified in lissencephalic patients. Because of the difficulty in creating
transgenic knock-ins to study the functional effects of individual point mutations in an animal model, we
sought to develop a system where we used a small-interfering RNA sequence to knockdown endogenous
Tuba1a and replace it with our mutated construct in neurons. By maintaining the endogenous intracellular
tubulin levels, this system allowed expression of exogenous tubulin in neurons. We will evaluate three
missense mutations: S140G, R402H, and R264C. The S140G mutation impairs tubulin heterodimer
formation through inhibition of GTP binding. The R402H mutation may affect the binding of DCX to
Tuba1a. It is unclear how the R264C mutation causes the defect. We will study the migration of neurons
expressing mutant tubulin, and also evaluate the axon and dendrite morphology of the neurons
transfected with each mutation to understand how TUBA1A mutations affect the development of the
cerebral cortex.
2857/L094
Mechanisms Underlying Centriole Elimination during C. elegans Oogenesis.
T. Mikeladze-Dvali, P. Gönczy; Swiss Institute for Experimental Cancer Research (ISREC), EPFL,
Lausanne, Switzerland
Centrosomes, the primary microtubule organizing centers of animal cells, consist of a pair of centrioles
surrounded by pericentriolar material (PCM). Centrosomes are crucial for many fundamental processes,
including cell polarity, nuclear positioning, spindle assembly and cell migration. Centrosome number must
strictly be regulated both in somatic cells and in the germline. In proliferating cells, a single centrosome
duplicates to give rise to two entities by the end of the cell cycle, one of which is inherited by each
daughter cell. Gametogenesis posses a unique challenge to this general rule because one of the parental
centrosomes must be eliminated so that only one centrosome is inherited by the zygote. In most
metazoans, including humans and C. elegans, centrioles are eliminated in the female germline, while they
are maintained during spermatogenesis and are thus contributed strictly paternally to the zygote. The
molecular basis underlying the differential inheritance of centrioles in the female and male germline is not
understood. The C. elegans germline provides a powerful system to investigate this question. The
hermaphrodite germline is a continuum of maturing oocytes that contains mitotically proliferating nuclei in
its distal end, which progressively transition into meiosis before maturing into cellularized oocytes. Here
we show that centrioles and the surrounding PCM disappear in the loop region of the gonad at the onset
of oocyte cellularization. We found that the PCM proteins ZYG-9 and TAC-1 are only present in the
mitotic region, whereas the PCM proteins SPD-2, SPD-5 and TBG-1, as well as the centriolar proteins
SAS-4, SAS-5 and SAS-6, are detectable throughout the gonad up to the loop region. Using a candidate
approach, we are in the process of testing which components are needed for centriole disappearance
during oogenesis.
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2858/L095
Uneven Distribution of Microtubule Stability in a Plant Cytokinetic Array.
1,2
1,2
3
3
1,2 1
T. Murata , S. Nonaka , T. Sano , S. Hasezawa , M. Hasebe ; National Institute for Basic Biology,
2
Okazaki, Japan, Department of Basic Biology, Graduate School for Advanced Studies, Okazaki, Japan,
3
Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan
Cytokinesis is an essential step of cell division. In land plants, the new cell wall, called the cell plate,
separates the parental cell into two. The cell plate initially appears as a disc between daughter nuclei and
then expands towards the cell periphery until it fuses with parental cell walls. Our goal is to understand
mechanism of the cell plate expansion. The periphery of cell plate is surrounded by a doughnut-shape
microtubule structure, called phragmoplast, which works for transportation of cell wall precursors to the
leading edge (outermost region) of the cell plate. Previous studies have shown that translocation of the
phragmoplast towards the cell periphery is the driving force of the cell plate expansion, and microtubule
polymerization/depolymerization is involved in the translocation. Spatial control of microtubule behavior in
the phragmoplast would be an important clue for understanding the mechanism of the phragmoplast
translocation. We analyzed distribution of growing ends of microtubules and microtubule lifespan in the
phragmoplast. We found that, GFP-EB1 labeled plus ends were evenly distributed and grew in various
directions in the phragmoplast while microtubules at the leading edge were more stable than other
microtubules. We propose that random elongation coupled with biased stabilization/destabilization leads
net movement of phragmoplast microtubules, which in turn causes redistribution of cell plate deposition
towards the cell periphery.
2859/L096
The Non-Catalytic Carboxyl-Terminal Domain of ARFGAP1 Inhibits Cell Spreading and
Antagonizes the Activation of Rac1.
S. Yu; School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong
Accumulating evidence suggests that regulation of the actin cytoskeleton and membrane trafficking is
coordinated in mammalian cells. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found
on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also
can affect the actin cytoskeleton. In cells transfected with a truncated ARFGAP1 that lacks catalytic
activity for ARF called GAP273, cell migration and spreading is inhibited. Overexpression of GAP273 also
caused appearance of foci of cortical actin for much longer periods than non-transfected cells. Live cell
imaging of GAP273 cells revealed that actin-rich membrane blebs were formed but these structures did
not make protrusions of actin spikes, suggesting that GAP273 interfered with the regulation of actin
dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1
suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of
RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely
downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to
produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci.
Therefore, ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In
addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in
the carboxyl-terminal portion of the protein.
2860/L097
Identification for the Requirement of Mitotic Motors in Stress Granule Dynamics.
1,2
2
2
1,2 1
K. M. Bartoli , J. Soe , J. Glasser , W. S. Saunders ; Biochemistry and Molecular Genetics, University
2
of Pittsburgh School of Medicine, Pittsburgh, PA, Biological Sciences, University of Pittsburgh,
Pittsburgh, PA
Stress granules (SGs) are cytoplasmic foci that contain RNA binding proteins, the 40S ribosomal
subunits, stalled translation initiation complexes, and RNA aggregates that form under stressful
conditions such as oxidative stress and UV damage. Microtubules have previously been demonstrated to
be required for SG formation, and we confirmed this in RPE1 cells treated with arsenite, to induce SG
formation. After nocodazole treatment, to inhibit microtubules, a 30% reduction in SG formation was
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observed. As SGs have dynamic properties, and microtubules are demonstrated to function in SG
formation, we investigated whether two mitotic microtubule motors, Eg5 and Kid, may function in the SG
pathway. Recent data has shown that kinesin 1 and dynein localizes to SGs and functions in the
assembly or disassembly process. Therefore, to investigate if Eg5 and Kid may localize to SGs, we first
used immunofluoresence and observed both Eg5 and Kid to localize to SGs. Next, to identify if Eg5 and
Kid functions in SG assembly, siRNA studies were pursued. Interestingly, a 17% and 32% reduction in
SG formation was observed after Kid or Eg5 knockdown, respectively. Furthermore, Eg5’s ATPase
activity may not be required for its function in SG assembly as monastrol, a small molecule Eg5 inhibitor,
did not interfere with SG formation. Additionally, by live cell imaging and immunofluorescence analysis,
Eg5 was shown to be required for SG coalescence, as in the absence of Eg5, SGs that formed did not
coalesce and remained as numerous small particles. Finally, SG dissolution was investigated after Eg5
was inhibited by monastrol revealing a significant lag in SG disassembly, demonstrating that Eg5’s
ATPase was required for the dissolution of SGs. In conclusion, the identification of two mitotic motors in
SG formation, coalescence, and/or dissolution demonstrates that these two motors are novel SG
components and paves the way for the identification of other cytoskeletal components in SG dynamics.
2861/L098
In Vivo Function of Galectin-3 in Motile Cilia of Airway Epithelium.
D. Delacour, E. Pichard, T. Dang, F. Poirier; Institut Jacques Monod CNRS, Paris, France
Galectins, a family of beta-galactoside binding lectins, participate in an exceptionally broad range of
biological processes. It has been established that galectin-3 localizes at the primary cilium in epithelial
cells, and that the absence of galectin-3 generates growth defects of primary cilia. Here, we aimed at
extending these observations to motile cilia. Using confocal and electron microscopy, we show that
endogenous galectin-3 is also detected in motile cilia in adult mouse trachea. Ultrastructural studies
revealed a large panel of cilium abnormalities in tracheas of galectin 3 null mutants, including swollen
cilia, compound cilia, and also abnormal axonemal organization (incorrect 9+2 microtubular organization).
Live imaging experiments performed on mouse tracheal explants demonstrated that the amplitude of the
movement was reduced in gal3-/- tracheas although cilium beating frequency was unchanged.
Consistently, the movement of fluorescent beads over tracheal epithelium is severely affected in gal3-/explants. Although gal3 mutant mice are alive and look healthy in SPF animal house conditions, we have
evidence for defects in mucociliary clearance. Collectively, these data establish that galectin-3 is required
for correct structure and function of motile cilia in adult mice.
2862/L099
Critical Role of Galectin-7 in the Control of Epidermal Homeostasis after Injury.
G. Gendronneau, S. Sanii, D. Delacour, T. Dang, S. Sidhu, T. Magnaldo, F. Poirier; Institut Jacques
Monod, Paris, France
Galectins, a family of β-galactoside binding lectins, have recently emerged as novel regulators of tissue
homeostasis. Galectin-7 is predominantly expressed in stratified epithelia, especially in epidermis.
Galectin-7-deficient mice are viable and do not display any overt defects in skin architecture or in
expression of epidermal markers in steady state conditions. In contrast, the control of cell apoptosis,
proliferation and migration is compromised when the animals are exposed to different types of skin injury.
Our results imply that galectin 7 acts as a multifunctional protein during skin repair in vivo. When we
performed wound-healing experiments, we found that galectin-7 is normally enriched in the protrusions of
leading edge keratinocytes where it colocalizes with the ruffle marker, cortactin. In mutant mice lacking
galectin 7, there is a delay in wound healing, the process of “re-epithelialization” is slower owing to a
defect in keratinocyte migration. In addition, cortactin polarization is perturbed in mutant leading edge
keratinocytes both in vivo and in vitro. Present experiments aim at deciphering the role of galectin 7 in
keratinocyte collective migration.
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2863/L100
Isoform Specific Function of the Formin FMNL2 in Colorectoral Cancer Cells, Required for
Metastasis.
C. Péladeau, J. Copeland, S. Copeland; Medicine, University of Ottawa, Ottawa, ON, Canada
Cancer cell metastasis is driven by actin-dependent cell migration and is thus dependent on the function
of actin cytoskeleton remodelling proteins. Formins are a highly conserved family of cytoskeletal
remodelling proteins which are thought to be required in vivo for invasion and metastasis. In particular,
the Diaphanous-Related Formin FMNL2 has been suggested to be important for invasion in a variety of
tumour models. RNAi-mediated knockdown of FMNL2 expression inhibits invasion by MDA-MB-435
melanoma cells in vitro. There is also a direct correlation between increased invasiveness in colorectal
cancer (CRC) cells and increased levels of FMNL2 expression. Alternative splicing of human FMNL2
mRNA is predicted to generate a variety of FMNL2 protein isoforms. Results from our lab, and others,
suggests that this is likely to affect the regulation, activity and subcellular localization of the encoded
proteins. The main objective of this study is to determine if the expression of a specific FMNL2 isoform is
required to drive invasion and metastasis in a CRC tumour model. Using reverse transcription polymerase
chain reaction (RT-PCR), we have identified a number of novel FMNL2 splice forms expressed in noninvasive (SW480) and highly invasive (SW620) colorectal cancer cells. The relative levels of expression
of these splice variants in invasive and non-invasive cell-lines will be determined by qPCR and the
requirement for a specific FMNL2 isoform to drive invasion will be tested in vitro and in vivo.
2864/L101
Evidence for a Modular Septin Complex at the Dendritic Spine Neck.
C. Galimow, H. Ewers; ETH Zurich, Zurich, Switzerland
Septins are a family of cytoskeletal GTPases that polymerize into higher-order structures in vivo. In
mammals, 14 septin isoforms exist that are further classified into groups by sequence homology. The
septins assemble into hexameric complexes in a stoichiometry of 2:2:2 each from a different septin group.
The complexes polymerize further into higher-order structures that are involved in neurotransmitter
release, neuronal development and migration. All members of the SEPT2 group are highly homologous in
their sequence and it has been shown that they can assemble with septins 6 and 7 into SEPTX/6/7
complexes, in vitro. While septin2 is not expressed in hippocampal neurons, a neuron specific isoform
called septin5 is found here at the neck of dendritic spines together with septin11 and septin7. However, it
was demonstrated that in septin5 knock-out mice the protein level of septin2 was upregulated. Here, we
ask whether the neuron-specific septin5 can be replaced by other septins from the SEPT2 group in the
SEPT5/SEPT7/SEPT11 complex located at the neck of dendritic spines. We downregulated septin5
expression by RNA-interference and overexpressed GFP-tagged SEPT2 group septins in the same
neuron. When we analyzed the endogenous expression of septin7 by immunofluorescence in septin5
downregulated cells, we found it to be reduced. The expression of septin2-GFP in such cells could rescue
septin7 expression to normal levels. At the same time, septin2-GFP that was overexpressed in septin5
downregulated cells colocalized with endogenous septin7 at dendritic spine necks. Our experiments show
that septin2 may replace septin5 in neuronal septin complexes. These findings may explain why different
septins are expressed in different tissues and provide a means to investigate specifically the function of
individual septins.
2865/L102
KIF9 Knockdown in Mammalian Cells Leads to Multiple Mitotic Defects.
A. M. Billow, N. Quintyne, L. Alsina; Florida Atlantic University, Jupiter, FL
The kinesin family of microtubule motors is divided into a number of subfamilies based upon structure and
function. While kinesins with amino-terminal motor domains are involved in directional transport, those
with centrally-located motor domains tend to have roles in functions other than motility. KIF9 is a centralmotor domain kinesin that is largely uncharacterized. It was originally identified by sequence homology to
other kinesins and shown to interact with the Ras-like GTPase Gem (Piddini et al., 2001). Subsequent
studies have indicated that KIF9 plays a role in proper microtubule organizing center (MTOC) positioning
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and timely mitotic entry in Dictyostelium (Tikhonenko et al., 2009). Furthermore, it has been shown in
Trypanasome brucei that KIF9 is required for flagellar movement, likely via interaction with dynein
(Demonchy et al., 2009). We have examined KIF9 function in mammalian cells by using siRNA-mediated
knockdown in COS-7 cells. By knocking down KIF9 expression in these cells, we have seen several
effects on normal cell cycle progression. First, a transfected, synchronized population of cells shows a
notable delay in mitotic entry. Second, we have observed a decreased mitotic index in unsynchronized
cells, suggesting cells either fail to enter mitosis or abort the process prior to completion. Of the cells in
that population that enter mitosis, we see an increase in the number of cells in prometaphase and
metaphase. We also observe a defect in spindle pole migration during anaphase. Taken together, we
propose that KIF9 is important for normal progression through mitosis at several stages during the
process.
2866/L103
Knockdown of Dynactin’s p24 Subunit Leads to Defects throughout the Cell Cycle.
L. McCullough, N. Quintyne; Wilkes Honors College, Florida Atlantic University, Jupiter, FL
The function of p24, a subunit of the shoulder/sidearm sub-complex of dynactin, is largely unknown.
Previous studies have shown that overexpression of p24 leads to microtubule disorganization and
centrosomal defects. This effect is also observed when the other subunits of the shoulder/sidearm (p150
and dynamitin) are overexpressed. In this study, we have used overexpression and siRNA knockdown
experiments to further clarify the function of the p24 subunit. When p24 is overexpressed, we see
microtubule disruption, centrosomal defects, and the absence of mitotic cells. These results, in
concordance with previous studies, show that cells overexpressing p24 fail to progress through mitosis, in
addition to the defects shown in microtubule and centrosome integrity. The Golgi complex appears to be
unaffected by the change in p24 expression level. When using siRNA knockdown, we see a more
profound effect on the frequency of microtubule disruption in the population, but not as great of an effect
on disruption severity, when compared to p24 overexpression. We also saw similar results as the
overexpression studies, with the cells containing centrosomal defects but normal Golgi complexes.
Additionally, we observed many mitotic defects in siRNA knockdown cells, suggesting that these cells do
enter into mitosis, but the process does not properly progress. This indicates that p24 may be integral to
dynactin’s function in mitotic progression and not interphase.
2867/L104
βH-Spectrin Binds to Annexin B9 and Is Required for Protein Turnover and Multivesicular Body
Function.
1
1
1
2
1
1 1
M. Tjota , S. Lee , J. Wu , J. Williams , M. Khanna , G. Thomas ; Biology/BMB, Penn State, University
2
Park, PA, Surgical Research, Vanderbilt University Medical Center, Nashville, TN
The Spectrin-Based Membrane Skeleton (SBMS) is best known for its contributions to cell shape and
integrity. However, recent results from several labs have pointed to novel roles for the SBMS in the
endomembrane system. Drosophila βHeavy-spectrin (βH) is an apical spectrin isoform that stabilizes cell
adhesion complexes in the apicolateral region of epithelial cells, and is also required to stabilize the apical
levels of a V-type ATPase in cuprophilic cells (CC) of the midgut. In CC, βH is a terminal web protein that
is required to maintain the integrity of Rab5-positive early endosomes, and over-expression of βH Cterminal segment 33 in the salivary gland causes membrane expansion through sequestration of
Dynamin. Thus, βH appears to have a significant role during endocytosis and in the endosome system
and suggests the hypothesis that the presence of the βH SBMS stabilizes integral membrane proteins by
modulating their recycling. Annexins are proteins with widespread roles in the endomembrane system.
Here we show that a subset of βH spliceoforms binds to the small Ca2+-binding protein Annexin B9. We
show that knockdown of AnxB9 results in an alteration in the polarized distribution of βH and its
appearance on vesicular cytoplasmic structures that label with markers for the multivesicular body (MVB).
A similar accumulation of βH is also caused by βH up-regulation in Pak mutations, and Minikarst (a
dominant negative βH protein) is also seen to accumulate in this way. This suggests that βH has a role in
the endosomal system wherein it modulates MVB function and therefore recycling/degradation decisions.
Other labs have shown that mutations in the ESCRT complexes (which are essential for MVB operation)
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leads to impaired cargo degradation and sustained signaling by receptors slowed or trapped in signaling
endosomes following endocytosis. Here we show that AnxB9 and βH knockdown leads to the
accumulation of ubiquitinated proteins and sustained EGFR signaling, consistent with a proposed role for
the AnxB9/βH complex in MVB modulation. Our results reinforce the notion that the SBMS is dynamically
associated with protein trafficking processes and is significant contributor to the fidelity of such systems.
2868/L105
Quantitative Analysis of Arp2/3 Regulator Function In Vivo.
B. J. Galletta, J. A. Cooper; Cell Biology and Physiology, Washington University School of Medicine,
Saint Louis, MO
Spatial and temporal regulation of Arp2/3 activity is proposed to be critical for efficient generation of force
and movement by actin filament networks. Many cells have several potential Arp2/3 regulators. An
outstanding question is how individual Arp2/3 regulators influence the function of actin networks, and how
the activities of several spatially and temporally colocalized regulators are coordinated in vivo. The yeast
actin patch, which corresponds to sites of endocytosis, contains five conserved proteins that activate
Arp2/3 in vitro. We previously found that Arp2/3 regulators have both distinct and overlapping roles in the
formation of the patch, the initiation of movement away from the membrane, and the movement of the
patch into the cytoplasm. However, the molecular mechanism by which Arp2/3 regulators are influencing
the assembly of actin networks at patches remains unclear. The prevailing simple hypothesis is that the
regulators will recruit and/or activate Arp2/3 via their Arp2/3 binding regions. To test this hypothesis in
vivo, we followed the composition of dendritic nucleation proteins within the patch, over time, using
quantitative, live cell, fluorescence microscopy. Using an internal reference, we determined the number of
molecules of patch proteins throughout the lifetime of actin patches in living cells. From these studies we
have determined the stoichiometry of many critical actin regulatory proteins within the patch. Using
mutants in Arp2/3 regulators lacking their acidic, Arp2/3 binding motif, we have determined how the
amount of actin, Arp2/3 and barbed ends, as well as the kinetics of their assembly and disassembly, are
altered when Arp2/3 regulators cannot bind to Arp2/3. Strikingly, our data does not support the simple
hypothesis that the primary role of the acidic, Arp2/3 binding regions, of the Arp2/3 regulators is to recruit
Arp2/3 to the patch or to activate it to nucleate actin filaments once there. Instead, these mutants recruit
equal or in some cases increased amounts of Arp2/3 and other actin binding proteins, arguing that the
defects in patch dynamics and motility are due to more subtle changes in the actin filament network
architecture.
2869/L106
Coarse-Grained Modeling of Organelle Motility in Living Cells.
1
1
2
2
2
1 1
K. Daly , K. Lemoi , Y. Liu , Z. Shen , L. Vidali , E. Tuzel ; Department of Physics, Worcester
2
Polytechnic Institute, Worcester, MA, Department of Biology and Biotechnology, Worcester Polytechnic
Institute, Worcester, MA
Organelle motility is essential for the functioning of the eukaryotic cell. Actively modifying intracellular
structures allows cells to change and adapt to different conditions. In plants, reorganization of the
chloroplasts is critical to adapt to changes in light quality and intensity. Our analysis of chloroplast motility
in moss Physcomitrella patens provides evidence that this active transport is facilitated by the microtubule
cytoskeleton and molecular motors. We will present detailed in vivo measurements of organelle
organization and motility, combined with coarse-grained mesoscale modeling. Our simulations take into
account both thermal fluctuations and the topology of the microtubule network, and shed light into the
molecular mechanisms at play.
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2870/L107
Differential Expression of Formins during Heart Development and Stem Cell Differentiation to
Cardiomyocytes.
1
2
3
1
1 1
C. Maulion , S. Blystone , W. Coetzee , G. Morley , K. Maass ; Leon H. Charney Division of Cardiology,
2
NYU School of Medicine, New York, NY, Cell and Developmental Biology, SUNY Upstate Medical
3
University, Syracuse, NY, Pediatric Cardiology, NYU School of Medicine, New York, NY
Formins dynamically regulate actin microfilament assembly, influencing processes such as cell division,
signal transduction, migration, and cell-cell contact formation. The actin cytoskeleton affects ion channel
activity and formation of mechanical cell-cell junctions. As in vitro differentiation of embryonic stem cells
(ESC) results in phenotypical immature cardiomyocytes, differences in formin expression might reflect
alterations to the actin cytoskeleton of adult cardiomyocytes. Objective: To compare formin expression
during heart development to the profile of in vitro differentiated cardiomyocytes. Methods: Samples: ESC
and cardiac tissue from embryonic day 14.5, neonatal day 2 or 2 month old mice of the 129P2 strain
(heart development); spontaneously contracting cardiomyocytes differentiated from 129P2 ESC (in vitro
differentiation). Transcript analysis performed by qRT-PCR analysis (15 mouse formin genes, 2 stemness
genes, 4 cardiomyocyte differentiation genes). Expression of candidate formin proteins was analyzed
using commercially available Diap1 and Fhod1 antibodies. Results: qRT-PCR analysis revealed
significant changes in formin expression for 9 of 15 formins during heart development. Four patterns of
transcript changes were observed: formins expressed in ESC and decreasing during development
(Diap1, Diap3); transcripts peeking during fetal heart development (Fhod1, Fmnl1, Grid2ip); transcripts
altered during heart development compared to ESC and adult heart (Daam1, Daam2, Fmnl2, Fmnl3);
transcript increasing during development (Fhod3). In vitro differentiated cardiomyocytes revealed
significant increases in expression of embryonic formins (Grid2ip, Fhod1, Fmnl2) and formins otherwise
not differentially expressed during heart development (Diap2, Fmn1, Fmn2, Inf2). Expression of Fhod1
and Diap1 protein was studied in ESC, neonatal cardiomyocytes and in vitro differentiated
cardiomyocytes, verifying the observed changes in transcript expression. Conclusion: Formins are
dynamically regulated during cardiac development. Modifying formin expression of in vitro differentiated
cardiomyocytes could improve functional maturity and therefore their potential use in cell replacement
therapy for cardiac repair.
2871/L108
Cytokine and MMP Expression in Zebrafish Keratocyte Motility.
K. Leyva, J. Reyes, A. Sumner, T. McDonald, E. E. Hull; Midwestern University, Glendale, AZ
Fish keratocytes, a widely-used system to study cell motility, are typically grown in primary explant
cultures for 1-2 days before replating for use in cell studies focusing on individually migrating cells.
Although it is a routine part of the experimental system, little is known about the initial explant culture
period and how changes in gene expression during this period might affect keratocyte motility.
Keratinocytes, the human analog of fish keratocytes, have a complex pattern of differential expression of
cytokines and MMPs during primary culture, a pattern that changes during culture and in vitro wound
healing assays. Therefore, we assayed the expression and role of cytokines during explant culture at ½,
1, and 3 days of culture compared to baseline. Data presented here suggests that TNFα mRNA
expression increases approximately 6 fold during the first 24 hours in culture and then decreases to
values comparable to initial levels at subsequent time-points but addition of TNFα cytokine decreases
sheet growth. In contrast, peak expression levels of TGFβ do not occur until approximately 3 days after
initiation of explant culture. Levels of MMP 2, 9, 13, and 14a are all differentially expressed at the mRNA
level and total MMP activity increases in 3 day explant cultures. Addition of either a broad spectrum or a
MMP 2/9 specific inhibitor to cell sheet assays results in decreased sheet migration while addition of
MMP2 increases sheet area. Therefore, we suggest that keratocytes modulate the expression of
cytokines during explant culture in a manner similar to the primary human keratinocytes and that these
compounds regulate fish keratocyte motility.
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2872/L109
Ndel1 Is a Regulated Scaffold That Coordinates the Interaction of LIS1 and Dynein.
1,2
1,2
2
2
2
1 1
E. Zylkiewicz , M. Kijanska , W. Choi , U. Derewenda , Z. Derewenda , T. Stukenberg ; Biochemistry
2
and Molecular Genetics, University of Virginia, Charlottesville, VA, Biophysics and Molecular Physiology,
University of Virginia, Charlottesville, VA
Ndel1 has been implicated in various dynein-related processes, but its role in the dynein regulatory
pathway is not clear. We have developed an assay to study function of Ndel1 in dynein-dependent
microtubule aster formation in Xenopus egg extracts. We demonstrate that Ndel1 is required to focus
microtubules into asters and the N-terminal coiled-coil domain is sufficient to rescue Ndel1-depleted
extracts. Two domains within the N-terminus are critical for this function of Ndel1: a LIS1 binding domain
and a novel dynein binding domain. In addition, we find that aster focusing in Ndel1-depleted extracts can
be rescued by increasing levels of LIS1. This suggests that Ndel1 recruits LIS1 to dynein. Finally, we find
that nonphosphorylatable mutants of Ndel1 block the interaction between Ndel1 and dynein. We propose
that Ndel1 acts as a regulated scaffold to bring together components of the dynein regulatory pathway.
2873/L110
Conditional Ablation of Nonmuscle Myosin II-A in Mice Results in Squamous Cell Carcinoma of
the Tongue.
1
2
3
3
1 1
M. Conti , L. R. Brinster , Z. Chen , C. Van Waes , R. S. Adelstein ; Laboratory of Molecular Cardiology,
2
3
NHLBI,NIH, Bethesda, MD, Office of Research Services, NIH, Bethesda, MD, Tumor Biology Section,
Head and Neck Surgery Branch, NIDCD, NIH, Bethesda, MD
Nonmuscle myosin (NM) IIs are ubiquitously expressed members of the myosin family of motor proteins.
They are hexamers composed of a pair of nonmuscle myosin heavy chains (NMHC) and two pairs of light
chains. The three NMHCs are encoded by separate genes and the myosins are designated II-A, II-B and
II-C according to the make up of their heavy chains. Since germline ablation of NM II-A in the earliest
mouse embryo leads to embryonic death before organ development, NM II-A was conditionally deleted by
crossing NMHC II-A floxed mice to Nkx2.5 Cre recombinase expressing mice. Nkx2.5 is expressed in the
early heart and later in the tongue epithelium and in a mosaic pattern in the pancreas, stomach, and
intestines. Mice homozygous for the deletion were born and survived for up to two years and there
appeared to be no defects in the heart or in early heart development. However, the mice were runted and
developed an invasive, differentiated squamous cell carcinoma (SCC) of the tongue. Microscope
evidence for SCC could be seen as early as four days after birth. Typically mice survived for 2-3 months
but in some cases the SCC progressed slowly and the mice survived up to 2 years of age. Mice
heterozygous for the deletion were normal but mice in which one allele was null for II-A and the other
allele conditionally ablated by Nkx Cre expression also developed SCC. Microarray analysis of primary
human keratinocytes and head and neck SCC cell lines indicates a decrease in mRNA encoding NM II-A
in the SCC cell lines while expression of mRNAs encoding NM II-C and II-B were more variable. Except
for widespread calcification of medium size blood vessels, all other organs of the affected mice appeared
normal. Our data suggest a tumor-suppressive function of NM II-A in tumorigenesis of tongue SCC.
2874/L111
Polarized Myosin Produces Unequal Size Daughters during Asymmetric Cell Division.
G. Ou, N. Stuurman, M. D'Ambrosio, R. D. Vale; UCSF/HHMI, San Francisco, CA
Asymmetric cell division can be caused by non-symmetric positioning of the mitotic spindle before
cytokinesis. Alternatively, a dividing cell with a centered spindle can also yield differently sized daughter
cells using largely unknown mechanisms. We found such an asymmetric division in the C. elegans Q
neuroblast lineage. In these cells, myosin II accumulated in the anterior part of the cell that became a
small daughter cell after division (eventually undergoing apoptosis), suggesting that asymmetric
contractile forces produce differently sized daughters. Indeed, partial inactivation of anterior myosin using
chromophore-assisted laser inactivation created a more symmetric division and allowed the survival and
differentiation of the anterior daughter cell. Our data suggest that the balance of myosin activity on both
sides of a dividing cell can govern the size and fate of the daughters. We are currently testing whether
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known polarity pathways contribute to myosin asymmetry and also performing RNAi screens to uncover
molecular mechanisms that polarize myosin during asymmetric cell division.
2875/L112
How Bacillus Subtilis Ftsz Works: Toward Understanding a Direct Interaction with B. Subtilis
MreB.
K. April, K. Amann; Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI
MreB and FtsZ are major cytoskeletal proteins in bacterial cells. MreB is an actin homolog and gives
nearly all non-spherical bacteria their defined shape, segregates DNA, coordinates cell wall deposition
and establishes cellular polarity. We are interested in understanding how MreB carries out its many roles.
In B. subtilis cells, MreB polymerizes into a helical structure, condenses into a pre-divisional ring and
exhibits dynamic behavior. However, in vitro biochemical work from our lab complicates our
understanding of how B. subtilis MreB functions dynamically in vivo[1]. We believe MreB relies on direct
interactions with other major cytoskeletal proteins to behave dynamically and carry out its critical cellular
roles. FtsZ is a potential direct binding partner of MreB. FtsZ, a tubulin homolog, self-assembles into the
cytokinetic ring responsible for division of nearly all prokaryotic cells. Studies suggest MreB and FtsZ
coordinate to control cell growth and division[2,3]. Additionally, FtsZ is required for the proper localization
of the MreB pre-divisional ring and the subsequent split of the ring for distribution of MreB to daughter
cells[3,4]. Preliminary studies in our lab demonstrate these proteins are capable of directly interacting in
vitro: B. subtilis FtsZ changes the light scattering profile of B. subtilis MreB and also exhibits increased
high-g pelleting activity in the presence of MreB. We are interested in determining the nature of this
interaction. However, little is known about the polymerization properties and polymer dynamics of B.
subtilis FtsZ. Our work is aimed at determining these properties. We have successfully purified native
FtsZ and have begun to study its light scattering and high-g sedimentation profiles, and to assess its
GTPase activity. Once B. subtilis FtsZ is better understood, we will study the nature of the interaction
between FtsZ and native B. subtilis MreB. We aim to better understand how MreB works dynamically in
bacterial cells to perform its various cellular roles. [1]Mayer and Amann 2009. Cell Motil Cytoskeleton
66:109-18. [2]Doi et al. 1988. J Bacteriol 170:4619-24. [3]Figge et al. 2004. Mol Microbiol 51:1321-32.
[4]Vats and Rothfield 2007. PNAS 104:17795-800.
2876/L113
Regulation of the Novel Microtubule-Associated Formin INF1.
1
1
1
2
1 1
W. Kulacz , S. Thurston , K. Young , R. Screaton , J. Copeland ; University of Ottawa, Ottawa, ON,
2
Canada, Apoptosis Research Center, Ottawa, ON, Canada
Cell polarity is an essential function required for a number of cellular events, including asymmetric cell
division, specialization of cellular function, and cell migration. In most contexts, cell polarity is controlled
by the coordinated actions of the actin cytoskeleton and microtubule (MT) network. Much effort has been
made to identify bridging factors that coordinate the dynamic regulation of MT and F-actin organization
which is thought to be essential for the initial breaking of cellular symmetry. Our laboratory has recently
identified the cytoskeletal remodeling protein Inverted Formin1 (INF1) as a novel microtubule-associated
formin. We showed that INF1 expression induces both actin stress fiber formation and microtubule
stabilization and that knockdown of INF1 causes a significant loss of acetylated α-tubulin, a marker for
stable microtubules. The ability of INF1 to regulate actin polymerization and induce MT stabilization
suggests it serves as an actin/MT “bridging factor” necessary for the establishment and maintenance of
cell polarity. Many of the signaling pathways that govern cell polarity ultimately converge on the regulation
of MT stability through the action of the effector kinases such as Microtubule Affinity Regulating Kinase 2
(MARK2). MARK2 is one of four human MARK proteins (MARK1-4) and is regulated by the Par-3/Par-6
polarity pathway. MARK2 phosphorylates a number of MT-associated proteins (MAPs) at consensus K-XG-S motifs reducing their affinity for MTs and leading to MT destabilization. We noted the presence of two
K-X-G-S motifs in INF1 adjacent to its microtubule-binding domain (MTBD) suggesting that INF1 activity
may be regulated by MARK2. Consistent with this hypothesis, we show that INF1 is a MARK2 substrate
in vitro and that co-expression of MARK2 inhibits INF1 activity in vivo. Thus MARK2 may serve as a
regulatory link between INF1 activity and the cell polarity machinery.
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2877/L114
A Novel Role for IFT88 in Dynein1-Mediated Transport during Mitotic Spindle Assembly.
1
1
2
2
1 1
B. Delaval , A. Bright , L. Covassin-Barberis , N. Lawson , S. Doxsey ; Program in Molecular Medicine,
2
University of Massachusetts Medical School, Worcester, MA, Program in Gene Function and Expression,
University of Massachusetts Medical School, Worcester, MA
Intraflagellar transport (IFT) is a bidirectional transport system required for generating primary and motile
cilia in noncycling cells. IFT proteins form multimeric complexes that transport ciliary components along
cilia using molecular motors. IFT88 is a conserved, core IFT protein required for ciliogenesis. Disruption
of IFT88 and other cilia proteins results in a host of disorders collectively termed ciliopathies. Interestingly,
IFT88 localizes not only to cilia and basal bodies but also to mitotic centrosomes/spindle poles. In mitotic
cells, duplicated centrosomes orient the mitotic spindle and organize astral microtubules. The localization
of IFT88 to spindle poles suggests that the protein functions in mitosis. To test this we depleted IFT88 in
human cells and zebrafish embryos. We found that IFT88 depletion leads to several mitotic defects
including disrupted spindle poles, reduced astral microtubules, misoriented spindles and misaligned
chromosomes. A more thorough analysis revealed mislocalization of the microtubule nucleating
components, γ tubulin and EB1, from spindle poles in IFT88 depleted cells. Consistent with this result
IFT88 depletion lead to delayed movement of microtubule bundles (which contain γ tubulin and EB1) to
spindle poles during spindle assembly. These results suggested a role for IFT88 in transport, potentially
in conjunction with a microtubule-associated, molecular motor. In fact, we found that IFT88’s spindle pole
localization is microtubule dependent and that IFT88 interacts with dynein1 (but not cilia-associated
dynein2) as part of a large mitotic complex. In conclusion we have identified IFT88 as a component of a
novel mitotic dynein1 complex involved in the transport of microtubule bundles and microtubule
nucleating proteins to spindle poles. This work also implicates a potential role for the mitotic defects
associated with IFT88 in cilia disorders, either in combination with, or as an alternative to, cilia-mediated
defects.
2878/L115
Superresolution Microscopy of the Septin Complex.
J. Ries, C. Galimow, H. Ewers; Nano-Optics Lab, ETH Zurich, Zurich, Switzerland
The septins are a conserved family of cytoskeletal GTPases that interact with membranes and the
cytoskeleton. All fungi and animals investigated so far have septin genes ranging from 2 in C. elegans to
14 in humans. All septins share a common unique and conserved GTPase domain at the N-terminus and
most of them bear a C-terminal coiled-coil domain. Several different septin molecules combine into
nonpolar, dimeric, rod-shaped complexes. These are thought to assemble end-over-end into higher order
structures that orchestrate complex cellular events at the interface of the cytoskeleton and the plasma
membrane. In this way, septin complexes organize the cleavage furrow in dividing cells, serve as a corset
to maintain cell shape in cell motility and control the morphological differentiation of nerve cells. The
septins have been implied in cancer and several human diseases, especially in the nervous system,
where many septin isoforms are highly expressed. The integrity of the septin complexes and higher-order
structures is essential for septin function. However, the architecture of septin polymers and the rules
guiding their assembly, while seemingly conserved from yeast to mammals, are not clear. We use here
photoactivated localization microscopy to investigate the ultrastructure of septin higher order structures in
cells. Individual septin rods are localized within higher-order structures with nanometer precision and
structures of complexes assembled from the molecular positions. Our data provide important insight into
the organization of septin complexes into higher order structures.
2879/L116
NAD+ Regeneration Regulates Vascular Smooth Muscle Cell Polarity and Directed Migration.
1
1
1
1
1
2
1 1
A. Watson , E. van der Veer , Z. Nong , M. Frontini , C. O'Neil , M. Chu , J. Pickering ; Robarts
2
Research Institute, London, ON, Canada, London Health Sciences Centre, London, ON, Canada
Directional cell migration typically requires a chemotactic source but also depends on the inherent ability
of cells to crawl in a straight line. We recently discovered that the NAD+ biosynthetic enzyme,
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nicotinamide phosphoribosyltransferase (Nampt; also known as PBEF and Visfatin) was substantially
upregulated as human vascular smooth muscle cells (SMCs) migrated into patterned, multilayered
aggregates. In the current study, we established by immunostaining that Nampt is expressed in the
endothelial cell and smooth muscle cell (SMC) layers of human internal thoracic artery, adventitial
microvessels of the aorta, and was upregulated during SMC accumulation following balloon injury to the
rat carotid artery. To determine if Nampt regulates SMC motility, human SMCs were infected with
retrovirus containing Nampt cDNA and motility parameters quantified by digital time-lapse video
microscopy. Compared to vector-infected SMCs, Nampt-overexpressing SMCs displayed an increase in
migration speed. More striking however was the directionally persistent migration path of Nampt-SMCs,
with an increase in the ratio of net displacement to total distance traveled (D/T). In contrast, inhibition of
Nampt activity with FK866 resulted in chaotic motility and lower D/T ratios. Lamellipodial protrusion rate
was increased 2.0-fold in Nampt-overexpressing SMCs (p<0.001). Furthermore, the location of the
protrusive lamellipod in Nampt-SMCs was restrained, but deviated substantially in control SMCs. This
anchoring of lamellipodia by Nampt was associated with activation of cdc42, and expression of dominantnegative cdc42 suppressed the lamellipodial anchoring effect of Nampt. CONCLUSION: Nampt imparts
SMCs with an increased ability to crawl in a straight line. This control over directional persistence may be
critical for rapid vascular stabilization and repair.
2880/L117
In Vivo Motor Stalling of Intracellular 1μm Paramagnetic Microbeads Undergoing Active Transport.
1
2
3
2 1
K. I. Ford , T. O'Brien , L. Mair , R. Superfine ; Biomedical Engineering, University of North Carolina
2
3
Chapel Hill, Chapel Hill, NC, Physics, University of North Carolina Chapel Hill, Chapel Hill, NC, Material
Science and Engineering, University of North Carolina Chapel Hill, Chapel Hill, NC
Paramagnetic nano and micro-scale particles are of current interest in use as non-viral vectors for gene
delivery. These vectors are intended for use in transfection of cell cultures as well as in gene therapy for
clinical application in patients. The application of magnetic forces provides the investigator with the ability
to exert control over the vectors once they are in the system of interest. The question that requires further
investigation is the force that must be applied to a vector to manipulate it in the desired manner. The
focus of this study is to measure the forces that are exerted on an endocytosed vector inside of a living
cell. When a nano or micro-sized particle is endocytosed by a cell it is initially enveloped in an early
endosome. Endocytosed particles can be observed undergoing active transport similar to organelles in
the cell. This transport is due to molecular motors associating to the vesicle encapsulating the bead and
causing transport along the cytoskeletal networks. The motors enable the bead to move bi-directionally
along microtubules to effect transport to different locations in the cytoplasm. 1μm paramagnetic beads are
dosed into HeLa cell cultures and undergo endocytosis. Once there, video is taken to capture the
displacement of the beads undergoing active transport. Displacements, velocities, and step sizes of the
transported beads are analyzed. Forces are exerted on the bead by means of a 3-dimensional force
microscope (3DFM) system. Forces are applied in a variety of ramp and step functions and the resulting
movements of the beads are tracked and analyzed. Based on the calibration of the 3DFM we determine a
stalling force for the transported beads. Also, by using experimentally determined cytoplasmic viscosity,
and the known forces exerted by individual molecular motors, we make estimations about the number of
motors that are attached to the bead.
2881/L118
Regulation of EGFP-Actin Dynamics in Living T Cells Stimulated with SDF-1β during Cell
Migration.
1
1
1
2
1
H. Zamudio-Meza , X. Ambriz-Peña , E. Garcia-Zepeda , I. Meza , G. Soldevila-Melgarejo ;
1
2
Inmunologia, Intituto de Investigaciones Biomedicas, UNAM, México City, Mexico, Biomedicina
Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México City, Mexico
Introduction. The actin cytoskeleton dynamics play an important role in T lymphocyte migration during the
basal traffic to secondary organs and the homing to sites of inflammation during the course of an immune
response.These fenomena are regulated by chemokines, through the activation of Rho family small
GTPases pathways, among others. SDF-1β (Stromal Derivate Factor 1-β) is considered an homeostatic
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chemokine that induces actin reorganization through CXCR4 (CXC chemokine sub-family member)
interaction expressed on T and B cells. Methods. In the present work, fixed and living Jurkat cells
expressing Enhanced Green Fluorescent Protein-actin (EGFP-actin) were monitored for actin
reorganization during SDF-1β stimulation, utilizing fluorescence and time-lapse microscopy. Also, we
analyzed F-actin levels and chemotaxis index. Results. SDF-1β induced morphological changes and
EGFP-actin reorganization in Jurkat cells to form lamella and focal adhesion structures. Specific drugs
that antagonize actin organization and regulate actin-signalling pathways were tested. Cytochalasin D (Factin inhibitor) and Worthmanin (PI3K inhibitor) treatment precluded morphological and actin changes.
Interestingly, WHI-P131 (Jak3 inhibitor) and Y-27632 (ROC kinase inhibitor), inhibited morphological
changes, actin dynamics and F-actin levels, but did not interfere with the formation of lamella. These
drugs, as well as the Rac1 inhibitor (NSC23766), also significantly decreased the migration index of
Jurkat cells in response to SDF-1β in the chemotaxis assays. Conclusions. Our study shows that actin
dynamics and morphological changes, induced by SDF-1β, promote the formation of specialized
structures required in phenomena such as cell polarization and migration. These data suggest, the
involvement of Jak3 in chemokine-mediated actin cytoskeleton reorganization.
2882/L119
Molecular Modeling of Tyrosine with the Threaded Molecular Model of Tubulin Tyrosine Ligase
(TTL) Offers a Novel Insights of Visualization of Tubulin Tyrosination In Vivo.
1
1
2
1 1
P. K. Naik , A. Banerjee , S. L. Bane , H. C. Joshi ; Cell Biology, Emory University School of Medicine,
2
Atlanta, GA, Chemistry, Binghamton University, Binghamton, NY
Cellular microtubules undergo many types of post-translational modifications of unknown functions. One
such modification is the site-specific removal of a genetically encoded tyrosine residue at the C-terminus
of alpha tubulin subunit by an alpha tubulin-specific carboxypeptidase (TCP). Following this, the tyrosine
residue is relegated back onto the detyrosinated protein. The tyrosination/detyrosination of tubulin is a
cyclic process and is required for proper cell growth. This mysterious re-tyrosination of alpha-tubulin is a
specific event and is mediated by a tubulin tyrosine ligase (TTL). Human genome analysis reveals that
this enzyme is one of the 14-membered family of enzymes collectively called tubulin tyrosine ligase like
enzymes (TTLLs). Some members of this family are already being associated with a variety of essential
divergent functions from histone modifying epigenetic information systems to centrosome function to
directed cell motility of metastatic invasive prostate cell carcinoma. In order to begin a real time analysis
of this enzyme in action in vivo, we have generated a threaded structure of the substrate-enzyme
molecular model with minimal free energy. We then designed a novel synthetic substrate that is
permissible with the docking site and is fluorescent. This novel substrate is capable of visualizing the
function of the enzyme in action in vivo. This novel reagent will be instrumental in unraveling the sites of
action of TTL involved in a variety of cellular functions due to this specific and conserved cyclic activity of
a novel enzyme family.
2883/L120
Tissue Elongation Requires Oscillating Contractions of a Basal Actomyosin Network.
L. He, X. Wang, H. Tang, D. Montell; Biological Chemistry, Johns Hopkins University, Baltimore, MD
The goal of tissue engineering is to create artificial organs and tissues in vitro, which requires
understanding not only how cell types re specified but also how cells cooperate to generate appropriate
organ shapes and tissue architectures. The molecular and mechanical mechanisms governing some
multicellular morphogenetic movements are beginning to be elucidated. For example, periodic contraction
of apical networks of actomyosin in Drosophila embryos drives mesoderm invagination and dorsal
closure; and apical accumulation of myosin specifically at dorsal/ventral (D/V) cell-cell boundaries drives
directional cell intercalation during germ band elongation. Here we use live imaging to show that the basal
surfaces of Drosophila follicle cells undergo a series of directional, oscillating contractions driven by
periodic myosin accumulation on a polarized actin network. Inhibition of the actomyosin contractions or
their coupling to extracellular matrix (ECM) blocked tissue elongation, whereas enhancement of the
contractions exaggerated it. Myosin accumulated in a periodic manner prior to each contraction and was
regulated by the small GTPase Rho, its downstream kinase ROCK and cytosolic calcium. Disrupting the
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link between the actin cytoskeleton and the ECM decreased, while enhancing cell-ECM adhesion
increased, the amplitude and period of the contractions. In contrast, disrupting cell-cell adhesions resulted
in loss of the actin network. Our findings reveal a novel mechanism controlling organ shape and a new
model for the study of the effects of oscillatory actomyosin activity within a coherent cell sheet.
2884/L121
Osteopontin as a Physiologic Substrate for PHEX in X-Linked Hypophosphatemia.
1
3
3
1
1
1
1
N. M. Barros , W. N. Addison , B. Hoac , R. L. Neves , L. Coppini , E. Alvarenga , J. B. Pesquero , A. K.
1
2,3 1
Carmona , M. D. McKee ; Department of Biophysics, Universidade Federal de Sao Paulo, Sao Paulo,
2
3
Brazil, Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada, Faculty of
Dentistry, McGill University, Montreal, QC, Canada
Mutations in the PHEX gene (phosphate-regulating gene with homologies to endopeptidase on the X
chromosome) cause X-linked hypophosphatemia (XLH), an inherited form of rickets with renal phosphate
wasting leading to hypophosphatemia and defective bone mineralization (osteomalacia). PHEX has
homology to members of the M13 metallopeptidase family, and while it is expressed predominantly by
bone and tooth cells, inactivating mutations in PHEX lead to renal phosphate wasting implying the
accumulation of a secreted, circulating phosphaturic factor whose distal actions in the kidney lead to
osteomalacia. Despite these observations, the physiologic substrates for PHEX remain unknown, and
other molecular actions of PHEX are poorly understood, including the intrinsic osteoblast defect where
inactivation of PHEX has direct local consequences on mineralization in the extracellular matrix. In the
present work, we investigated osteopontin (OPN) - a member of the SIBLING (small integrin-binding
ligand, N-linked glycoprotein) protein family prominently secreted by osteoblasts and also found in plasma
- as a potential physiologic substrate for PHEX. Using cleavage assays, gel electrophoresis and mass
spectrometry (MS), we demonstrate enzymatic cleavage of OPN by PHEX, with cleavage products having
reduced mineralization-inhibiting potency in MC3T3-E1 osteoblast cultures. Similar assays showed no
cleavage of mineralization-regulating fibroblast growth factor-23 and bone sialoprotein, thus
demonstrating selective PHEX activity for OPN. PHEX hydrolysis of OPN resulted initially in two major
fragments of 37 and 22 kDa as detected by SDS-PAGE/Stains-All staining and MS analysis. With
increased incubation times or using higher concentrations of PHEX, the 22 kDa fragment was completely
degraded. Importantly, Western blotting of Hyp (the murine homolog of human XLH) mouse bone extracts
clearly showed accumulation of an OPN fragment of approximately 22 kDa that was not present in wildtype mouse bone having active PHEX. In conclusion, these results show that OPN is a substrate for
PHEX, and that accumulation of a small OPN fragment may contribute to the osteomalacic XLH/Hyp
phenotype. Funded by CIHR, CNPq and Fapesp.
2885/L122
Localization of Obscurin Kinases in the Extracellular Matrix of Striated Muscle Cells.
L. R. Hu, J. Valenti, A. Kontrogianni-Konstantopoulos; Department of Biochemistry and Molecular
Biology, University of Maryland Baltimore, Baltimore, MD
Obscurins are giant muscle proteins implicated in myofibrillogenesis and linked to hypertrophic
cardiomyopathy. They contain a tandem array of adhesion, i.e. immunoglobulin (Ig) and fibronectin-III
(Fn-III) domains, and signaling motifs, including an isoleucine-glutamine (IQ) repeat, a Src homology 3
(SH3) domain, a pleckstrin-homology (PH) domain, a Rho-guanine nucleotide exchange factor (RhoGEF) motif, and two serine/threonine kinase domains, namely SK1 and SK2. Three out of the four
reported obscurin isoforms contain single or tandem kinase domains. Using immunofluorescence
combined with confocal microscopy and antibodies specific to the kinase-bearing obscurin isoforms, we
found that these are present in diverse locations in striated muscle cells, including the sarcolemma, the
Z/I junction and the M-band. To identify potential binding partners or substrates of SK1 and SK2 in
+ +
cardiac cells, we employed the yeast two-hybrid system. Our screening demonstrated that Na /K ATPase and N-cadherin, both of which are major components of adherens junctions, are potential
interacting partners of SK1 and SK2, respectively. Detailed deletion analyses revealed that the
+ +
extracellular domain of the β-subunit of Na /K -ATPase (NKA) is sufficient to interact with the catalytic
portion of SK1, whereas both the extracellular and intracellular regions of N-cadherin are required to
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interact with the catalytic portion of SK2. Given the extracellular localization of the interacting portions of
NKA and N-cadherin, we next examined their co-distribution with SK1 and SK2, as a means of validating
their physiological binding in the context of the cell. To this end, we used non-permeabilized primary
cultures of skeletal and cardiac muscle cells and labeled them with antibodies to NKA, N-cadherin and
the kinase domains of obscurin. Our results demonstrated that at least some portions of the obscurinkinase isoform(s) are localized extracellularlly. Currently, we are investigating the roles of SK1 and SK2 in
regulating cardiac function by manipulating their expression levels through adenoviral-mediated gene
delivery.
2886/L123
Crawling Cells Can Close Wounds without Purse Strings or Signaling.
1,2
1,2 1
P. Lee , C. Wolgemuth ; Cell Biology, University of Connecticut Health Center, Farmington, CT,
2
Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT
When a gash or gouge is made in a confluent layer of epithelial cells, the cells move to fill in the “wound”.
In some cases, such as in wounded embryonic chick wing buds, the movement of the cells is driven by
cortical actin contraction (i.e., a purse string mechanism). In adult tissue, though, cells apparently crawl to
close wounds. At the single cell level, this crawling is driven by the dynamics of the cell’s actin
cytoskeleton, which is regulated by a complex biochemical network, and cell signaling has been proposed
to play a significant role in directing cells to move into the denuded area. However, wounds made in
monolayers of Madin-Darby canine kidney (MDCK) cells still close even when a row of cells is deactivated
at the periphery of the wound, and recent experiments show complex, highly-correlated cellular motions
that extend tens of cell lengths away from the boundary. These experiments suggest a dominant role for
mechanics in wound healing. Here we present a biophysical description of the collective migration of
epithelial cells during wound healing based on the basic motility of single cells and cell-cell interactions.
This model quantitatively captures the dynamics of wound closure and reproduces the complex cellular
flows that are observed. These results provide the mechanism by which wounds can close without purse
strings and suggest that wound closure is predominantly a mechanical process that is modified, but not
produced, by cell-cell signaling.
2887/L124
The Requirement for R-SNARE Proteins in Granule Exocytosis: VAMP4 and VAMP7 Are
Indispensable for NK Cell Cytotoxicity.
1,2
1
1
2
1 1
K. Krzewski , A. Gil-Krzewska , J. Watts , J. E. Coligan , J. L. Strominger ; Molecular and Cellular
2
Biology, Harvard, Cambridge, MA, Receptor Cell Biology Section, Laboratory of Immunogenetics, NIAID,
NIH, Rockville, MD
Natural killer (NK) cells kill cancer and virus-infected cells through their cytolytic activity. The last step in
NK cell cytotoxicity, fusion of lytic granules with the plasma membrane resulting in exocytosis of granule
content, is still poorly understood. Proteins from the SNARE family mediate all membrane fusion events in
the cell. Here we show that NK cells express all seven members of the R-SNARE subgroup. Two of these
R-SNARE proteins, VAMP4 and VAMP7, are associated with lytic granules during cytotoxic interactions.
However, only VAMP7 co-localizes with granules in non-activated cells. The differences in co-localization
of these two R-SNARE proteins, with respect to perforin-containing cytotoxic granules, indicate that they
have different functions in exocytosis. Using the tumor NK cell line, YTS, we show that RNAi-mediated
disruption of expression of either VAMP4 or VAMP7 inhibits release of lytic granules and severely impairs
NK cell cytotoxic activity. Furthermore, VAMP7 but not VAMP4 is involved in cytokine secretion and
endocytosis in NK cells, indicating that VAMP7 is involved in many fusion processes and thus plays a
more general function in NK cell activity than VAMP4.
SUNDAY
2888/L125
Alpha-Parvin Phosphorylation Is Necessary for Matrix Degradation.
J. Pignatelli, C. E. Turner; SUNY Upstate Medical University, Syracuse, NY
The mechanisms controlling tumor metastasis, the major cause of cancer related fatalities, are poorly
understood, although disregulation of cell adhesion and motility are important factors in tumor
progression. We have previously described alpha-parvin (actopaxin) as a paxillin and F-actin binding
focal adhesion protein. The amino-terminus of actopaxin contains several serine/threonine residues that
when phosphorylated contribute to the regulation of cell spreading and migration. Herein, we have found
that serine phosphorylation of alpha-parvin is upregulated in invasive MDA-MB-231 breast cancer cells
when compared to their non-invasive MCF10A counterparts, and that alpha-parvin phosphorylation
contributes to the regulation of matrix degradation, a pivotal step of invasion. Using U2OS osteosarcoma
cells stably expressing WT, phosphomemetic (S4/8D) and non-phosphorylatable (Quint) alpha-parvin we
have determined that alpha-parvin phosphorylation is necessary for efficient degradation of fluorescent
gelatin matrix. Treatment of the WT and S4/8D cells with Src and MMP inhibitors (PP2 and GM6001
respectively) blocks matrix degradation. It has been suggested that cell contractility contributes the
process of matrix degradation. To address this in our system, we treated WT, S4/8D, and Quint
expressing cells with Rac 1 and MLCK inhibitors. Addition of the Rac 1 inhibitor NSC23766 to WT or
S4/8D cells prevented matrix degradation, while treatment of Quint cells with the MLCK inhibitors ML-7 or
blebbistatin rescued degradation to control levels. In addition, Rac 1 inhibition converted the phenotype of
WT and S4/8D cells from a highly protrusive well spread morphology to the more Rho-like contractile
morphology seen in the Quint expressing cells. These data indicate a possible role for alpha-parvin in
regulating Rho GTPase signaling and contractility to control matrix degradation and motility. A causative
role for alpha-parvin phosphorylation in tumor cell invasion is currently under investigation.
2889/L126
ADAMTS-1 Stimulates Migration, Invasion and Invadopodia Formation in Breast Cancer Cells.
1,2
2
2
2
2 1
V. M. Freitas , J. B. Amaral , L. P. Mariano , R. Jaeger , G. M. Machado-Santelli ; Natural Sciences
2
and Humanities Center, Federal University of ABC, Santo Andre, Brazil, Department of Cell and
Developmental Biology, University of Sao Paulo, Institute of Biomedical Sciences, Sao Paulo, Brazil
ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin motifs) is a member of the ADAMTS
family of metalloproteases. This enzyme is related to pathological processes such as inflammation and
cancer. In spite of ADAMTS-1 biological relevance, the mechanisms underlying its involvement in tumor
biology remain elusive. Here we studied the role played by ADAMTS-1 regulating migration and invasion
of a malignant mammary cell line (MDA-MB-231). For time-lapse fluorescence microscopy, cells had
ADAMTS-1 silenced by shRNA-GFP. For migration and invasion assays ADAMTS-1 was silenced by
siRNA. MDA-MB-231 cells transfected with scrambled interference RNA served as controls. Immunoblot
confirmed silencing efficiency. Migration was investigated by time-lapse fluorescence microscopy in an
Olympus IX 81 inverted microscope equipped with Orca R2 CCD camera. Video recording was carried
out using Cell Observer image software (Olympus). MDA-MB-231 treated and control cells were
maintained at 37 °C in a temperature-controlled chamber. Images were collected every 5 minutes (total of
4 hours and 30 minutes). Velocity measurements (MTrack J plugin, Image J software) showed that
silencing of ADAMTS-1 enhanced migratory activity of MDA-MB-231 cells compared to controls. Cells
with reduced expression of ADAMTS-1 also exhibited increase in migration and invasion, as shown in
Transwell assay and in Boyden chambers, respectively. Cancer cells depend on invadopodia to degrade
and invade surrounding extracellular matrix. Invadopodia are membrane protrusions with enzymes
required for ECM degradation. Silencing of ADAMTS-1 by siRNA increased invadopodia activity in MDAMB-231 cells, as shown by fluorescent degradation substrate assays. Cortactin and MT1-MMP are
important invadopodia proteins. Immunoblot of cells transfected with ADAMTS-1 siRNA showed
augmentation of cortactin and MT1-MMP compared to controls. We suggest that ADAMTS-1 stimulates
migration, invasion and invadopodia formation in breast cancer cells. Support: FAPESP (06/54963-0 and
10/07699-1) and CNPq (470779/2007-1).
SUNDAY
2890/L127
E-Cadherin Cooperates with the Neuregulin-ErbB Receptor Signaling to Promote Schwann Cell
Myelination.
S. Basak, D. Desai, R. Ramos, P. Maurel, H. A. Kim; Biological Sciences, Rutgers University, Newark,
NJ
E-cadherin, a member of the classical cadherin family of calcium dependent adhesion proteins has been
implicated in a variety of biological processes including cell development and maintenance of cell shape,
motility and cell-cell interactions. In epithelial cells, E-cadherin plays a role as a signaling molecule
working in conjunction with receptor tyrosine kinases (RTKs). There is now increasing evidence
suggesting that the molecular crosstalk between E-cadherin and RTKs regulates growth and
differentiation of the cells. Myelination in the peripheral nervous system (PNS) is regulated by axonSchwann cell interaction mediated by the Nrg1-erbB RTK signaling. In vivo, E-cadherin expression
increases in post-natal Schwann cells at the onset of myelination. Therefore, we tested the hypothesis
that E-cadherin cooperates with Nrg1-erbB signaling to regulate PNS myelination. Our data showed that
E-cadherin associated with erbB2 during the early stages of myelination. Down-regulation of E-cadherin
expression using shRNA or Cre-mediated gene deletion decreased Nrg1-mediated erbB signaling in
Schwann cells. Furthermore, overexpression of E-cadherin in Schwann cells increased Nrg1-induced
erbB signaling. When these Schwann cells were co-cultured with DRG neurons, there was a marked
increase in myelination, accompanied by an increase in erbB2 and Akt activation. Interestingly, Ecadherin overexpression inhibited Erk activation in the co-cultures. Our study also showed that Nrg1 type
III expressed on PNS axons is required for inducing E-cadherin expression in Schwann cells prior to
myelination. Altogether, our data indicate that a crosstalk between E-cadherin and Nrg1-erbB signaling
regulates myelination in the PNS.
2891/L128
Three-Dimensional Hyaluronic Acid-Based Hydrogel Systems for Studies of Cancer Invasion.
1,3
1,3
1,3
2,3
2,3
1,3
2,3
L. A. Gurski , L. N. Labrada , N. T. Nguyen , X. Xu , A. K. Jha , K. L. van Golen , X. Jia , M. C.
1,3,4 1
2
Farach-Carson ; Biological Sciences, University of Delaware, Newark, DE, Materials Science and
3
Engineering, University of Delaware, Newark, DE, Center for Translational Cancer Research, University
4
of Delaware, Newark, DE, Biochemistry and Cell Biology, Rice University, Houston, TX
To address the limitations of currently available invasion assays, we developed a novel system based on
a three-dimensional (3D) hyaluronic acid (HA) hydrogel that provides a more flexible, quantifiable, and
physiologically relevant method to study cancer cell invasion. This invasion assay reflects the prevalence
of HA in the bone marrow microenvironment and its role in the promotion of cancer cell motility and tissue
invasion, making our system ideal for studying invasion through bone marrow or other HA-rich tissues.
The HA hydrogel crosslinks via interaction of aldehyde and hydrazide groups, releasing water as the only
by-product. This bio-compatible cross-linking process allows for direct encapsulation of cells within the
gel. For construction of the invasion assay, the cell/gel construct is plated into a 12 mm tissue culture
insert. The cells are situated on a single plane such that individual cell movement from that plane can be
tracked and measured. Here, we show that the 3D HA hydrogel invasion assay allows for cell
migration/invasion in response to a chemoattractant, and that this movement is easily quantified using
confocal microscopy techniques over a distance of up to 1mm. We also show that culture in the HA
hydrogel causes prostate cancer (PCa) cells to differentially express, localize, or activate HA receptor
proteins and downstream effectors, all of which contribute to PCa motility and metastasis. These
observed changes may reflect the ability of the HA receptors, cluster of differentiation 44 (CD44) and/or
receptor for hyaluronan-mediated motility (RHAMM), to interact with the HA hydrogel components.
Additionally, PCa cell invasion through the HA hydrogel depends on their expression of hyaluronidases;
more highly invasive cell lines express hyaluronidases at higher levels. Ongoing work aims to utilize the
3D HA hydrogel invasion assay to study HA receptor signaling in bone metastatic PCa cells. Specifically,
we plan to examine the role that RHAMM plays in PCa metastasis, studying how it activates Rho
GTPases that increase cell motility. This information will provide new avenues for interfering with
processes that enhance cell motility during metastasis.
SUNDAY
2892/L129
DDR1 Triggers Epithelial Cell Differentiation by Promoting Cell Adhesion through Stabilization of
E-Cadherin.
1
2
3
4 1
Y. Yeh , C. Wu , Y. Wang , M. Tang ; Institute of Basic Medical Sciences, National Cheng Kung
2
University, Tainan, Taiwan, Department of Cell Biology and Anatomy, National Cheng Kung University,
3
Tainan, Taiwan, Department of Medicine, Skeleton-Joint Research Center, National Cheng Kung
4
University, Tainan, Taiwan, Department of Physiology, National Cheng Kung University, Tainan, Taiwan
Discoidin domain receptor 1 (DDR1) promotes E-cadherin-mediated adhesion. However, the underline
mechanism and the significance have not been elucidated. Here, we showed that DDR1 overexpression
augmented, whereas dominant negative mutant (DN-DDR1) or shRNA of DDR1 inhibited E-cadherin
localized in cell-cell junctions in epithelial cells. DDR1 changed the localization and abundance of Ecadherin, as well as epithelial plasticity, as manifested by enhancement of microvilli formation and
alteration of cytoskeletal organization. DDR1 also reduced protein abundance of mesechymal markers,
while DN-DDR1 and sh-DDR1 showed opposite effects. These results suggest that activation is required
for DDR1-induced cell differentiation. Expression of DDR1 augmented E-cadherin protein levels by
decreasing its degradation rate. Photobleaching and photoconversion of E-cadherin conjugated with Eos
fluorescence protein demonstrated that DDR1 increased the stability of E-cadherin on the cell membrane,
whereas sh-DDR1 decreased it. Pull-down assay and expression of constitutive active or dominant
negative Cdc42 showed that DDR1 stabilized E-cadherin through inactivation of Cdc42. Altogether, our
results show that DDR1 promotes cell-cell adhesion and differentiation through stabilization of Ecadherin, which is mediated by Cdc42 inactivation.
2893/L130
Early Responses of Mammary Epithelial Cells to Integrin Engagement on An EHS-Matrix Predict
Their Potential to Organise in 3-D.
S. Mc Nally, S. Mc Carthy, J. Whyte, F. Martin; SBBS, The Conway Institute, Dublin, Ireland
MCF10A cells [‘normal’ human mammary epithelial cells], supported on a laminin-rich extracellular matrix
[ECM] form acini; by day 8 of culture they exhibit 2 distinct cell populations,the one in contact with the
ECM which have become polarised and exited the cell cycle, the other,the luminal cells,are
disorganised,committed to die and so facilitate lumen clearance. The objectives of our study are to
employ a range of disturbances of intracellular signalling cascades to disrupt acinus formation, for
instance, TGF-beta treatment drives formation of spheroids of unpolarised cells expressing mesenchymal
markers; or expression of a kinase-dead PKC-zeta, or a constitutively active vav2 [GEF] induce the
formation of large lumen-filled ‘multi-acinar’ spheres. However,we noted that the latter dysmorphic
outcome could be reversed by inhibition of EGFR at a very early time point during acinus
development.This suggested that early responses of the MCF10A cells to integrin engagement on the
EHS-matrix would predict their eventual potential to form acini.We have observed normal MCF10A cells
and cells subjected to signalling defects, in culture, in the immediate period after their exposure to EHS
ECM. We find differences in the behaviour of the cultured single cells and in the nature of the early 3-D
assemblies that they form, and these differences are predictive of outcome at maturity.
2894/L131
TIMP-2 Tail Peptide Disrupts the MMP-2 Activation Complex.
X. Xu, Z. Chen, M. Mikhailova, S. Pal, E. M. Lafer, S. Baber, B. Steffensen; Departments of Periodontics
and Biochemistry, University Texas HSC, San Antonio, TX
Cellular activation of latent matrix metalloproteinase-2 (proMMP-2) requires formation of a cell
membrane-associated activation complex involving proMMP-2, tissue inhibitor of metalloproteinases-2
(TIMP-2), and membrane type 1 matrix metalloproteinase (MT1-MMP). Disruption of the complex may
inhibit proMMP-2 activation and therefore may serve as a strategy for specific inhibition of MMP-2. Our
hypothesis was that TIMP-2 derived binding site peptides can inhibit activation of proMMP-2 by
competing the interactions between proMMP-2 and TIMP-2 in the activation complex. In the present
study, we synthesized TIMP-2 tail peptides P175L, which consisted of the last 20 residues of the TIMP-2
SUNDAY
tail region, and P175S, which corresponded to P175L but was missing the last 9 residues. Our
experiments showed that P175L bound to the hemopexin domain of MMP-2 (PEX) in surface plasma
-6
resonance assays with a KDapp of 1.6 x 10 M. Moreover, in assays measuring enzyme activation of
proMMP-2 by membrane preparations from human fibrosarcoma cells (HT1080), P175L inhibited the cell
membrane mediated activation of proMMP-2 in a concentration-dependent manner with IC50 at 30 μM.
The interaction of shorter P175S with 10 μM PEX peptide only reached 97 RUs, which was about six fold
lower than that of P175L (579 RUs), and the binding of P175S to PEX was consistently very weak and
non- saturable over a concentration range of PEX (10 - 0.1 μM). Moreover, P175S did not inhibit
activation of proMMP-2 at concentration of 1 mM. These observations indicated that the last 9 residues of
the TIMP-2 tail are critical for the interactions of TIMP-2 with PEX in the activation complex. In summary,
proMMP 2 activation can be inhibited by a synthetic peptide derived from TIMP-2 tail that competes the
binding between PEX and TIMP-2 in the proMMP-2 activation complex. This research has provided
evidence for a potential novel strategy to specifically inhibit MMP-2. (Supported by NIDCR grants DE
018135, DE 014318 (COSTAR), and DE17139).
MONDAY
Late Abstracts 2
2895/L001
Substrate Positioning Residues Are Essential for Collagenolysis by MMP-2.
M. Mikhailova, X. Xu, S. Pal, B. Steffensen; Departments of Periodontics and Biochemistry, University
Texas HSC, San Antonio, TX
Interactions of matrix metalloproteinase-2 (MMP-2) with native and denatured (gelatin) forms of collagen
are mediated by the collagen-binding domain (CBD), which consists of three in tandem fibronectin-like
type II modules (FN II). We identified previously the collagen-binding residues in each FN II module by
NMR spectroscopy and demonstrated that alanine substitution of these residues altered the affinity of
CBD for native and denatured type I collagen. The present study investigated the contributions of the
CBD binding site residues to: 1) the interactions of full-length MMP-2 with gelatin, and 2) the enzymatic
activities of MMP-2. We introduced the same alanine substitutions into one, two, or three CBD modules of
full length MMP-2 concurrently using a PCR-based approach for site-specific mutagenesis. Recombinant
2+
wild type (WT) and MMP-2 variants were expressed in E. coli and purified by Ni -affinity
chromatography. Among the three FN II modules, modification of residues in module 3 had greatest effect
on the affinity of MMP-2 to gelatin compared to WT. Substitutions in two or three modules simultaneously
further reduced the binding. Enzymatic assays showed that MMP-2 with single binding site mutations had
20-40% lower activities than WT MMP-2. Simultaneous changes in two CBD modules reduced the
activities by 60-75%, and concurrent modifications in all three CBD modules reduced the MMP-2 activity
by >80%. CD analyses verified that changes in activities among the MMP-2 did not result from structural
perturbations. Moreover, the alanine in MMP-2 did not reduce the hydrolysis of the short peptide
substrate NFF-1, which does not require CBD-mediated binding for degradation. In conclusion,
modifications in the CBD of full-length MMP-2 that abrogated gelatin binding also reduced the activities of
MMP-2 on collagen α-chains. This demonstrated that positioning of the substrate by the CBD is required
for collagenolysis. (Supported by NIDCR grants DE016312 and DE17139).
2896/L002
Knockdown and Knockout of the Damm-1 Gene in Dictyostelium Discoideum Suggests It Is an
Essential Gene Required in Cell Adhesion.
S. D. Burrows, D. A. Larochelle; Biology, Clark University, Worcester, MA
We have identified a novel gene (Damm-1) in Dictyostelium discoideum that functions in cell adhesion.
Over-expression of Damm-1 leads to increased surface adhesion (ACSB 2009, Abs #1248). Multiple
attempts to disrupt the Damm-1 locus by homologous recombination proved unsuccessful, suggesting
that Damm-1 is an essential gene. To circumvent the apparent lethality of the knock out (KO) construct,
two approaches were undertaken. The first, using RNAi to suppress the Damm-1 protein levels, was
pursued with the understanding that RNAi typically does not reduce target protein levels to zero. Two
RNAi constructs, one construct to each of the two exons of the Damm-1 gene, were inserted into the
doxycycline-inducible vector pDM310 and transformed into AX2 parental cells. Cells were induced with
doxycycline (10μg/ml) and cell growth studies show a 50-60% reduction in growth from the uninduced
controls at 5 days. The uninduced cell lines show the same growth rates as the AX2 parental cells. A
second, more novel approach was to generate a Damm-1 gene disruption by homologous recombination
in a cell line also expressing exogenous Damm-1 behind the same doxycycline-inducible promoter used
for the RNAi. Cells were induced with 10μg/ml of doxycycline before transformation and this dose was
maintained throughout the selection process. This allowed for suppression of the KO as long as
doxycycline levels were maintained. Recently a KO cell line has been identified using PCR analysis to
confirm the insertion of the KO construct at the Damm-1 locus. The isolation of a KO strain by this
approach confirms that Damm-1 is indeed an essential gene.
MONDAY
2897/L003
Molecular Pathways Leading to Integrin aIIbb3-Dependent Proplatelet Formation in Human
Megakaryocytes.
1
1
1
1,2
3 1
N. Morin , G. Bompard , G. Rabeharivelo , C. Delsert , N. Kieffer ; CRBM UMR5237, CNRS/UNIV
2
3
MTP 1 ET 2, Montpellier, France, Ifremer, La Tremblade, France, CNRS-LIA124, Shangai, China
We have recently described a new, autosomal platelet disorder with a clinical phenotype of
macrothrombocytopenia, and shown that a mutation in the b subunit cytoplasmic tail of the platelet
fibrinogen receptor, integrin aIIbb3, was responsible for the physiopathology of the disease. Expression of
the mutant receptor in CHO cells unexpectedly induced the formation of long, microtubule-driven
protrusions, mimicking megakaryocyte proplatelet extensions, thus underlining the role of integrin aIIbb3
in initiating proplatet formation. We use the CHO cells model as source of proplatelet-like forming cells, to
identify the integrin aIIbb3-dependent signaling pathway initiating proplatelet formation. We show here,
using biochemical as well as fixed and live cell imaging that the induction of protrusion closely resembles
the initiation of neurite outgrowth. The process requires the activation of Rac/Cdc42 pathways and the
regulation of the activity of p21-activated kinase. We analyze the consequences of inhibiting these
pathways on the relocalization of focal adhesion proteins and the reorganization of both the actin and
microtubules networks. We are currently investigating the potential target of PAK on the microtubule
network.
2898/L004
FoxQ1 Exerts Major Influences on Epithelial Differentiation and TGF-β1-Mediated Cell Plasticity.
1
1
1
1
1
2
3
A. Feuerborn , P. K. Srivastava , S. Küffer , W. A. Grandy , T. P. Sijmonsma , N. Gretz , B. Brors , H.
1 1
Gröne ; Cellular and Molecular Pathology, German Cancer Research Centre, Heidelberg, Germany,
2
3
Department of Medical Research, Medical Research Centre, Heidelberg, Germany, Department of
Theoretical Bioinformatics, German Cancer Research Centre, Heidelberg, Germany
Transcription factors are crucial mediators of epithelial cell differentiation and cell plasticity. We identified
several Forkhead transcription factors (Fox-factors) that are affected in expression upon TGF-β1
(Transforming Growth Factor-β1)-dependent changes in epithelial cell differentiation and TGF-β1mediated cell plasticity. NMuMG cells (Namru Mouse Mammary Gland), a common cell culture model to
study molecular mechanisms of cytokine-induced changes in epithelial differentiation were used. RNAi
mediated functional analyses of FoxQ1, a Fox-factor increased in expression upon TGF-β1 stimulation,
revealed that FoxQ1 regulates cell morphology and epithelial cell characteristics. Confocal as well as
transmission electron microscopy demonstrated an increase in cell size and the establishment of
pronounced cell-cell contacts upon the repression of FoxQ1. This was paralleled by the increased
expression of several junction proteins (e.g. E-cadherin and Occludin) as well as changes in cytoskeletal
dynamics, including an increased expression of the actin binding and epithelial specific protein Villin-1. In
addition, FoxQ1 expression was necessary for maintaining the expression of the Cyclin-dependent
kinases CDK4/CDK6 and regular G1/S-phase cell cycle progression. Gene expression profiling of TGFβ1 induced cells with an impaired induction of FoxQ1 expression suggested that FoxQ1 is a relevant
downstream mediator of TGF-β1 induced gene expression changes. This included the differential
expression of transcription factors known to be involved in epithelial plasticity, e.g. Ets-1, Zeb1 and Zeb2.
In summary, this study has elucidated the functional impact of FoxQ1 on epithelial differentiation and
suggests that TGF-β1 mediated epithelial cell plasticity is partially regulated via FoxQ1.
2899/L005
cdGAPL Inhibits Cell Migration by Suppressing Focal Adhesion Dynamics.
D. B. Wormer, C. E. Turner; Cell and Developmental Biology, SUNY Upstate Medical University,
Syracuse, NY
Integrin transmembrane receptors signal to the Rho family of GTPases to coordinate cell-extracellular
matrix (ECM) interactions and regulate cell migration. However, the diverse and complex mechanisms
which couple integrins to Rho family signaling during cell migration are incompletely understood. In our
current study we evaluated the role of cdGAPL, a Rac1 and Cdc42 GTPase activating protein (GAP) in
MONDAY
the migration of an osteosarcoma cell line (U2OS). To determine the effects of cdGAP’s GTPase activity
on cell migration and morphology, we used a combination of biochemistry and live cell imaging including
total internal reflection, (TIRF), confocal, and phase contrast microscopy, as well as transwell migration
assays employing expression of wild type and GAP dead mutant forms of cdGAPL. We also monitored
cell-ECM interactions by observing Focal Adhesion (FA) turnover and calculated FA assembly and
disassembly rates by tracking the fluorescence intensity of individual adhesions containing GFP tagged
FA markers. cdGAPL localized to FAs and expression of cdGAPL inhibited both random cell migration on
fibronectin and collagen, as well as chemotaxis towards serum. A GAP dead mutant of cdGAPL
increased migration velocity above that of cells expressing wild type cdGAPL. Expression of cdGAPL
increased FA lifetimes, slowing both FA assembly and disassembly, while cells expressing a GAP dead
mutant of cdGAPL had decreased FA lifetimes. We conclude that cdGAPL and its GAP activity are
important for mediating multiple modes of cell migration, and that expression of cdGAPL suppresses cell
migration by inhibiting FA dynamics.
2900/L006
Rho-GAP ARHGAP21 Increases Cell-Cell and Cell-Matrix Adhesion of Epithelial Cells.
1
1
1
2
2
2
1 1
K. Barcellos , M. Lazarini , C. L. Bigarella , D. M. Staley , G. Call , M. Hansen , S. T. Saad ; The
2
Hematology and Hemotherapy Center, University of Campinas, Campinas, Brazil, Department of
Physiology and Developmental Biology, Brigham Young University, Provo, UT
Objective: Cell adhesion has many important aspects in cell biology, including cell differentiation,
development, and migration. Defective cell adhesion is one the cancer’s hallmarks. The cadherin and
integrin mediated cell adhesions may be regulated by Rho-GTPases. ARHGAP21 has been described as
a negative regulator of RhoGTPase, preferentially upon Cdc42 and RhoA, and as a partner of alphacatenin that controls alpha-catenin recruitment to the adherens junctions. The aim of this work was to
evaluate the expression of ARHGAP21 in cell-cell adhesion formation and to analyze cell aggregation,
cell proliferation and cell-matrix adhesion of cells lacking ARHGAP21 expression. Methods: ARHGAP21
localization was evaluated by imunofluorescence in MDCK and SW480 cells undergoing cell-cell contact
formation. Cell aggregation and proliferation were measure by Hanging Drop Aggregation and MTT Cell
Proliferation assays, respectively, in SW480 cells with ARHGAP21 inhibition (performed through siRNA
transfection). LNCaP cells depleted of ARHGAP21 were used to analyze cell-matrix adhesion and
expression of genes involved in cell adhesion. Results: ARHGAP21 is localized in the nucleus and
cytoplasm of MDCK and SW480 cells; however, ARHGAP21 was detected at cell-cell junctions four hours
after initiation of cell-cell adhesion, with a subsequent decrease in localization in subsequent hours.
SW480 cells lacking ARHGAP21 expression showed weaker cell-cell adhesions and a decrease in cell
proliferation. In LNCaP cells, ARHGAP21 knockdown reduced cell adhesion to fibronectin and matrigel,
and resulted in an increased expression of genes involved in cell adhesion; including TGF-beta-induced
and podocalyxin-like. Conclusions: ARHGAP21 is a Rho-GAP protein involved in cell-cell and cell-matrix
adhesion. The abnormal adhesion and proliferation observed in cells lacking ARHGAP21 suggest that
this Rho-GAP could be related with tumor development, especially in metastasis and invasiveness of
tumor cells. Supported by: FAPESP.
2901/L007
Animal Models of Retinal Detachment and Age-Related Macular Degeneration: RPE-Specific
Depletion of Chloride Intracellular Channel 4.
J. Chuang, S. Chou, C. Sung; Ophthalmology/Cell & Dev. Biology, Weill Cornell Medical College, New
York, NY
The retinal pigment epithelium (RPE) is sandwiched between the photoreceptors and choriocapillaries.
The molecular mechanism underlying the inter-relationship between RPE cells and their neighboring cells
is poorly understood because the intimate interactions between these cell types are impossible to model
and study in vitro. The cell biology of RPE has a recent growing interest because RPE has been identified
as the primary site in the etiology of several blindness diseases including age-related macular
degeneration (AMD) and retinal detachment (RD). We have established two independent approaches —
(1) a novel plasmid-based transfection and (2) RPE-specific knockout mice — to cell-autonomously
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deplete chloride intracellular channel 4 (CLIC4) in RPE in situ. CLIC4 is a peripheral membrane protein
that is abundantly expressed in apical RPE microvilli (MV). Our comprehensive electron microscopic
analysis showed that CLIC4 is critical for the morphogenesis of apical MV, and basal infoldings. It is also
important for the adhesion between RPE and photoreceptor outer segment, proper basal extracelluar
deposits, and the structure/function of both neural retinas and choriocapillaries. These rodent models
exhibited several morphological features resemble those described for AMD and RD. Mechanistic studies
showed that ectopically expressing ezrin (also known to be important for MV morphogenesis) failed to
rescue the changes exerted by CLIC4 silencing. CLIC4 suppressed RPE also had altered expressions of
several cell surface channels/transporters, which may be attributable to subretinal fluid accumulation in
these animals.
2902/L008
Syndesmos, a New Effector of Syndecan-4 Mediated Signaling.
M. Roulet, S. Wilcox-Adelman; Boston Biomedical Research Institute, Watertown, MA
Precise integration of environmental cues is critical for adhesion-dependent cells to receive and respond
to the surrounding environment in both physiological and pathological processes. Signals from
extracellular matrix (ECM) proteins and/or growth factors can modify cell adhesion, migration, proliferation
or differentiation. Syndecan-4, uniquely positioned to regulate both types of pathways, modulates cell
responsiveness to ECM signaling by acting as a co-receptor with the integrin alpha5beta1 to generate
stress fibers and focal adhesions (FA). Cells isolated from syndecan-4 null mice show impaired cell
migration and syndecan-4 upregulation has been observed in certain metastatic cancers, indicating a
more complex role in cell-substrate interactions. Selective recruitment of FA associated proteins,
mediated by syndecan-4, could serve to modulate signaling pathways involved in cell-matrix adhesion,
proliferation and migration. The cytoplasmic protein syndesmos specifically binds syndecan-4 but also the
adaptor protein paxillin. A syndecan-4-syndesmos-paxillin complex could represent a novel tertiary level
of regulation in the syndecan-4 adhesion- and migration- mediated signaling pathway. Syndesmos
function during cell adhesion, migration and proliferation was studied by using shRNA to knock down its
expression in wild-type (WT) and syndecan-4 null (KO) fibroblasts. Syndesmos loss leads to actin stress
fiber disorganization and to the modification of paxillin recruitment in FA in WT cells, mimicking in this way
syndecan-4 loss. Contrary to syndecan-4 loss, syndesmos loss increases both cell migration and
proliferation in WT but also in KO cells, indicating that syndesmos also has functionality independent of
syndecan-4. In summary, a syndecan-4-syndesmos-paxillin complex could be considered as a new level
of syndecan-4 mediated signaling pathway regulation. A better understanding of the role syndecan-4
plays in promoting cell migration and proliferation via syndesmos can contribute to the development of
new therapies in treating chronic wounds, such as dermal ulcers, or cancer metastasis.
2903/L009
Collagenase-Resistant Type I Collagen Promotes Vascular Smooth Muscle Cell Senescence.
1
1
1
1
2
1
1 1
C. O'Neil , A. Watson , F. Vafaie , Z. Nong , M. Chu , D. Holdsworth , J. Pickering ; Robarts Research
2
Institute, London, ON, Canada, London Health Sciences Centre, London, ON, Canada
Senescent vascular smooth muscle cells (SMCs) have been identified in atherosclerotic lesions. SMCs in
atherosclerotic arteries reside within a milieu of type I collagen fibrils that, over time, can undergo
progressive intermolecular cross-linking rendering the collagen resistant to proteolysis. We hypothesized
that vascular SMC longevity depends on the extent to which the surrounding type I collagen can be
proteolytically edited. To test this, we studied mice with a targeted mutation of the ¾-¼ collagenase
cleavage site in type I collagen (Col1a1r/r). Mice harboring the Col1a1r/r mutation appeared normal up to
3 months of age but subsequently developed impaired weight gain, decreased adipose tissue content,
and increased kyphosis, increased blood pressure, and shortened lifespan. Furthermore, SMCs in the
aortic wall of aged, Col1a1r/r mice responded to the stress of angiotensin II infusion by displaying
senescence-associated beta galactosidase activity. To determine if collagenase-resistant collagen
directly impacted SMC longevity, primary human SMCs were cultured on collagen harvested from the tails
of wild-type or Col1a1r/r mice. SMCs on mutant collagen displayed a 67% decrease in cumulative
population doublings, which was associated with increased senescence-associated beta-galactosidase
MONDAY
(SA-beta-gal) activity and increased p21 and p16 expression. Furthermore, stress-induced premature
senescence, initiated by serum-deprivation and assessed by SA-beta-gal activity and p21/p16
expression, was also amplified in cells cultured on collagenase-resistant collagen. Mouse embryo
fibroblasts derived from Col1a1r/r mice likewise underwent accelerated aging. Conclusions: Collagenaseresistant type I collagen induces a premature aging-like syndrome in mice and accelerates replicative and
stress-induced senescence of vascular SMCs. These findings identify extracellular matrix control over
vascular cell senescence, which may contribute to vascular aging.
2904/L010
Lung Vascular Smooth Muscle Cell Differentiation Controlled by Elastic Matrix.
1
1
1
1
2
2
K. Ihida-stansbury , S. Sanyal , C. Navarro , P. A. Janmey , H. Sundararaghavan , J. Burdick , P. L.
1 1
Jones ; Instiute for Medicine & Engineering, University of Pennsylvania, Philadelphia, PA,
2
Bioengineering, University of Pennsylvania, Philadelphia, PA
Matrix property is an important modulator of lung morphogenesis, postnatal lung development, and
compensatory lung regeneration yet the effect of matrix biophysical and biochemical properties on stem
or progenitor cells in lung morphogenesis is unclear. In this study, we found that deletion of homeobox
gene transcription factor, Prx1, in mouse downregulated expression of elastic matrix components:
tropoelastin, fibrillin1/2 and in developing lungs and vascular smooth muscle cell (SMC) differentiation
was suppressed. Furthermore, we found that TGF-beta localization in the Prx1-null lungs was
disorganized compare to wild type lungs. Since elastin and its associated proteins control tissue stiffness
(biophysical) and localization of TGF-beta (biochemical), we hypothesized that Prx1-dependent elastic
matrix properties control lung vascular SMC differentiation. The matrix stiffness of the lungs was
measured using newly developed decellularized lung scaffolds derived from Prx1-wild type and -null mice
and showed that the stiffness of the Prx1-null lung matrix was softer than wild type counterparts.
Mesenchymal precursor cells cultured on stiff synthetic 2D and 3D substrate showed more SM
differentiation than cells cultured on softer substrate, consistent with increased TGF-beta stimulation.
Mesenchymal precursor cells cultured in the dicellularized scaffolds differentiated and re-constructed the
in vivo characteristics of vascular SMC in Prx1-wild type and -null lungs respectively. Stiffening the Prx1null lung scaffold restore the colonization and differentiation of mesenchymal cells as wild type scaffold in
some extent. These results suggest that Prx1-dependent lung elastic matrix regulates lung vascular
development via control of matrix stiffness and TGF-beta signaling. Furthermore, decellularized scaffolds
are a novel and promising approach to the study the role of lung matrix in cell differentiation.
2905/L011
Lumen Formation in HGF-Induced MDCK Tubulogenesis Is Regulated by ROCK-MyosinII Pathway.
M. Kim, K. M. Mostov; Anatomy, University of California San Francisco, San Francisco, CA
Hepatocyte growth factor (HGF), a large multifunctional polypeptide growth and motility factor, is known to
play important roles during embryonic development and tissue repair. In a three-dimensional type I
collagen model, HGF induces Madin-Darby canine kidney (MDCK) epithelial cysts to form long, branching
tubules (tubulogenesis). To investigate the cellular events that occur during HGF-mediated remodeling,
we examined the real-time behavior of cells during HGF-induced tubulogenesis. Time-lapse imaging
revealed that basal protrusions from MDCK cysts by HGF treatment transform into tubes. However,
increased sprouting and cell protrusion did not necessarily result in increased tubule formation. We also
observed that single cell shape change (elongation) followed by migration leads to generate the luminal
surface, which is connected with the cyst lumen. While cells in a cord established de novo apical domains
and created lumens at the apical region, the inhibition of Rho kinase or myosinII by pharmaceutical drug
after chain formation induced abnormal tubules with discontinuous and multi lumens, which was not
caused by the loss of (apicobasal) polarity or apical junction assembly. Furthermore, cell apoptosis is not
required for luminal clearance in tubular structure. We propose that HGF induces MDCK tubulogenesis
via the combined mechanism of cell movement and rearrangement and that lumen formation in tubules is
regulated by ROCK-myosinII pathway.
MONDAY
2906/L012
Inhibition of Cellular Adhesion Mediated by Aminopeptidase N (CD13).
C. A. Garay, E. Ortega; Immunology, IIB, UNAM, Mexico city, Mexico
CD13 is a glycoprotein considered as a myeloid lineage marker. It has aminopeptidase activity and it
plays a key role as a viral receptor, in angiogenesis, tumor cell invasion, and metastasis. Our group
described the ability of a reduced number of monoclonal antibodies against CD13 to induce homotypic
aggregation in the monocytic cell line U-937; the phenomenon seems to be related to binding to a specific
site on CD13 molecule. We decided to block aggregation to study cell-cell interactions. We generated two
different hybridomas, which produce the monoclonal antibodies C and E. These antibodies block
aggregation of U-937 cells in a time and dose-dependent manner, and induce detachment of cells already
aggregated. Binding of C and E antibodies does not abrogate binding of mab 452 (anti-CD13) to
monocytic cells, indicating that the epitopes recognized by these antibodies are different. We used C and
E antibodies to immunoprecipitate their antigen in U-937 lysate, and revealed a 160 kDa protein by
western blot using the corresponding antibody. The immunoprecipitated protein was identified as CD13 or
Aminopeptidase N as determined by mass spectrometry. Reduced CD13 expression by siRNAs in U-937
cells, diminished binding of inhibitory antibodies C and E to the cells, supporting that the antigen
recognized by these antibodies is CD13. Regarding the mechanism of inhibition, we observed that, a)
membrane expression of CD13 is not modified by the presence of inhibitory antibodies determined by
flow cytometry, b) Fab fragments of mabs C and E are not sufficient to inhibit aggregation, crosslinking by
C or E alters the signal transduction induced by mAb 452. Although there are several antibodies that
recognize CD13, the C and E antibodies bind to a different epitope involved in cell adhesion. The
antibodies obtained may represent an important tool to study the cell-cell interactions mediated by CD13
in physiological and pathological conditions such as tumor progression and metastasis in cancer.
2907/L013
β1 Integrins and N-Cadherin Cooperate to Control the Invasion of the Caecum and Hindgut by
Enteric Neural Crest Cells.
F. Broders; Institut Curie CNRS, Paris, France
Integrins and cadherins are transmembrane receptors that mediate cell-matrix and cell-cell adhesion,
respectively, and play important roles during development. We used the Ht-PA-Cre conditional promoter
to target β1-integrin and N-cadherin gene disruption specifically in migratory neural crest cells and
analysed the crosstalk between these receptors during enteric neural crest cell migration at different
stages of enteric nervous system development. The β1-integrin conditional mutant exhibited major
alterations in the structure of the enteric nervous system caused by an increased intercellular adhesion
and changes in the migratory modes of β1-integrin null neural crest cells during gut colonization (Breau et
al., 2006, 2009). Here we show that N-cadherin-null neural crest cells failed to colonize the distal part of
the hindgut but to a lesser extent than that observed in β1-integrin null cells. In contrast, double mutants
exhibited a drastic aganglionosis of the gut starting at the level of the proximal hindgut. The trajectories
and speed of β1-integrin and N-cadherin-null enteric neural crest cells were affected. The abnormal cell
aggregation observed in β1-integrin null cells was abrogated when the N-cadherin gene was also
disrupted in the double mutants and the organization of the enteric nervous system was partly rescued.
This indicates that this process is mainly driven by an N-cadherin-dependent mechanism. Finally, we
observed that the ganglia network displayed specific alterations in the single mutants compared to
controls and to double mutants. Our results indicate that a crosstalk between β1-integrins and N-cadherin
occurs in enteric neural crest cells to control gut colonization and enteric nervous system ontogenesis.
2908/L014
Deregulation of Rab5 and Rab4 Proteins in p85R274A-Expressing Cells Alters PDGFR Trafficking.
1,2
2,1
2,1
2,1
2,1
1
2,1
D. Anderson , D. Chamberlain , J. Oberg , L. Furber , S. Poland , A. Hawrysh , S. Knafelc , H.
3 1
McBride ; Cancer Research Unit, Saskatchewan Cancer Agency, Saskatoon, SK, Canada,
2
3
Biochemistry, University of Saskatchewan, Saskatoon, SK, Canada, University of Ottawa Heart Institute,
Ottawa, ON, Canada
MONDAY
Activated receptor tyrosine kinases recruit many signaling proteins to activate downstream cell
proliferation and survival pathways, including phosphatidylinositol 3-kinase (PI3K) consisting of a p85
regulatory protein and a p110 catalytic protein. We have recently shown the p85α protein also has in vitro
GTPase activating protein (GAP) activity towards Rab5 and Rab4, small GTPases that regulate vesicle
trafficking events for activated receptors. Expression of a GAP-defective mutant, p85R274A, resulted in
sustained levels of activated platelet-derived growth factor receptors (PDGFRs) and enhanced
downstream signaling. Our objective was to characterize Rab5- and Rab4-mediated PDGFR trafficking in
cells expressing wild type p85 and GAP-defective mutant p85R274A. Wild type p85 overexpressing cells
had slower PDGFR trafficking consistent with enhanced GAP activity deactivating Rab5 and Rab4 to
block their vesicle trafficking functions. Mutant p85R274A expression increased the internalization rate of
PDGFRs, a Rab5-dependent process, without preventing PDGFR ubiquitination. Immunofluorescence
studies further demonstrated that p85R274A-expressing cells showed Rab5 accumulation at intracellular
locations. Pull-down and FRAP (fluorescence recovery after photobleaching) experiments indicate this is
likely membrane-associated Rab5-GTP, sustained due to decreased p85 GAP activity for the p85R274A
mutant. These cells also had substantial amounts of activated PDGFRs in Rab4-positive recycling
endosomes, a compartment that usually contains primarily deactivated/dephosphorylated receptors. Our
results suggest that the PDGFR-associated GAP activity of p85 regulates both Rab5 and Rab4 functions
in cells to influence the movement of activated PDGFR through endosomal compartments. Disruption of
this regulation by p85R274A expression impacts PDGFR phosphorylation/dephosphorylation,
degradation kinetics and downstream signaling by altering the time receptors spend in specific
intracellular endosomal compartments. These results demonstrate that the p85α protein is an important
regulator of Rab-mediated PDGFR trafficking, which significantly impacts receptor signaling and
degradation. Supported by the CCS (grant #019040).
2909/L015
KIF5C, a Kinesin Motor Involved in Apical Trafficking of MDCK Cells.
R. Jacob, K. Astanina; Philipps-University, Marburg, Germany
Polarized traffic in epithelial cells depends on well organised pathways that direct secretory cargo to the
apical or basolateral plasma membrane. In MDCK cells, apical trafficking can further be divided into a
lipid raft-dependent and a raft-independent route, which separate biosynthetic cargo in a post Golgi
endosomal compartment. We have now identified KIF5C as a kinesin motor for apical trafficking of both,
raft-associated sucrase isomaltase and raft-independent neurotrophin receptor. KIF5C was identified by
mass spectrometry in vesicle enriched fractions and on immunoisolated post Golgi vesicles carrying
apical cargo. The amount of vesicle associated KIF5C was highest on material isolated directly after
TGN-release and declined thereafter. Altogether, our data suggest that KIF5C is involved in the passage
of apical cargo molecules to a post Golgi endosomal compartment, where further segregation into distinct
vesicle populations proceeds.
2910/L016
Structural Basis for Clamping of Membrane Fusion by Complexin.
1
1
1,2
1
1
1 1
D. Kuemmel , S. Krishnakumar , D. Radoff , C. Giraudo , J. Rothman , K. M. Reinisch ; Cell Biology,
2
Yale School of Medicine, New Haven, CT, Department of Biochemistry and Molecular Biophysics,
Columbia University, New York, NY
Complexin, a component of the membrane fusion machinery at neuronal synapses, interacts with soluble
N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes during regulated
exocytosis and inhibits fusion prior to neuronal stimulation. To investigate how complexin arrests fusion,
we have determined the crystal structure of complexin bound to a modified SNARE complex designed to
mimic the pre-fusion form. In the structure, the complexin central helix is anchored to the half-zippered
SNAREpin, and its accessory helix interacts with a second SNARE complex in the site occupied by the
VAMP2 C-terminus post-fusion. The structure suggests that complexin locks pre-fusion SNARE
complexes into a semi-rigid lattice, with the accessory helix blocking SNARE assembly. This lattice
precludes formation of a hemi-fusion stem and suggests a mechanism how synaptotagmin relieves
complexin inhibition.
MONDAY
2911/L017
Gene Transduction in the Salivary Glands as a Tool to Study Membrane Trafficking in Live
Animals.
M. Sramkova, L. Parente, R. Weigert; IMTU, NIDCR/NIH, Bethesda, MD
The ability to express genes in specific cell population of living animal provides a unique tool to address
many biological questions in the proper physiological context. We explored the use of alternative gene
transfer with the goal to address key questions on the molecular machineries regulating membrane
trafficking. We injected plasmid DNA encoding fluorescent proteins into the rat submandibular glands
(SG) and observed their in vivo expression. When plasmid DNA is injected alone, the reporter molecule is
expressed in approximately 0.05% of the cells of the parenchyma, which we have identified as
intercalated ducts. The addition of adenoviral (rAd5) particles increased the level of transduction to 0.52% of the cells with the expression primarily in the large striated and granular ducts and, to a lesser
extent in the acinar cells. We reasoned that plasmid DNA might be internalized by the acinar cells via the
endocytic pathways and for this reason, compensatory endocytosis was stimulated by activating the betaadrenergic receptors during plasmid DNA injection. Under these conditions, the reporter molecule was
expressed in ~1% of the cells of the parenchyma, primarily in acinar cells. Finally, we have utilized this
approach to study the trafficking and sorting of two water channel proteins that are expressed in SG,
aquaporin 3 (AQP3) and aquaporin 5 (AQP5), as well as beta-adreneric receptor (β-AdrR). After 16 hrs,
all molecules were expressed at the plasma membrane (PM) of both large ducts and acini in proper
domain of the PM, with their patterns closely resembling those of the endogenous molecules.
Furthermore, AQP5 was also localized in a population of vesicles that was closely associated with the
trans-Golgi network (TGN). Co-expression of YFP-AQP5 and mCherry-TGN38 and time-lapse imaging in
the live animal, revealed a transient co-localization between these two molecules, suggesting that the
AQP5-containing vesicles might be the transport intermediates involved in the sorting of this molecule. In
conclusion, we showed how the expression of fluorescently tagged molecules enables the study of the
dynamics of various organelles in live animals at a resolution comparable to that achieved in cell cultures.
2912/L018
Syt1p Promotes Activation of Arl1p at the Late Golgi to Recruit Imh1p.
1
1
1
1
1
1
1
2
1 1
K. Chen , P. Tsai , J. Hsu , H. Hsu , C. Fang , L. Chang , Y. Tsai , C. Yu , F. Lee ; Molecular Medicine,
2
National Taiwan University, Taipei, Taiwan, Cell and Molecular Biology, Chang Gung University, TaoYuan, Taiwan
In yeast, Arl3p recruits Arl1p GTPase to regulate Golgi function and structure. However, the molecular
mechanism involved in regulating activation of Arl1p at the Golgi is unknown. Here, we show that Syt1p
promoted activation of Arl1p and recruitment of a golgin protein, Imh1p, to the Golgi. Deletion of SYT1
resulted in the majority of Arl1p being distributed diffusely throughout the cytosol. Overexpression of
Syt1p increased Arl1p-GTP production in vivo and the Syt1-Sec7 domain promoted nucleotide exchange
on Arl1p in vitro. Syt1p function required the N-terminal region, Sec7 and PH domains. Arl1p, but not
Arl3p, interacted with Syt1p. Localization of Syt1p to the Golgi did not require Arl3p. Unlike arl1Δ or arl3Δ
mutants, syt1Δ did not show defects in Gas1p transport, cell wall integrity or vacuolar structure. These
findings reveal that activation of Arl1p is regulated in part by Syt1p, and imply that Arl1p activation, by
using more than one GEF, exerts distinct biological activities at the Golgi compartment.
2913/L019
Phosphorylation of a Membrane Curvature-Sensing Motif Switches Function of the HOPS Subunit
Vps41 in Membrane Tethering.
1
1
2
1
3
1
1
2
M. Cabrera , L. Langemeyer , M. Mari , R. Rethmeier , I. Orban , A. Perz , C. Bröcker , J. Griffith , D.
3
3
2
1
1 1
Klose , H. Steinhoff , F. Reggiori , S. Engelbrecht-Vandré , C. Ungermann ; Department of Biology and
2
Chemistry, University of Osnabrueck, Osnabrueck, Germany, Department of Cell Biology and Institute of
3
Biomembranes, University Medical Centre Utrecht, Utrecht, Netherlands, Department of Physics
Experimental Physics section, University of Osnabrueck, Osnabrueck, Germany
MONDAY
Tethering factors are organelle-specific multisubunit protein complexes that identify, along with Rab
GTPases, transport vesicles and trigger their SNARE-mediated fusion of specific transport vesicles with
the target membranes. Little is known about how tethering factors discriminates between different
trafficking pathways which may converge at the same organelle. Here we describe a phosphorylationbased switch mechanism, which allows the HOPS effector subunit Vps41 to operate in two distinct fusion
events, namely endosome-vacuole and AP-3 vesicle-vacuole fusion. Vps41 contains an amphipathic lipid
packing sensor (ALPS) motif, which recognizes highly curved membranes. At endosomes, this motif is
inserted into the lipid bilayer and masks the binding motif for the δ-subunit of the AP-3 complex, Apl5,
without affecting the Vps41 function in endosome-vacuole fusion. At the much less curved vacuole, the
ALPS motif becomes available for phosphorylation by the resident casein kinase Yck3. As a result, the
Apl5-binding site is exposed, and allowings AP-3 vesicle binding s to bind to Vps41, followed by specific
fusion with the vacuolar membrane. This multifunctional tethering factor thus discriminates between
trafficking routes by switching from a curvature-sensing to a coat recognition mode upon phoshorylation.
2914/L020
Simulations of the P-Glycoprotein Transport Network in Confluent Monolayers to Identify the
Signature Plots for Drug-Drug Interactions Due to Competitive and Uncompetitive Binding.
D. A. Silverman, E. Martinez, M. O'Connor, J. Bentz; Drexel University, Philadelphia, PA
Objective: Steady-state single-binding-site Michaelis Menten equations have been successfully used to
analyze kinetic mechanisms of water-soluble enzymes for nearly 100 years. However, these equations do
not adequately predict the kinetics for membrane transporters in confluent cell monolayers, whose
binding sites lie within the membrane, e.g. P-glycoprotein (P-gp) and other related multidrug resistance
transporters. Inhibition of drug-binding to P-gp by other substrates is responsible for many clinically
important drug-drug interactions. We use simulations to construct the signature plots needed to identify
the types of inhibition mechanisms that occur between two different P-gp substrates, based upon
experimentally fitted kinetic parameters of P-gp transport. Methods: Simulations utilizing MATLAB are
used to create the “Eadie-Hofstee” equivalent plots for confluent monolayers of P-gp expressing cells.
The effect of binding mechanism (e.g. competitive versus uncompetitive versus mixed competition),
experimental error, and P-gp surface density on the shape of the signature plots has been developed.
Results: We have found that these signature plots are curved for the confluent cell monolayers, rather
than straight lines, as is found for water-soluble enzymes. Thus, different criteria are needed to evaluate
the binding mechanism within the P-gp binding site, which can accommodate several substrates
simultaneously and which may vary with different substrates. Conclusions: IC50 determination is the
primary experiment used in the pharmaceutical industry to rank-order inhibitors. Conventionally this
analysis is assumed to follow purely competitive inhibition, which need not be correct. Quantitative
evaluation of inhibitors must start with the identification of the mechanism of binding inhibition, which our
analysis accomplishes through these signature plots.
2915/L021
The I-BAR Protein MIM Senses Membrane Curvature Generated by Cellular Dewetting to Control
Line Tension and Arp2/3-Driven Actin Polymerization.
1
2
3
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4
2
1
M. Maddugoda , D. Gonzalez-Rodriguez , J. Saarikangas , F. Prodon , S. Janel , D. Cuvelier , A. Doye ,
4
2
3
2
1 1
2
F. Lafont , P. Bassereau , P. Lappalainen , F. Brochard , E. Lemichez ; INSERM, Nice, France, Institut
3
4
Curie, Paris, France, Institute of Biotechnology, Helsinki, Finland, Institut Pasteur de Lille, Lille, France
Our aim is to understand the mechanisms by which cells perceive newly formed curved membranes
during the formation of transcellular tunnels (macroapertures), to control their opening and closure.
Transcellular holes in the endothelium control body fluid balance, particle exchange, and are a risk factor
of bacterial dissemination in tissues. Recently, exciting new data from our lab has shown that the exotoxin
EDIN, isolated from a strain of S. aureus triggers the formation of large transcellular tunnels, termed
macroapertures, in endothelial cells (Boyer et al, 2006). Here we have identified the opening of
macroaperture tunnels, as a cellular form of dewetting. Dewetting is a physical phenomenon where dry
patches nucleate and grow within a thin liquid film. In cells, unlike in liquid films, macroaperture formation
is reversible, i.e. cells are able to sense and block cellular dewetting. By screening for proteins that sense
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and regulate changes to membrane curvature we have identified Missing in Metastasis (MIM) as a master
regulator of macroaperture sensing as well as closure. We find that the I-BAR-domain of MIM senses the
newly curved edge of MAs, a few milliseconds after the opening event. By combining biophysical
modeling for dewetting with experimental measurements we provide evidence that the MIM I-BAR domain
bears intrinsic scaffolding properties that actively increase line tension at MA edges and controls
macroaperture size. In addition, full length MIM triggers Arp2/3 driven actin polymerization, which leads to
MA closure by extension of actin rich membrane waves. Collectively we find that MIM is an essential
regulator of cellular dewetting and that MIM is able to sense the opening, limit the size, and drive the
active closure of macropertures by the complimentary use of several protein domains.
2916/L022
Cdc42 Promotes Exocytotic Fusion Pore Dilation.
M. Bretou, O. Jouannot, I. Fanget, N. Larochette, C. Desnos, F. Darchen; CNRS UMR 8192, Université
Paris Descartes, Paris, France
Rho GTPases control actin dynamics and cell polarity. Among them, Cdc42 has been implicated in
hormone secretion and Glut4 translocation. Using enterochromaffin BON cells (a cell line derived from a
human carcinoid tumor) as a secretory model, we have investigated the mechanisms underlying the
effect of Cdc42 on exocytosis. Total Internal Reflection Fluorescence Microscopy (TIRFM) experiments
revealed that Cdc42 silencing strongly reduced the number of NPY-mRFP labelled secretory granules
(SGs) undergoing exocytosis. In contrast, Cdc42 silencing had little effect on the recruitment and docking
of SGs at the plasma membrane. SG exocytosis induces the release of serotonin that can be detected by
carbon fibre amperometry as current spikes, each spike reflecting a single fusion event. Cdc42 silencing
severely decreased the occurrence of high amplitude spikes, in agreement with TIRFM data, but only
slightly reduced that of small spikes. The distribution of spike amplitude, spike charge and maximal spike
rise velocity was shifted toward lower values by Cdc42 silencing, suggesting reduced dilation of the fusion
pore. In addition, the occurrence of very small-sized events was increased suggesting that fusion pores
often fail to enlarge upon Cdc42 silencing. Overall, fusion probability was not affected. The effect of
Cdc42 silencing on fusion pores was confirmed by TIRFM using the pH-sensitive NPY-pHluorin to label
SGs. Aborted events were detected as transient elevation of granule fluorescence due to the efflux of
protons through a narrow fusion pore without release of NPY-mRFP. Finally, increasing the membrane
tension by bathing the cells in hypo-osmotic medium partially rescued the effects of Cdc42 silencing. We
conclude that Cdc42 controls fusion pore dilation and thereby the rate and extent of secretory product
release. This effect is likely mediated by changes in membrane tension.
2917/L023
The L355P Diabetic Mutation in Kir6.2 Causes Loss of PKC Dependent down Regulation of the
KATP Channel.
C. J. Cockcroft, J. Mankouri, R. Karnik, T. K. Taneja, P. T. Manna, A. Sivaprasadarao; Multidisciplinary
Cardiovascular Research Centre, Institute of Membrane and Systems Biology, University of Leeds,
Leeds, United Kingdom
The pancreatic ATP sensitive potassium (KATP) channel is an octomeric complex comprising four subunits
each of Kir6.2 and SUR1. It plays a crucial role in glucose stimulated insulin secretion by coupling insulin
secretion to ATP/ADP ratio. Genetic mutations in the channel subunits cause diseases of abnormal
insulin secretion either by altering the sensitivity of the channel to the ATP/ADP ratio, or by affecting
expression at the plasma membrane. We examined the effect of the L355P mutation in Kir6.2, reported in
a patient with type-2 diabetes, on the surface expression of the channel. For this, we co-expressed either
wild-type (WT) or L355P Kir6.2 constructs containing an extracellular HA epitope with SUR1 in HEK293
cells and followed trafficking of the channel using chemiluminescence and immunocytochemistry
approaches. We found that surface levels of the mutant channel (relative to the total channel) are ~4-fold
higher compared to WT. Since the mutation is located within an endosomal trafficking motif
352
356
( DSRLL ), we examined the effect of the mutation on endosomal trafficking. Importantly, PKC
activation has been shown to downregulate the cell surface expression of KATP channels by inhibiting
recycling. We found that compared to WT channels the L355P mutation results in increased trafficking to
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the cell surface in the presence of the PKC activator, phorbol-12-myristate-13-acetate (PMA). In contrast
to WT channels, mutant channel recycling is un-inhibited during PKC activation. Moreover, in PKC
induced cells, internalised mutant channels are largely distributed to the periphery of the cell, whereas
WT channels are more heavily localised to the perinuclear region. We conclude that the L355P mutation
prevents the ability of PKC to downregulate the delivery of channels to the cell surface via a recycling
pathway. Since glucose and several insulin secretogoues stimulate PKC, the loss of PKC downregulation
may contribute to type-2 diabetes in the patient with the L355P mutation.
2918/L024
Characterization of the RalBP1 and Reps2 Complex in Endocytosis.
D. Bezabih, G. Henry, J. Baleja; Tufts Medical School, Boston, MA
Endocytosis is a fundamental process that regulates nutrient uptake, intracellular signaling and
morphogenesis. Dysfunction leads to severe metabolic, neurodegenerative and proliferative diseases.
RalBP1 and Reps2 are important proteins of endocytosis that interact with each other. The proteins are
discovered as downstream targets of the Ral/Ras signaling pathway. They are involved in endocytosis of
epidermal growth factor (EGF), insulin and transferrin. In addition, RalBP1 effluxes chemotherapeutic
drugs through the transport of their glutathione conjugates. The interacting region of RalBP1 and Reps2
has been mapped to the C-termini 150 amino acids of each protein and is predicted to form a coiled-coil.
In this study, we used coiled-coil and disordered region prediction tools to identify the C-termini core
interacting region of each protein and used site directed PCR mutagenesis according to the predictions to
create different constructs. We showed that the C-terminal fragment of RalBP1 exists as a trimer in
solution using gel filtration and cross-linking. The structures of the RalBP1 fragment and the various
constructs of Reps2 were characterized by circular dichroism. The circular dichroism ellipticity value
indicated that RalBP1 is 80% α-helix. Similar experiments showed that the longest construct of Reps2 is
mostly unstructured whereas the shortest is significantly structured. From gel filtration and affinity
precipitation assays we found that each construct of Reps2 bound the RalBP1 fragment. So far we have
identified the core region of Reps2 that interacts with the C-terminal region of RalBP1. These results
provide the reagents to define the core complex of RalBP1/Reps2 for three-dimensional structure
determination and to create peptide and other inhibitors useful for studying the role of RalBP1 in
controlling endocytosis.
2919/L025
The Par1 Receptor Posseses a Functional Signal Peptide Essential for Receptor Biosynthesis.
1
2
1 1
D. Zampatis , N. Tsopanoglou , R. Schuelein ; Protein Trafficking, Leibniz Institut für Molekulare
2
Pharmakologie, Berlin, Germany, Pharmacology, Medicine School of Patras, Patras, Greece
Protease-activated receptors or PARs are a subfamily of G-protein coupled receptors (GPCRs) that are
activated by cleavage of their extracellular domain. PARs are activated by the action of serine proteases
such as thrombin (acts on PARs 1, 3 and 4) and thrypsin (PAR 2). These enzymes cleave the N tail of the
receptor, which in turn acts as a tethered ligand. In the cleaved state, part of the receptor itself acts as the
agonist, causing a physiological response. There are four (4) known PARs numbered from one to four
(PAR1, PAR2, PAR3 and PAR4). Purpose:In the case of the PAR1 receptor, it is known that the receptor
has a cleavage site in position 41-42 in its N tail where thrombin cleaves and activates the receptor but
nothing is known whether the receptor possesses or not a functional signal peptide.Methods: According
to prediction programs, the PAR1 receptor also possesses a cleavable signal peptide in its N tail
(residues 1-21) which might mediate targeting of the receptor to the membrane of the endoplasmic
reticulum (ER), the first step of the intracellular transport of the receptor. We have answered these
questions using marker protein secretion studies, immunoprecipitation, ELISA and IF experiments.
Results: Our results indicate that the signal peptide mediates ER targeting and insertion of the receptor
and is cleaved off following the ER insertion process and the signal peptide is essential for the receptor
biosynthesis. Also the N35 does not inhibit the function of the signal peptide even though increases the
retention of the receptor into the ER but does not significantly affect the expression of the receptor.
Conclusion: Taken together, these results indicate that the signal peptide is indeed functional. And
essential for receptor biosynthesis
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2920/L026
Exit of LDL-Derived Cholesterol from Lysosomes.
1
1
3
3
2
1 1
R. Uronen , T. Blom , Z. Li , R. Bittman , J. Peränen , E. Ikonen ; Institute of Biomedicine, University of
2
Helsinki, Helsinki, Finland, Institute of Biotechnology, University of Helsinki, Helsinki, Finland,
3
Department of Chemistry and Biochemistry, Queens College of The City University of New York, New
York, NY
Cells acquire cholesterol through receptor mediated endocytosis of low density lipoprotein (LDL) particles. LDL is transported to lysosomes and cholesteryl esters are hydrolyzed by the enzyme acid
lipase. How cholesterol then leaves lysosomes has so far been unclear, except that two proteins,
Niemann-Pick type C1 and 2 (NPC1 and NPC2) are involved in the process. Here we establish a new tool
to study this pathway: LDL-particles labeled with esterified BODIPY-cholesterol. We show that the
hydrolysis of the BODIPY-cholesteryl ester endocytosed in LDL is dependent on acid lipase and its exit
from lysosomes is dependent on NPC1 and NPC2, as it is for natural cholesterol. We then provide
evidence that the exit of LDL-derived cholesterol from lysosomes is dependent on vesicular trafficking;
the process is inhibited by disruption of the microtubule network or by depletion of distinct Rab GTPases.
Of the 14 candidate Rabs tested, the strongest inhibition on LDL-cholesterol exit from lysosomes was
observed upon Rab8 depletion. We are in the process of characterizing the Rab8 interactome and its
mode of action in this transport process.
2921/L027
Role of Filamentous Actin in Multigranular Exocytosis.
J. A. Valentijn, M. J. Mourik, S. Kooijman, A. J. Koster, K. M. Valentijn; Molecular Cell biology, Leiden
University Medical Center, Leiden, Netherlands
Multigranular exocytosis (ME) is a form of regulated exocytosis whereby multiple secretory granules
coalesce prior to fusion with the plasma membrane. We have recently shown that ME represents a
significant pathway for the release of the blood clotting protein, von Willebrand Factor (VWF), from
endothelial-cell specific secretory granules termed Weibel-Palade bodies (WPBs). An intermediate
structure consisting of coalesced WPBs was identified and named secretory pod. Here, we investigated
whether filamentous actin (F-actin), which plays a role in the exocytosis of single secretory granules, is
also involved in ME. Using fluorescence and electron microscopy applied to human umbilical vein
endothelial cells, we found that either stabilization of F-actin with jasplakinolide or disruption of F-actin
with cytochalasin D, both caused a dramatic increase in the number of secretory pods. These pods were
mostly inaccessible to extracellular anti-VWF antibodies, indicating that they had not yet undergone
exocytosis and that jasplakinolide as well as cytochalasin D inhibited the release of VWF. Conversely,
latrunculin B, which disrupts F-actin via a different mechanism, did not affect secretory pod number but
enhanced VWF release. While secretory pods usually displayed clathrin-coated profiles, these were
absent from the pods of jasplakinolide-treated cells. Labeling of F-actin with fluorophore-conjugated
phalloidin revealed that secretory pods were frequently tethered to actin stress fibers, and that a
subpopulation of pods was coated with F-actin. The actin tethering and coating was counteracted by
latrunculin B, and by NSC23766, an inhibitor of the GTP-binding protein Rac1. Actin-coated secretory
pods also contained VWF Proregion, suggesting that the Proregion and F-actin positive pods had not yet
undergone exocytosis because Proregion rapidly dissipates upon exocytosis. We propose that F-actin is
involved in ME by regulating the life span of secretory pods in a twofold fashion: firstly via modulation of
the rate of exocytosis of secretory pods, and secondly via modulation of clathrin-mediated retrieval of
secretory pod membrane. In addition, F-actin may stabilize secretory pods via tethering and coating.
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2922/L028
Structural and Biochemical Analysis of the Exomer Complex from S. Cerevisiae.
J. Paczkowski, B. C. Richardson, A. Strassner, M. Smolka, C. Fromme; Weill Institute of Cell and
Molecular Biology, Cornell University, Ithaca, NY
The transport of transmembrane cargo proteins between membrane-bound compartments via membraneenclosed vesicles is a hallmark of eukaryotic cells. Though the structure, function, and molecular
mechanism of recruitment and cargo specificity are known and well-studied for a number of coat proteins
that mediate these various steps, the mechanism behind the direct transport of transmembrane cargo
from the trans-Golgi network (TGN) to the plasma membrane (PM) is relatively unknown. A recently
discovered, Arf1p-dependent coat protein complex in Saccharomyces cerevisiae named “exomer” has
provided insights into the mechanism and role of coat proteins in this process. Exomer is a complex made
up of Chs5p and four paralogous interacting proteins known as the ChAPs (Chs6p, Bud7p, Bch1p,
Bch2p), which act as cargo adaptors. Exomer has a known role in trafficking Chs3p and Fus1p, with
known subunit specificities, to the PM, but there remain unidentified exomer-dependent cargo based on
phenotypic evidence. It is also unknown how exomer interacts with these cargo proteins. Using structural
and proteomic tools we are beginning to understand these fundamental questions. Using stable isotope
labeled amino acids in cell culture (SILAC) mass spectrometry, we have identified a number of previously
unknown transmembrane cargo whose trafficking appears to be exomer-dependent. Additionally, we
have obtained crystals of Chs5p-Chs6p and Chs5p-Bch1p complexes and we have collected an initial Xray dataset at 3.6 Å. Consistent with our biochemical data, the diffraction data reveals that exomer forms
a dimer of heterodimers. Work will continue in order to improve the resolution as well as overcome the
final challenge of solving the phase problem.
2923/L029
Identification and Analysis of the HOPS-SNARE Interface during Yeast Vacuole Fusion.
L. Krämer, C. Ungermann; Biochemistry, University of Osnabrück, Osnabrück, Germany
The evolutionary conserved SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein
(SNAP) receptor)-proteins are crucial factors in intracellular membrane trafficking. Their primary role is to
control membrane fusion of cellular transport vesicles with cell membranes. Most SNAREs are integral
membrane proteins with a transmembrane helix at their C-terminus. SNAREs assemble into stable
bundles of four-helices. The low SNARE specificity gives an indication of down-stream actors which are
important for tethering. The hexameric HOPS tethering complex is one of the potential factors which can
affect the specificity of the SNARE assembly. The interaction of HOPS with the single SNAREs Vam7 and
Vam3 has been observed but the functional importance is not completely clear. We identify here novel
interactions between HOPS and the vacuolar SNAREs and provide evidence for their functional
relevance using in vitro fusion and in vivo assays.
2924/L030
Fast Anodotropic Expansion of Cell Membrane under Exposure to High-Rate Nanosecond
Duration Electric Pulses (nsEP).
M. Rassokhin, A. Pakhomov; Frank Reidy Research Center for Bioelectrics, Old Dominion University,
Norfolk, VA
Partial permeabilization of cell plasma membrane by different means, e.g., by electroporation, often leads
to cell deformation and volume changes. A typical consequence of membrane permeabilization by nsEP
is water uptake due to a colloid osmotic pressure gradient resulting in cell swelling and blebbing. Blebs
form in an apparently random fashion within seconds after nsEP exposure, indicating recruitment of
membrane reserves that were concealed within plasma membrane invaginations. However, the exact
mechanisms determining the location of blebs, their initiation and growth have not been well understood.
We have identified nsEP exposure conditions that enable bleb initiation at the anodic pole of the cell,
followed by maximum recruitment of the membrane reserves and growth of a giant bleb-like protrusion(s)
towards anode. Within just 40-75 sec of continual exposure to high-rate high-intensity nsEP (e.g., 60-ns
pulses at 6.3 kV/cm, 20Hz) such protrusions can extend up to several multiples of the cell diameter. The
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formation of giant blebs is accompanied by just modest propidium entry into cells and was observed only
in Ca2+-free buffers. The blebs “deflate” within 10-20 sec after cessation of exposure by integration of the
bleb membrane back into the plasma membrane of now considerably swollen cell. At present, giant
anodotropic blebs (GABs) were observed in U937 cells but not in CHO or Jurkat cells. This difference
possibly indicates the lack of considerable membrane reserves in the latter two cell lines, and may
explain their much higher sensitivity to nsEP. Ongoing experiments using ceramide and sphingomyelin
fluorescent probes are intended to investigate GAB composition and growth mechanisms. In addition, fast
externalization of plasma membrane invaginations into GAB makes them accessible for investigation
using standard fluorescence and patch clamp. Supported by R01CA125482 from the National Cancer
Institute and R01GM088303 from the National Institute of General Medical Sciences
2925/L031
Parkin Functions in a Novel Vesicular Pathway Governing Mitochondrial Quality Control.
1
2
1
1
1
2,3
2
V. Soubannier , G. McLelland , R. Zunino , E. Braschi , P. Rippstein , B. Kaufman , E. Shoubridge , E.
2
1 1
2
Fon , H. McBride ; Cell dynamics, Ottawa Heart Institute, Ottawa, ON, Canada, Montreal Neurological
3
Institute, McGill University, Montréal, QC, Canada, Department of Animal Biology, School of Veterinary
Medicine, Philadelphia, PA
Interference with the mechanisms that govern mitochondrial quality control have been linked with
neurodegenerative diseases. However, the mechanisms that regulate mitochondrial protein and lipid
turnover are poorly understood. We have identified a novel mitochondrial quality control pathway
characterized by mitochondria derived vesicles (MDVs). MDVs were induced by oxidative stress and
delivered to lysosomes. Their biogenesis can be reconstituted in vitro and is independent of DRP1induced mitochondrial fission. Remarkably, the amount of cargo removed by the MDV pathway is
comparable to the rates of degradation mediated by mitochondrial proteases. MDVs are selectively
enriched for oxidized proteins, whose identity was dependent upon the type of mitochondrial stress. In
particular, the induction of MDVs by intrinsic mitochondrial damage required the selective recruitment of
Parkin to the vesicles. These data are the first to characterize a novel vesicle transport route between the
mitochondria and lysosomes, providing new insights into the pathogenesis of Parkinson's disease.
2926/L032
Multiple Modes of Endophilin-Mediated Conversion of Lipid Vesicles into Coated Tubes.
1
2
2
1 1
N. Mizuno , C. Jao , R. Langen , A. C. Steven ; LSBR, NIAMS, National Institutes of Health, Bethesda,
2
MD, Zilkha Neurogenetic Institute, University of Southern California, Los Angeles, CA
Endophilin is an N-BAR protein involved in synaptic endocytosis. It has been proposed to act in neck
formation during the budding of pre-synaptic vesicles. Endophilin dimer, the functional unit, is a crescentshaped molecule whose concave surface is an interaction surface that, together with 4 amphipathic
helices, docks on to lipids. Endophilin has been shown to remodel vesicles into tubes. Although its crystal
structure has been solved, the molecular mechanism of tubulation has been unclear. We have used cryoEM in combination with EPR spectroscopy to investigate endophilin-mediated tubulation. After adding
endophilin to preparations of vesicles, we observed tubular coated bilayers. These tubes vary in diameter,
even on a given tube. Tube diameter correlates with the protein:lipid ratio, with more protein resulting in
thinner tubes. We performed three-dimensional reconstructions of tube segments with two different radii,
which revealed two different packings of the BAR domains. Both packings may be described as strands
of BAR domains in end-to-end association, with lateral association of the strands to complete the helical
lattices. On tubes with different radii, endophilin appears to adjust its packing by tilting. At high protein
concentrations, we observed exceptionally thin tubes, with only ~ 35 Å between the two density peaks
seen in projection. Protein attachment is less regular on these tubes but endophilin molecules appear to
orient with their long axes approximately aligned with the tube axes. We interpret these structures as
decorated cylindrical micelles. These observations depict the endophilin BAR domain dimer as a versatile
building-block that can induce membrane curvature over a wide range of tube radii. We propose a model
for the role of endophilin in endocytosis, that invokes a high local concentration of endophilin, whereby
the hemi-fission stage of vesicle budding is recapitulated in the cylindrical micelles observed in vitro.
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2927/L033
GOLPH3 Relates Golgi Morphology to Its Function.
1
1
1
1
1
2
1
1
S. Farber-Katz , M. Ng , H. C. Dippold , M. C. Peterman , S. Lee , M. L. Kerr , R. Sim , P. A. Wiharto ,
1
2
3
4
4
3
1
K. A. Galbraith , S. Madhavarapu , G. J. Fuchs , T. Meerloo , M. G. Farquhar , H. Zhou , S. Field ;
1
Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, CA,
2
3
Division of Signal Transduction, Beth Israel-Deaconess Medical Center, Boston, MA, Ludwig Institute
4
for Cancer Research, University of California, San Diego, La Jolla, CA, Cellular and Molecular Medicine,
University of California, San Diego, La Jolla, CA
To better understand the Golgi we have investigated the role of the abundant Golgi-specific lipid,
phosphatidylinositol-4-phosphate (PtdIns(4)P). We found that the Golgi protein GOLPH3 binds to
PtdIns(4)P both in vitro and in vivo. In fact, GOLPH3 is highly conserved from yeast to humans, and we
found that its ability to bind to PtdIns(4)P is also conserved. We showed that binding to PtdIns(4)P is
required for GOLPH3 to localize at the Golgi, and we created point mutants that abolish the ability of
GOLPH3 to bind to PtdIns(4)P. In addition to binding to PtdIns(4)P, we discovered that GOLPH3 also
binds to the unconventional myosin, MYO18A, which binds to F-actin. We showed that depletion of
PtdIns(4)P, siRNA knockdown of GOLPH3 or MYO18A, and depolymerization of F-actin all cause the
Golgi ribbon to condense at one end of the nucleus. Similarly, siRNA knockdown of GOLPH3 or MYO18A
or depolymerization of F-actin sharply reduces trafficking from the Golgi. Our data indicate that this
complex serves to link the Golgi membrane to the actin cytoskeleton, and to create a tensile force that
functions to pull vesicles and tubules off the Golgi. A consequence of this tensile force is the stretching of
the Golgi ribbon around the nucleus, as seen by immunofluorescence, and the familiar flattened Golgi
stacks, as seen by electron microscopy.
2928/L034
Isoform Specific Tissue Distribution of URATv1/GLUT9 and Their Trafficking in the Polarized
Cells.
T. Kimura, N. Anzai, H. Sakurai; Kyorin University School of Medicine, Tokyo, Japan
We have recently identified SLC2A9 (also known as GLUT9) as a voltage-driven urate transporter,
URATv1 (Anzai et al. JBC 2008). URATv1 is likely to mediate urate reabsorption because patients with
loss of function URATv1 mutations result in renal hypouricemia. SLC2A9 gene is reported to have 2
splice variants: short isoform (URATv1-S) with 512 amino acids and long isoform (URATv1-L) with 540
amino acids. Their S terminal 490 residues are identical and only difference lies at their N-termini. We
examined expression pattern of these 2 isoforms in the kidney and placenta and found that their
apical/basolateral membrane domain localization was different. By isoform specific antibody staining,
URATv1-S was expressed at the apical side of the distal tubule in the kidney and maternal side of the
syncytiotrophoblast and endothelial cell in the placenta while URATv1-L was expressed at the basolateral
side of the proximal tubule in the kidney and the endothelial cell in the placenta. Based on these results, it
is hypothesized that N -terminal residues of URATv1 are critical for membrane sorting and we
investigated this possibility further in a MDCK cell culture system. Fluorescent-tagged URATv1 isoforms
were transfected into MDCK cells. URATv1-S was expressed both at the apical and basolateral
membrane while URATv1-L was expressed only at the basolateral membrane. Removal of the N-terminal
16 amino acids from URATv1-S did not affect its sorting. Removal of the N-terminal 30 amino acids, but
not N-terminal 25 amino acids, from URATv1-L resulted in both apical and basolateral expression. To our
surprise, replacement of the N-terminal 25th to 30th amino acids of URATv1-L with alanine did not affect
its basolateral localization. In conclusion, we found that 2 isoforms of URATv1 have unique intra tissue
distribution both in the kidney and placenta and that basolateral membrane localization of URATv1-L
appeared to be determined by N-terminal residues. We are now investigating detailed mechanism of its
sorting.
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2929/L035
The pH Domain Proteins IPIP27A and B Link OCRL1 to Receptor Recycling in the Endocytic
Pathway.
C. Noakes, M. Lowe; Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom
Mutation of the inositol polyphosphate 5-phosphatase OCRL1 results in two disorders in humans, namely
Lowe syndrome, characterized by ocular, nervous system and renal defects, and Type 2 Dent’s disease,
in which only the renal symptoms are evident. The disease mechanisms of these syndromes are poorly
understood. In an effort to better understand how Lowe syndrome is caused we have identified two novel
OCRL1-binding proteins, termed inositol polyphosphate phosphatase interacting protein of 27kDa
(IPIP27) A and B. The IPIPs bind to the C-terminal region of OCRL1 via a conserved motif which and can
compete with the binding of another known OCRL1 binding partner, the signaling adaptor protein APPL1,
to this region of OCRL1. The IPIPs localize to early and recycling endosomes and the trans-Golgi network
(TGN). We have studied the function of these proteins using a variety of techniques, including
overexpression of wild type and mutant IPIP constructs and RNAi mediated depletion of endogenous
IPIPs in cell-based functional assays. Our studies show that the IPIPs are required for efficient receptor
recycling from endosomes to the TGN and plasma membrane. Our results identify IPIP27A and B as key
players in endocytic trafficking and strongly suggest that defects in this process are responsible for
pathology of Lowe syndrome and Dent’s disease.
2930/L036
Sec1p and Mso1p C-Terminal Tails Co-Operate with the Snares and Sec4p in Polarized
Exocytosis.
N. K. Aro, M. Weber-Boyvat, J. Jäntti; Institute of Biotechnology, University of Helsinki, Helsinki, Finland
The Sec1/Munc18 (SM) protein family members are evolutionary conserved proteins that perform an
essential, albeit poorly understood function in SNARE complex regulation in membrane fusion.
Saccharomyces cerevisiae Sec1p is a homologue of the mammalian Munc18-1 that has been shown to
bind syntaxin 1 in a closed conformation, but also syntaxin 1 containing SNARE complexes. The S.
cerevisiae Sec1p has a C-terminal tail that is missing in its mammalian homologues. The function of this
tail is currently unknown. Here we show that deletion of the Sec1p tail renders cells temperature-sensitive
for growth, reduce sporulation efficiency and result in loss of Sec1p-SNARE complex interaction. Our
results suggest that this tail is especially important for binding to Sso1p. The Bimolecular fluorescence
complementation (BiFC) assay results suggest that in the SNARE deficient sso2-1 Δsso1 cells, Mso1p, a
Sec1p binding protein, helps to target Sec1p lacking the C-terminal tail to the sites of secretion. In
addition, the results suggest that the Mso1p C-terminus is important for Sec1p targeting. We show, that in
addition to Sec1p, Mso1p interacts with the Rab-GTPase Sec4p in vitro. The BiFC results suggest that
Mso1p acts in close association with Sec4p on intracellular membranes in the bud. This association
depends on the Sec4p guanine nucleotide exchange factor Sec2p. Our results reveal a novel binding
mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a novel role of Mso1p as
an effector of Sec4p.
2931/L037
Regulation of Vascular Endothelial Growth Factor Receptor 2 Trafficking and Angiogenesis by
Golgi Localized T-SNARE Syntaxin 6.
1
1
1
2
3
2
1
V. Manickam , A. Tiwari , J. Jung , R. Bhattacharya , A. Goel , D. Mukhopadhyay , A. Choudhury ;
1
2
Anatomy and Cell Biology, University of Iowa, Iowa City, IA, Biochemistry and Molecular Biology, Mayo
3
Clinic College of Medicine, Rochester, MN, Radiation Oncology, University of Iowa, Iowa City, IA
Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in physiological and pathological
angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by endocytosis and secretory
transport through the Golgi apparatus. To date the mechanism whereby the VEGFR2 traffics through the
Golgi apparatus remains incompletely characterized. Here we show in human endothelial cells that
binding of VEGF to the cell surface localized VEGFR2 stimulates exit of intracellular VEGFR2 from the
Golgi apparatus. Brefeldin A treatment reduced the level of surface VEGFR2, confirming that VEGFR2
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traffics through the Golgi apparatus en route to the PM. Mechanistically, we show that inhibition of
syntaxin 6, a Golgi-localized target membrane-soluble N-ethylmaleimide attachment protein receptor (tSNARE) protein, interferes with VEGFR2 trafficking to the PM and facilitates lysosomal degradation of the
VEGFR2. In cell culture, inhibition of syntaxin 6 also reduced VEGF-induced cell proliferation, cell
migration and vascular tube formation. Furthermore, in a mouse ear model of angiogenesis, an inhibitory
form of syntaxin 6 reduced VEGF-induced neovascularization and permeability. Our data demonstrate the
importance of syntaxin 6 in the maintenance of cellular VEGFR2 levels, and suggest that the inhibitory
form of syntaxin 6 has good potential as an anti-angiogenic agent.
2932/L038
Molecular and Functional Characterization of Cargo-Binding Sites on µ-Subunits of Adaptor
Protein Complexes.
1,2
1,2
2
2
3
2 1
G. A. Mardones , P. Burgos , Y. Lin , J. Magadán , J. Hurley , J. S. Bonifacino ; Physiology,
2
3
Universidad Austral de Chile, Valdivia, Chile, CBMP/NICHD, NIH, Bethesda, MD, LMB/NIDDK, NIH,
Bethesda, MD
Protein trafficking in the secretory and endocytic pathways is mediated by vesicular transport in which the
adaptor protein (AP) complexes, AP-1, AP-2, AP-3 and AP-4, play key roles in the vesicle formation and
cargo selection. These complexes are heterotetramers with two large subunits (γ and β1 in AP-1; α and
β2 in AP-2; δ and β3 in AP-3; ε and β4 in AP-4), a medium-sized subunit (µ1-µ4), and a small subunit
(σ1-σ4). The functions of AP complexes are essential for many physiological processes, as underscored
by the embryonic lethality of mutations in several AP subunits and the occurrence of genetic disorders
due to AP subunit defects such as the MEDNIK syndrome for AP-1, and the Hermansky-Pudlak
syndrome for AP-3. The µ subunit of AP-1, AP-2 and AP-3 recognize sorting signals fitting the YXXØ
consensus motif (Ø is a bulky hydrophobic residue). Recently we discovered that the µ4 subunit of AP-4
recognizes a different aromatic-amino-acid-based motif, YX[FYL][FL]E, in the tail of Alzheimer's disease
amyloid precursor protein (APP). X-ray crystallographic analyses showed that both types of signal bind to
different sites on the surface of μ2 and μ4, respectively. The aim of this study was to determine which of
these mechanisms is used by the other AP complexes. Resolution of the crystal structure of the Cterminal domain of the µ3A subunit of AP-3 bound to YXXØ signals showed that these signals bind to a
site similar to that on μ2 and distinct from that of μ4. Mutations at the signal-binding site on µ3A
abrogated both binding in Y2H and ITC experiments, and the normal transport of reporter proteins in
RNAi/rescue experiments. Our structural, mutational, biochemical and cell biological analyses support the
functionality of the YXXØ-signal binding site on AP-3. FONDECYT 1100896.
2933/L039
Cell-Contact Induced Ephrin Reverse Endocytosis Is Regulated by Src Family Kinases.
M. Sakkou, R. Klein; Max-Planck Institute of Neurobiology, Munich, Germany
Cells have self-organizing features that control their behavior. Ephrins are membrane tethered guidance
cues that bind to Eph receptor tyrosine kinases (RTKs). During embryonic development the Eph/ephrin
signaling system controls a large variety of cellular responses including contact-mediated attraction or
repulsion, adhesion or de-adhesion and migration. Moreover, unlike other RTKs, Eph/ephrin signaling
dynamics are regulated by a large number of components like proteolytic cleavage, trans-endocytosis, bidirectional signaling, higher order clustering and cis-interactions of Ephs and ephrins. Their relative
contribution though during morphogenesis is not yet known. Ongoing work indicates that bidirectional
Eph/ephrin endocytosis is a critical regulator and in cell culture is required to convert an adhesive force to
a repulsive signal. My aims are to study the role of endocytosis in Eph/ephrin signaling, to characterize
the endocytic pathways that are activated by Ephs and ephrins and to investigate their relative functional
role in various cellular responses. In a candidate search for proteins required for Eph/ephrin transendocytosis we found that Src family kinases (SFKs) are specifically required for ephrin “reverse”, but not
Eph “forward” endocytosis, despite the fact that Src is activated downstream of both Ephs and ephrins.
Furthermore, we show that the Src requirement is exclusive for trans-endocytosis of Ephs after cell-cell
interaction but not for internalization of soluble Eph-Fc fusion proteins (reagents which are widely used in
Eph/ephrin signaling studies). To further assess the functional role of SFKs in ephrin “reverse”
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endocytosis we blocked SFK activation in primary neurons and observed that the de-attachment between
ephrinB-positive neuronal growth cones and EphB2-expressing cells was significantly slower. Taken
together these data indicate that Eph/ephrin complexes are internalized via a phagocytic -like mechanism
which specifically requires the activation of SFKs in the ephrin-positive cell. Moreover, SFKs appear to
activate a novel pathway downstream ephrinBs which is independent of ephrinB tyrosine phosphorylation
and PDZ domain protein engagement.
2934/L040
Proteomic Analysis of Human TRAPP Identifies Three Novel Subunits.
1
1
1
2
1
2
1,2 1
P. Scrivens , B. Noueihed , N. Shahrzad , M. Bostina , S. Hul , I. Rouiller , M. Sacher ; Concordia
2
University, Montreal, QC, Canada, McGill University, Montreal, QC, Canada
The multisubunit tethering complex TRAPP is conserved throughout Eukarya. Saccharomyces cerevisiae
TRAPP is organized in discrete subassemblies, TRAPPI and -II, implicated in ER-to-Golgi and late Golgi
trafficking, respectively. No such subassemblies have yet been reported in mammalian cells, and little is
known about the architecture of mammalian TRAPP. We recently reported a novel TRAPPII-associated
protein, YEL048c/TCA17, and its mammalian orthologue TrappC2L (C2L). YEL048c-p/Tca17p and C2L
are ancestrally related to Trs20p and TrappC2 (C2). Given that Trs20p is found in both TRAPPI and -II,
whereas YEL048c-p/Tca17p appears to be a TRAPPII component, we hypothesized that their
mammalian orthologues C2 and C2L might precipitate distinct complexes. Contrary to our hypothesis, we
found that TAP-C2 and -C2L complexes appeared indistinguishable by SDS-PAGE and mass
spectrometry. Further, both TAP-C2 and -C2L precipitated several novel proteins which we here report as
TrappC8, TrappC11, and TrappC12. TrappC8 is the mammalian orthologue of the yeast TRAPP protein
Trs85p, whereas TrappC11 and -C12 have no orthologues in S. cerevisiae. RNA interference of any of
the novel TRAPP proteins results in Golgi fragmentation, indicating that they are indeed involved in
membrane trafficking. Depletion of C11 or C12 resulted in accumulation of VSV-G ts045-GFP in punctae
colabeling with ERGIC53, but apposed to Sec31a, suggesting that exit from the ER was not impeded. In
order to better understand the organization of mammalian TRAPP, we have mapped all binary
interactions between the known and novel TRAPP proteins, and have performed single particle electron
microscopy on TAP-C11 complexes precipitated from mammalian cells. What emerges is a portrait of
mammalian TRAPP where the fundamental unit is larger than S. cerevisiae TRAPPI, and where
functional diversification may be achieved by the inclusion of these novel TRAPP subunits.
2935/L041
The First Transmembrane Domain of LCB1 Is Essential for Regulation of Serine
Palmitoyltransferase by the ORMs.
1
1
1
2
2
1 1
N. Sengupta , G. Han , K. Gable , D. Bacikova , J. M. Harmon , T. M. Dunn ; Biochemistry, USUHS,
2
Bethesda, MD, Pharmacology, USUHS, Bethesda, MD
Serine palmitoyltransferase (SPT) catalyzes the committed and rate limiting step of sphingolipid
biosynthesis, condensation of serine with an acyl-CoA to form 3-ketosphinganine. Yeast SPT is
comprised of three ER-associated transmembrane proteins, Lcb1p, Lcb2p and Tsc3p. Basal activity of
the Lcb1-Lcb2p heterodimer is stimulated several fold by the small Tsc3p subunit, which binds the
heterodimer and alters the acyl-CoA substrate preference. Although there are no homologs of Tsc3p in
higher eukaryotes, we have recently discovered the ssSPT family of functional orthologs, evolutionarily
conserved from S. pombe to humans. In studies aimed at elucidating the mechanism by which these
small subunits activate SPT, we found that they interact with the first transmembrane domain (TMD1) of
LCB1. However, this interaction is not critical for heterodimer activation. Recently the yeast ORM proteins
were reported to be in a complex with SPT and to negatively regulate its activity (Breslow et al (2010)
Nature, 463, 1048-53; Han et al (2010) PNAS, 107, 5851-6). Interestingly, we find that the ORMs also
bind to TMD1 of LCB1 and that this interaction is critical for their function. For example, whether or not
the ORMs are present, yeast LCB1ΔTMD1 mutants are cold and tunicamycin sensitive and accumulate
high levels of sphingolipids comparable to those observed in the orm1Δorm2Δ mutants.
Immunoprecipitation assays reveal that the ORMs bind to the heterodimer even in the absence of the
small subunit, but whether or not they have the potential to regulate activity of the heterodimer is not yet
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known. To resolve whether the ORMs inhibit by blocking small subunit activation or by directly modulating
the heterodimer, we are investigating their effect on basal activity of LCB1-LCB2 heterodimer by a
number of approaches.
2936/L042
Arl1p-Drs2p-Gea2p Cooperate to Recruit Golgin Imh1p to the Golgi.
P. Tsai, K. Chen, F. Lee; National Taiwan University, Taipei, Taiwan
The golgin proteins are thought to be involved in the structural organization of Golgi and protein
trafficking. Imh1p is a GRIP-domain containing golgin protein in yeast and its Golgi recruitment involves
interaction with a small GTPase Arl1 protein. Here we show that the recruitment of Imh1p to the Golgi
relies on interactions between the flippase Drs2p and the ArfGEF Gea2p. Deletion of either Drs2p or
Gea2p abolished the recruitment of Imh1p to the Golgi, but did not affect the Golgi targeting of Arl1p. The
recruitment of Imh1p to the Golgi was not affected in gea1 mutant and overexpression of Gea1p in gea2
mutant could not rescue the recruitment of Imh1p to the Golgi, suggesting that Gea2p plays a specific
role in Arl1p-mediated Golgi recruitment of Imh1p. Furthermore, the Golgi recruitment of Imh1p was
dependent on Drs2p activity and Gea2p binding to the C-terminal domain of Drs2p was required for the
localization of Imh1p. Consistent with previous report that Gea2p plays a role in the Drs2p-mediated
membrane dynamics at TGN, our study provides further evidence that Drs2p-Gea2p interaction may
cooperate with Arl1p to recruit golgin Imh1p to the Golgi membrane.
2937/L043
The ERAD Component Edem1 Undergoes Selective Autophagy by Endoplasmic ReticulumDerived Phagophore.
J. Roth, S. Park, I. Jang, Y. Lee, J. Cho, B. Guhl, C. Zuber; Department of Integrated Omics for
Biomedical Science, Yonsei University, Seoul, Republic of Korea
EDEM1 is an ERAD component, which appears to be involved in the dislocation process from the lumen
of the endoplasmic reticulum (ER) to the cytoplasm of not correctly folded glycoproteins. Previously, we
have shown that endogenous EDEM1 in HepG2 cells becomes itself dislocated to the cytoplasm where it
forms detergent-insoluble aggregates and is subsequently degraded by basal autophagy. We have
investigated the details of the autophagic degradation of endogenous EDEM1. By double confocal
immunofluorescence, EDEM1 exists in complexes with the cargo receptors for selective autophagy p62,
Nbr1 or Alfy, respectively. Inhibition of autophagy by wortmannin resulted in accumulation and increase in
size of aggregates composed of EDEM1 and p62, Nbr1 or Alfy, respectively, as revealed by confocal
double immunofluorescence. Presence of EDEM1 and p62 in cytoplasmic aggregates was directly shown
by double immunogold labeling of ultrathin frozen sections. Three out of the 26 lysine residues of EDEM1
are predicted to become ubiquitinated and by combined immunoprecipitation and Western blot analysis,
we detected ubiquitinated EDEM1. Under steady state conditions, we observed by immunoelectron
microscopy that cytoplasmic EDEM1 is engulfed by double membrane structures characteristic of
phagophores (isolation membrane). By serial section analysis, the origin of the phagophore for selective
autophagy of EDEM1 could be identified as modified parts of rough ER cisternae. Together, these
observations demonstrate that endogenous EDEM1 in HepG2 cells that are not expressing foreign
misfolded glycoproteins is degraded by selective autophagy. Supported by by the World Class University
program through the National Research Foundation of Korea funded by the Ministry of Education,
Science and Technology (R31-2008-000-10086-0) and a grant from the National Research Foundation of
Korea by the Ministry of Education, Science and Technology (2010-0027736).
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2938/L044
Tight Control of the Arf Activator Arno by a Positive Feedback Loop and Competition with
Effectors.
1
1
1
2
1
1 1
D. Stalder , R. Gautier , E. Macia , C. Jackson , B. Antonny , H. Barelli ; IPMC-CNRS UMR6097,
2
Valbonne, France, LEBS, Gif-sur-Yvette, France
The guanine nucleotide exchange factor Arno has been proposed to promote Arf cascades : one GTPbound Arf subtype binds to Arno PH domain, relieving inhibition of the adjacent Sec7 domain, which then
promotes nucleotide exchange on a different Arf subtype. We tested this model by reconstituting
exchange reactions using full-length Arno and myristoylated Arf1 and Arf6 subtypes on artificial liposomes
of defined composition. We observed that Arf6-GTP is about 100-fold more efficient in stimulating Arno on
a membrane system than in solution and acts in a synergistic manner with anionic lipids. Surprisingly, we
also observed that in the absence of Arf6-GTP, Arno is activated by its own product, Arf1-GTP, through a
positive feedback loop. Notably, abolishing Arf1/Arf6 stimulation of Arno by a mutation in its PH domain or
by adding Arf effectors sets Arno activity to basal level. In contrast, weakening the inhibitory contact
between the PH and Sec7 domains makes Arno less dependent on the positive feedback loop leading to
uncontrolled events in cells. We conclude that Arno can behave as a bistable switch. When active Arf
subtypes vary within usual limits, Arno remains dormant because Arf effectors efficiently compete with
Arno for GTP-bound Arf molecules. In contrast upon an unusual burst of Arf6-GTP, Arno can switch to a
self-activating state thanks to the feedback effect of Arf1-GTP. Overall, the positive feedback loop
between Arno and Arf proteins assures that the reaction moves forward decisively and should permit
massive and sustained Arf cascades under specific circumstances.
2939/L045
Stability of Luciferase Activity during Autophagy.
I. Tanida, K. Hanada; Department of Cell Biology and Biochemistry, National Institute of Infectious
Diseases, Tokyo, Japan
Luciferase is a well-known reporter protein, and is also used to monitor an activity of replication of a viral
replicon. Autophagy (macroautophagy) is a dynamic process for degradation of cytosolic components,
and induced by many cellular stress including starvation, low energy state, oxidative stress, and viral and
bacterial infections. It is possible that an activity of luciferase is destabilized during autophagy, since
autophagy is one of major protein-degradation systems. To investigate the issue of whether or not
autophagy influences on an activity of luciferase, we measured an activity of firefly luciferase under the
control of CMV IE promoter under the starvation conditions in HEK293 cells using luc2 gene that is
optimized for mammalian expression. Under the starvation conditions, its activity decreased in a timedependent manner. During autophagy, proteins are degraded by lysosomal cathepsins. E64d and
pepstatin A, inhibitors for lysosomal cathepsins, inhibit autophagic flux. To investigate the contribution of
lysosome on the decrease in activity of luciferase, we measured its activity in the absence and presence
of the inhibitors. Interestingly, the inhibitors, E64d and pepstatin A, didn’t inhibit the decrease of luciferase
activity, suggesting that little lysosomal cathepsins contribute the decrease of luciferase activity. These
results indicate that an unknown mechanism inhibits luciferase activity during autophagy, and suggested
that an activity of luciferase as a reporter is underestimated under the autophagy-inducible conditions like
viral infection.
2940/L046
Lipase Maturation Factor 1: Elucidation of Structural Features.
S. Neher, D. S. Booth, Y. Cheng, P. Walter; Biochemistry & Biophysics, UCSF, San Francisco, CA
Lipoprotein lipase (LPL) plays a critical and complex role in lipid metabolism. LPL catalyzes the hydrolysis
of the triacylglycerol component of very low density lipoproteins. Deficient levels of LPL can cause
hypertriglyceridemia and associated disorders, such as diabetes and atherosclerosis. LPL cannot
efficiently exit the ER and achieve appropriate levels without a transmembrane protein whose identity was
only recently discovered, lipase maturation factor (LMF1)(1). In cells lacking LMF1, inactive LPL is
retained in the ER as large, intermolecular disulfide bonded aggregates that are subject to
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degradation(2). Indeed, a form of familial hypertriglyceridemia maps to LMF1, directly establishing its role
in human disease(1). LMF1’s structure is essentially uncharacterized; in fact it belongs to a family of
proteins with a similar “domain of unknown function” (Duf1222). Here, we begin to elucidate the structure
and function of LMF1 in order to better understand its role in lipase folding and assemble. First, we
characterize the membrane topology of LMF1 and find that it spans the membrane five times and its
large, C-terminal domain is located in the endoplasmic reticulum. Additionally, we present images of
purified LMF1 obtained with electron microscopy. In combination, these results begin to uncover the
structural features of a novel membrane protein maturation factor. 1. M. Peterfy et al., Nat Genet 39, 1483
(Dec, 2007). 2. O. Ben-Zeev, H. Z. Mao, M. H. Doolittle, J Biol Chem 277, 10727 (Mar 22, 2002).
2941/L047
Investigation of the Role of PtdIns(4,5)P2 in G Protein-Coupled Receptor Endocytosis.
1
1
1
2
1
1 1
D. J. Tóth , J. Tóth , A. Balla , T. Balla , L. Hunyady , P. Várnai ; Department of Physiology,
2
Semmelweis University Faculty of Medicine, Budapest, Hungary, Section on Molecular Signal
Transduction, National Institute of Child Health and Human Development, National Institutes of Health,
Bethesda, MD
Receptor endocytosis plays an important role in regulating the responsiveness of the cell to a certain
ligand. Since phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) has been reported to participate in the
endocytosis of some plasma membrane receptors (e.g. EGF receptor and transferrin receptor) and many
of the endocytic proteins bind PtdIns(4,5)P2, it seemed plausible that this lipid plays a role in the
internalization of GPCRs, too. In this study we investigated the PtdIns(4,5)P2 dependence of the
internalization of two GPCRs, type 1 angiotensin II receptor (AT1R) and type 2C serotonin receptor
(5HT2CR). We used confocal microscopy as well as the highly sensitive method of bioluminescence
resonance energy transfer (BRET) which allows the detection of molecular proximity between two
appropriately labeled proteins. By fusing the receptors to Renilla luciferase and applying fluorescently
tagged proteins of the endocytic machinery (β-arrestin 2, PM targeted Venus, Rab5) we were able to
follow the process as a sequence of molecular interactions in HEK293 cells. For the depletion of
PtdIns(4,5)P2 in the PM we used the previously developed rapamycin-inducible heterodimerization
system, in which rapamycin induces the recruitment of a 5-phosphatase enzyme domain to the PM where
PtdIns(4,5)P2 is then degraded. Lipid depletion was verified by BRET measurements with the PH domain
of PLCδ1 which specifically binds PtdIns(4,5)P2. Next, we tested the effect of PtdIns(4,5)P2 depletion on
certain stages of GPCR endocytosis. We found that for both examined receptors the interaction with βarrestin 2 remained unaffected by the depletion of the lipid. The signal showing the arrival of the receptor
in the early endosome was abolished in the absence of PtdIns(4,5)P2, whereas the signal corresponding
to the receptor leaving the PM was reduced but not eliminated. Our subsequent confocal studies revealed
that after lipid depletion stimulated AT1Rs cluster along the PM but do not enter the cells. These data
suggest that the internalization of AT1R and 5HT2CR is dependent on PtdIns(4,5)P2, and the lack of this
lipid prevents the fission of their endocytic vesicles from the PM.
2942/L048
Adenosine A2A Receptor Is Involved in Cell Surface Expression of A2B Receptor.
K. Moriyama, M. V. Sitkovsky; New England Inflammation and Tissue Protection Institute, Northeastern
University, Boston, MA
The A2A and A2B adenosine receptors (A2AR and A2BR) are implicated in many physiological
processes. However, the mechanisms of their intracellular maturation and trafficking are poorly
understood. In comparative studies of A2AR versus A2BR expression in transfected cells, we noticed that
levels of cell surface expression of A2BR are significantly lower than those of A2AR. The large portion of
the A2BR is degraded by the proteasome. Studies of cell surface expression of A2BR chimeric molecules
in transfectants suggested that A2BR does not have the dominant forward transport signal for export from
the endoplasmic reticulum (ER) to the cell surface. The A2BR surface expression was increased in A2BR
chimeras where the A2BR carboxyl terminus (CT) was replaced or fused with A2AR CT. Co-transfection
of A2AR with A2BR enhanced surface expression of A2BR though F(X)6LL motif in A2AR CT. The
requirements of A2AR expression for better A2BR cell surface expression was established not only in
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transfectants but also confirmed by observations of much lower levels of A2BR- induced intracellular
cAMP accumulation in response to A2BR-activating ligand in splenocytes from A2AR-/- mice than in wild
type mice. Results of mechanistic studies suggest that poor A2BR expression at the cell surface might be
mainly accounted for by the lack of dominant forward transport signal from the ER to the plasma
membrane, and it is likely that A2BR forms a hetero-oligomer complex for its better function.
2943/L049
Lipidomics in Epithelial Cells.
1
1
1
2
1
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M. J. Gerl , C. Klose , A. Schevchenko , H. Beug , K. Simons , J. L. Sampaio ; MPI-CBG, Dresden,
2
Germany, Research Institute of Molecular Pathology, Vienna, Austria
The lipid raft concept was proposed as a model to explain the asymmetric lipid and protein distribution in
epithelial cells, whose plasma membrane (PM) is divided into two distinct surface domains: an apical
domain enriched in sphingolipids and cholesterol facing the lumen and a basolateral domain enriched in
glycerophospholipids facing neighboring cells. According to the hypothesis, lipids and proteins destined
for the apical or basolateral PM segregate in a domain formation process at the trans-Golgi Network
(TGN), one of the major sorting stations in the exocytotic pathway. To validate some predictions of this
model, we have set up an improved methodology that relies on an optimized lipid extraction coupled to
high mass accuracy mass spectrometric detection that resolves in an absolute quantitative manner
glycerophospholipids, cholesterol and (glyco-)sphingolipids to the species level. We use this technique to
quantify changes in global lipid composition during polarization and the concomitant formation of apical
and basolateral membranes in MDCK cells, an epithelial cell culture model. We find significant
remodeling of the lipidome during epithelial polarization with a major shift from sphingomyelin to
glycosphingolipid dominated membranes. During this process sphingolipids become longer, more
saturated and hydroxylated; however these tendencies in the individual sphingolipid classes seem to be
specifically regulated. In analogy with inversion of polarization, induced epithelial-to-mesenchymal
transition of MDCK cells changes the lipidome towards the non-polarized state. Finally, using a novel
preparation procedure, we quantify the lipid composition of the apical PM and find a strong enrichment of
cholesterol and sphingolipids. Therefore the lipidome changes during MDCK polarization are consistent
with the creation of a glycolipid-rich apical surface.
2944/L050
PtdIns 4-Kinase Signalling and Sphingolipid Biosynthesis Are Required for Efficient Retention of
Mannosyltransferases in the Golgi Apparatus of S. Cerevisiae.
1
2
1 1
C. S. Wood , J. Liu , C. G. Burd ; Cell and Developmental Biology, University of Pennsylvania,
2
Philadelphia, PA, Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia,
PA
A longstanding question in secretory cell biology regards how resident glycosyltransferases of the Golgi
apparatus are retained as secretory cargo moves in the anterograde direction. Multiple factors are
proposed to mediate segregation of secretory cargo from retrograde cargo, including packaging of Golgi
residents into COPI retrograde vesicles, lipid-based mechanisms which exclude Golgi residents from
secretory domains enriched in sphingolipids/sterols, and interactions between Golgi residents and
cytoplasmic coat proteins. We show that in cells lacking the yeast protein Vps74 multiple Golgi
mannosyltransferases fail to be retained within Golgi cisternae and are delivered to the lysosome-like
vacuole and degraded. Vps74 binds the cytosolic regions of these enzymes and maintains them in the
early secretory pathway. Interestingly, overexpression of Vps74 results in mislocalization of these
enzymes to the endoplasmic reticulum suggesting that Vps74 may facilitate their retrograde transport.
Targeting of Vps74 to the Golgi apparatus requires ongoing synthesis of phosphatidylinositol 4phosphate, (PtdIns4P) a signalling lipid that promotes export from the Golgi. When synthesis of PtdIns4P
is disrupted by inactivation of Pik1, Vps74 is localized to the cytosol and the medial Golgi enzyme Kre2 is
delivered to the vacuole. These data show that Golgi localization of a subset of glycosyltransferases is
facilitated by targeting of Vps74 to the Golgi membrane through binding PtdIns4P and Golgi residents. To
identify additional factors required for retention of Golgi residents, we used genetic interaction profiling to
identify gene deletions that result in synthetic growth defects when combined with the vps74Δ mutation.
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Many of the genes identified encode components of the sphingolipid biosynthetic pathway. In a subset of
these mutants Kre2 fails to be efficiently retained in the Golgi, yet Vps74 localization is unaffected. We
speculate that perturbations to sphingolipid synthesis and organization in the Golgi result in inefficient
sorting of Golgi residents by Vps74.
2945/L051
Determining the Role of the Aminopeptidase 1 C-Terminus.
K. Long, M. Morales Quinones, P. E. Stromhaug; University of Missouri-Columbia, Columbia, MO
Autophagy is a process in which cellular elements are sequestered and sent to the lysosome in mammals
or the vacuole in yeast for degradation. Aminopeptidase 1 (Ape1) is a hydrolase that resides in the
vacuole but is made as a precursor (prApe1) in the cytosol and quickly forms a dodecamer. After
aggregation Ape1 utilizes the non-classical cytoplasm-to-vacuole targeting (Cvt) biosynthetic pathway.
The propeptide plays a role in Ape1 aggregation in the cytosol, and deleting the entire propeptide
sequence inhibits vacuolar localization of Ape1. Preliminary data suggests that the C-terminus of Ape1 is
also involved with aggregation and dodecamerization of Ape1, and that both the N-terminal propeptide
and the C-terminus may interact with the Atg19 receptor protein in order to form the Cvt vesicle. Since the
protein must aggregate in order to be transported to the vacuole, this study investigates the role that the
C-terminus of Ape1 plays in vacuolar localization. We created several C-terminal amino acid mutations
and deletions and analyzed them with various methods including Western blotting, two-hybrid assays,
protein pull-downs, and examination in vivo under microscopy. Results indicate that deleting only one
amino acid prevents vacuolar transport, and deleting over three inhibits aggregation in vivo. All deletions
show decreased interaction with the Atg19 receptor, eventually ceasing entirely. Many deletions show
degenerate amounts of protein, which may signify a C-terminal function in the stability of Ape1. Efforts
have also been promising in determining the Atg19 binding site, which would allow us to better predict the
structure of Ape1 required for interaction. These results suggest that the C-terminus plays an important
part in vacuolar localization through Ape1 stability and aggregation, Atg19 binding and Cvt vesicle
formation, or some combination of these mechanisms.
2946/L052
Ape1 Aggregation and Binding to Atg19 Is Mediated by Its Propeptide.
M. Morales Quinones, P. E. Stromhaug; Biological Sciences, University of Missouri - Columbia,
COLUMBIA, MO
The aggregation of misfolded proteins causes disease and ageing; autophagy counteracts this by
eliminating damaged components and enabling cells to survive starvation. Impaired autophagy
contributes to disease, including the two leading causes of death in the US, heart failure and cancer; it
also helps eliminate bacteria and faulty protein aggregates. Furthermore, in animal models of
Huntington’s and Parkinson’s disease, enhanced autophagy has improved clearance of faulty protein
aggregates and hence reduced the symptoms of neurodegeneration. The Cytoplasm-to-vacuole-targeting
(Cvt) pathway in Saccharomyces cerevisiae encompasses the aggregation of the premature form of
Aminopeptidase 1 (prApe1) in cytosol, and its subsequent sequestration by autophagic proteins into a
vesicle for transport to the vacuole, where it hydrolyses peptides. Although the autophagic proteins
involved in the Cvt pathway have been identified, for many their functions have not been fully elucidated.
We are studying the molecular mechanism of Ape1 aggregation, and show that the propeptide of Ape1
mediates aggregation and vesicle formation, via binding to other Ape1 proteins and to the receptor Atg19.
The molecular mechanism of Ape1 aggregation involves hydrophobicity while Atg19 binding requires
additional electrostatic interactions. Furthermore, aggregation facilitates subsequent Atg19 binding for
vacuolar transport. Future studies could determine whether Ape1 dodecamerization positions propeptides
close together so they form trimeric structures, with sufficient affinity for propeptides from other
dodecamers to form propeptide hexamers, and consequently causing aggregation. We also show that
Ape1 can aggregate and bind Atg19 and Atg8 in vitro, using buffers with ions that promote
hydrophobicity. Hence the Cvt complex could be used as a scaffold for an in vitro assay of autophagic
vesicle formation to elucidate the functions of key autophagic proteins, which have not been fully resolved
by classical genetic approaches. A similar assay could be used to study how faulty protein aggregates
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can be targeted by the autophagic machinery, to further our understanding and develop better treatments
for neurodegenerative diseases.
2947/L053
Cargo Flux Determines ER Exit Site Organisation during B Cell Differentiation.
1
1
1,2
1 1
E. McCarthy , J. M. Cliff , S. J. Kirk , T. H. Ward ; Immunology and Infection, London School of
2
Hygiene & Tropical Medicine, London, United Kingdom, Biopharm R&D, Domantis Ltd, Cambridge,
United Kingdom
To form a crucial line of defence against infectious diseases, activated B cells differentiate into plasma
cells. These are efficient cellular factories dedicated to the production and secretion of antibodies into the
bloodstream. At the ultrastructural level, plasma cells are characterised by a massive proliferation of the
endoplasmic reticulum (ER) to accommodate immunoglobulin synthesis and assembly. Using an in vitro
differentiation assay, we have been investigating the changes that take place in components of the early
secretory pathway upon B cell activation and how ER exit site assembly and organization is regulated in
response to such an increased secretory burden. Over a 7 day period of differentiation, B cells undergo
major morphological changes, increasing in size, and, in response to a need to migrate, polarising with
formation of a trailing uropod. ER exit sites increase in number concurrent with lymphocyte polarization,
and their spatial distribution is dictated by movement of the MTOC into the uropod. When the cell rounds
up again, correlating with diminishment of the migration phase and the onset of immunoglobulin
secretion, the ER exit sites are redistributed throughout the cell and are no longer clustered at the MTOC.
The involvement of different isoforms of COPII components have been investigated and Sar1B is
specifically upregulated as ER exit sites proliferate to cope with cargo load. The results suggest
differential roles for Sar1A and Sar1B in the organisation of ER exit sites during B cell differentiation.
2948/L054
Rab GTPase Regulation of Retromer-Mediated Sorting in the Endosomal System.
T. Liu, B. K. Sackey, C. G. Burd; Department of Cell and Developmental Biology, School of Medicine,
University of Pennsylvania, Philadelphia, PA
Cargo in the endosomal system has two possible fates: It can be retained and ultimately degraded in
lysosomes, or it can be exported to other organelles. ‘Retromer’ is a complex of proteins that mediates
cargo export by sorting it into tubules that fission from endosomes and fuse with the Golgi apparatus.
Yeast retromer is composed of five proteins that can be dissociated into two sub-complexes: One subcomplex is composed of the sorting nexins, Vps5 and Vps17, and a second sub-complex, which has been
implicated in cargo selection (the ‘cargo selection complex’ or CSC), contains the Vps26, Vps29, and
Vps35 proteins. Vps5 and Vps17 each possess a PtdIns3P-binding PX domain and a BAR domain and
they have been implicated in membrane recruitment of the CSC. Surprisingly, fluorescence microscopy of
GFP-tagged CSC subunits shows that they localize to endosomes and to the membrane of the lysosomelike vacuole, whereas the Vps5 and Vps17 retromer subunits localize only to endosomes. Recruitment of
retromer CSC to vacuole membranes requires activated (ie, GTP-bound) Ypt7, a Rab7 ortholog that
localizes to the vacuole membrane at steady state. In vitro and in vivo binding experiments demonstrate
that yeast CSC binds to GTP-bound Ypt7. Vacuole targeting and Ypt7 binding is disrupted by deletion of
a small (approx. 30 amino acids) region of Vps35 that is distinct from the binding sites for Vps26 and
Vps29, thus identifying a putative Ypt7 binding site on the Vps35 CSC subunit. Conversely, recruitment of
CSC to endosomes is ablated by deletion of VPS5 and VPS17, but is unaffected by deletion of the
putative Ypt7 binding site or by deletion of the YPT7 gene. Cargo sorting assays demonstrate that
recognition of Ypt7 is essential for the sorting function of retromer, even for cargo that is thought to be
exported from endosomes. These results indicate that retromer CSC is recruited by distinct mechanisms
to endosomes and to the vacuole, that the Ypt7 Rab GTPase is essential for retromer function, and raise
the possibility that CSC has functions that are independent of the Vps5 and Vps17 retromer sorting
nexins.
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2949/L055
The Binding Site Crevice of CB2 Cannabinoid Receptor.
P. Kumar, Z. Qiao, Z. Song; Pharmacology and Toxicology, University of Louisville School of Medicine,
Louisville, KY
We have used the substituted-cysteine accessibility method (SCAM) to map the residues in the second
extracellular loop (ECL2) of the CB2 cannabinoid receptor that contribute to the surface of the wateraccessible binding-site crevice. Using a background of the mutant C2.59 which is insensitive to
methanethiosulfonate (MTS) reagents, we mutated to cysteine, one at a time, 14 consecutive residues in
the ECL2 of the CB2 receptor. These mutant receptors were expressed in HEK293 cells and treated with
3
the thiol-specific reagent methanethiosulfonate ethylammonium (MTSEA). [ H] CP55940 binding was
significantly inhibited by MTSEA for six mutant receptors (T173C, P176C, S180C, L185C, E181C, and
N188C), which indicate that the MTSEA reaction occurred within the binding site. Six of the mutants
(T173C, P176C, S180C, E181C, L185C, and N188C) were protected from the inhibitory effects of
MTSEA when pretreated with the cannabinoid agonist WIN55212-2. From these data, we concluded that
these six residues form part of the binding site in the ECL2 of CB2 cannabinoid receptor.
2950/L056
Dynamics of Protein Expression and Membrane Recruitment of the Rab GAP Gyp7.
D. P. Nickerson, A. J. Merz; Biochemistry, University of Washington, Seattle, WA
Rab GTPases cycle between active GTP-bound and inactive GDP-bound states in order to regulate
membrane traffic. Rabs exhibit slow intrinsic rates of GTP hydrolysis and depend upon GTPase activating
proteins (GAPs) as negative regulators of their signaling and localization. The Rab Ypt7 regulates
membrane dynamics at the yeast vacuole/lysosome, which performs critical cellular functions including
disposal and recycling of macromolecules, osmotic stress response, and nutrient sensing and growth
signaling. Mechanisms regulating the function of the Ypt7 GAP Gyp7 are largely uncharacterized. We
report calcineurin-dependent upregulation of Gyp7 protein in response to calcium stress, consistent with
mRNA microarray analyses (Yoshimoto et al., 2002). While Gyp7 localization appears predominantly
cytoplasmic at basal expression levels, overexpression results in Gyp7 accumulation at several
subcellular compartments, including the vacuole membrane and cellular bud neck. The amino-terminal
domain of Gyp7 mediates its localization to specific sites. While in vitro studies indicate that the Gyp7
amino-terminus is not required for triggering nucleotide hydrolysis on Ypt7 and other substrate Rabs
(Albert et al., 1999), it is required for Gyp7 to function in endolysosomal cargo sorting in vivo. These data
suggest that Gyp7 is regulated in vivo to control both its abundance and sites of activity during normal cell
function and as part of the calcineurin stress-response regulon.
2951/L057
Endocytosis, Trafficking and Degradation of the Pore-Forming Toxin SLO during Plasma
Membrane Repair.
M. Corrotte, M. C. Fernandes, C. Tam, N. Andrews; Cell Biology and molecular genetics, University of
Maryland, College Park, MD
Plasma membrane repair after mechanical injury or permeabilization with pore forming toxins is an
important cellular survival mechanism. Previous work identified calcium dependent exocytosis of
lysosomes, followed by rapid endocytosis, as the mechanism for removal of Streptolysin O (SLO) pores
from the plasma membrane (1, 2). However, the fate of the toxin pores after cellular internalization
remained unknown. In this study we investigated whether mammalian cells could intracellularly process
and eliminate membrane-inserted SLO pores, and by what mechanism. Our results show that endosomes
formed during plasma membrane permeabilization with SLO follow the endosomal pathway to lysosomes.
Furthermore, we found that SLO is progressively degraded in a lysosomal dependent manner.
Quantitiative immuno-electron microscopy revealed that GFP-tagged SLO gradually moves away from the
plasma membrane and enters lysosomes, with a kinetic similar to that seen for SLO-induced endosomes
labeled with fluid phase tracers. Ubiquitination of membrane proteins is a well described mechanism to
promote internalization and degradation of membrane proteins by the endo/lysosomal pathway. Following
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endocytosis, membrane proteins are sorted on early endosomes and processed by the ESCRT complex
for internalization into multivesicular bodies (MVB) for degradation. We show that upon internalization,
SLO is rapidly ubiquitinated and interacts with Hrs, a member of the ESCRT-0 complex responsible for
sorting ubiquitinated proteins. Degradation of SLO is dependent on Vps24, a member of the ESCRT-III
complex, indicating that MVB formation is necessary for SLO processing and elimination. Overall, we
provide evidence that SLO, upon endocytosis during plasma membrane repair, is ubiquitinated,
processed by the ESCRT complex, and degraded in MVBs/Lysosomes. These findings indicate that an
endogenous, lysosomal degradation pathway can be used by mammalian cells to eliminate exogenous
pore-forming proteins. (1) Repair of injured plasma membrane by rapid Ca2+-dependent endocytosis.
Idone V et al. JCB, 2008 (2) Exocytosis of acid sphingomyelinase by wounded cells promotes
endocytosis and plasma membrane repair. Tam C et al. JCB, 2010
2952/L058
IRAP Is a Key Regulator of Insulin-Dependant GLUT4 Trafficking by Controlling the Sorting of
GLUT4 from Endosomes to GLUT4-Specialized Compartments.
1
1
1
2
1 1
I. Jordens , D. Molle , W. Xiong , S. R. Keller , T. E. McGraw ; Department of Biochemistry, Weill
2
Cornell Medical College of Cornell University, New York, NY, Division of Endocrinology, department of
Medecine, University of Virginia, Charlottesville, VA
Insulin stimulates glucose uptake by regulating the transport of the facilitative glucose transporter GLUT4
from intracellular compartments to the plasma membrane. In the absence of insulin, GLUT4 is actively
sequestered away from the constitutive recycling general endosomes into GLUT4-specialized
compartments (GSVs), thereby controlling the amount of GLUT4 at the plasma membrane. We
investigated the role of IRAP (Insulin-Regulated AminoPeptidase) in GLUT4 trafficking. We demonstrated
that IRAP is not only a cargo of the insulin-dependant GLUT4 trafficking pathway but is required for a
proper intracellular retention of GLUT4 and then glucose homeostatis. Indeed, in unstimulated 3T3-L1
adipocytes, IRAP knockdown leads to an increase of plasma membrane level of GLUT4, due to an
elevated exocytosis and unaffected endocytosis. Moreover, IRAP functions at a distinct step than the Rab
GTPase AS160, an essential effector of GLUT4 retention mechanism regulating the translocation of
GLUT4 from GSVs to the plasma membrane. Indeed, IRAP is required for the sorting of GLUT4 from
general endosomes to the GSVs, as revealed by the redistribution of GLUT4 to endosomes in IRAP
knockdown cells. Finally, we demonstrate that IRAP trafficking is independent of GLUT4. In conclusion, in
this study, we demonstrated that IRAP is both cargo and a key regulator of the insulin-regulated pathway.
2953/L059
The Clock Protein Cryptochrome Mediates Nuclear Localization of PP5.
N. A. Amborn, K. E. Marz; Biology, Gustavus Adolphus College, Saint Peter, MN
Vertebrate Cryptochromes (CRYs) are circadian clock proteins that shuttle between the nucleus and the
cytoplasm; protein phosphatase 5 (PP5, or Ppp5) is an enzyme that is generally cytoplasmic but can be
recruited elsewhere by binding partners. Partch et al. (2006) demonstrated that CRYs bind to PP5 and
inhibit its activation of the kinase casein kinase I epsilon (CKIε), which phosphorylates several circadian
clock proteins that have functions in the nucleus (Eide et al., 2002). Here we show that PP5 is recruited to
the nucleus in the presence of mouse CRY1 (mCRY1). HEK-293 cells expressing only PP5 exhibit
cytoplasmic localization of PP5, but cells expressing PP5 and mCRY1 exhibit nuclear localization of both
proteins. This altered PP5 localization was used to characterize PP5 binding by mCRY1 site-directed
mutants in order to characterize the binding interface between the two proteins.
2954/L060
A Novel Role for SEH1 and MTC5 in the Intracellular Trafficking of the General Amino Acid
Permease Gap1p in Saccharomyces cerevisiae.
E. D. Spear, C. Kaiser; Biology, MIT, Cambridge, MA
For optimal growth and survival, cells must be able to sense the nutritional environment and respond
appropriately. In Saccharomyces cerevisiae, intracellular trafficking of the general amino acid permease
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Gap1p is regulated by the quality of growth conditions. When cells are grown on a good nitrogen source,
such as glutamine, newly synthesized Gap1p gets sorted to the vacuole where it is degraded.
Conversely, cells grown on a poor nitrogen source, such as ammonium or proline, have high permease
activity due to increased delivery of Gap1p to the plasma membrane. Here we show that a null mutant of
SEH1, encoding a nuclear pore-associated protein, has very low Gap1p activity even when cells are
grown in ammonium medium. A role for Seh1p within the Nup84 nuclear pore complex has been
previously characterized. Consistent with those studies, we observe Seh1-GFP predominantly localized
to the nuclear rim. In addition to nuclear staining, we also observe a pool of Seh1-GFP on the vacuolar
membrane. By mutating the Seh1-interacting domain of the nucleoporin Nup85p, we were able to
selectively disrupt Seh1-GFP localization at the nuclear rim and cause accumulation of Seh1-GFP at the
vacuolar membrane. Intriguingly, it is not the amount, but rather the distribution of Seh1p within the cell
that impacts Gap1p activity. Cells disrupted for nuclear pore-associated Seh1p, but retaining vacuolar
Seh1p exhibit normal Gap1p permease activity. This is in contrast to the low Gap1p activity observed in
seh1Δ cells growing in ammonium medium. Additionally, we find that deletion of MTC5, a gene encoding
an uncharacterized protein localized to the vacuole membrane, decreases the vacuolar pool of Seh1GFP, which could explain the low Gap1p activity observed in the mtc5Δ mutant. We further show that
unique domains within Mtc5p are required to bind to Seh1p and Npr2p, a phosphoprotein recently shown
to be necessary for adaptation from a good nitrogen source to poor nitrogen source. We are currently
exploring the mechanisms by which Seh1p, Mtc5p and Npr2p ultimately control the regulated trafficking of
Gap1p to the cell surface.
2955/L061
Intracellular Trafficking of Membrane Impermeable Cargoes to the Cytosol.
1
1
2
1 1
D. T. Jacobs , R. L. Logan , T. Begay , J. P. Krise ; Pharmaceutical Chemistry, University of Kansas,
2
Lawrence, KS, Environmental Sciences, Haskell Indian Nations University, Lawrence, KS
The cytosolic delivery of membrane impermeable compounds can have important implications on the
therapeutic efficacy of anti-cancer drugs, gene delivery systems, and antibiotics. Aminoglycosides are
clinically useful antibiotics that target Gram-negative bacteria and, unfortunately, also can exhibit toxicity
toward eukaryotic cells and tissues. Although the mechanisms are unclear, it is well documented that
Gentamicin (and a close analog, G418), can exert strong toxicity towards the proximal tubule epithelium
in the kidneys, and the hair cells of the inner ear. (Prayle A et al., Thorax. 2010 Jul;65(7):654-8)
Aminoglycoside antibiotics and bacterial toxins (such as Cholera toxin and Shiga toxin) have been shown
to undergo retrograde transport from the cell surface to the endoplasmic reticulum (ER), and are released
into the cytosol where they exert toxicity. (Sandoval and Molitoris, Am J Physiol Renal Physiol. 2004
Apr;286(4):F617-24) We are exploring the mechanisms that allow membrane impermeable cargoes to
undergo cytosolic delivery. Our strategy is to use known inhibitors of retrograde trafficking to trace the
transport of fluorescently-conjugated G418 in human cells. Our results indicate that G418 associates with
an EEA1-positive endosomal compartment, a transferrin-positive recycling compartment, and a LAMP1positive lysosomal compartment. Also, consistent with following a retrograde transport route,
fluorescently-labeled G418 localized to the ER, but not the Golgi apparatus. Knockdown of GCC185 and
Golgin97 does not change the cellular toxicity profile of G418, suggesting that they are not involved in the
retrograde transport of G418. Cells treated with AlF- significantly retain cellular G418 conjugate
fluorescence, whereas treatment with Brefeldin A did not alter the distribution or trafficking of
fluorescently-labeled G418. Our results suggest that retrograde trafficking of G418 can occur through a
novel, non-Golgi mediated mechanism. Future studies will include using dominant-negative trafficking
proteins and pharmacological inhibitors in conjunction with biochemical fractionation to identify the
subcellular distribution of membrane impermeable cargoes.
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2956/L062
Visualisation of Four Different Cell Membrane Associated Proteins on Epithelial Cells of Ileum: A
Confocal Microscopic Study.
R. G. Aktas; Histology and Embryology, Koc University, Istanbul, Turkey
OBJECTIVE: The aim of the study was to examine the localization of four different cell membrane
associated proteins on ileal epithelial cells: i)CD10/CALLA(neutral endopeptidase, a cell surface enzyme),
ii) hCLCA 1 (calcium dependent chloride), iii) Alpha 5(integrin), iv) Alpha6F (Na-K ATPase). METHOD:
Specimens from normal rat ileum was fixed in 2%paraformaldehyde and embedded either in BMMA or in
K4M. Semithin sections were stained overnight with the primary antibodies which are specific to the
proteins mentioned above. Secondary antibody was Alexa 488 for all sections. Pictures were taken by
using Zeiss LSM510 META confocal microscope. RESULTS: i)CD10/CALLA has been located on the
apical surfaces of enterocytes , ii) hCLCA 1 location was clear in goblet cells. No positive staining was
observed in enterocytes, paneth cells or M cells. iii) Staining with Alpha 5 was clear between the
neighboring sides of epithelium , iv) Alpha6F was obviously located on the lateral surfaces of enterocytes
and goblet cells. CONCLUSION: i) Microscopic localization of CD10 is consistent with the results of
several studies about intestine. ii) CLCA is related with mucin expression. The relation of this protein with
the goblet cell hyperplasia was reported previously. We state that this protein is also in goblet cells which
are normal in size and number. iii)alpha 5 is important in adhesion development in intraperitoneal organs.
This study reports morphologic evidence showing its potential for the adhesion between epithelial cells at
morphologic level. iv)Alpha6F has been demonstrated around goblet cells as well as between enterocytes
at morphologic level.
2957/L063
A New Dibasic ER Retention Motif in a GIRK Potassium Channel from Xenopus laevis oocytes.
B. Díaz-Bello, C. Rangel, C. Salvador, L. Escobar; Physiolgy, UNAM, Mexico City, Mexico
G protein-activated inwardly rectifying potassium channels (GIRKs) belong to the Kir family. GIRKs in
mammals (GIRK1-GIRK4) form heterotetramers of GIRK1/GIRKx. GIRKs subunits contain two
transmembrane segments with cytoplasmic N- and C- terminal domains. GIRKs are activated by G
protein-coupled receptors, causing hyperpolarization and then, producing inhibitory synapses in the
central nervous system. GIRK5 is an endogenous channel in Xenopus laevis oocytes. In contrast to
GIRKs from mammals, GIRK5 is functional like a homotetramer (Heding 1996, Salvador 2001). Traffic of
GIRK5 to the oocyte plasma membrane depends on the phosphorylation of a unique tyrosine (Y16) at the
N-terminal domain (Mora y Escobar 2005). Diacidic motifs like KKXX or RRXX are related to ER retention
(van Vliet et al, 2003). In this study we localized WT EGFP-GIRK5 in the endoplasmic reticulum (ER). A
functional study was carried out after an alanine scanning in the Y16 downstream sequence K13R14L15.
Voltage clamp recordings and confocal microscopy assays were performed in oocytes previously injected
with each of the GIRK5 mutants. GIRK5K13A and GIRK5R14A but not GIRK5L15A displayed potassium
currents. Accordingly, GFP-GIRK5K13A and GFP-GIRK5R14A were observed at the plasma membrane;
in contrast, GFP-GIRK5L15A was localized at the ER. These results suggest that the KRXY at the NH2
terminus of GIRK5 is a new diacidic ER retention motif.
2958/L064
Membrane Protein Association with Lipid Rafts and Cytoskeleton Quantified in Real Time during
Signaling and Perturbation.
1,2
1 1
2
A. Pralle , H. Huang ; Physics, University at Buffalo SUNY, Amherst, NY, Biophysics and Physiology,
University at Buffalo SUNY, Amherst, NY
A central challenge to understanding cell signaling is to quantify how the membrane ultra-structure
modulates signaling dynamics and how vice-versa signaling influences the membrane ultra-structure.
Spatial membrane domains, such as created by lipid rafts and the membrane cytoskeleton, influence
membrane protein mobility and hence membrane bound processes. Not only are these domains modified
in a range of pathologies but varies drugs are also thought to influence them. However, direct
visualization of these lipid domains in intact cells is challenging because of their small dimension and
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dynamics. We present a non-destructive camera based FCS method for quantifying the membrane
protein association with lipid domains in intact cells and with membrane cytoskeleton domains. Most
importantly, our method provides continuous monitoring of changes in the protein domain interactions
over time. We investigated the modulation of GPI-anchored GFP with lipid domains over time in response
to external perturbation and stimulation. As example for signaling induced changes, we studied the
changes in the interaction of temperature sensitive ion channel, TRPV2, with lipid domains and
membrane cytoskeleton upon activation by 2-APB.
2959/L065
Localization of HGSNAT in Lysosomal Membrane Compartment.
1
1
1
1
2
1
1 1
F. Majer , J. Urinovska , J. Sikora , E. Svobodova , D. Cmarko , J. Lukas , M. Hrebicek ; Institute of
2
Inherited Metabolic Disorders, Prague, Czech Republic, Institute of Cellular Biology and Pathology,
Prague, Czech Republic
Human Acetyl-Coenzyme A: Alpha-Glucosaminide N-Acetyltransferase (HGSNAT) is a lysosomal
membrane enzyme localized in microdomains [Taute et al. 2002]. Defects of HGSNAT are associated
with lysosomal storage of heparan sulfate and results in a rare autosomal recessive disorder
Mucopolysaccharidosis IIIC (Sanfilippo syndrome C) [Hrebicek et al., 2006; Fan et al. 2006]. Our aim was
to find membrane partners and spatial distribution of HGSNAT within the cell. We have obtained a
polyclonal rabbit antibody against HGSNAT peptide epitope. Using confocal microscopy the anti-Hgsnat
antibody signal has been found in human fibroblasts only in a subpopulation of LAMP-2-containing
vesicles. The signal did not co-localize either with cell compartment markers COX1 (mitochondria), PDI
(endoplasmic reticulum), Golgi58K (Golgi apparatus), EEA1 (early endosomal compartment) or mannoso6-phosphate receptor (M6PR). Furthermore, anti-HGSNAT did not co-localize with lipid microdomain
markers flotillin 1, GM1 ganglioside or globotriaosylceramide. Anti-HGSNAT partially co-localized with
sortilin and showed microdomain lysosomal localization. The presence of the signal in only a subset of
lysosomal vesicles may suggest existence of specialized subpopulation of lysosomes. This study was
funded by the research project 0021620806 from the Ministry of Education, Youth and Sports of the
Czech Republic
2960/L066
Adaptor Protein Complex Interactions with the Delta-2 Glutamate Receptor.
1
2
1 1
Y. Lin , P. Burgos , J. S. Bonifacino ; Cell Biology and Metabolism Program, NICHD, NIH, Bethesda,
2
MD, Department of Physiology, Faculty of Medicine, Universidad Austral de Chile, Valdivia, Chile
Transmembrane proteins in neurons are specifically targeted to pre- and post-synaptic sites, but the
mechanisms underlying these processes remain unclear. In this study we have characterized the
existence of molecular determinants that could mediate polarized distribution of the ionotropic glutamate
receptor delta-2 (d2), an orphan subunit essential in cerebellar synapse formation. In this study we found
that the cytoplasmic domain of d2 binds by yeast-2-hybrid to all known mu-subunits of Adaptor Protein
(AP) complexes. APs mediate intracellular protein trafficking by recognizing sorting signals (amino acid
motifs) in target proteins and connecting them to cell sorting machinery. Site-directed mutagenesis
revealed two potential novel kinds of sorting signals: a non-canonical tyrosine-based motif and an
unusual disparate motif consisting of sets of aromatic amino acids. Ongoing experiments are elucidating
the role of these novel motifs in the sorting of glutamate receptors in neurons.
2961/L067
Ang2/Fat-Free Is a Conserved Subunit of the Golgi-Associated Retrograde Protein Complex.
1
1
1
1
2
2
2
C. Schindler , F. Perez-Victoria , J. Magadán , G. A. Mardones , C. Delevoye , M. Romao , G. Raposo ,
1 1
2
J. Bonifacino ; NICHD, NIH, Bethesda, MD, Centre National de la Recherche Scientifique-Unité Mixte
de Recherche 144, Institut Curie, Paris, France
The Golgi-associated Retrograde Protein (GARP) complex mediates tethering and fusion of endosomederived transport carriers to the trans-Golgi network (TGN). In the yeast Saccharomyces cerevisiae,
GARP comprises four subunits named Vps51p, Vps52p, Vps53p and Vps54p. Bioinformatics and
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biochemical analyses have shown that other eukaryotes have orthologs of these subunits, except for
Vps51p. A yeast two-hybrid screen of a human cDNA library identified a phylogenetically conserved
protein, Ang2/Fat-free, which interacts with Vps52, Vps53 and Vps54. Human Ang2 is larger than yeast
Vps51p, but exhibits significant homology in the N-terminal coiled-coil region that mediates assembly with
other GARP subunits. Biochemical analyses show that human Ang2, Vps52, Vps53 and Vps54 form an
obligatory 1:1:1:1 complex that strongly interacts with the regulatory Habc domain of the TGN SNARE
Syntaxin 6. Depletion of Ang2 or the GARP subunits similarly affects protein retrieval to the TGN,
lysosomal enzyme sorting and autophagy. These findings indicate that Ang2 is an integral component of
the GARP complex in most eukaryotes.
2962/L068
Roles for Rtg2p in Retrograde Signaling in Saccharomyces Cerevisiae: A Complementation Based
Approach.
E. S. Ünlü, D. M. Gordon; Biological Sciences, Mississippi State University, Mississippi State, MS
In Saccharomyces cerevisiae, RTG2 has been identified as a key regulator of mitochondria to nuclear
communication, or retrograde signaling. In retrograde signaling mitochondrial dysfunction is sensed by
cytosolic Rtg2p which transmits the signal to the Rtg1p/Rtg3p transcription factor triggering its nuclear
translocation. Central to this signaling pathway is the reversible interaction between Rtg2 and the
negative regulator Mks1p. When bound to Rtg2p, Mks1p is inactive and retrograde signaling is on. When
interaction with Rtg2p is prevented, Mks1p associates with Bmh1p which inhibits Rtg1p/Rtg3p nuclear
localization turning retrograde signaling off. Putative RTG2 homologues have been identified in four other
yeast including A. gossypii, C. glabrata, V. polyspora and K. lactis. We have found that the ability of
Rtg2p homologs to rescue a Δrtg2 strain, as measured by Cit2p and Aco1p protein levels, varied from
partial to complete complementation. To determine whether reduction in retrograde signaling was due to
reduced interaction between Rtg2p homologs and Mks1p, co-immunoprecipitation assays were carried
out. We found the interaction status between Rtg2p and Mks1p did not parallel the complementation data.
Most interestingly, the expression of the Rtg2p homologs from C. glabrata and K. lactis completely
complemented all mutant phenotypes tested but showed significantly reduced interaction with Mks1p.
These results suggest that control of gene expression via retrograde signaling involves an additional
pathway that is independent of Mks1p interaction. Recently Rtg2p has been shown to localize to the
nucleus where it assembles with other proteins to form the SLIK complex. The SLIK complex is involved
in gene regulation including that of the retrograde response gene CIT2. To determine the role of SLIK in
the retrograde signaling observed for each Rtg2p homolog, co-immunoprecipitation and ChIP assays will
be carried out. Comparison of Rtg2p homologs provides insight into domains responsible for its diverse
roles in retrograde signaling activities.
2963/L069
LAMMER Kinases Dsk1p and Kic1p Are Required for Efficient Pre-Mrna Splicing and Proper
Localization of Poly(A)+ RNA.
1
1
1
1
2
3 1
Z. I. Tang , M. Luca , J. Portillio , A. Guven , R. Lin , A. Carr ; W.M. Keck Science Center, Claremont
2
Colleges, Claremont, CA, Molecular and Cellular Biology, Beckman Research Institute of the City of
3
Hope, Duarte, CA, Genome Damage and Stability Center, University of Sussex, Falmer, United Kingdom
Dsk1p and Kic1p are the orthologues of human SRPK1 and mammalian CLK1, respectively in fission
yeast Schizosaccharomyces pombe. They belong to a class of dual-specificity protein kinases, the
prototypes of which are featured with the “LAMMER” signature motif. Both protein kinases are implicated
in the cell cycle regulation and pre-mRNA splicing. In addition to the involvement of Dsk1p in splicing, we
previously showed that Dsk1p kinase phosphorylates SR (serine-arginine rich) splicing factors, Srp1p and
Srp2p, and regulates their cellular localization in S. pombe. However, not much is known about the
functions of the two kinases in the mRNA processing pathway. The purpose of this study is to examine
the possible roles of Dsk1p and Kic1p in mRNA processing such as splicing and mRNA localization. We
have tagged dsk1+ and kic1+ genes at their genomic loci with TAP (tandem affinity purification) and
successfully isolated Dsk1p-TAP complex. We also used a minigene system to investigate the
involvement of both kinases in pre-mRNA splicing. Finally, we determined the mRNA distribution in dsk1-
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and kic1-deletion strains by detecting poly(A) in the cells with in situ hybridization and
immunefluorescence microscopy. In agreement with our previous results of SR protein phosphorylation
by Dsk1p, the TAP-isolated Dsk1p complex in vivo contains Srp1p, Srp2p, Rsd1p and Prp2p splicing
factors, in addition to several other proteins involved in mRNA processing. Moreover, Kic1p is also
required for efficiently splicing of nda3+ minigene transcripts. Interestingly, dsk1- and kic1-deletion
mutants display temperature-dependent phenotypes in poly(A) RNA distribution. These data provide the
first evidence for the function of Kic1p in mRNA processing and demonstrate that Dsk1p is a component
of a complex containing factors mediating multiple steps in mRNA processing. Furthermore, our results
indicate that Dsk1p and Kic1p may have distinct roles in regulating mRNA processing and cell-cycle
progression.
2964/L070
5'-End Surveillance by Xrn2 Acts as a Shared Mechanism for Mammalian Pre-rRNA Maturation and
Decay.
M. Wang, D. G. Pestov; Cell Biology, UMDNJ-SOM, Stratford, NJ
Ribosome biogenesis requires multiple nuclease activities to process pre-rRNA transcripts into mature
rRNA species and eliminate defective products of transcription and processing. We find that in
mammalian cells, the 5' exonuclease Xrn2 plays a major role in both maturation of rRNA and degradation
of a variety of discarded pre-rRNA species. Precursors of 5.8S and 28S rRNAs containing 5' extensions
accumulate in mouse cells after siRNA-mediated knockdown of Xrn2, indicating similarity in the 5'-end
maturation mechanisms between mammals and yeast. Strikingly, degradation of many aberrant pre-rRNA
species, attributed mainly to 3' exonucleases in yeast studies, occurs 5' to 3' in mammalian cells and is
mediated by Xrn2. Furthermore, depletion of Xrn2 reveals pre-rRNAs derived by cleavage events that
deviate from the main processing pathway. We propose that probing of pre-rRNA maturation
intermediates by exonucleases serves the dual function of generating mature rRNAs and suppressing
suboptimal processing paths during ribosome assembly.
2965/L071
Expression of the Anemonea Majano GFP-Like Gene Amfp486 in Response to Temperature
Changes.
P. J. Griffith, J. Prempeh; Drexel University, Philadelphia, PA
We're considering a biomarker for the current changes in the climate, in terms of global warming
especially. We're looking at one species to determine the effect of temperature change in terms of
quantitative expression of the green fluorescent proteins (GFP). In particular, we have considered a
particular protein that in one of these species provides fluorescence and in another provides merely a
bluish green coloration. This protein is highly conserved between many different species. The change in
this protein between these species is negligible, but makes a large difference, and its expression is highly
regulated by environmental factors- which is what we're considering. This same protein, when folded
incorrectly in mice causes the skin to stop developing in utero and causes the mice to die at birth, so it's a
highly relevant protein. It's also one with stringent internal controls, which we can use a control ourselves;
by looking at the RNA and then the protein (to verify that the translation and folding is occurring correctly),
we can then be assured that any change in phenotypic expression will be the result of our changes in
environmental stimuli.
2966/L072
Probing the P-Site for Functionality during Maturation of the 60S Ribosomal Subunit.
C. Bussiere, S. Arora, A. Johnson; Molecular Genetics and Microbiology, UT Austin, Austin, TX
In eukaryotic cells, ribosomes are largely preassembled in the nucleus. The subunits are matured in the
cytoplasm by the release of export and assembly factors and the addition of ribosomal proteins to
generate functionally active subunits. We previously showed that the large (60S) ribosomal subunit
protein Rpl10 is required for the release of the nuclear export adaptor Nmd3 from nascent 60S subunits.
More recently we ordered the events of cytoplasmic 60S maturation and found that the release of Nmd3
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requires the prior release of the subunit anti-association factor Tif6 by the GTPase Efl1. This prompted us
to ask if Rpl10 was required for any steps upstream of Nmd3 release. Rpl10 is located in a cleft between
the central protuberance and the GTPase stalk and contains an internal unstructured loop that embraces
the P-site tRNA. Deletion of the P-site loop of Rpl10 trapped Tif6 in the cytoplasm, indicating a novel role
for Rpl10 in release of Tif6, prior to its role in Nmd3 release. Mutagenesis of the P-site loop yielded
mutants that specifically affect 60S biogenesis. Surprisingly, these mutants could be suppressed by a
mutant allele of Tif6 that has weakened affinity for the ribosome, bypassing the need for Efl1. Because
Efl1 is normally required for the release of Tif6, this result implicates Efl1 in signaling between Rpl10 and
Tif6. Efl1 is closely related to the translation elongation factor eEF2/EF-G, the GTPase that is responsible
for translocation. Based on the crystal structure for eEF2, the suppressing mutations in Efl1 map to
domain interfaces of eEF2 where the antibiotics sordarin and fusidic acid bind. These drugs trap
eEF2/EF-G in a post translocation conformation, suggesting that the suppressing mutations shift the
equilibrium of Efl1 toward a post-translocation-like conformation. These results identify a molecular
signaling event from the P-site to Tif6, 90Å away. Correct signaling activates the GTPase of Efl1,
triggering the release of Tif6 via a pseudo-translocation event. We suggest that interrogation of the P-site
of the ribosome represents a quasi-functional quality control check of the large subunit, prior to its first
round of bona fide translation.
2967/L073
Interferon Stimulated Response Elements (ISRE) in Human Chromosome 21 Genes.
L. E. Maroun, A. Jagadeesh, K. Sampson, H. North, B. Robson, A. Rehemtula, I. Dhanoya, M. Khalid, K.
Parikh, M. Siddique, V. Tegeltija, J. Husni; Saint Matthew's University School of Medicine, Grand
Cayman, Cayman Islands
Transgenic animals and patient treatment experience suggests that interferon-alpha (IFN-α) can be
neurotoxic. Both components of the IFN-α receptor are encoded on human chromosome 21 (Ch21).
However, the observed 50% increase in receptor numbers alone cannot explain the 5-7 fold IFN
supersensitivity of cells from people with Down syndrome (trisomy 21). The objective of our study is to
identify Ch21 genes that are potentially regulated by IFN-α, and which might explain the higher-thanexpected IFN supersensitivity. We used online search engines to search for IFN-α control sites (ISRE) in
or near Ch21 genes. A functional ISRE site could be located quite far (>100,000 bp) from the transcription
start site (TSS).We executed a whole chromosome search using the ISRE consensus sequence
(AGTTTCNNTTTCNC/T) which provided a short list (13 of the estimated 235-400 genes on Ch21) that
met our arbitrarily restrictive criteria of containing at least two ISRE sites within the gene and/or upstream
of its TSS. This list contained a number of genes considered candidates for Down syndrome
neuropathology that have not previously been associated with interferon action. For example, the PCBP3
gene was found to have two ISRE sites, one 8,386 bp upstream, and one 4,948 bp internal, of its TSS. In
addition, in the same region, it contained the ISRE sites reported for the HLA-A, BF, and Factor-B genes.
The PCBP3 gene is important in Tau related dementia (e.g. Alzheimer’s and Down syndrome). Our
observations suggest that there are potentially a number of interferon regulated genes on Ch21 which
could augment the action of interferon and partly explain the 5 to 7 fold IFN-α supersensitivity of trisomy
21 cells.
2968/L074
New Components of Heat-Induced Stress Granules in Saccharomyces cerevisiae.
1
2
1
1
1
1
1 1
T. Grousl , P. Ivanov , I. Malcova , I. Frydlova , P. Vasicova , L. Novakova , J. Hasek ; Inst.
2
Microbiology AS CR, v.v.i., Prague 4, Czech Republic, Faculty of Biology, Moscow State University,
Moscow, Russian Federation
In stressed cells, cytoplasmic ribonucleoproteins accumulate in distinct assemblies of variable
composition. We have recently referred in Saccharomyces cerevisiae that the stress granules (SGs)
containing translation initiation factor eIF3 and 40S ribosomal subunits are transiently assembled under
the robust heat shock. We have identified the main components of these SGs but the links between the
translational arrest, polysome dissassembly and formation of SGs are still unknown. Here we refer on
new, yet unknown components of SGs which include some translation elongation and translation
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termination factors. Some of these proteins accumulated even under a milder heat stress. In addition, we
found that heat shock also induced disruption of F-actin bundles. Moreover, F-actin accumulations were
distinct from SGs. Our data suggest that disruption of F-actin bundles in heat-shocked cells of S.
cerevisiae may result from sequestering of elongation factors in SGs. This work was financed by grants
CSF 204/09/1924, LC545 and IRCAV0Z50200510.
2969/L075
Barrier-To-Autointegration Factors Nuclear Localization and LAP2α Mislocalization Are a Common
Features in Laminopathies with Defective Prelamin A Processing.
1
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3
4
5
1
2
C. Capanni , M. Columbaro , R. Foisner , G. Novelli , M. Wehnert , S. Squarzoni , N. M. Maraldi , G.
1 1
2
Lattanzi ; Institute of Molecular Genetics unit of Bologna c/o IOR, CNR, Bologna, Italy, Laboratory of
3
Musculoskeletal Cell Biology, IOR, Bologna, Italy, Department of Medical Biochemistry, Max F. Perutz
4
Laboratories, Medical University of Vienna, Vienna, Austria, Dept. of Biopathology and Diagnostic
5
Imaging, University of Rome Tor Vergata, Rome, Italy, Institute of Human Genetics, Ernst- Moritz- ArndtUniversität Greifswald, Greifswald, Germany
Lamin A is produced as a protein precursor (prelamin A) which is post-transcriptionally modified at its Cterminal region. It has been demonstrated that accumulation of different intermediates of prelamin A
processing induces chromatin remodeling. This effect is consistent with chromatin disorganization
described in laminopathies with prelamin A accumulation. In this regard, we have recently suggested the
involvement of barrier-to-autointegration factor (BAF), a LEM binding protein linking DNA, in prelamin A
chromatin remodeling functions. In particular, we demonstrated that in prelamin A accumulating cells,
obtained through different experimental approaches, BAF is predominantly located in the nucleus where it
colocalizes and interacts with different prelamin A forms including progerin. In order to understand the
pathophysiology of laminopathies with prelamin A accumulation we wondered if BAF nuclear localization
is a common event in these diseases. Our preliminary results obtained by immunofluorescence analysis
showed that the BAF heterogeneous localization between nucleus and cytoplasm described in control
cells changed in Familial Partial Lipodystrophy (FPLD), Mandibuloacral Dysplasia (MADA) and Restricted
Dermopathy (RD) cells. In particular, we observed BAF nuclear recruitment in prelamin A positive skin
fibroblast cells from FPLD and MADA patients, where it colocalized with prelamin A either at intranuclear
aggregates or at the nuclear rim. In RD cells, in which the last proteolytic cleavage step of prelamin A
processing is completely defective, BAF localized in the nucleus in all nuclei, where it could be detected
at nucleoplasmic level or at the nuclear rim. However, the evaluation of BAF protein amount by Western
Blotting analysis showed that prelamin A accumulation did not affect BAF protein levels. Analogous
results were also obtained when the localization and expression of LAP2α was studied, suggesting that
the BAF/LAP2α protein complex involvement could be considered as a common pathophysiological event
in laminopathies with prelamin A accumulation.
2970/L076
Single-Molecule Studies of Nucleocytoplasmic Transport: from 1D to 3D.
W. Yang; Bowling Green State University, Bowling Green, OH
The nuclear pore complex (NPC), the sole gateway for nucleocytoplasmic exchange in eukaryotic cells,
allows for the passive diffusion of small molecules (< 20-40 kDa) and the transport-receptor-facilitated
translocation of larger molecules. However, the precise transport mechanism as to how these two
transport modes pave their pathways through the NPC remains in dispute among numerous transport
models. By a newly developed single-molecule speedy microscopy and a deconvolution algorithm, we
advanced the single-molecule imaging of nucleocytoplasmic transport from 1D (one-dimension) to 3D.
The 3D routes for both passive and facilitated transport through human NPCs under real-time trafficking
conditions have been obtained with a spatial resolution of 9 nm at 0.4 ms. Our data strongly support a
transport mechanism in which transport receptors or transport-receptor-cargo complexes pave their
pathways by interacting with the FG repeats at the periphery around a single primary central axial
channel for passive diffusion of small molecules.
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2971/L077
Sodium Selenate Enhances Endothelial Cell STAT3 Tyrosine Phosphorylation and DNA Binding.
3
1
1
1,2
1 1
H. J. Alturkmani , C. Zgheib , F. A. Zouein , M. Kurdi , G. W. Booz ; Pharmacology and Toxicology,
2
University of Mississippi Medical Center, Jackson, MS, Chemistry and Biochemistry, Lebanese
3
University, Hadath, Lebanon, Alfaisal University, College of Medicine, Riyadh, Saudi Arabia
Sodium selenate has been shown to lessen tau pathology in several animal models of Alzheimer's
disease ostensibly through activation of protein phosphatase 2A (PP2A) suggesting that selenate may
have therapeutic value; however, the cellular actions of selenate are not well defined. Since PP2A is
known to dephosphorylate STAT3 S727 and DNA binding of STAT3 is enhanced by S727
phosphorylation, we reasoned that sodium selenate would also reduce STAT3 activity. The transcription
factor STAT3 is important in fever and the acute phase response of inflammation and has been
implicated in some cancers, atherosclerosis, and rheumatoid arthritis. Experiments were performed using
human microvascular endothelial cells (HMEC-1) and the IL-6 type cytokine leukemia inhibitory factor
(LIF). Cells were pretreated for 2 h with 100 µM sodium selenate, a concentration effective in stimulating
PP2A with no adverse effect on cell viability. In contrast to expectations, pretreatment of HMEC-1 for 2 h
with 100 µM sodium selenate had no effect on LIF-induced S727 phosphorylation, which was in any case
modest. Sodium selenate, however, markedly increased LIF-induced STAT3 Y705 phosphorylation in
nuclear extracts at 1 h by 70.9 ± 10.0% (n=7; P<0.01). Consistent with the role of Y705 phosphorylation in
STAT3 dimerization and transcriptional activity, sodium selenate enhanced binding activity of nuclear
extracts from cells treated 1 h with LIF to an oligonucleotide containing a STAT3 consensus binding site
by 79.0 ± 21.5% (n=7; P<0.01). Sodium selenate had no significant effect on basal STAT3 tyrosine
phosphorylation or LIF-induced p42/p44 MAP kinase activity over 60 minutes. These findings are
consistent with sodium selenate inhibiting the tyrosine dephosphorylation of STAT3, which has not been
previously reported. Studies are underway to identify the tyrosine phosphatase targeted by sodium
selenate. In conclusion, the beneficial actions of sodium selenate in PP2A activation and treating
Alzheimer's disease may be offset by a pro-inflammatory action in stimulating STAT3 activity.
2972/L078
Positioning Drosophila Centromeric Heterochromatin Sequences by Comparative Genomic
Hybridization Analysis.
1
2
1
1,3 1
B. He , A. Caudy , S. Raj , E. Wieschaus ; Molecular Biology, Princeton University, Princeton, NJ,
2
3
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, Howard Hughes
Medical Institute, Princeton, NJ
About one third of the Drosophila melanogaster genome is heterochromatic. To date, the majority of the
Drosophila heterochromatin have not been mapped due to the difficulties in assembling repetitive
sequences that constitute the majority of the heterochromatin. Here we developed a strategy combining
deep sequencing with comparative genomic hybridization (CGH) to position the unmapped Drosophila
heterochromatin sequences to specific chromosomal regions. In order to construct a genomic library for
Drosophila heterochromatin, we sequenced the entire fly genome by deep sequencing of DNA extracted
from blastoderm embryos, followed by excluding sequences that can be mapped to the euchromatic
reference genome. Custom microarrays containing probes corresponding to these unmapped sequences
were manufactured and used for CGH analysis. By analyzing DNA isolated from embryos deficient for
each chromosome or chromosome arm, we were able to position most of the unmapped heterochromatic
sequences to specific chromosome arms. In addition, these sequences could be further positioned to
more specific chromosomal regions by using translocations with breakpoints at different locations of the
centromeric heterochromatin. Subsequent analysis indicated that sequences located at different
heterochromatin regions have diverse sequence composition and expression profile. Overall, our study
provides a useful approach to localize unmapped heterochromatic sequences to specific chromosomal
locations, which will facilitate the generation of a complete map of Drosophila heterochromatin.
Combining functional analysis with positional information of heterochromatic sequences also helps to
improve our understanding of the organization and function of heterochromatin.
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2973/L079
Localization of Suns and LAP2alpha in the Human Myocardium.
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4
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2
M. Columbaro , O. Leone , C. Capanni , S. Squarzoni , R. Foisner , N. M. Maraldi , G. Lattanzi ;
1
2
Laboratory of Musculoskeletal Cell Biology, Istituto Ortopedico Rizzoli, Bologna, Italy, Institute of
3
Molecular Genetics c/o IOR, CNR, Bologna, Italy, Department of Pathology, S.Orsola-Malpighi Hospital,
4
University of Bologna, Bologna, Italy, Department of Medical Biochemistry, Max F.Perutz Laboratories,
Medical University of Vienna, Vienna, Austria
While the distribution of nuclear envelope proteins in skeletal muscle has been widely described, a
comprehensive picture of nuclear envelope organization and dynamics in cardiac tissue is still lacking.
However, mutations in the nuclear lamina constituent lamin A/C cause severe forms of dilated
cardiomyopathy with conduction defects (CMD-CD). To start addressing the biological mechanisms
causing CMD-CD, we investigated three lamin binding partners, implicated in muscle functionality, namely
SUN1, SUN2 and LAP2alpha, and related their expression and localization in the human myocardium to
the post-translational processing of the lamin A precursor. SUNs are known to localize in the inner
nuclear membrane of most cell types and are involved in positioning of nuclei through interaction with
nesprins. They also co-ordinate positioning of chromatin domains within the nucleus. LAP2alpha is a
nucleoplasmic chromatin-binding protein implicated in the balance between cellular proliferation and
differentiation through its interaction with A-type lamins and the retinoblastoma protein. LAP2alpha is also
capable of binding prelamin A and its recruitment to the nuclear periphery has been associated with the
presence of farnesylated prelamin A. Our data show that SUN1 and SUN2 are located at the nuclear rim
of human myocardium nuclei, co-localizing with farnesylated prelamin A.Unexpectedly, we find that also
LAP2alpha localizes at the nuclear rim of cardiomyocytes, but not in the nucleoplasm. Importantly, we
find that SUN1, but not SUN2, is also localized in the intercalated discs. We hypothesize that farnesylated
prelamin A might drive recruitment of SUN1 and Lap2alpha to the nuclear envelope of cardiomyocytes,
thus contributing to the organization of the nuclear membrane, as well as to the interplay between
cytoskeleton and chromatin domains mediated by SUN1. SUN1 localization at both the nuclear
membrane and the intercalated discs, further suggests that SUN1 might mediate mechanosignaling
mechanisms implicated in myocardium functionality.
2974/L080
The Expression of Pluripotent Genes in Normal Human Endometrium.
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3
3 1
K. Y. Pérez , J. Hyun-Park , P. S. Tanwar , J. Teixeira ; Biology, Interamerican University of Puerto Rico
2
3
at San German, San Germán, Stem Cell Institute, Harvard Medical School, Boston, MA, Vincent Center
for Reproductive Biology, Massachusetts General Hospital, Boston, MA
Induced pluripotent stem (iPS) cells are a promising tool for the treatment of degenerative diseases since
they could provide ample supplementation for deficient tissues. An adult cell type suitable for pluripotency
induction should have a differentiated state amenable for reprogramming and ideally their acquisition from
patients should be minimally invasive. Endometrial cells of the uterus are constantly proliferating, which
suggests higher pluripontency capacity, and can be obtained with relative simplicity from donors.
However, an endometrial gene expression profile necessary to determine their reprogramming fitness has
not been reported. The OCT4, SOX2, NANOG and LIN-28 genes have all been associated in
pluripotency. If any of these genes are naturally expressed in the endometrium, the possibility of
developing iPS cells from this tissue source would be high. By performing RTPCR on RNA purified from
human endometrial cells, we determined that the OCT4, SOX2, NANOG and LIN-28 genes are all
significantly expressed in the endometrium. Immunohistochemical analysis confirmed nuclear expression
of these pluripotent genes in normal human endometrial epithelial cells. These results suggest that
endometrial tissue should be further explored as a cellular source for reprogramming into pluripotent stem
cells, which in turn could be used in future regenerative medicine therapies.
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2975/L081
Pcg Bodies Studied by Correlative Light-Electron Microscopy.
1,2
1,2
1,2
1,2 1
J. Smigova , P. Juda , D. Cmarko , I. Raška ; Charles University, First Faculty of Medicine,
2
Institute of Cellular Biology and Pathology, Prague, Czech Republic, Institute of Physiology, ASCR, v.v.i.,
Prague, Czech Republic
The cell nucleus has a compartmentalized structure built up by chromatin areas and the interchromatin
compartment (IC). Whereas the chromatin consists of the active (euchromatin), temporarily inactive
(facultative heterochromatin) and inactive (constitutive heterochromatin) form, the IC is enriched in the
number of different subdomains called nuclear bodies. Generally, the term nuclear body is used for a
region within the nucleus that is morphologically distinct from its surroundings when observed by
transmission electron microscopy. In this respect, fine structural information about the nuclear Polycomb
body (PcG body), that in fluorescence microscopy appears to be highly enriched in Polycomb proteins,
has not yet been established. To reveal the ultrastructure of the PcG body in cells stably expressing
recombinant Polycomb BMI1-GFP protein, we used correlative light electron microscopy (CLEM) with
implemented high-pressure freezing and cryosubstitution. This approach enabled to identify PcG bodies
not with the nuclear bodies but with the domains enriched in the heterochromatin fascicles. Importantly,
the quantitative evaluation of the immunogold on section labelling of BMI1 protein in the chromatin
fascicles corresponding to "PcG body" did not differ from chromatin fascicles outside of the PcG domains.
We also performed two approaches mimicking the processing of cells for fluorescence. First consisted of
conventional pre-embedding labeling and the second consisted of extraction with the cytoskeleton buffer
containing Triton that preceded fixation and immunolabeling. Both approaches confirmed the structure of
condensed chromatin behind the PcG body fluorescence signal, however, the ultrastructure of chromatin
was altered. This work was supported by grants MSM0021620806, LC535, AVOZ50110509 and for P.J.
also the Grant 260501 from the Charles University in Prague.
2976/L082
An Artificial Chromatin Modulator for Stable Transgene Expression.
J. Tanase, K. Udagawa, J. Nishikawa, T. Ohyama; Waseda University, Shinjuku-ku, Japan
Stable and efficient expression of transgenes requires the surrounding chromatin to be appropriately
organized. Recently, we found that a curved DNA segment of 180 bp (T20) could highly activate
transcription of a reporter gene in the genome chromatin context of HeLa cells, irrespective of the locus of
reporter. Thus, T20 seemed to be a useful tool for high-level and stable expression of transgenes. With
these backgrounds of T20 research, we established several sets of mouse ES cell lines each harboring a
single copy of the reporter construct, in which each set comprised a T20-harboring cell line and a T20less control cell line. In the ES cells and in the hepatocytes originating from these cells, transcriptional
activation by T20 occurred only in transcriptionally active loci in the genome. Thus, the effect of T20 was
different between HeLa and the mouse ES cells. However, transcriptional activation by T20 was also
remarkable in yeast cells as in HeLa cells. Even in the presence of nucleosomes, T20 activated
transcription from a UAS-deleted CYC1 promoter that is usually silenced in the presence of nucleosomes.
Analyses of the chromatin structures suggested that functioning as a dock for the histone core and
allowing nucleosome sliding are the possible mechanisms underlying the transcriptional activation by
T20. The potential of T20 or other similar structures for chromatin engineering will be discussed.
2977/L083
Replication Timing of Pseudo-Nors.
1
1
1
1
1
1
1
1
E. Smirnov , D. Cmarco , L. Kováčik , G. Hagen , A. Popov , B. Ryabchenko , F. Amim , O. Raška , J.
2
2
1 1
2
Prieto , B. McStay , I. Raška ; Charles University in Prague, Prague, Czech Republic, National
University of Ireland, Galway, Ireland
In mammalian cells, transcriptionally active ribosomal genes are replicated in the early S phase, and the
silent ribosomal genes in the late S phase, though mechanisms of this timing remain unknown. UBF
(Upstream Binding Factor), a DNA binding protein and component of the pol I transcription machinery, is
considered to be responsible for the loose chromatin structure of the active rDNA. Here we question
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whether such structure alone can ensure early replication of DNA. We investigate this problem on the
model of pseudo-NORs, the tandem arrays of heterologous DNA sequence with high affinity for UBF,
introduced into human chromosomes. Such arrays are not transcribed, yet efficiently bind UBF and mimic
the chromatin structure of active rDNA. Transfecting these cells with UBF-GFP and PCNA-RFP, we found
that replication of pseudo-NORs is not restricted to the early S phase, but continues in the late S phase at
a significant level. These results were confirmed in the experiments with incorporation of thymidin analog
EdU. Our data indicate that the specific loose structure of chromatin, produced by the architect protein
UBF, is not sufficient for the early replication. The work was supported by the grants from the Ministry of
Education, Youth and Sports of the Czech Republic MSM0021620806 and LC535, the Academy of
Sciences of the Czech Republic AV0Z50110509, the Grant Agency of the Czech Republic 304/09/1047,
and the Science Foundation Ireland Principal Investigator award 07/IN.1/B924.
2978/L084
Synergistic Interactions between Mre11-A470T and Ctf18Δ in Telomere Recombination.
H. Gao, A. J. Lustig; Biochemistry, Tulane University, New Orleans, LA
In Saccharomyces cerevisiae, the Mre11/Rad50/Xrs2 (MRX) complex plays multiple and essential roles
at the telomere in protecting the genome stability. The MRX complex is required for telomerase-mediated
telomere elongation. However, in the absence of telomerase, the MRX complex is necessary for one form
of telomere recombination. In contrast, Ctf18, an alternative component of RFC1, is involved in multiple
cellular processes including sister chromatid cohesion, homologous recombination, DNA replication,
telomere silencing and telomere length regulation. Our lab recently identified an mre11 mutation (mre11A470T) that dramatically induces telomere recombination both between the telomeric tracts adjacent to
subtelomeric sequences (Type 1A) and between telomeric tracts to give rise to highly elongated
telomeres (Type II). The high rate and type of recombination permits the bypass of senescence observed
in telomerase-negative mre11-A470T cells We have found several genetic lines of evidence for the
interaction between the these proteins as indicated by their the synergism in cold, HU and MMS
sensitivity. Interestingly, both mutations create truncated telomeres in a common pathway, with mre11A470T acting epistatic to ctf18Δ. Given this evidence for genetic interaction, we compared the
senescence and recombination properties of these mutants in telomerase negative cells. After an initial
loss in viability in both ctf18Δ and ctf18Δ mre11-A470T, the double mutant regained viability at a rate
higher than ctf18Δ cells. In initial analysis of recombinants formed in ctf18Δ tlc1 and ctf18Δ mre11-A470T
tlc1, we found that ctf18Δ tlc1 exclusively gave rise to Type II forms, while the double mutant gave rise
only to Type 1A in the absence of Type II recombination. Hence, as in the telomere size pathway, mre11A470T and ctf18Δ fall into the same recombination pathway with mre11-A470T being epistatic to ctf18Δ.
Our results support a role for Ctf18 as a negative regulator of Type IA recombinants facilitated by the
mre11-A470T mutation, suggesting a role for the Ctf18 factor in telomeric recombination.
2979/L085
Characterization of the Role of Utp22p in Nuclear Trna Export in Saccharomyces cerevisiae.
A. R. Clayton, M. B. Eswara, D. Mangroo; Molecular and Cellular Biology, Univeristy of Guelph, Guelph,
ON, Canada
Previous studies have identified Utp8p to be involved in nuclear tRNA export in Saccharomyces
cerevisiae. Utp8p belongs to a family of U three proteins that are involved in pre-ribosomal RNA
processing and maturation in the nucleolus. It has been shown, however, that Utp8p functions as an intranuclear factor that collects mature tRNAs from aminoacyl-tRNA synthetases in the nucleolus, and delivers
them to the nuclear tRNA export receptors, Los1p and Msn5p, at the nuclear pore complex. Furthermore,
Utp8p was shown to be involved in the export of mature tRNAs derived from both intronless and introncontaining pre-tRNA transcripts. To further understand the tRNA export process in S. cerevisiae, Tandem
Affinity Purification coupled with Mass Spectrometry was conducted to identify proteins that co-purify with
Utp8p. Utp22p, an essential 140 kDa protein, is one of the proteins that were identified in this screen.
Utp22p, like Utp8p, is known to be involved in the processing and maturation of pre-18S ribosomal RNA
in the nucleolus of S. cerevisiae. However, since Utp8p functions in nuclear tRNA export in S. cerevisiae,
we hypothesize that Utp22p may also play a role in the nuclear tRNA export process. The goal of this
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study was to investigate whether Utp22p participates in nuclear tRNA export in S. cerevisiae. Depletion of
Utp22p caused nuclear accumulation of mature tRNAs derived from both intronless and intron-containing
precursors. Utp22p does not bind tRNA directly, but does co-purify with Utp8p, Utp9p, which has recently
been characterized as another nucleolar component that functions with Utp8p in nuclear tRNA export, the
nuclear tRNA export receptor Los1p and the tyrosyl-tRNA synthetase Tys1p. Co-purification with Utp22p
was not observed with the GTPase Gsp1p or Cex1p, which unloads tRNAs from the export receptors at
the cytoplasmic face of the nuclear pore complex. Utp22p interacts directly with Utp8p, Utp9p and Los1p
in vitro, as well as Tys1p in a tRNA-dependent manner. We propose that Utp22p may recruit Utp8p to the
aminoacyl-tRNA synthetases, such as Tys1p, and act together with Utp8p and Utp9p in the translocation
of aminoacylated tRNA from the nucleolus to the nuclear tRNA export receptors.
2980/L086
Gamma (Interferon) Activated Sequences (GAS) in Human Chromosome 21 Genes.
L. E. Maroun, A. Jagadeesh, K. Sampson, H. H. North, B. Robson, A. Tzonkov, R. M. Faugue, D.
Elyaman, S. Sampson, P. Aghajafari, R. Valentino Mackey, R. Chugh; Saint Matthew's University School
of Medicine, Grand Cayman, Cayman Islands
Interferon-gamma (IFN-γ) is associated with demyelization in transgenic animals and Multiple sclerosis
where IFN-γ inhibition is thought to be the underlying mechanism in IFN-beta therapy. One of the two
components of the IFN-γ receptor is encoded by a gene on human chromosome 21 (Ch21) and cells from
people with Down syndrome (Trisomy 21) are supersensitive to IFN-γ activity. We have used online
sequence search engines to find genes on Ch21 that may be modulated by IFN-γ and, thus, contribute to
this supersensitivity. As the Gamma (interferon) Activated Sequence (GAS) consensus sequence is too
general to be useful, we searched Ch21 genes using GAS examples known to be functional in IFN-γ
modulated genes. Although functional GAS sites can be located in unexpected locations, genes chosen
for detailed analysis were from a group containing two or more GAS sites in the vicinity of their
transcription start site (TSS). We found that a number of Ch21 genes met these criteria including genes
previously reported to be under IFN-γ control and genes not previously associated with interferon action.
As an example of the latter, we found that the RCAN1 gene contained a number of different known GAS
sequences close to its TSS. These included the reported IFN-γ control sites for the c-fos(wt), GBP1, and
ICAM-1 genes. The RCAN1 gene is considered to be a candidate for involvement in Down syndrome
neuropathology because both its overexpression and underexpression are associated with altered
neuronal vesicular trafficking. The data suggest that the IFN-γ supersensitivity of trisomy21 cells is due in
part to both increased receptor numbers and the presence of genes on Ch21 that are potentially
modulated by IFN-γ.
2981/L087
Comprehensive RNAi Analysis for the Discovery of Novel UPR-Associated Genes in
Caenorhabditis elegans.
1,2
1
1
2,3 1
K. Sakaki , S. Yoshina , S. Mitani , R. J. Kaufman ; Physiology, Tokyo Women's Medical University,
2
3
Tokyo, Japan, Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI, Internal
Medicine, University of Michigan Medical School, Ann Arbor, MI
Unfolded protein response (UPR) is the intracellular stress signaling to counteract the stress derived from
the accumulation of misfolded proteins in the endoplasmic reticulum (ER), referred as ER stress. The
regulation of UPR signaling has been getting into the spotlight for its tight association with the proliferation
of the secretive tissues and the symptoms in the diseases such as diabetes and neurodegenerative
diseases. Herein, we report the results of our comprehensive RNAi analysis to identify a set of UPRassociated-genes in the nematode Caenorhabditis elegans. In UPR signaling of worms, three of ER
stress sensing proteins (IRE1, PERK and ATF6) and the basic mechanism to activate UPR signaling are
highly conserved. Then, the double deficiency of ire-1 with the either of atf-6 or pek-1 causes the
synthetic lethal and intestinal degeneration at the larval stage. We examined the feeding-mediated RNAi
analysis about 16,757 clones that cover approximately 87% of C.elegans genome. As the result, we
found that 10 genes associated with the regulation of ER function, protein biosynthesis, signal
transduction and RNA metabolism caused the synthetic severe growth defect and intestinal degeneration
MONDAY
by RNAi treatment in ire-1(v33) mutant, but not in atf-6(tm1533) and pek-1(ok275). Especially, we found a
couple of genes concerning RNA metabolism were elevated their basal level expression in ire-1(v33) in
comparison with wild-type and the other UPR mutants. Then, reporter analysis using zcIs4 strain
harboring hsp-4 promoter-driven GFP expression revealed that ER stress was induced by RNAi-mediated
knockdown of these genes. These observations indicate that these genes are required for ER function
homeostasis in the normal development of the worms lacking ire-1 gene. Now, we will present the results
of our latest genetical and cell biological analysis, and hope to discuss about the roles of these genes on
ER function homeostasis.
2982/L088
Nuclear Transport of Dp71.
1
1
1
2
1
M. R. Suarez-Sanchez , P. Gomez , J. A. Hernandez-Ibarra , M. A. Bermudez-de-Leon , B. Cisneros ;
1
2
Genetics and Molecular Biology, Cinvestav-IPN, Mexico, Mexico, Division of Genetics, Molecular
Biology Laboratory, Northeast Biomedical Research Center, IMSS, Monterrey, Mexico
Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin, the DMD gene product. DMD
gene encodes different distrophin isoforms, being Dp71 the main dystrophin in the nervous system; in fact
it is thought that Dp71 is involved in the mental retardation exhibited by 30% of DMD patients. Although
Dp71 is a component of the dystrophin-associated protein complex (DAPC), which is associated to
plasmatic membrane, we have recently reported the presence of Dp71 in the nucleus of different cell
lines; however, the mechanisms regulating its nuclear localization remained to be revealed. In silico
analysis shows that Dp71 has no classical nuclear localization signal (NLS). Therefore, in order to
delineate the nuclear transport pathway of Dp71, we created fusion proteins between different domains of
Dp71 and GFP. By transient transfection of fusion proteins-encoded vectors and confocal microscopy
analysis, we found that the ZZ domain promotes the nuclear import of Dp71. Additionally, by using a
lectin that blocks the nuclear import through the nuclear pore as well as a Ran negative dominant mutant,
we showed that Dp71 nuclear import is mediated by importins in a Ran-dependent manner. As Dp71 is a
cytoskeleton-associated protein, we analyzed whether the integrity of both actin and microtubule
cytoskeleton influences the nuclear transport of Dp71. We found that integrity of microtubules plays an
important role in the nuclear transport of Dp71. In silico analysis revealed several classical nuclear export
signals (NES) in the Dp71 sequence. The use of a Crm1 inhibitor increase the nuclear signal of Dp71,
suggesting the functionality of the NES. We conclude that nuclear transport of Dp71 is mediated by the
importin pathway with additional participation of microtubules and with no involvement of a classical NLS.
Instead, the ZZ domain of Dp71 was mapped as a key regulator of nuclear transport.
2983/L089
Positioning of the Cohesin-Loading Factor NIPBL in the Human Genome Yields Insights in
Cornelia De Lange Syndrome.
1
1
2
2
3
4 1
K. S. Wendt , J. Zuin , J. Bryne , B. Lenhard , W. van Ijcken , J. Peters ; Cell biology, Erasmus Medical
2
Center, Rotterdam, Netherlands, Computational Biology Unit-Bergen Center for Computational Science
3
and Sars Centre for Marine Molecular Biology, University of Bergen, 5008 Bergen, Norway, Biomics
4
Department, Erasmus Medical Center, 3015GE Rotterdam, Netherlands, Institute of Molecular
Pathology, 1030 Vienna, Austria
The cohesin complex is fundamental to ensure genomic stability during cell divisions but it has also
important roles during DNA damage repair and for transcriptional regulation. Genome-wide studies of
cohesin revealed that it localizes together with the chromatin insulator CTCF and to a smaller extend with
other transcription factors. Association of cohesin with chromatin is controlled by the NIPBL/MAU-2
complex and it is still controversial whether cohesin is directly recruited to its binding-sites or re-localizes
from dedicated loading sites. To address this question we analyzed the genome-wide distribution of
NIPBL and cohesin in the diploid human breast epithelial cell line HB2 by chromatin immunoprecipitation
and high-throughput sequencing. We identified more than 3000 specific NIPBL binding-sites, which
localize mostly to the 5’ ends of genes and overlap only to a small extend with cohesin. We analyzed
these binding sites in detail and found that that NIPBL might be important to maintain the activity of these
genes. This has important implications for the human developmental syndrome, Cornelia de Lange
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syndrome, which was linked in more than 50% of the cases to mutations in the NIPBL gene. We will
present our analysis of NIPBL binding-sites in context with the syndrome and show that the expression of
important developmental transcription factors might be controlled by NIPBL-binding.
2984/L090
Nesprins Form Higher Molecular Weight Complexes at the Nuclear Envelope.
1
2
1
2
2
1 1
S. Taranum , W. Lu , S. Neumann , V. Jaeger , I. Karakesisoglou , A. Noegel ; Institute of Biochemistry,
2
Medical Faculty, University of Cologne, Cologne, Germany, School of Biological and Biomedical
Sciences, Durham University, Durham, United Kingdom
Nesprins-1 and -2 are giant tail-anchored nuclear envelope proteins that bind F-actin via their actin
binding domains and connect the nucleus to the actin cytoskeleton. Nesprin-3, a further member of the
Nesprin family, connects the nucleus to the intermediate filament network. Here we report oligomerization
of an N-terminal spectrin repeat of Nesprin-1. We also show direct interaction between Nesprin-1 with
Nesprin-3 in which the actin binding domain and a part of the rod domain of Nesprin-1 can bind to
Nesprin-3. Our findings suggest novel functions for Nesprins by which they form a network through
mutual interactions at the nuclear envelope which interlinks cytoskeletal networks.
2985/L091
Novel Nuclear Import Receptor for Hsp70s Is Required for Attenuation of Heat-Shock Response
and Protecting Cell Damages from Stress.
S. Kose, M. Furuta, N. Imamoto; ASI, Cellular Dynamics Lab., RIKEN, Wako, Japan
Many molecular chaperones are expressed to protect cells against protein damages, which occur in
various cellular compartments leading to lethal effects under environmental stresses. Hsc70/Hsp70
belongs to a major family of molecular chaperones involved in highly conserved protective systems.
Previous studies suggest that the conventional nucleocytoplasmic transport is down-regulated whereas
nuclear import of Hsc70/Hsp70 is up-regulated during stress response. Properties of Hsc70/Hsp70 as
chaperones in protein homeostasis are well investigated, however, their nuclear function and mechanism
of nuclear migration under stress remain elusive. Here we report that a heat-shock-induced nuclear
import of Hsp70s is mediated by a novel receptor, which does not belong to well-characterized members
of importin β family. Using in vitro transport assays, a soluble protein was biochemically purified as an
essential factor for heat-shock-induced nuclear import of Hsp70s. This molecule is an evolutionarily
conserved protein from yeast to human with unknown function. This molexcule binds directly to ATP-form
of Hsc70/Hsp70, but likely dissociate from its ADP-form, indicating the import is coupled with an ATPase
cycle of Hsc70/Hsp70. Importantly, this molecule binds directly to FG-Nups and translocate through the
nuclear pore complex (NPC) by itself, showing characteristic features common to all known nuclear
transport receptors. In living cells, depletion of this molecule potently inhibits heat-shock-induced nuclear
migration of Hsp70s, reduces cell viability after heat-shock, and causes significant delay in attenuation of
heat-shock response. Taken together, our data show that this molecule is a novel nuclear import receptor
for Hsp70s required for attenuation of heat-shock response and protecting cell damages from the stress.
We thus propose to name this novel receptor, “Hikeshi”, a traditional Japanese compound word for
trouble-shooter, fire fighter or smoke jumper.
2986/L092
MNK1 Regulates the Phosphorylation and the Subcellular Distribution of hnRNP A1 in Senescent
Fibroblast Cells.
S. Ziaei, N. Shimada, H. Kucharavy, K. Hubbard; Department of Biology, The City College of New York,
New York, NY
Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is an RNA-binding protein that modulates
splice site usage, general splicing factors, polyadenylation, and cleavage efficiency. The protein has also
been implicated in mRNA stability and transport from the nucleus. We have previously demonstrated that
hnRNP A1 has diminished protein levels and shows cytoplasmic accumulation in senescent human
diploid fibroblasts. Furthermore, we have shown that the altered protein expression and localization of
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hnRNP A1 was affected by inhibition of p38 MAPK activity, which plays key roles in senescence and in
stress response. In this study, we explored molecular mechanisms responsible for the regulation of
hnRNP A1 downstream of p38 MAPK. We show that inhibition of MNK1, a substrate for p38 MAPK,
activity with CGP 57380 decreased the phosphorylation levels of hnRNP A1 in protein in young cells and
blocked the cytoplasmic accumulation of the protein in senescent fibroblast cells. MNK1 formed a
complex with hnRNP A1. The expression levels of MNK1, phospho-MNK1, and phospho-eIF4E proteins
were also elevated as a consequence of aging. These data suggest that MNK1 regulates the
phosphorylation and the subcellular distribution of hnRNP A1 and that MNK1 may play a role in the
induction of senescence.
2987/L093
The Structural and Functional Analysis of the Orc6 Protein.
1
2
2
1 1
M. Balasov , S. Liu , Y. Liu , I. Chesnokov ; Biochemistry and Mol Genetics, University of Alabama,
2
Birmingham, AL, Institute of Biophysics, Beijing, China
The Origin recognition complex (ORC) is a six-subunit protein important for the DNA replication in
eukaryotic cells. ORC is also involved in other cell functions. The smallest metazoan ORC subunit, Orc6,
consists of two functional domains. Larger N-terminal domain directly involved in DNA binding and is
important for DNA replication. Smaller C-terminal domain is important for cytokinesis. The structural
analysis revealed that the N-terminal domain of both Drosophila and human Orc6 has an overall fold
similar to the transcription factor TFIIB. A model of Orc6 binding to DNA is produced. Amino acids of Orc6
which are directly involved in DNA binding are identified. Alterations of these amino acids abolish DNA
binding ability of Orc6 and also result in reduced levels of DNA replication in vitro and in cultured cells.
Furthermore, we have shown that the expression of human Orc6 in Drosophila Orc6 mutant cells rescued
DNA replication. The ability of human Orc6 protein to support DNA replication in Drosophila cells
indicates that two proteins are homologous in replication function and also provide an opportunity for
further molecular dissection of human Orc6 in vivo, using Drosophila as a model system. We propose that
Orc6 is a DNA binding subunit of ORC and may position ORC at the origins of DNA replication similar to
the role of TFIIB in positioning transcription pre-initiation complex at the promoter.
2988/L094
Chromatin Signatures of Gene Promoters Associated with Cell Identity in Bone Marrow Derived
Multipotent Stromal Cells during In Vitro Expansion.
P. J. Lynch, K. Steers, B. Stultz, D. Hursh; CBER, FDA, Bethesda, MD
Multipotent mesenchymal stromal cells (MSCs) are attractive candidates for cell-based therapies due to
their multi-lineage differentiation potential and their capacity to modulate the host immune response.
Currently the use of MSCs in clinical settings will require culture amplification, which may introduce
biological variability. Considerable evidence has emerged suggesting that dynamic, specialized chromatin
structures play an important role in determining cell identity and fate. In particular, gene promoters are
often packaged into domains of transcriptionally active, silenced, or poised chromatin. In this study,
chromatin immunoprecipitation (IP) was used to assess whether time in culture altered the chromatin
structure at gene promoters important to cell identity and function of human bone marrow-derived MSCs.
As expected, in early passage MSCs cell surface markers CD73 and CD105, which are expressed in the
undifferentiated state, were highly enriched in active chromatin structures. Tissue-specific gene
promoters along the adipogenic (LPL, PPARG2, FABP4) and osteogenic (SPP1) were found to be
enriched in both active and silenced chromatin structures, suggesting that these promoters are either
poised, or heterogeneous in the MSC population. Furthermore, the IDO1 gene, which has been
implicated in the immunomodulation function of MSCs, was enriched in both active and silenced
chromatin structures. The cyclin dependent kinase inhibitor 2A (p16INK4A), whose expression is
positively correlated with replicative senescence, was also associated with active and silenced chromatin
markings. The chromatin structures assayed did not change significantly between early and middle
passage cell cultures. In conclusion, these data are consistent with an MSC culture epigenetically poised
to differentiate along the osteo- and adipogenic lineages. Importantly, these structures were maintained
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between two different passages, implying that MSCs can be amplified in culture to some degree without
significantly altering their epigenetic identities at key gene promoters.
2989/L095
Identification of Rox1 RNA Sequences That Contribute to Drosophila X Chromosome Recognition.
Y. Kong, V. H. Meller; Department of Biological Sciences, Wayne State University, Detroit, MI
roX RNAs are involved in the chromosome-wide gene regulation that occurs during dosage compensation
in Drosophila. Dosage compensation equalizes expression of X-linked genes in males and females. In
Drosophila, this occurs by two-fold up-regulation of transcription from the single male X chromosome.
This is mediated by the Male Specific Lethal (MSL) complex, composed of five proteins and the two noncoding RNA on the X (roX) RNAs, roX1 and roX2. Elimination of any of these proteins, or simultaneous
mutation of both roX genes, is male lethal during the third larval instar. The MSL complex binds to
transcribed X-linked genes, where it modifies chromatin to facilitate expression. roX RNAs are required
for X-localization of the MSL complex. Integration of roX into the MSL complex enables binding to X
chromatin. Intriguingly, both roX genes are X-linked, and capable of recruiting MSL binding to chromatin
near sites of roX transcription. Over 1 kb at the 5' end of roX1 functions in X-localization. Deletion of
portions of this region show no phenotype, but elimination of the entire 5' end destroys MSL Xlocalization, suggesting multiple redundant elements. To investigate the function of the 5' end of roX1, I
created roX1 transgenes containing different fragments from this region. All of these transgenes partially
rescue male survival and MSL recruitment to the X chromosome, but only one 5' roX1 fragment directs
MSL binding to chromatin flanking the site of transcription. I conclude that the 5' end of roX1 has some
redundancy, but also contains regions with unique activity. Intriguingly, roX1 has many transcription start
sites that are developmentally regulated. Recruitment to chromatin flanking the transcription site is
promoted by a part of roX1 that is absent in most roX1 transcripts, suggesting that differential regulation
of the roX1 transcription start site may be important for X-recognition.
2990/L096
Functional Screening for Genes That Regulate Androgen Signaling in Androgen Deprived Prostate
Cancer Cells.
1
1
1
2
3
2
1
1
E. Levina , M. Baig , P. Ohouo , S. Carmack , T. Harkins , Y. Ding , I. Roninson , R. Buttyan , M.
1 1
2
Shtutman ; Ordway Research Institute, Albany, NY, Wadsworth Center, New York State Department of
3
Health, Albany, NY, 454 Life Sciences, a Roche company, Branford, CT
Background and objectives: We have enzymatically generated libraries of Transdominant Genetic
Inhibitors (TGI), including short hairpin RNA (shRNA) inducible lentiviral vectors. Our goals were to use
these libraries to identify novel gene targets that regulate androgen receptor (AR) activity in the absence
of androgen. Brief methodology description: Cells transduced with shRNA libraries are selected for the
desired phenotype. Massive parallel sequencing of shRNAs recovered from transduced cells before and
after selection is followed by identification of genes that give rise to shRNAs enriched by selection. A
shRNA libraries prepared from cDNA of breast or prostate cancer cell lines were screened for altered
expression of fluorescent reporters from the androgen-responsive probasin promoter in PC cells growing
in androgen-depleted media. Phenotypic effects of genes targeted by shRNAs that were most highly
enriched by selection were individually validated by siRNA transfection. Results: shRNA selection was
used to identify genes that regulate androgen receptor activity in PC cells cultured in androgen-depleted
media. Genes targeted by enriched shRNAs were individually analyzed by siRNA transfection, measuring
the expression of endogenous androgen-responsive genes. Knockdown of >30 genes activated or
inhibited androgen-responsive genes under hormone-free conditions. Knockdown of some of these genes
also stimulated LNCaP cell growth in androgen-free media. The strongest effects on activation of
androgen receptor were observed with IGSF8, a negative regulator of tetraspanin, knockdown of which
activated AR, and with CD9, member of tetraspanin family, knockdown of which inhibited AR.
Conclusions: We have identified new genes androgen receptor activity. Of special interest, inhibition
member of tetraspanin family protein CD9 and IGSF8 (EWI-2), CD9 binding protein that significantly
affects androgen receptor activity.
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2991/L097
Molecular Population Genetics of Inversion Breakpoint Regions in Drosophila pseudoobscura.
A. Wallace; the Pennsylvania State University, State College, PA
Inversions are common in numerous organisms and have been linked to several diseases in humans.
Inversions can have a profound effect on the pattern and organization of nucleotide variability because
they affect levels of nucleotide polymorphism. Consequently, chromosomal inversions may alter gene
order and can result in the elimination of genes or the introduction of new genes. The pattern and
organization of variability on the chromosome can provide a means of understanding some genetic
mutations and the diseases associated with them. The 3rd chromosome of Drosophila pseudoobscura
has been shown to have more than 30 gene arrangements resulting from paracentric inversions and is
therefore a dependable model system to study variability. This study examined nucleotide diversity of 11
genetic markers near six pairs of inversion breakpoints to test hypotheses about the molecular
evolutionary genomics of various chromosomal arrangements in D. pseudoobscura. Approximately 100
3rd isochromosomal lines of D. pseudoobscura collected from four populations in the Southwestern
United States were sequenced for each marker. To determine the impact of marker position on variability,
nucleotide heterozygosity was estimated at each breakpoint region based on the neutral mutation
parameter. Nucleotide heterozygosity was different for each breakpoint region and the level of
heterozygosity within each arrangement varied for every marker. Overall, markers closer to the distal
segment of the chromosome had higher levels of nucleotide heterozygosity than markers within the
proximal segment. Also, our results rejected the hypothesis that newer inversions have lower levels of
nucleotide variability near the breakpoints than within the inverted segment. Intra- and inter-locus linkage
disequilibrium (LD) was estimated to assess whether the inversion leads to an elevation of LD and to test
whether sites in breakpoint pairs are in LD. High levels of LD were observed within all 11 regions and also
between most breakpoint pairs. The central region of the chromosome had the highest levels of LD
compared to the proximal and distal regions because this is the region that experiences the highest level
of recombination suppression.
2992/L098
Chromatin Organization during Yeast Sporulation.
1,2
2,3
1,2 1
L. Zhang , H. Ma , B. Pugh ; Center for Eukaryotic Gene Regulation, Pennsylvania State University,
2
University Park, PA, The Integrative Biosciences Program in Cell and Developmental Biology,
3
Pennsylvania State University, University Park, PA, Biology, Pennsylvania State University, University
Park, PA
The plasticity of chromatin organization as chromosomes undergo a full compendium of transactions
including DNA replication, synapsis, recombination, chromatin compaction, and changes in transcription
during a developmental program is unknown. We generated genome-wide maps of individual nucleosome
organizational states (positions, occupancy, modifications) in Saccharomyces as they progressed through
these stages. The resolution of the study was sufficiently high to definitively determine that the
chromatin’s primary organizational state remained essentially fixed at all times. Targeted changes did
occur. An acute starvation response involved repositioning of nucleosomes over transcription start sites,
whereas a developmental program (gametogenesis) involved antisense control. The remarkable
constancy of the genome extended to histone modification states, where three-quarters of all genes, most
of which are rarely transcribed, contained position-specific “activating” marks. The marks appear to be
positional subfeatures of most transcription units, where they might convey positional information that
includes distance from the transcription start site and direction of transcription. Our findings suggest that
many features of genomic chromatin organization are constant and common, but tweaked in a programspecific manner.
2993/L099
Oxygen Regulates Expression of Fibronectin and αvβ3 Integrin in Fetal Rat Lung Fibroblasts.
1,2
1,2
3
2,3
1
1,2 1
A. DeCoux , A. Menard , J. Chaplin , J. Benjamin , G. Wilson , S. A. Gebb ; Cell Biology and
2
Neuroscience, University of South Alabama, Mobile, AL, Center for Lung Biology, University of South
3
Alabama, Mobile, AL, Neonatology, University of South Alabama, Mobile, AL
MONDAY
Fibronectin (FN) is an extracellular matrix (ECM) protein that plays a key role in lung alveolarization. In
the preterm infant, exposure to increased levels of oxygen (O2) disrupts alveolarization. The increased
oxidative stress likely targets alveolar fibroblasts, leading to altered ECM production. However, it is
unclear whether O2 tension directly regulates ECM expression in these cells as they transition from the
fetal to the air-breathing lung or in the preterm lung exposed to hyperoxia. We tested the hypothesis that
O2 regulates the synthesis and deposition of both FN and its cellular receptor integrin αvβ3 in primary
cultures of fetal lung fibroblasts. Lungs from fetal day-18 Sprague-Dawley rat pups were isolated, minced,
digested in 1% collagenase-I in HEPES buffered DMEM, and mechanically dispersed. Fibroblasts were
purified from the cell suspension by differential adherence and cultured for 24 or 48 hrs at O2 tensions
representing fetal (3%), adult (21%), or supplemental (65%) O2. Protein and mRNA expression of FN and
the αv and β3 integrin subunits were quantified by western and RT-PCR. Basal levels of FN mRNA and
protein were detected in fibroblasts cultured at fetal O2 tension. When transitioned to adult O2 tension, FN
mRNA and protein increased. In contrast, exposure to hyperoxia elicited a marked decrease in FN mRNA
and protein in these cells. Similarly, the protein expression of both the αv and β3 integrin subunits were
increased at adult O2 tension and decreased in hyperoxic cultures. While a decrease in the protein
expression of the αv subunit was observed in cells cultured in hyperoxic conditions, it was less dramatic
than the change seen in the β3 subunit. These data demonstrate that O2 regulates expression of FN and
its receptor in primary fetal lung fibroblasts. During the normal transition to adult O2 tension, FN and
integrin αvβ3 expression increased. Conversely, pathologic levels of O2 decrease expression of these
ECM components. Future studies will investigate the mechanism through which O2 regulates the
synthesis and depositon of fetal lung FN and its role in physiologic and pathophysiologic alveolarization.
2994/L100
Decision Making at a Subcellular Level Determines the Outcome of Bacteriophage Infection.
1
1
1
2
2
1,3 1
L. Zeng , S. O. Skinner , C. Zong , J. Sippy , M. Feiss , I. Golding ; University of Illinois at Urbana2
3
Champaign, Urbana, IL, University of Iowa, Iowa City, IA, Baylor College of Medicine, Houston, TX
When the process of cell-fate determination is examined at single-cell resolution, it is often observed that
individual cells undergo different fates even when subject to identical conditions. This “noisy” phenotype
is usually attributed to the inherent stochasticity of chemical reactions in the cell. Here we demonstrate
how the observed single-cell heterogeneity can be explained by a cascade of decisions occurring at the
sub-cellular level. We follow the post-infection decision in bacteriophage lambda at single-virus resolution,
and show that a choice between lysis and lysogeny is first made at the level of the individual virus. The
decisions by all viruses infecting a single cell are then integrated in a precise (noise-free) way, such that
only a unanimous vote by all viruses leads to the establishment of lysogeny. By detecting and integrating
over the sub-cellular “hidden variables”, we are able to predict the level of noise measured at the singlecell level (Zeng et al., 2010). Zeng, L., Skinner, S.O., Zong, C., Sippy, J., Feiss, M., and Golding, I.
(2010). Decision making at a subcellular level determines the outcome of bacteriophage infection. Cell
141, 682-691.
2995/L101
Genetic Mosaicism among iPSC-Derived Human Neurons.
1,2
1
3
1
1,2
3
1
M. J. McConnell , K. J. Brennand , T. Voet , A. Mahendran , J. C. Piper , J. Vermeesch , F. H. Gage ;
1
2
Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, CA, Crick-Jacobs Center for
3
Theoretical and Computational Biology, Salk Institute for Biological Studies, La Jolla, CA, Center for
Human Genetics, Katholieke Universiteit, Leuven, Belgium
Genetic mosaicism is the presence of more than one version of an individual’s genome within that
individual (i.e. somatic mosaicism), or within cell lines derived from a single individual. Retrotransposition
by L1 elements creates neuron-to-neuron genetic differences in human brains; however, the diversity and
prevalence of unique genomes is unknown. Moreover, the relationship between retrotransposition and
other genomic changes (e.g. aneuploidy, copy number variation) is unexplored; limited by the necessity of
single genome analysis and the availability of acutely isolated human neurons. We circumvent these
limitations, and leverage the clonality inherent in human induced pluripotent stem cell (hiPSC) derivation,
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by first amplifying single hiPSC-derived neuronal genomes, then using quantitative PCR and microarray
assays to identify genomic differences in individual human neurons. Most of the iPSC-derived neurons
analyzed here had unique genomes.
2996/L102
The Drosophila CK1 Gamma Homolog, Gilgamesh, Controls Tissue Morphogenesis through
Regulation of Vesicle Trafficking.
W. Gault, U. Weber, M. Mlodzik; Developmental and Regenerative Biology, Mount Sinai School of
Medicine, New York, NY
In addition to apicobasal polarity, epithelia are polarized along a perpendicular plane through a
phenomenon referred to as Planar Cell Polarity (PCP). The PCP pathway coordinates tissue orientation
in vertebrates and invertebrates by defining a polarity axis through the asymmetric localization of a set of
core proteins, including the frizzled (fz) receptor. In the Drosophila wing, PCP is evident in the
establishment of a single distally oriented hair (trichome) from each wing cell. While a bias in Fz transport
along microtubules has been proposed as a mechanism for trichome site selection along the proximodistal axis of wing cells, the mode by which a single trichome is refined within the distal vertex is not well
understood. From a modifier screen to identify PCP regulators, we have identified and characterized the
function of the Drosophila Casien Kinase 1 γ (CK1γ) homolog, gilgamesh (gish), in the regulation of wing
hair morphogenesis. gish is conserved between species and orthologs in yeast have been described to
affect bud morphogenesis and may regulate polarized cell growth through an effect on membrane
trafficking. The phenotype of gish included multiple distally oriented trichomes, and genetic interactions
with gish and the PCP effector multiple wing hairs (mwh) suggests a genetically redundant relationship.
Localization studies identified Myc-Gish at the plasma membrane, within intracellular puncta and enriched
around the base of the developing trichome. Furthermore, our analysis also resulted in a strong genetic
interaction between gish and the Rab11 GTPase. In gish mutant wings, Rab11 immunostaining revealed
abnormal recycling endosomal localization during trichome formation. Overall, data presented here
support a model by which gish, through regulation of Rab11 localization, effects vesicle recycling to
promote single trichome formation.
2997/L103
IGF1 Role in VEGF-Dependent Angiogenesis of Bovine Retina Endothelial Cells (BRECs).
1,3
1,3
2,3 1
J. Tachjadi , J. F. Blaize , W. J. Lamoreaux ; Biology, The Graduate Center - City University of New
2
York, New York, NY, Advanced Imaging Facility, College of Staten Island - City University of New York,
3
Staten Island, NY, Biology, College of Staten Island - City University of New York, Staten Island, NY
Retinopathy of prematurity (ROP) has abnormal temporal and spatial regulations of VEGF and IGF1
resulted in abnormal retinal neovascularization which requires the activation of receptor tyrosine kinases
(RTKs) by VEGF and IGF-1 ligands. Signaling proteins of activated RTK (Akt/PKB, MEK/ERB, IP3, DAG,
and Src) are promoting cell survival, cell proliferation, vascular permeability and cell migration. The goals
of this study are to demonstrate in vitro tube formation assay model of ROP using BREC; and to
determine the signaling proteins involved for angiogenesis affected by VEGF and IGF1 synergy. BREC
primary cultures were seeded to collagen mixture gel containing bovine collagen, fibronectin, laminin and
RPMI medium on well plate and let it grow for 3 days; after aspirating the medium and add another layer
of gel on top of the cells for 1 hour in the incubator, adding growth medium for 24 hours. Serum free
medium with VEGF, IGF1 or both were added to the wells under normoxic or hypoxic incubator
environment and photographs were taken for 48 - 72 hours. In separate experiments, specific signaling
proteins (MAPK or Akt/PKB) were inhibited, treated with same treatments under normoxic or hypoxic
culture environment and the effects on angiogenesis were observed with time-lapse microscopy,
confirmed with conditioned media’s MMP2 and MMP9 quantified using ELISA; VEGF receptor (VEGFR2)
upregulation was also observed using confocal microscopy. The tube formation assay showed the
decrease in the number of branch formations and length of tube with signal protein blockers compared to
control. This study demonstrates the crucial roles of IGF1, and the signaling proteins involved in VEGFdependent angiogenesis of BREC.
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2998/L104
Notch Signaling Regulates Late-Stage Epidermal Differentiation and Postnatal Hair Cycle
Homeostasis.
1
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2
4
1 1
H. Lin , C. Kao , K. Lin , V. Kaartinen , L. Yang ; Institute of Cellular and System Medicine, National
2
Health Research Institutes, Miaoli County, Taiwan, Division of Medical Engineering, National Health
3
Research Institutes, Miaoli County, Taiwan, Center of General Education, Chang Gung University,
4
Taoyuan, Taiwan, Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor, MI
Notch signaling regulates a variety of processes such as differentiation, proliferation, apoptosis, and cell
fate choice. Ligand binding leads to conformational changes in the Notch receptor and leads to NICD
release from the membrane. NICD translocates into the nucleus and binds to Rbpj and Mastermind,
thereby activating downstream target genes. Notch signaling is modulated by glycosylation of the
extracellular domain of Notch receptors and O-fucose modification of mammalian Notch receptors by
Protein O-fucose transferase-1 (Pofut1) is required for efficient ligand-receptor binding and subsequent
signal transduction. Multiple Notch receptors and ligands are expressed in the epidermis and hair follicle
during embryonic development and adult stage. Although Notch signaling has been shown to regulate
differentiation of the epidermis and hair follicles, it remains unclear how Notch signaling participates in
late-stage epidermal differentiation and postnatal hair cycle homeostasis. Here, we conditionally
inactivated Notch signaling by deletion of Rbpj and Pofut1 using Tgfb3-Cre which induces gene
recombination in hair follicle lineages and suprabasal layer of the epidermis. Rbpj conditional knockout
(RbpjcKO) mice displayed an earlier onset of epidermis and hair follicle defects than Pofut1 conditional
knock (Pofut1cKO) mice. RbpjcKO mice exhibited granular parakeratosis early in life and Pofut1cKO mice
developed defects in granular layer differentiation later in life. Compared with RbpjcKO mice which did not
develop pelage after birth, Pofut1cKO mice displayed epidermal hyperplasia, progressive hair loss, and
epidermal cysts in the second hair cycle. Pofut1 deletion in follicular lineages resulted in fewer bulge stem
cells and a concomitant increase of K14-positive keratinocytes in the isthmus. Interestingly, Pofut1cKO
hair follicles displayed delayed anagen progression and dysregulation of proliferation and apoptosis
during the hair cycle transition. Taken together, our data demonstrate that Notch signaling has roles in
maintaining tissue homeostasis of the skin.
2999/L105
FGF Control of Basal and Apical Cytoskeletal Activities during Inner Ear Invagination.
X. Sai; sensory development, riken kobe institution, Kobe, Japan
The inner ear contains the sensory organs for the hearing and balancing and is embedded within the
cephalic mesoderm of the embryo. However it is induced by fibroblast growth factor signals as an
epithelial placode in the surface ectoderm adjacent to the posterior hindbrain. In the chick, the inner ear
placode first becomes morphologically apparent as a thickening of the ectoderm. It then gradually
invaginates in a two-step process to form an otocyst within the head mesenchyme. Initial invagination is
driven by basal expansion. We had previously showed that basal fibroblast growth factor (FGF) signalling
acting through phospholipase Cγ activates basal myosin II. Myosin II exhibits a non-canonical activity that
results in the local depletion of actin filaments and thus causes the basal expansion of the otic placode.
Subsequent to basal expansion of the inner ear placode, apical constriction serves to complete
invagination. We find that Cdc42 is localized apically and that it is necessary to drive the purse-string
apical actin contractions that drive later inner ear invagination. Thus, FGF signalling exerts profound
cytoskeletal effects on otic cells, coordinating the morphogenesis of the inner ear. It exerts a direct, nontranslational effect on the basal cytoskeleton and likely, a translational effect on apical actin constriction.
3000/L106
Epigenetic Memory in Induced Pluripotent Stem Cells.
1
2
1
3
4
3
2
1 1
A. Doi , K. Kim , H. Ji , L. Ehrlich , M. J. Aryee , I. L. Weissman , G. Daley , A. P. Feinberg ; Center for
Epigenetics and Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD,
2
Division of Pediatric Hematology/Oncology, Children’s Hospital Boston and Howard Hughes Medical
3
Institute, Boston, MA, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University
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4
School of Medicine, Stanford, CA, Department of Biostatistics, Johns Hopkins Bloomberg School of
Public Health, Baltimore, MD
Induced pluripotent stem cells (iPSCs) are generated by the epigenetic reprogramming of somatic cells
by the ectopic expression of a small number of transcription factors. Here we used Comprehensive Highthroughput Array-based Relative Methylation (CHARM) analysis, which interrogates ~4.6 million CpG
sites including almost all CpG islands and nearby sequences termed shores, to assess the methylome of
stem cells derived by transcription-factor-based reprogramming and somatic cell nuclear transfer. We
show that low-passage iPSCs from adult murine tissues retain DNA methylation signatures characteristic
of their tissue of origin, directing their differentiation towards their donor cell. Comparing DMRs in bloodderived iPSCs to those that define hematopoietic progenitors identified in a study using highly purified
multipotent and lineage-specific hematopoietic progenitors, we observed that iPSCs derived from Kit1,
lineage-negative myeloid marrow precursors (B-iPSCs) cluster alongside common myeloid progenitors
(CMPs), while B-lymphocyte-derived iPSCs (Bl-iPSCs) cluster with progenitors of the lymphoid lineage
(CLPs). Moreover, at hematopoiesis-related loci such as the Gcnt2 gene, which is hypermethylated and
transcriptionally silent in the lymphoid lineage, the lymphoid-derived Bl-iPSCs showed hypermethylation
relative to ESCs while the myeloid-derived B-iPSCs did not. Thus, the residual DNA methylation signature
can discriminate the precise lineage for blood derivatives, further supporting the phenomenon of
epigenetic memory in iPSCs. In contrast, the methylome and differentiation propensity of stem cells
derived by nuclear transfer were more similar to classical ESCs than were iPSCs. Our data indicate that
reprogramming by nuclear transfer establishes the pluripotent ground state more readily than
transcription-factor-based reprogramming, which can leave an epigenetic memory characteristic of its
tissue of origin.
3001/L107
The Secreted Frizzled Related Protein 1 Regulates Wnt Signaling in C. elegans.
M. Harterink, T. Doan, T. Middelkoop, H. Korswagen; Hubrecht Institute, Utrecht, Netherlands
The Wnt family of secreted signaling proteins is responsible for important developmental and homeostatic
processes throughout the animal kingdom. Wnts often play important roles in anteroposterior patterning,
where Wnts are predominantly expressed in the posterior (Petersen and Reddien, 2009). Interestingly,
secreted Wnt inhibitors are often expressed in the anterior, and are proposed to form a counteracting
gradient to carefully regulate signaling. The importance is illustrated by the fact that defects in secreted
Wnt inhibitors are often associated with cancer as well as other diseases. The Secreted Frizzled Related
Proteins (SFRPs) are the largest family of secreted Wnt inhibitors. They contain a domain similar to the
Wnt binding domain of the Wnt receptor Frizzled and therefore were quickly recognized as an
extracellular Wnt antagonists. However, SFRPs have also been proposed to inhibit Wnt signaling the
level of the Wnt receptor. Furthermore a report from Mii and Taira (2009) suggest that SFRPs can
enhance the diffusion of Wnt ligand and expand their signaling range, when ectopically expressed in the
Xenopus embryo. Since C. elegans only has one sfrp gene, it represents the perfect model to carefully
analyze SFRP function in Wnt signaling as well as its effect on Wnt gradient formation.
3002/L108
The Role of p21 in Precise Control of Proliferation of Mouse Hair Follicle Stem Cells.
J. Lee, K. C. Lilja, B. S. White, T. Tumbar; Molecular Biology & Genetics, Cornell University, Ithaca, NY
P21 (Cdkn1a) is a cyclin-dependent kinase inhibitor that regulates cell cycle progression at G1-S
transition. P21 also plays an important role in maintaining mouse hematopoietic and neural stem cells by
preventing their over-proliferation and exhaustion. In mouse skin, a homozygous null p21 mutation results
in increased skin tumor susceptibility together with hyper-proliferation and increased clonogenecity of in
vitro epithelial-derived keratinocytes in short-term assays. However, the cellular dynamics in the absence
of p21 during normal skin homeostasis as well as its role in long-term stem cell maintenance is not fully
understood. Interestingly, our data suggest that hair follicle stem cells show increased proliferation in the
absence of p21 during the normal hair cycle by failure to enter quiescence in a timely manner. Increased
apoptosis seems to compensate for over-proliferation in the niche, to maintain a normal pool size. In
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addition, p21 knockout hair follicle stem cells lose their stem cell marker CD34 during early anagen, when
these cells are known to self-renew. Moreover, when removed from their niche, p21 knockout hair follicle
stem cells show altered ability to proliferate and form colonies in short-term assay and a differential
HDAC-mediated proliferation response. Our data provides new insights in the cellular dynamics regulated
by p21, and suggests that its level and activity in regulating stem cell self-renewal is more complex than
previously recognized.
3003/L109
BMP4/Noggin Contribute to Abnormal Neurogenesis in the Adult Hippocampal of APPswe/PS1 E9
Transgenic Mouse Model of Alzheimers Disease.
1
2 1
J. Tang , H. Xu ; Division of Developmental Biology, Cincinnati Children's Hospital Medical Center,
2
Cincinnati, OH, Center for Nuclear Receptors and Cell Signaling, University of Houston, Houston, TX
[Introduction:] DG area of hippocampus in the brain is where neurogenesis process continues into
adulthood. Perturbation of neurogenesis can alter hippocampal function. Bone morphogenetic protein-4
and its antagonist Noggin are crucial to both embryonic development and the adult nervous system.
However, it is not known whether BMP4 /Noggin could contribute to the regulation of abnormal
neurogenesis in APPswe/PS1DeltaE9 dtg mouse models. [Methods:] BrdU labeled is used for the
analysis of adult hippocampal neurogenesis. Pathological and behavioral test were also applied. In situ
hybridization and immonuhistochemistry are used for the expression of BMP4/Noggin in hippocampus at
mRNA and protein levels,and the Noggin/Fc chimera were injected into lateral ventricle to investigate the
fuction of Noggin in the proliferation and differentiation of NSCs in hippocampus. [Results:] 1) BrdUlabelled cells in DG of 9- and 12-month-old dtg(+/+) mice was markedly reduced compared with agematched dtg(-/-) littermates. There exist significant negative correlation between the BrdU cells and Abeta
plaques in the brain. 2) higher expression of BMP4 and lower expression of Noggin are proved to be age
dependent,and negatively correlated with each other. 3) decreased neurogenesis in AD dtg mice was
significantly correlation with the increased expression of BMP4 and decreased expression of Noggin at
both mRNA and protein levels; 4) Intraventricular administration of a chimeric Noggin/Fc protein resulted
in a significant increase in the number of BrdU-labeled cells and the NSC-derived differentiated cells in
APP/PS1 mice.
3004/L110
Using Video Bioinformatics Tools to Study Stem Cell Dynamics.
2,1
2,1
2,1 1
S. Fonteno , S. C. Lin , P. Talbot ; Cell Biology & Neuroscience, University of CA, Riverside, CA,
2
Stem Cell Center, Unviersity of California, Riverside, CA
Our purpose was to develop a method for rapidly assaying dynamic cellular endpoints of human
embryonic stem cells (hESCs) using video bioinformatics (VB) tools coupled with the novel video
acquisition and cell incubation technology. Our progress relied on both the principles of VB, wherein data
from microscopic time lapse imagery is automatically extracted, processed, and analyzed for endpoint
parameters and on the ability to grow hESCs in a stable environment that enables video imaging. To
develop and demonstrate a robust tool for quantification of a biological process, we observed the
dynamics of hESCs colony growth. Our initial steps in development of the assay were done using control
data collected from hESCs plated on Matrigel coated 12-well plates that were imaged at 7minute intervals
in a BioStation CT for 48 hours at 37C and 5% CO2. We utilized CL-Quant software to develop a VB
algorithm to automate extraction of data pertinent to colony growth. The algorithm included 3 subset
routines: (1) initial segmentation code that utilized a channel mask to separate the colony and
background, (2) an enhancement code that filtered out any remaining image noise (e.g. cellular debris),
and (3) a measurement code that quantified the number of pixels in each colony. The subsets were
applied in sequence to video data of individual hESC colonies collected over 48 hours. To ensure
accuracy of the VB data, the same control colonies were manually analyzed using Adobe Photoshop, and
the subsequent data were compared with that of the automated CL-Quant analysis. The data obtained
from both methods were virtually identical, indicating the VB analysis was accurate. Analysis of control
data with VB showed that the rate of colony growth is independent of initial colony size. These initial steps
in the development and calibration of a stem cell dynamics assay using fast and reliable VB methods
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demonstrates the potential for this colony growth assay and is the first step toward future development of
automated assays for efficient quantitative analysis of other dynamic processes including cell migration,
attachment, proliferation, and apoptosis.
3005/L111
Probing the Molecule Mechanism for Symmetry Breaking in Mammalian Meiotic Oocytes.
H. Li; Stowers institute for medical research, Kansas City, MO
Oocyte meiotic divisions are highly asymmetric, and Fmn2 plays an essential role in the initiation of
symmetry breaking in mouse meiotic oocytes. In order to explore the molecules which are involved in the
symmetric breaking process, we obtained proteomic profiles from different maturation stages of oocytes;
established a mouse oocyte cDNA library; and applied two hybrid system screening. Through
immunoprecipitation and confocal microscopy approaches, our primary results suggest that some
molecules from ribosome biogenesis, vesicle traffic, and cytoplasmic mRNA decapping processes are
associated with symmetric breaking. We are investigating how these molecules directly contribute to
symmetric breaking by using various functional analyses.
3006/L112
Effects of Boar Semen Separation Technique on Sperm Telomere Length.
T. Han, J. Kim, H. Park, Y. Hwang, D. Kim; Biological Science, Gachon University of Medicine & Science,
Incheon, Republic of Korea
Telomere length is considered a new parameter of semen quality, and of important value as predictor of
artificial insemination (AI)-success rate. Sorting highly motile spermatozoa is one of the way to increase
the AI-success rate, which contains swim-up and density gradient separate. Telomeres are DNA
segments made up of short guanine-rich repeats that are manifold kilobases long at the chromosomes
end. Here we investigate optimal way to reduce telomere damage by using diverse separation technique
and measure each sample telomere length by quantitative PCR (qPCR). Boar semen aliquots used to
evaluate techniques were treated by the following ways: centrifuge (600 x g for 10 min) and washed on
media, swim-up in washed sperm pellet, density gradient separation, density gradient followed by swimup and measure telomere length by qPCR with control samples. Our study can find how to increase AIsuccess rate in livestock industry and develop separation technique for clinical use.
3007/L113
Evaluation of Boar Sperm ROS Concentration and Apoptosis Levels According to Freezing
Method.
H. Park, J. Kim, T. Han, Y. Hwang, D. Kim; Biological Science, Gachon University of Medicine & Science,
Incheon, Republic of Korea
It is generally accepted that sperm apoptosis levels and reactive oxygen species (ROS) concentration are
very important in artificial insemination (AI) because these factors affect to fertility. Also sperm ROS and
apoptosis levels reflect in sperm motility and quality because these factors are mainly influenced by
sperm mitochondrial dysfunctions. The aim of this study is to investigate the relevance among boar sperm
ROS concentration, apoptosis and freezing method. Each aliquot is stored in the following ways; slow
frozen with several buffer, snap frozen by liquid nitrogen, kept on ice and kept at room temperature. After
thawing out these samples, we measure ROS concentration and apoptosis levels of each sample using
FACS. These results suggest how much affect sperm ROS concentration and apoptosis levels by
freezing methods. And it can be useful to confirm that which factor is more sensitive by comparison.
3008/L114
Generation of Conditional Null Allele for PLC-γ1 in Mice.
1,2
3
3
3
3
1
2,1 1
Y. Yang , K. Hamada , K. Kawahara , M. Nishio , A. Suzuki , S. Ryo , P. Suh ; POSTECH, Pohang,
2
3
Republic of Korea, UNIST, Ulsan, Republic of Korea, Kyushu University, Fukuoka, Japan
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Phosphoinositide-specific phospholipase C is an enzyme that hydrolyzes phosphatidylinosito 4,5bisphosphate(PIP2) to generate 2nd messengers, inositol-1,4,5-triphosphate and diacylglycerol. These
are necessary in a variety of the signal transduction process. Among the thirteen mammalian PLC
isozymes, PLC-γ1 is ubiquitously expressed and regulates a multitude of cellular functions in various
tissues. Many studies have suggested that PLC-γ1 regulates cell proliferation, differentiation and survival
programs. Furthermore, in vivo data support important roles for PLC-γ1 in tumor metastasis. PLC-γ1
deficient mice showed embryonic lethal due to the defect of vasculargenesis. To further characterize the
role of PLC-γ1 in vivo, we have generated conditional alleles of the plc-g1 gene in which two loxp sites
flank exon 3-5. Homozygous germline deletion of PLC-γ1 caused embryonic lethal identical to those
previously described for PLC-γ1 null embryos. We crossed PLC-γ1 flox mice with hepatocyte-specific
albumin-Cre transgenic mice and confirmed the deletion of PLC-γ1 in liver. This PLC-γ1 conditional null
allele will provide a valuable method to gain insight into the tissue- specific function of PLC-γ1.
3009/L115
Loss of the Histone Demethylase Lsd1 Non-Autonomously Causes Stem Cell Tumors in the
Drosophila Ovary.
S. Eliazer, N. Shalaby, M. Buszczak; Molecular Biology, UT Southwestern Medical Center, Dallas, TX
Specialized microenvironments called niches keep stem cells in an undifferentiated and self-renewing
state. Dedicated stromal cells form niches by producing a variety of factors that act directly upon stem
cells. The size and signaling output of niches must be finely tuned to ensure proper tissue homeostasis.
While advances have been made in identifying factors that promote niche cell fate, the mechanisms that
restrict niche cell formation during development and limit niche signaling output in adults remain poorly
understood. Here we show that the histone demethylase Lsd1 limits the size of the germline stem cell
(GSC) niche in Drosophila ovaries. GSC maintenance depends on BMP signals produced by a small
cluster of cap cells located at the anterior tip of the germarium. Lsd1 mutants display GSC-like tumor
formation, marked by ectopic BMP signal responsiveness throughout much of the germline. Clonal
analysis and cell-type specific rescue experiments demonstrate that Lsd1 functions within the escort cells
(ECs) that reside immediately adjacent to cap cells and prevents them from ectopically producing niche
specific signals. Temporally restricted gene knock-down experiments suggest that Lsd1 functions both
during development, to specify EC fate, and in adulthood, to prevent ECs from forming ectopic niches
independent of changes in cell fate. The role of Lsd1 in repressing niche specific signals may have
important implications for understanding how disruption of its mammalian homolog contributes to cancer
and metastasis.
3010/L116
Species-Specific Mechanisms for Scavenging Reactive Oxygen Species in Mammalian and Fish
Spermatozoa.
1
2
2
3
4
5 1
T. Harumi , T. Andoh , T. Ichikawa , N. Shimizu , Y. Hayashi , R. Yanagimachi ; Anatomy, Asahikawa
2
Medical University, Asahikawa, Japan, Akkeshi Station, Hokkaido National Fisheries Research Institute,
3
4
Akkeshi, Japan, Animal Laboratory, Asahikawa Medical University, Asahikawa, Japan, Life Science,
5
Asahikawa Medical University, Asahikawa, Japan, Institute for Biogenesis Research, University of
Hawaii, Honolulu, HI
In aerobic organisms, mitochondria produce the energy needed for biological functions mainly via the
electron transport chain, concomitantly generating reactive oxygen species (ROS). Because ROS have
the potential to cause cellular damage, cells defend against ROS through either enzymatic and/or nonenzymatic ROS eliminating systems abundant in cytoplasm. In the most of spermatozoa, mitochondria
exist near nucleus within minimal cytoplasm. Therefore, to protect nucleus from ROS, spermatozoa must
be equipped with any effective mechanism for scavenging ROS, some of which possibly allow them to
swim with high respiration rate. In this study, we examined ROS eliminating mechanisms in mammalian
and fish spermatozoa, in relationship to their respiration activities during swimming. As mammalian
spermatozoa are surrounded with seminal plasma, we tested the H2O2 degradation activity in both
seminal plasma and spermatozoa from rat. Both of them were found to have high H2O2 degradation
activity. Addition of a catalase inhibitor, aminotoriazole (ATZ) significantly repressed the activity in seminal
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plasma, whereas both ATZ and heat treatment (>100°C) did not completely the activity in spermatozoa.
Rat spermatozoa contained glutathione only in a trace amount. In conclusion, rat spermatozoa scavenge
ROS through both of the catalase-mediated system and some non-enzymatic mechanisms. Spermatozoa
of most of fish species examined, such as flounder, herring and tuna, showed high activities to degrade
hydrogen peroxide (H2O2) added exogenously, while the spermatozoa of a flounder, Liopsetta obscura,
exceptionally showed low activity. In the fish spermatozoa showing high respiration rates, ATZ inhibited
the H2O2 degradation activity in flounder and herring but not in tuna. These results suggest that ROS
metabolism system in fish spermatozoa acts with species-specific manner.
3011/L117
Smyd3 Is Required for Development of Heart and Skeletal Muscle in Zebrafish.
1
1
1
2
1 1
T. Fujii , S. Tsunesumi , K. Yamaguchi , S. Watanabe , Y. Furukawa ; Division of Clinical reserch, The
2
university of tokyo, Tokyo, Japan, Division of Molecular Developmental Biology, The University of Tokyo,
Tokyo, Japan
OBJECTIVE: SMYD3 is a histone H3 lysine-4-specific methyltransferase whose expression is frequently
elevated in colorectal, hepatocellular and breast carcinomas. Although SMYD3 plays a crucial role in the
proliferation and survival of cancer cells, its physiological function remains to be clarified. In our previous
study, we uncovered that Smyd3 plays a crucial role in the development of heart and skeletal muscle in
zebrafish. To clarify the precise role of Smyd3 in cardiac and skeletal muscle development, we further
investigated the importance of two forms of smyd3 transcripts, and expression of cardiac and skeletal
muscle markers in Smyd3 morphants. METHODS: Smyd3 morphants were generated by the injection
with smyd3-specific antisense morpholino-oligonucleotides into zebrafish eggs. We performed rescue
experiments using two forms of smyd3 mRNA. In addition, we analyzed expression of anterior lateral
plate mesoderm markers including gata4, scl, nkx2.5 and hand2, three heart chamber-specific markers
including cmlc2, amhc, and vmhc, and five skeletal muscle markers, including mck, mylz2, smyhc1,
myod, and myog by in situ hybridization. RESULTS: The Smyd3 morphants showed pericardial edema
and curved trunk. Although co-injection with mRNA of Smyd3 variant2 decreased the defects, that with
variant1 did not complement the defects. In situ hybridization revealed that expression of gata4, scl,
nkx2.5, hand2, myod and myog was almost normal in Smyd3 morphants. However expression of cmlc2,
amhc, and vmhc was deregulated in the morphants. In addition, expression levels of myod and myog
were reduced at early developmental stages but their elevated expression persisted at late stages in the
morphants. CONCLUSION: These data suggest that Smyd3 may be involved in the heart and trunk
development through the deregulation of chamber-specific as well as muscle-specific gene expression.
3012/L118
Alpha 2-Macroglobulin Regulation of Axial and Gut Morphogenesis in Xenopus laevis.
L. L. Pineda Salgado, D. Kessler; Cell and Developmental Biology, University of Pennsylvania School of
Medicine, Philadelphia, PA
α2-macroglobulin (α2M) is a major serum protein with diverse functions, including inhibition of protease
activity and binding of growth factors and cytokines. In Xenopus laevis, two α2M genes, Endodermin
(Edd) and Panza, have been isolated. Edd is expressed in endoderm and dorsal mesoderm cells from
gastrula to neurula stage and with the onset of gut coiling is restricted to the liver. Panza is expressed in
the dorsal gut endoderm, from neurulation through the late tadpole stage, and during gut coiling Panza is
localized to the liver. The distinct and overlapping expression of Edd and Panza in gut endoderm
suggests that α2M function may be important for differentiation and morphogenesis of the gut. To
examine the role of Edd and Panza in gut development, loss-of-function experiments using morpholino
oligos (MO) were performed. Panza knockdown resulted in a ventral bending of the posterior axis, lack of
gut coiling, kinking of the notochord and somites defects. Edd knockdown resulted in a shortened body
axis, a failure of tail outgrowth, lack of gut coiling, notochord shortening and somites defects. Knockdown
of Edd and Panza together resulted in a dramatically shortened body axis, absence of tail structures,
head and eyes defects, and lack of gut coiling, notochord degeneration and disorganized somites.
Mesodermal and endodermal patterning were unaffected by Edd or Panza knockdown from the gastrula
through the coiled gut stage. EM analysis of the notochord sheath revealed a dramatic reduction of the
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thickness and density of the collagen fibers, and these defects were more severe in the double
knockdown. These results suggest that α2M function is essential for axial and gut morphogenesis during
development and suggest a possible role in maintaining the formation and assembly of extracellular
matrix components.
3013/L119
The Putative Lipid Transporter, Arv1, Is Required for Activation of the Pheromone Response
Pathway in Saccharomyces cerevisiae.
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1
1,2 1
M. Villasmil , A. Ansbach , J. T. Nickels ; Dept. of Biochemistry and Molecular Biology, Drexel
2
University College of Medicine, Philadelphia, PA, Division of Pharmacogenomics, Medical Diagnostic
Laboratories, LLC, Hamilton, NJ
We are using the budding yeast Saccharomyces cerevisiae as a model to study cell surface polarized
growth. S. cerevisiae exhibit three types of polarized growth: budding, mating, and filamentous growth.
Membrane microdomains, also known as lipid rafts, are sphingolipid and sterol-rich regions of the plasma
membrane, which are capable of mediating membrane sorting, cell adhesion, and signal transduction. To
gain a better understanding of the role of sphingolipids and sterols during polarized growth, we chose to
study the mating response because it is an inducible system. Pheromone binding to the G-protein
coupled pheromone receptor Ste2/3 leads to the activation of pheromone response MAPK signaling
cascade. Activation of the pheromone response pathway results in cell cycle arrest in G1 phase, matingspecific gene transcriptional induction, and changes in cytoskeletal structure, which allows for polarized
cell growth and alterations in nuclear architecture, ultimately leading to cell fusion and formation of an a/α
diploid cell. The pheromone activated Gβγ dimer recruits the MAPK cascade scaffold protein Ste5 to the
plasma membrane. Membrane recruitment of Ste5 is important for mating pathway signaling. In this
study, we are focusing on the role of the putative lipid transporter, Arv1, in yeast mating. Arv1 function is
essential in yeast mutants unable to esterify sterols and arv1 null cells have been shown to have defects
in sphingolipid metabolism, sterol trafficking, and GPI biosynthesis. Here we demonstrate that arv1 cells
are mating defective because of a failure to effectively recruit the MAPK scaffold Ste5 to the plasma
membrane. Failure to polarize phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) likely contributes to the
mislocalization of Ste5 during the mating response in arv1. These defects ultimately lead to the inability of
arv1 cells to generate shmoos or mate. Our Arv1 structure/function studies demonstrate that the
conserved Arv1 homology domain (AHD) is required for Arv1 function and proper mating.
3014/L120
Understanding the Functions of Cephalic Hedgehog during Xenopus laevis Neural Development.
1
2 1
2
A. R. Morris , L. L. Pineda Salgado ; Biology, Haverford College, Haverford, PA, Cell and
Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA
In 1995, Ekker and colleagues identified three distinct hedgehog gene homologues in Xenopus - banded
hedgehog (Xbhh), cephalic hedgehog (Xchh) and sonic hedgehog (Xshh). While the functions of Xshh
have been well studied, considerably less is known about Xbhh and Xchh. During the earliest stages of
neural tube formation in Xenopus laevis, Xchh is strongly expressed near the presumptive central
nervous system, particularly at the anterior end of the embryo. Based on this expression pattern, we
propose that Xchh is involved in neurulation. To examine the role of Xchh in early Xenopus laevis neural
development, loss-of-function experiments using morpholino oligos (MO) at the one-cell stage were
performed. Xchh knockdown resulted in various abnormal phenotypes, including slowed neural fold
formation, incomplete neural tube closure, and reduced head size. The observed anterior neural tube
closure defects may be due to disruptions in the normal organization of cytoskeletal actin and
microtubules, and suggest a novel role for Xchh in Xenopus laevis neurulation.
MONDAY
3015/L121
Zic Transcription Factors Regulate Multiple Cellular Processes during Zebrafish Neural Tube
Morphogenesis.
1
1
2
1 1
M. Nyholm , J. TeSlaa , S. Burgess , Y. Grinblat ; Departments of Anatomy and Zoology, University of
2
Wisconsin, Madison, WI, Developmental Genomics Section, Genome Technology Branch, NHGRI,
Bethesda, MD
Vertebrate embryos develop a neural tube, the rudiment of the central nervous system, through the
process of neurulation. A key morphogenetic step in neurulation is dorsolateral hingepoint (DLHP)
bending, which requires an apical acto-myosin network. The genetic mechanism of DLHP formation is
poorly understood, although several essential genes have been identified in amniotes. One of these is
Zic2, which encodes a zinc-finger transcription factor. Using morpholino knockdown, we demonstrated a
conserved role for zebrafish zic2a in cranial neural tube morphogenesis and found that zic2a and zic5
regulate apical acto-myosin contraction, apical junction integrity and the cell cycle during DLHP bending.
To better understand how Zic transcription factors regulate these cellular processes, we sought to identify
downstream Zic targets. As a first step toward this goal, we used microarray technology to isolate genes
that are differentially expressed in wild-type embryos versus embryos with depleted zic2a and zic5 gene
products (zic morphants). Genes involved in Retinoic Acid and Wnt signaling, as well as genes that
control the cytoskeleton, vesicle trafficking and the cell cycle were mis-regulated in zic morphants. For
example, ctsc and lgmn, which participate in endosomal degradation, are upregulated in zic morphants,
as are the cytoskeletal genes rsph9 and arp5c. Several cell cycle genes including aspm and gap43 are
downregulated in zic morphants. To identify direct transcriptional targets of Zics we are developing a
chromatin immunoprecipitation method using in vivo biotinylation (bioChIP). These studies address the
question of how gene transcription can result in cellular changes that drive morphogenesis, particularly
during epithelial tube formation.
3016/L122
Nuclear Dynamics of Pluripotency Genes during Embryonic Stem Cell Differentiation.
M. S. Bodnar, D. L. Spector; Cold Spring Harbor Laboratory, Watson School of Biological Sciences, Cold
Spring Harbor, NY
Mammalian embryonic development proceeds in a highly organized fashion, orchestrating the creation of
a complex multi-cellular organism from just one cell. Embryonic Stem (ES) cells, which are derived from
the inner cell mass (ICM) of a blastocyst stage embryo, are an excellent in vitro model for the study of
pre-implantation mammalian development. During the process of differentiation, ES cells undergo
dramatic changes in gene expression/repression, chromatin structure, and histone modifications. Little is
known about how such drastic changes are coordinated in time and space in the complex nuclear
environment. Chromosomal movements/interactions may be an integral event in the transition from the
ES cell transcriptional program to cell-type specific gene expression. In addition, gene interactions with
nuclear bodies may somehow mediate the observed changes in chromatin organization and gene
expression. We were therefore interested in investigating the nuclear position of critical ES cell genes
during the transition of ES cells from a pluripotent to differentiated state. Murine ES cells were
differentiated by withdrawal of leukemia inhibitory factor (LIF) or with retinoic acid (RA) and fixed on glass
coverslips at 12-hour intervals following the onset of differentiation, utilizing methods to preserve the 3-D
structure of the nucleus. Fluorescence in situ hybridization (FISH) was used to examine the 3-D nuclear
position of genes including Nanog, Sox2, Oct4, and Tcf3 in pluripotent and differentiating ES cells.
Interestingly, we identified changes in the position of the Oct4 gene during early ES cell differentiation
induced with both RA and withdrawal of LIF. We are currently investigating interactions of the Oct4 gene
locus with other gene loci and/or nuclear bodies, as well as the impact of Oct4 gene position on the
repression of Oct4 transcription.
MONDAY
3017/L123
Activation of Wnt Signaling by Reactive Oxygen Species.
1
1
1,2 1
J. T. Hwang , J. Wen , G. M. Kelly ; Biology, University of Western Ontario, London, ON, Canada,
2
Paediatrics, Children's Health Research Institute, University of Western Ontario, London, ON, Canada
Differentiation of primitive endoderm from cells of the inner cell mass is one of the earliest cell fate
decisions made in the developing mouse embryo. The molecular mechanisms driving differentiation are
poorly understood and difficult to study in vivo. Mouse F9 embryonic carcinoma cells have been used
extensively as a model for recapitulating extraembryonic endoderm differentiation in vitro. When F9 cells
are treated with retinoic acid (RA), they differentiate into primitive endoderm. This differentiation involves
many signaling events, including the canonical Wnt/β-catenin and planar cell polarity pathways. We found
that RA-induced differentiation was accompanied by a sustained increase in reactive oxygen species
(ROS), which are known to positively regulate Wnt signaling. F9 cells were treated with hydrogen
peroxide to test the hypothesis that this would activate the Wnt signaling required for primitive endoderm
formation. Hydrogen peroxide treatment resulted in the appearance of biochemical, molecular and
morphological markers of primitive endoderm. Furthermore, Tcf reporter analysis indicated that canonical
Wnt/β-catenin signaling had been activated. In contrast, F9 cells pre-treated with antioxidants N-Acetyl
Cysteine or Trolox reduced the ability of RA to induce differentiation. The expression of Nox genes, as a
source of the ROS, was examined over the course of differentiation. Nox4 was found to be up-regulated
in response to RA and treatment with DPI, a NOX inhibitor, attenuated RA-induced differentiation.
Together, these in vitro results suggest that ROS signaling, converging on the Wnt/β-catenin pathway
positively regulates the formation of extraembryonic endoderm in the developing mouse embryo.
3018/L124
A Mathematical Model of Non-Muscle Myosin Function in the Cellularization of Primordial Germ
Cells in C. elegans.
3
1
2 1
T. Kachur , A. Dawes , D. Pilgrim ; Mathematical and Statistical Sciences, University of Alberta,
2
3
Edmonton, AB, Canada, Biological Siences, University of Alberta, Edmonton, AB, Canada, Biology,
Grant MacEwan Community College, Edmonton, AB, Canada
Prior to complete cellularization, primordial germ cells in the gonad of the nematode worm Caenorhabditis
elegans have a small circular opening, the ring channel, through which proteins and other factors are
delivered as the germ cells mature. It has been demonstrated that the non-muscle myosins NMY-1 and
NMY-2 are localized near the ring channel suggesting they modulate the diameter of the opening. In this
poster, I will present a mathematical model of of the forces exerted by NMY-1 and NMY-2 on the ring
channel opening. The model suggests the antagonistic forces applied by NMY-1 and NMY-2 are required
to maintain the ring channel opening, and the model successfully predicts a variety of mutant phenotypes.
3019/L125
Bit1-Null Mice Present with Aberrant Heart Morphology and a Decrease in Cardiac Function.
G. S. Griffiths, A. Leychenko, S. Reiter, M. Matter; Cell and Molecular Biology, John A, Burns School Of
Medicine, Honolulu, HI
The Bcl-2 transcriptional regulator, Bit1, is an integrin effector protein that plays a role in integrinmediated signal transduction. Bit1-null mice are born smaller than wild type littermates and die within 8-12
days after birth by an unknown mechanism. These mice are phenotypically characterized by stunted
growth, trembling and hand-clasping. We show that Bit1 is expressed in the murine heart, specifically in
cardiomyocytes. In the heart there is enhanced expression of Bit1 during the first week after birth, a
crucial developmental period. Cardiomyocytes use integrin receptors to interact with the extracellular
matrix, a vital step for heart development and cardiac function. In conjunction with the high level of Bit1
expression during this critical cardiac developmental stage, this led to our hypothesis that Bit1 plays an
important role in heart development and heart function. To investigate our hypothesis, we utilized Bit1-null
mice which were obtained by breeding Bit1 heterozygotes. At day 8 after birth, heart function was
determined by echocardiography while general heart morphology was determined by H&E staining and
microscopy. In addition, WGA staining was performed to characterize cardiomyocyte morphology and
MONDAY
APOPtag staining was used to determine percentage apoptosis. While the Bit1-null mice are smaller and
weaker than age matched littermates, their heart to body ratios are similar. Echocardiograms
demonstrated decreased cardiac function and aberrant heart cytoarchitecture in the null mice when
compared to age matched littermates. H&E sections of null heart tissue indicate that it is denser, contains
more nuclei and has smaller myofibrils than WT littermates. In addition, cardiomyocyte staining
demonstrated reduced cardiomyocyte length and an overall increase in apoptotic cells in the Bit1-null
hearts. These findings provide the first evidence that loss of Bit1 results in altered heart cytoarchitecture
and decreased cardiac function, and suggest that the Bit1-null mice may die in general from cardiac
dysfunction, and specifically by decreased cardiac output induced by aberrant cardiomyocyte
morphology.
3020/L126
Chloride Intracellular Channel 4 in Lumen Formation.
S. Chou, J. Chuang, C. Sung; Weill Cornell Medical Collage, New York, NY
Lumen formation is the base of tubulogenesis and organ genesis which involves complex mechanisms
including apical membrane biogenesis, cytoskeleton rearrangement, and vesicles fusion and secretion.
Chloride intracellular channel 4 (CLIC4), one of Chloride Intracellular Channel proteins, has been proofed
contributed to tubulogenesis in C. elegan and angiogenesis in mouse, but the mechanism remains
unknown. Methods and results: By using short hairpin RNA, we generated CLIC4-knock down (KD) stable
MDCK cell line to study the mechanism of CLIC4 in lumen formation. Studies were performed in a
matrigel system, in which, single MDCK cell growth in 3-dimemtion system and form cyst with
proliferation. Immunostaining results showed large amount of CLIC4 expressing around the preapical
plasma membrane in early cysts. Cells with reduced CLIC4 would develop multi-lumen cysts. These
multilumens cysts remain most polarity characters such as gp135 and PKC-lambda on apical membrane,
and ZO-1 and Par3 on tight junction. To investigate the possible mechanism, studies were carried out by
transfecting different phospholipid reporters into cells- GFP conjugated PLC-PH domain and AKT-PH
domain which represents PI(4,5)P2 for apical membrane and PI(345)P3 for basalaterl membrane. The
results showed incomplete lipid segregation in CLCI4-KD cysts. One of PI(45)P2 generating enzymes PIPK1r showed an accumulation under apical actin belt in CLIC4-KD cysts by immunostaining. Further
study found an apical complex protein - Annexin 2, which carry PI(45)P2 vesicles, was unable to target to
apical membrane in CLIC4-KD MDCK, however, another apical complex molecule - cdc42 could rescue
CLIC4-KD phenotype. Conclusion: In this study, we identify CLIC4 contributes to central lumen
generation. Cells without sufficiency CLIC4 would form de novo lumens by interfering Anx2 mediated
enosome and exosome.
3021/L127
Isoform-Specific Dephosphorylation of Dynamin1 by Calcineurin Couples Neurotrophin Receptor
Endocytosis to Axonal Growth.
M. Ascano, D. J. Bodmer, R. Kuruvilla; Biology, Johns Hopkins University, Baltimore, MD
Endocytic events are critical for neuronal survival in response to extracellular neurotrophic cues, but
whether local axon growth is mediated by endocytosis-dependent signaling mechanisms remains unclear.
We find that Nerve Growth Factor (NGF) promotes endocytosis of its TrkA receptors and axon growth by
Calcineurin-mediated dephosphorylation of the endocytic GTPase, Dynamin1. Calcineurin signaling is
required locally in axons to support growth, in a manner independent of transcriptional responses.
Calcineurin associates with Dynamin1 via a PxIxIT interaction motif that is found only in specific spliced
variants. Dynamin1 variants with and without the PxIxIT box exhibit distinct patterns of sub-cellular
localizations, and perform discrete functions with phosphoregulation of only PxIxIT box-containing
isoforms being required for NGF-dependent TrkA internalization and axon growth. Thus, NGF signaling
actively modulates core components of the endocytic machinery to influence its own trafficking, and
phosphoregulation of Dynamin1 is a critical event coordinating neurotrophin receptor endocytosis and
axonal growth.
MONDAY
3022/L128
Local Dynamin-Independent Cycling of AMPA Receptors.
1,2,3
1,2 1
O. Glebov , J. M. Henley ; School of Biochemistry, University of Bristol, Bristol, United Kingdom,
2
3
MRC Centre for Synaptic Plasticity, University of Bristol, Bristol, United Kingdom, Trinity College,
University of Cambridge, Cambridge, United Kingdom
Fast excitatory transmission in the mammalian brain is carried out by the AMPA-subtype glutamate
receptors (AMPARs). While there has been much progress in elucidating the role of inducible AMPAR
trafficking in synaptic plasticity, the mechanism of their constitutive trafficking is poorly understood. Here,
we show that in dendritic spines, AMPARs may constitutively cycle via a novel endocytic pathway. In
contrast to agonist-induced AMPAR endocytosis, this pathway is independent of clathrin and dynamin
function and bypasses early and recycling endosomes. It is distinct from the previously characterized
dynamin-independent pathways since it does not require actin polymerization, as well as Arf1, Arf6 and
Cdc42 activation. Acute inhibition of Rac1 activation positively regulates dynamin-independent AMPAR
endocytosis. We conclude that constitutive cycling of AMPA receptors may operate using mechanisms
fundamentally different from conventional membrane trafficking.
3023/L129
LIM Kinase 1 and ADF/Cofilin Regulate Presynaptic Forms of Plasticity at the Drosophila
Neuromuscular Junction.
1,2
1 1
J. B. Machamer , G. Marqués ; Cell Biology, University of Alabama at Birmimgham, Birmingham, AL,
2
Neurology, Johns Hopkins School of Medicine, Baltimore, MD
Regulation of the actin cytoskeleton by LIM Kinases (LIMK1 and LIMK2) and their downstream targets
Actin Depolymerization Factor/Cofilin (ADF/Cofilin) in the mouse forebrain modulates dendritic spine
morphology, synaptic function, long-term potentiation, and learning and memory, but appear dispensable
for the regulation of most forms of presynaptic plasticity. However, these analyses were performed in
mice mutant for single members of these gene families, and thus their interpretation is confounded by
potential compensatory activity of homologous genes. Our objective is to characterize the role of the
single Drosophila homolog of LIMK (dLIMK1) and the single neuronally-expressed Drosophila homolog of
ADF/Cofilin (Twinstar; Tsr) in morphological and functional presynaptic plasticity of the larval NMJ using
fluorescent microscopy and electrophysiological analyses. We found that dLIMK1 acts via Tsr to
downregulate growth of the presynaptic terminal, especially by restraining the formation of immature
2+
branching boutons. Functionally we found that Tsr is required for the proper Ca -cooperativity of vesicle
fusion and normal paired-pulse facilitation, and that both dLIMK1 and Tsr are necessary for proper
tetanus-induced potentiation. These results demonstrate that dLIMK1 and Tsr modulate developmentallyregulated morphological and activity-dependent functional plasticity of Drosophila motoneurons and
illustrate the conservation of mechanisms regulating neuronal branching between postsynaptic excitatory
neurons in mice and presynaptic motoneurons in Drosophila. Furthermore, these results suggest that the
lack of alterations in presynaptic forms of plasticity in mice mutant for LIMK1 or non-muscle cofilin (n-cof)
could be a consequence of compensation by LIMK2 and ADF, respectively.
3024/L130
Functional Alterations in Epm2a-/- Mutant Mice, an Animal Model of Lafora Disease.
1
1
1
2
1
A. M. García-Cabrero , R. Guerrero , A. Marinas , S. Rodriguez de Cordoba , J. M. Serratosa , M. P.
1 1
2
Sanchez ; Neurology, IIS-Fundacion Jimenez Diaz-Capio, Madrid, Spain, Centro de Investigaciones
Biologicas, CSIC, Madrid, Spain
Lafora disease (LD) is a rare form of progressive myoclonus epilepsy caused by recessive mutations in
the EPM2A gene encoding laforin, a dual-specificity phosphatase, or in the EPM2B gene encoding malin,
an E3 ubiquitin ligase, and characterized by the presence of PAS-positive intracellular inclusions of
polyglucosans (Lafora bodies) in several tissues. Targeted disruption of the Epm2a gene was used to
-/generate a mouse line deficient for the expression of laforin, the Epm2a mouse line (Ganesh et al,
-/Human Mol Genet 11:1251-1262, 2002). Neuronal degeneration was widely observed in Epm2a
mutants beginning at two months of age and Lafora bodies developed in neuronal and non-neuronal
MONDAY
tissues soon afterwards. Objectives: To analyze the effects of laforin deletion on motor behavior, memory
-/and electroencephalographic activity in Epm2a mutant mice and the alterations in the sensitivity to the
-/epileptogenic substance pentylenetetrazole. Methods: Two groups of Epm2a mutants (4-7 months and
9-17 months of age) were analyzed on a computerized actimeter to measure motor activity, and on an
accelerating rotarod to assess motor coordination and balance. To measure episodic memory retention
we performed the object recognition task (ORT). Electroencephalographic activity was also analyzed with
-/or without pentylenetetrazole at subthreshold doses. Results: Epm2a mutant mice showed important
memory deficits, impaired motor coordination and balance, and altered increase in rearing, stereotyped
-/and deambulation movements, compared with the control groups. Epm2a mice also had a lower
-/pentylenetetrazole seizure threshold. Conclusions: The absence of laforin in Epm2a mutant mice results
-/in important behavioral deficits and alterations on the electroencephalographic activity. Epm2a mice
present some of the pathological aspects showed in patients with LD and may serve as a valuable
experimental tool for the study of this fatal disorder.
3025/L131
Clustering of Eph Receptors Is the Central Integrator to Elicit Appropriate Cellular Signaling
Responses.
1
2
2
1 1
A. Schaupp , O. Sabet , P. I. Bastiaens , R. Klein ; Molecular Neurobiology, Max-Planck-Institute of
2
Neurobiology, Martinsried, Germany, Systemic Cell Biology, Max-Planck-Institute of Molecular
Physiology, Dortmund, Germany
Eph receptor tyrosine kinases (RTKs) and their corresponding membrane-bound ephrin ligands are
essential regulators of cell movement and positioning required in establishing and stabilizing patterns of
cellular organization. They have been described as ‘graded molecular tags’ that monitor the abundance
or density of their binding partner on opposing cells and relay this information to elicit correspondingly
graded cellular responses. In contrast to the conventional concept of ligand induced RTK activation with
receptor dimers being fully signaling competent, Eph receptors require the formation of multimeric
Eph/Ephrin complexes to induce robust signaling. However, a clear mechanistic to functional link of Eph
clustering has, as yet, not been described. To investigate the function of higher-order clustering in Ephmediated cell responses we established a system using chemical dimerizer-inducers in order to generate
defined cluster size distributions of Ephs independent of ligand contact. Using fluorescence anisotropy
and live-cell imaging techniques as a readout, we found that ligand-independent, dimerizer-induced Eph
clustering is sufficient to activate Eph kinase activity and signaling, to trigger internalization and cell
contraction, mimicking the physiological ephrin-induced signal activation process. Moreover, cluster size
distributions positively correlate with cellular responses and can be altered by intracellular determinants
causing changes in clustering propensity. Our results suggest that clustering is the central integrator to
elicit appropriate cellular Eph-dependent signaling responses, which can possibly be modulated by the
binding of clustering determinants like intracellular adaptor proteins.
TUESDAY
Late Abstracts 3
3026/L001
LAP (AP180) Regulation of Synaptic Vesicle Protein Sorting and Retrieval in Drosophila.
P. Vanlandingham, H. Bao, C. Springer, B. Zhang; University of Oklahoma, Norman, OK
Synaptic Vesicles (SVs) act as principal functional units that store and release neurotransmitters from
presynaptic terminals. The precise regulation of SV trafficking is fundamental to communication across
chemical synapses. To sustain neurotransmission, clathrin-mediated endocytosis (CME) reconstitutes
and recycles SVs following exocytosis and neurotransmitter release. However, many question remain
regarding SV endocytosis. SVs most often completely fuse with the plasma membrane during exocytosis.
Therefore, to faithfully reconstitute functional SVs, CME must mediate at least two processes: 1) correct
recognition of SV components amid the myriad proteins and lipids in the PM; and, 2) sort and retrieve the
SV proteins on a rapid timescale, as SV assembly occurs on the order of seconds. Failed retrieval in one
or more of these component proteins could have a severe impact on synaptic function. We test a model in
which the Drosophila protein LAP (Like AP180) functions as a core component of the clathrin-mediated
endocytic machinery by coupling the recruitment of SV components to the reformation of SVs during
endocytosis. To test this model we are using a combination of quantitative biochemical analsysis and
imaging of SVs in lap mutants. LAP is shown to regulate SV size, synaptic transmission, and the
localization of a subset of SV proteins, including CSP, Synaptobrevin, and Synaptotagmin I. We have
also generated a LAP transgene expressed in its full genomic context and amenable to light-mediated
acute inactivation. We show that this construct is capable of rescuing both the viability and the synaptic
transmission defects in lap mutant flies, and are exploring the specific consequences of the acute
inactivation of LAP on SV recycling. The molecular identity of SVs is now well-known, but the
mechanisms underlying their reconstitution during SV recycling remain poorly understood. In addition to
understanding the basic cell biology of this process, this study has important disease implications as the
human homologue of LAP was recently shown in a genome wide association study to be significantly
associated with late onset Alzheimer’s disease.
3027/L002
Gap Junction Channels: Two Voltage Dependent Gates in Series Should Allow Voltage Induced
Steady State Cycling around a Circular Reaction Scheme.
M. V. Bennett; Neuroscience, Albert Einstein College of Medicine, Bronx, NY
Connexin gap junction channels exhibit varying degrees of dependence on transjunctional voltage (Vj)
with little or no dependence on the potential across non-junctional membrane. Physiological data and
symmetry suggest that each hemichannel has its own voltage gate(s) and that the voltage across each
gate depends on the state of gate(s) in series with it. For a symmetrical channel with one gate in each
hemichannel, gate 1 and gate 2, a given Vj will tend to close one gate; let it be gate 1. Then, the potential
across gate 2 will be reduced to zero, assuming complete closure of gate 1. Gate 2 will close
spontaneously at some rate, which will reduce the potential across gate 1, causing it to have an increased
probability of opening. When it opens, the voltage across gate 2 will cause it to open rapidly,
reestablishing the voltage drops as initially applied. Thus, the channel will cycle through open (O) and
closed (C) states as follows: O1O2, C1O2, C1C2, O1C2, O1O2, etc. This circular reaction scheme is
dissipative, driven by the applied voltage. Calculations with typical parameters indicate a small but
definite effect.
3028/L003
Role of Microtubules in Osmosensory Transduction in Supraoptic Nucleus Neurons.
M. Prager-Khoutorsky, C. W. Bourque; Centre for Research in Neuroscience, McGill University Health
Centre, Montreal, QC, Canada
Maintenance of constant plasma osmolality in mammals is vital, as changes in cell volume caused by
severe acute hyperosmolality or hypoosmolality can irreversibly damage organs and cause lethal
TUESDAY
neurological trauma. The osmotic control of vasopressin secretion plays a key role in osmoregulation
because this peptide stimulates water reabsorption by the kidney. Magnocellular neurosecretory cells
(MNCs) in the hypothalamic supraoptic nucleus are intrinsically osmosensitive. MNCs transduce osmotic
signals into spiking, which stimulates vasopressin release from axon terminals located in the
neurohypophysis. This transduction is mediated by stretch-inhibited nonselective cation channels which
are activated during hypertonicity-evoked shrinking, and inhibited during hypotonicity-evoked swelling.
Recent studies imply that osmosensation is a mechanical process, and sensitivity of transduction
increases in proportion to actin filament density. Here, we investigated the role of microtubules in this
process using confocal imaging and patch clamp recordings from supraoptic nucleus MNCs acutely
isolated from adult rats. Images of brain sections and isolated MNCs demonstrated that these neurons
feature a prominent microtubule network compared to other neurons. Using whole-cell current-clamp and
voltage-clamp recordings from acutely isolated MNCs, we tested the hypothesis that this unique
microtubule structure is important for intrinsic osmo- and mechanosensation. We found that disruption of
microtubules in MNCs with 0.5 uM nocodazole activates an inward cation current, consistent with the
hypothesis that these filaments mediate an inhibitory effect on the basal activity of MNCs that express the
transduction channels. In addition, we demonstrated that disruption of microtubules with nocodazole
abolishes suction- and hypertonicity-evoked activation of cation channels. We conclude that MNCs from
supraoptic nucleus exhibit a unique microtubule network that is an essential component for mechano- and
osmotransduction.
3029/L004
Regulated Trafficking of a Glutamate Receptor Subunit by Akt at the Drosophila Neuromuscular
Junction.
H. Lee, N. Zhao, M. Nguyen, M. M. Rolls, S. Selleck; Biochemistry and Molecular Biology, Pennsylvania
State University, State College, PA
Akt is a serine-threonine kinase that integrates a myriad of signals to govern cell growth, apoptosis,
glucose metabolism, cytoskeletal organization and exocytosis. We have explored the function of Akt in
synapse assembly and plasticity at the Drosophila neuromuscular junction (NMJ). Using RNAi-mediated
cell-type specific knockdown we have found that Akt is required for the expansion of a specialized
postsynaptic membrane compartment called the subsynaptic reticulum (SSR). Reduction of Akt function
1
phenocopies mutants in Gtaxin , a t-SNARE-encoding gene required for postsynaptic membrane growth.
Reduction of Akt function dramatically reduced the levels of a membrane marker, mCD8-GFP, associated
with the postsynaptic specialization as well as the size and complexity of the SSR observed directly with
transmission electron microscopy of synaptic boutons. Akt also affected the composition of the glutamate
receptor by regulating the trafficking of one receptor subunit, GluRIIA, to the synapse. Inhibition of Akt
produced an accumulation of GluRIIA within an intracellular membrane compartment, while leaving the
delivery of other glutamate receptor subunits (GluRIIB and GluRIIC) and synapse-localized proteins
(Discs-large, Basigin and Syndapin) intact. Our data provide evidence for Akt-mediated regulation of
secretion at the NMJ, affecting both the composition of a neurotransmitter receptor and the extent of
postsynaptic membrane expansion. These cellular functions provide a basis for Akt-mediated control of
synapse development and activity. Reference 1. Gorczyca D, Ashley J, Speese S, Gherbesi N, Thomas
U, Gundelfinger E, Gramates LS and Budnik V (2007) Postsynaptic membrane addition depends on the
Discs-Large-interacting t-SNARE Gtaxin. J.Neurosci 27(5):1033-1044.
3030/L005
Measurement of Leaf Phenotype Differences in Maize by In Situ Hyperspectral Confocal
Fluorescence Imaging.
1
2
2
2 1
M. C. Ubach , H. D. Jones , M. B. Sinclair , D. M. Haaland ; Crop Analytics, Monsanto Company, St.
2
Louis, MO, Sandia National Laboratories, Albuquerque, NM
Accurate measurement of changes in native fluorescent components in plant leaves, cells and organelles
as a result of mutation, environmental stress or treatment is critical to complement plant phenomics
screens. We used hyperspectral confocal fluorescence microscopy to overcome the limitations of
commercial filter-based instruments, namely, cross-talk and autofluorescence, which frequently prevent
TUESDAY
the generation of meaningful quantitative results. Seven maize (Zea mays) leaf mutants, characterized by
different amounts of leaf pigmentation ranging from white to dark green, were used to evaluate
hyperspectral fluorescence (HSF) imaging for measurement of pigment content in mesophyll and bundle
sheath chloroplasts of maize leaf. Leaves from plants at V3 stage were harvested for HSF imaging,
transmission electron microscopy and determination of pigment content by high performance liquid
chromatography. HSF imaging followed by multivariate curve resolution analyses revealed the presence
of six main pure spectral components in maize leaf (carotenoids, chlorophylls a/b, photosystem I and II,
and cell wall autofluorescence), and significant changes in chloroplast pigment concentration, pigment
ratios and spatial distribution as a function of mutant and chloroplast type. Results showed significant
changes in chlorophyll-b and photosystem I for lower yield mutants, and a significant increase in
photosystem I in bundle sheath chloroplasts for greener leaf, higher yield mutants. HSF revealed absence
or significant degeneration of leaf chloroplasts in mutants with white and yellow leaf phenotypes,
observations corroborated by electron microscopy. HSF imaging enabled accurate measurement of
changes in the amount and distribution of native fluorescent components in plant leaves, some not
detected by the naked eye or wet chemistry. HSF results correlated with leaf phenotype, chloroplast
ultrastructure and mutant performance. Acknowledgments.Sandia National Laboratories is a multiprogram laboratory operated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin
Company, for the U.S. Department of Energy’s National Nuclear Security Administration under contract
DE-AC04-94AL85000.
3031/L006
A Novel Peroxisomal Protein Involved in Peroxisome Dynamics in Yeast.
R. Tower, A. Fargarasanu, R. A. Rachubinski; Cell Biology, University of Alberta, Edmonton, AB, Canada
Peroxisomes are ubiquitous organelles involved in a diversity of metabolic processes, most notably the
oxidation of lipids and the detoxification of reactive oxygen species. In the yeast Saccharomyces
cerevisiae, peroxisome growth and division are controlled by the import of matrix proteins and a
specialized divisional machinery that includes both peroxisome-specific factors such as members of the
Pex11 protein family and general divisional factors like the dynamin-related protein Vps1p. Global yeast
2-hybrid analysis has demonstrated interactions between the products of a S. cerevisiae gene of
unknown function, YCL056c, and genes acting in peroxisome dynamics, including PEX11. Because our
morphological and biochemical analyses have demonstrated a role for YCL056c in peroxisome
biogenesis, we have tentatively renamed this gene PEX34. Confocal microscopy showed that GFPtagged Pex34p localized to punctate structures that costained with the known peroxisomal protein, 3ketoacyl-CoA thiolase (Pot1p). Pex34p localized preferentially to a 20,000 × g pellet fraction enriched for
peroxisomes and cofractionated with Pot1p and not with the mitochondrial protein Sdh2p by density
gradient centrifugation. Treatment of peroxisomes with dilute Tris and alkali sodium carbonate showed
that Pex34p exhibits the characteristics of a peripheral membrane protein. Microscopic analyses of cells
harboring single or double deletions of PEX34 and the PEX11 family of peroxisome divisional genes,
implicate Pex34p in regulating peroxisome size and abundance. Overexpression analyses support a role
for Pex34p in peroxisome dynamics upstream of the proteins of the Pex11 family. Quantification of
peroxisome numbers under typical peroxisome induction (medium containing oleic acid, which is
metabolized exclusively by peroxisomes) and noninduction (medium containing the fermentable carbon
source, glucose) conditions suggests that Pex34p has a primary role in regulating peroxisome abundance
under non-inducing conditions. Our results suggest that distinct pathways exist to control peroxisome
abundance under inducing and non-inducing peroxisome conditions in yeast, with Pex34p having a role
primarily in the peroxisome noninduction pathway.
3032/L007
ARL4A and GCC185 Cooperate in Regulating Structural Integrity of the Golgi Apparatus and
Endosome-to-Golgi Transport.
1,2
1
1
1
1,2
1,2 1
Y. Lin , T. Chiang , Y. Liu , Y. Tsai , L. Jang , F. Lee ; Institute of Molecular Medicine, College of
2
Medicine, National Taiwan University, Taipei, Taiwan, Department of Medical Research, National Taiwan
University Hospital, Taipei, Taiwan
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ARL4A is a developmentally-regulated member of the ARF/ARL GTPase family. Although the primary
structure of ARL4A is very similar to that of other ARF/ARL molecules, its function remains unclear. The
trans-Golgi network golgin GCC185 is required for maintenance of Golgi structure and endosome-toGolgi transport. We show here that interaction between ARL4A and GCC185 is required for structural
integrity of the Golgi and endosome-to-Golgi transport. Depletion of ARL4A in cells leads to Golgi
fragmentation and marked reduction of Shiga toxin B-subunit and mannose 6-phosphate receptor
internalization from endosomes to the Golgi. Ectopic expression of the wild-type or constitutively active
ARL4A rescues Golgi fragmentation in ARL4A-depleted cells. ARL4A interacts with the N-terminal subcoiled-coil regions of GCC185 in a GTP-dependent manner. Deletion of the ARL4A interacting sub-coiledcoil region of GCC185 results in loss of function in the maintenance of the Golgi structure and endosometo-Golgi transport. Furthermore, ARL4A-depleted cells are impaired in GCC185-mediated Golgi
recruitment of microtubule plus end-binding proteins, CLASPs. Together, our data demonstrate that
ARL4A and GCC185 cooperate in regulating structural integrity of the Golgi apparatus and endosome-toGolgi transport.
3033/L008
Novel Role of Aminomethyl Transferase-Like Protein in Blood Stage Plasmodium Parasites.
1
1
2
1
1
2
S. Maddur Ganesan , J. Morrisey , M. Spalding , S. Venu , P. Balabaskaran nina , S. Prigge , M.
1
1 1
Mather , A. Vaidya ; Microbiology and Immunology, Drexel University College of Medicine, Philadelphia,
2
PA, Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public
Health, Baltimore, MD
Biochemical reactions involving the transfer of one carbon units are collectively designated “One Carbon
Metabolism” (OCM). One carbon transfers have been shown to play essential roles in several major
cellular processes including DNA synthesis, mitochondrial and chloroplast protein synthesis, methyl group
biogenesis, amino acid and vitamin metabolism. In malarial parasites, the glycine cleavage complex
(GCV) was proposed to be involved in folate-mediated OCM within the mitochondrion. Generally, GCV is
made up of 4 proteins; P (glycine decarboxylase), T (aminomethyl transferase), H and L (lipoamide
dehydrogenase), which are nuclearly encoded and targeted to the mitochondrial matrix. There are 3
aminomethyl transferase-like proteins in the Plasmodium genome database. Here we investigated the
role of the putative mitochondrially targeted aminomethyl transferase (AMT1; the putative GCV T protein).
Gene disruption experiments revealed the nonessential nature of this gene in blood stage malaria
parasites, both in vitro (P. falciparum) and in vivo (P. berghei). P. falciparum knock out parasites exhibited
no growth deficiency in vitro. Surprisingly, the AMT1 knock out parasites were found to be deficient in
lipoylation of the pyruvate dehydrogenase (PDH) E2 subunit, which is located in the apicoplast and
depends on de novo biosynthesis of lipoic acid, while salvage-based lipoylation of mitochondrial E2
subunits was unaffected. Complementation of the AMT1 knock out appeared to rescue PDH lipoylation,
suggesting that this protein is involved in the lipoic acid pathway. Localization studies revealed that AMT1
is not targeted to the mitochondrion or the apicoplast. We have cloned AMT1 knock out P. berghei
parasites for further investigations. Currently we are exploring the role of this protein in mosquito and liver
stages.
3034/L009
Genetic Networks of Phenol-Stress Response in Fission Yeast Schizosaccharomyces pombe.
1
2
1
1
1
1
1 1
A. G. Moyzis , R. Wang , L. Taggart-Murphy , J. Portillio , A. Elkabti , T. Wen , Z. I. Tang ; W.M. Keck
2
Science Center, The Claremont Colleges, Claremont, CA, Engineering Department, Harvey Mudd
College, Claremont, CA
Living organisms in variable natural environments must respond to environmental changes; the ability of
species to adapt to such environmental stresses is necessary for their survival under natural selection.
This project is a genetic investigation of response networks to several phenol derivatives as
environmental stress factors. The selected phenol derivatives have been widely used as pesticides, food
preservatives, and high volume industrial chemicals. Their extensive use has raised concern for their
impact on global health and ecosystem. Intriguing biological effects of individual phenol derivatives
include protective anti-oxidation, prooxidation, DNA-damage and apoptosis. However, little is known
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about the principal cellular processes affected and how eukaryotes cope with the stress at a molecular
level. The purpose of this study is to identify the genes and decipher the pathways required for
responding to the phenol stress using fission yeast Schizosaccharomyces pombe as a model organism.
We have employed cell-based and genetic analysis and tested the effects of three phenol derivatives,
Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and Bisphenol-A (BPA), on the growth
of a collection of protein kinase gene-deletion mutants. In general, BHA resulted in more potent inhibitory
effects on cell growth than BHT. The analysis revealed that out of 85 mutant strains, 33 were BHAsensitive and 1 was BHA/BHT-resistant, consistent with studies on the growth inhibition of BHA in other
organisms. In addition, of the 85 deletion strains, 10 appeared to be BPA sensitive and 11 appeared to be
BPA resistant. Interestingly, the data indicate that BHA and BPA not only share common response
elements but also act on distinct targets in fission yeast. We now extend our study to the genomic scale in
order to provide comprehensive information about the molecular mechanism of environment-organism
interaction.
3035/L010
Development of a Method to Culture Endothelial Cells in the Presence of Cyclic Waveforms.
C. G. Conant, M. Schwartz, J. Nevill, C. Ionescu-Zanetti; Fluxion, S. San Francisco, CA
Development of in vitro models that allow simulation of in vivo conditions are now recognized to be
important to studying many disease paradigms such as hepatic cell biology, cancer biology, and
atherosclerosis. In some cases, growing cells ex vivo in a 3D culture model is sufficient to maintain tumor
markers or other biological markers seen in vivo. However in the case of endothelial cell biology, the ever
present shear force, exerted by blood flow, is a major factor in gene expression and subsequent
pathologies. Here, we describe a method of specialized endothelial cell culture using controlled shear
flow to generate cyclic waveforms to mimic athero-prone and athero-protected sites in the vasculature
(Dai G. et al 2004). We grew endothelial cells under different flow regimens for a period of 24 hours. We
then evaluated temporal changes in protein expression and morphology during application of
programmed shear and found significant differences in actin localization, cell density, and connexin 37
and 43 expression over time. This method is extensible to atherosclerosis modeling as well as
investigation of other aspects of endothelial cell biology.
3036/L011
Cytotoxic and Mutagenic Analysis of World Trade Center Particulate Matter on In Vitro Systems.
M. N. Hernandez, B. Soares, C. Lambroussis, A. DiLorenzo; Biology and Molecular Biology, Montclair
State University, Montclair, NJ
Particle matter (PM) is termed as a mixture of solid and liquid droplets found in the air. Composition and
size of PM can be made up of many elements (organic chemicals, acids, metals, solid, and dust particles)
while the size of particles (<10micrometers) are directly linked to their potential for causing health
problems. In this study, we attempt to define the parameters of cytotoxic and mutagenic effects that World
Trade Center (WTC) dust may have on male MRC-5 and female WI-38 Human Pulmonary Fibroblast.
Previous MSU studies have indicated that the MRC-5 and WI-38 fibroblasts undergo apoptosis
logarithmically in comparison to increased dust concentration. By measuring Cellular Membrane Integrity
LDH release we were able to demonstrate how increased dust exposure inhibits normal cellular function
and incites cellular degradation. Two Salmonella typhimurium strains carrying point mutations prohibiting
histidine synthesis were exposed to WTC dust, gypsum, and household dust. Mutagenicity of materials
were measured based on the ability of these materials to induce reverse mutations and enable colonies
of this bacteria to synthesize histidine. Conclusively, our data suggests that an increase in WTC dust
concentrations is associated with decreased membrane integrity of lung fibroblasts. Further data also
suggests that 25ppm & 250ppm doses may have mutagenic properties.
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3037/L012
Alveolar Type I Cells Suppress Cigarette Smoke Extract Induced Hydrogen Peroxide Produced by
Cultured Endothelial Cells.
1,2
1,2
1,2 1
2
C. Downs , C. J. Merkle , D. Montgomery ; University of Arizona, Tucson, AZ, The Southern
Arizona VA HealthCare System, Tucson, AZ
Cigarette smoke contains numerous oxidants that alter the redox state of the lung leading to oxidative
stress and lung injury. The responses of alveolar epithelial type I (AT I) cells and microvascular
endothelial cells (MVECs) to cigarette smoke extract (CSE) have not been fully elucidated. The purpose
here was to begin examining the effects of CSE on the redox state of AT I and MVECs cultured
separately and grown as co-cultures. Primary AT I and MVECs were obtained from the lungs of male
Fischer 344 rats. Cells were studied as separate cultures and as co-cultures with AT I and MVECs
seeded on opposites sides of Transwell Fluroblok™ filters with 1 micron pores. The concentration of CSE
was standardized by spectrophotometry from the smoke from one research grade (1R5F) cigarette
dissolved in 10 mL of RPMI 1640 with 10% FBS. Cells were exposed to CSE (1-100%) or vehicle control
for either 1 hour or 24 hours. The redox state was determined by measuring intracellular reactive oxygen
species (ROS), extracellular ROS, and total antioxidant capacity in AT I and MVECs in separate and cocultures using commercially available kits. The results showed ROS production in AT I cells did not
change even when exposed to 100% CSE. When grown separately AT I cells did not produce significant
H2O2 after 24 hours exposure to CSE, while MVECs demonstrated a 2 to 2.5 fold increase. Interestingly,
when AT I and MVECs were grown as co-cultures, H2O2 levels were reduced 1 to 2.5 fold compared to
MVECs cultured alone. Measures of the total antioxidant capacity of primary AT I cells suggest AT I cells
produce and release antioxidants into the surrounding culture medium which may explain the reduction of
H2O2 levels. Studies are in progress to test a “stop-or-mop” hypothesis to determine if the AT I cells
induce MVECs to stop H2O2 production or if antioxidants in the culture medium “mop-up” MVECproduced H2O2. The results of these studies may provide insight into mechanisms of CSE-induced injury
and possible therapeutic strategies to reduce oxidant burden and lung injury secondary to smoking and
related environmental exposures.
3038/L013
A Comparative Study of Osteoblasts and Osteosarcoma Cells in Conditioned Media.
J. Horne, K. Dorst, Y. Meng; Stony Brook University, Stony Brook, NY
Osteosarcoma is a malignant bone tumor that has a high incidence among children and adolescents. Due
to the tumor's ability to spread to other parts of the body and the relatively low survival rate, it is of interest
to further investigate the fundamental mechanisms of the disease. The morphology, proliferation,
differentiation and motility of rat osteosarcoma cells (ROS) and murine osteoblasts (MC3T3-E1) are
compared. Morphology is observed using Alexa Fluor 488 Phalloidin (for F-Actin), Propidium Iodide (for
cytoplasm), and DAPI (for nuclei). A proliferation study is conducted over 9 days by calculating the area
density of nuclei stained with DAPI. For differentiation assays, cells are maintained in induction medium
containing 50µg/ml ascorbic acid and 4mM β glycerol phosphate. A colorimetric assay for alkaline
phosphatase (ALP), an early differentiation marker, is carried out on days 0 (undifferentiated), 4 and 10.
SEM/EDXS is conducted on cells that are differentiated for 21 and 28 days to measure the production of
hydroxyapatite (HAP), a late differentiation marker. Another set of experiments is repeated with cells
cultured in conditioned medium that is generated by collecting the supernatant from cell cultures that
differentiated for either 7 days (immature conditioned medium, ICM) or 28 days (mature conditioned
medium, MCM). Newly seeded ROS cells are exposed to either 100% MC-conditioned medium and MC
cells are exposed to 100% ROS-conditioned medium. In addition, both cell types are exposed to a 50:50
mixture of conditioned media from both cell sources. The differentiation potential of all the samples is
measured by the production of ALP (up to 10 days) and HAP (up to 28 days) using the methods
described above. Preliminary data show that although the growth rates for both cell types are similar,
ROS cells in unconditioned medium produce significantly more ALP but fail to biomineralize, whereas, the
MC3T3 cells, also in unconditioned medium, produce HAP as early as 21 days.
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3039/L014
Effects of Increased Levels of TIMP-2 and ALA+TIMP-2 in MCF-7 Breast Cancer Cells.
M. Cepeda, S. Damjanovski; Biology, University of Western Ontario, London, ON, Canada
Background: The tissue inhibitors of metalloproteinases (TIMPs) are the natural inhibitors of matrix
metalloproteinases (MMPs), a family of proteins primarily involved in extracellular matrix turnover and
remodeling. The four members of the TIMP family directly inhibit the catalytic activity of MMPs but they
also possess unique biological activities independent of MMP inhibition. Apart from MMP inhibition, TIMP2 induces expression of the MMP inhibitor RECK through an integrin signaling pathway, as well as aids in
the activation of proMMP-2. Objective: To measure changes in gene expression and MMP activity in
MCF-7 breast cancer cells after treatment with increased levels of TIMP-2 or ALA+TIMP-2, a TIMP-2
mutant devoid of MMP binding activity. Methods: To obtain TIMP-2 or ALA+TIMP-2 conditioned media,
MCF-7 cells were transfected with constructs containing full length TIMP-2 or ALA+TIMP-2. After 24
hours of expression, the media from the transfectants was collected and quantified for levels of TIMP-2 or
ALA+TIMP-2. Untransfected MCF-7 cells were then treated with the conditioned media containing
increased levels of TIMP-2 or ALA+TIMP-2. After 24 hours of treatment an invasion assay was performed
and changes in gene expression and MMP activity were measured. Results: TIMP-2 treatment caused an
increase in MMP activity and invasiveness, while ALA+TIMP-2 treatment did not change MMP activity but
did decrease invasiveness. TIMP-2 treatment increased expression of RECK, MMP-2, and MMP-9 while
no change in gene expression was measured after ALA+TIMP-2 treatment. Conclusion: TIMP-2 exhibits
MMP dependent and independent activities that modulate MMP activity and expression. However, these
biological activities may be linked as ALA+TIMP-2 did not induce RECK expression.
3040/L015
Roles of Caveolin-1 and Caveolin-3 in Vascular Smooth Muscle Cell Phenotype.
J. Gutierrez-Pajares, S. Pavlides, M. P. Lisanti, P. Frank; Thomas Jefferson University, Philadelphia, PA
Vascular smooth muscle cells (vSMC) are characterized for their ability to acquire a contractile or
synthetic phenotype that involves a quiescent cellular state or a proliferative and migratory state,
respectively. In this respect, Krüppel-like factor 4 (KLF4) is considered a repressor of vSMC contractile
phenoype. vSMC express the caveolin family of proteins that includes caveolin-1, caveolin-2 and
caveolin-3. While the majority of differentiated cells express caveolin-1 (and caveolin-2), caveolin-3 is
mainly expressed in muscle cells. Interestingly, while vSMC express caveolin-1 and caveolin-3 in the
aorta, in vitro culture of vSMC induce a de-differentiation process characterized by the sole downregulation of caveolin-3. Thus, the aim of this work is to evaluate the role of caveolin-1 and caveolin-3 in
vSMC differentiation. For that purpose the vSMC A7r5 cell line was stably transduced with caveolin-1
(A7r5-C1), caveolin-3 (A7r5-C3), or vector alone (A7r5-v) and were characterized with respect to their
proliferation, migration, and response to culture conditions for inducing a contractile phenotype. We
observed that A7r5-C3 cells cultured on plastic dish showed reduced cellular proliferation rate as assayed
by BrdU incorporation and MTS. Besides, a reduced migration in wound-healing assay was also
observed in A7r5-C3 cells. Western blot analysis showed no differences in KLF4 expression under subconfluent culture conditions. However, confluent cultures in the absence of serum showed that KLF4 was
down-regulated in A7r5-v and A7r5-C3 cells, while persistent expression of KLF4 was observed in A7r5C1. To further investigate the role of caveolin-1 and -3 in vSMC, A7r5 cells were seeded on growth-factorreduced matrigel and their morphology was recorded. After 6 h of culture on matrigel, A7r5-C3 cells selforganized into a cellular network that re-organized 24 h later into a nodular morphology. Although
delayed, these morphological changes were also observed in A7r5-v and A7r5-C1. In conclusion, our
results point to a relevant role of caveolin-3 in vSMC differentiation while caveolin-1 may promote a
synthetic phenotype.
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3041/L016
Inhibition of Lkb1 Kinase Activity Induces Hyperproliferation and Cyst Formation in Mouse
Embryonic Pancreas.
B. Lo, G. Strasser, S. Couto, I. Mellman; GENENTECH, South San Francisco, CA
During pancreatic organogenesis, proliferating epithelial cells polarize and coordinate tubulogenesis
before forming differentiated ductal and acinar structures. A fundamental question is whether any of the
molecular mechanisms underlying these complex developmental processes continue to be important in
suppressing cancer formation in the adult organ. To address this question, we have studied the role of
Lkb1, a well established tumor suppressor, that has been implicated as a crucial regulator of cell cycle
and polarity, in the context of a developing pancreas. Using a chemical genetic approach we have
generated a mouse model in which wild-type Lkb1 has been replaced by a mutant Lkb1 that is
susceptible to pharmacological inhibition. By culturing embryonic pancreas from this mouse ex vivo, we
find that acutely inhibiting Lkb1 kinase activity leads to hyperproliferation of ductal epithelial cells and
dramatic formation of epithelial lined cystic structures. The induction of cysts appears to be limited to late
stage embryonic tissue in which acinar structures have already started to form. Mechanistically, the
development of cystic structures is suppressed by rapamycin and other mTOR inhibitors, suggesting that
Lkb1 inhibition leads to dysregulated mTOR activity. The use of the AMPK activator, A769662, however,
can only partially suppress the cystic phenotype leading to us to speculate that Lkb1 inhibition exerts
some of its effects in the embryonic pancreas via other downstream targets besides AMPK.
3042/L017
A Novel Role for Nitric Oxide/cGMP in Regulating TNFR1 to Inhibit TNF-Induced Hepatocyte
Apoptosis.
R. S. Chanthaphavong, Y. T. Lee, P. A. Loughran, M. J. Scott, T. R. Billiar; Surgery, University of
Pittsburgh, Pittsburgh, PA
Introduction: Hepatocyte (HC) cell death after TNF contributes to acute/chronic liver disease. TNF binds
TNFR1 to recruit DISC proteins, cleave caspase-8 and initiate apoptosis. Nitric oxide(NO) and cGMP are
known to inhibit apoptosis through NFκB-dependent gene expression + direct inactivation of caspases.
We hypothesized that NO/cGMP can also inhibit DISC formation + apoptosis by regulation of TNFR1
expression at the cell surface. Objective: To determine the role of NO/cGMP in regulating TNFR1
expression, DISC formation and apoptosis in mouse HC and liver Methods: Primary cultured mouse HC
were stimulated with TNF(2000U/mL) for up to 2h to induce apoptosis. Some cells were pretreated with
SNAP(NO-donor;200μM) + 8Br-cGMP(800μM) for 30min. Other cells were primed with
LPS(10ng/mL)+IFNδ(50U/mL) to increase TNFR1 expression, +/- TNFα-converting enzyme(TACE)
inhibitor(TAPI-1;10μM) for 24h. Cell viability, apoptosis + TNFR1-level were measured. TACE activity was
determined using Sensolyte TACE assay. FADD and caspase-8 were immunoprecipitated with TNFR1,
and visualized by colocalizing confocal immunofluorescence. In vivo, C57BL/6 mice injected
intraperitoneally with LPS(5mg/kg) +/- 30min pretreatment with 1400W(iNOS inhibitor;5mg/kg). Liver
TNFR1 expression was measured at 12h. Results: HC cell surface levels of TNFR1 decreased
significantly after treatment with NO/cGMP. DISC formation occurred in HC treated with TNF for 15min, +
FADD/caspase-8 coimmunoprecipitated with TNFR1 at 30min. Treatment with NO/cGMP significantly
inhibited DISC formation and apoptosis at 2h, also decreased significantly after NO/cGMP treatment.
TACE activity was significantly increased by NO/cGMP treatment and inhibition of TACE significantly
increased apoptosis level. TACE cleaved full length TNFR1 into segments, which were then ubiquitinated
(shown by ubiqutitin pull-down assay) and degraded after TNF or cGMP treatment. Full length TNFR1
was not ubiquitinated. In vivo, LPS treatment decreased liver TNFR1 level and this was inhibited by iNOS
inhibition with 1400W. Conclusions: Our results suggest NO/cGMP regulates TNF-induced apoptosis
through the activation of TACE to decrease levels of cell surface TNFR1 + decrease DISC formation.
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3043/L018
Cyclic AMP Regulates Lumen Formation in Mammary Acini by Accelerating Apoptosis.
P. I. Nedvetsky, S. Kwon, J. Debnath, K. M. Mostov; UCSF, San Francisco, CA
Epithelial cells form tubular and acinar structures notable for the presence of a hollow lumen. During
three-dimensional (3D) culture of MCF10A mammary epithelial acini, an established in vitro model for
glandular epithelial morphogenesis, lumen formation occurs through the apoptosis of central cells lacking
direct contact with surrounding extracellular matrix (ECM). Here, we identify the second messenger cAMP
as a previously unrecognized signal required for efficient lumen formation in MCF10A acini. When grown
in a laminin-rich extracellular matrix (Matrigel), MCF10A form spheroids or acini with a hollow lumen;
lumen formation is due to the death of centrally-located cells during morphogenesis. The standard culture
medium for MCF10A contains cholera toxin, a reagent that elevates cellular cAMP level by direct
interaction with cAMP-producing enzymes, adenylyl cyclases. Upon omitting cholera toxin from the
medium, a hollow lumen is not formed. Since cAMP is known to accelerate proliferation of mammary
epithelial cells, we assessed if the effects of reduced cAMP activations were due to reduced cell
proliferation. However, in MCF10A acini expressing HPV 16 E7, an oncoprotein that maintains high levels
of proliferation of cells, lumen formation still required cAMP. Strikingly, we observed that the presence of
cAMP strongly increased luminal apoptosis in MCF10A acini. This was concomitant with elevated
expression of the pro-apoptotic proteins BIM. Because BIM has been proposed as an essential mediator
of luminal cell death in MCF10A 3D acini, we hypothesize that cAMP regulates lumen formation via
induction of BIM and possibly other apoptotic proteins. Although previous work indicates that the
detachment of MCF10A cells from extracellular matrix is the primary regulator of BIM, our results indicate
that this induction at least partially depends on signals downstream of cAMP. In summary, cAMPdependent signaling represents a parallel, detachment-independent pathway for induction of apoptotic
proteins in MCF10A spheroids and thus contributes to lumen formation in mammary epithelial 3D culture.
3044/L019
Epigenetic Differences between Human Tissue and Corresponding Transformed and NonTransformed Cultured Cell Lines.
V. Devgan, S. Huang, S. Bao, G. Wilt, Q. Jiang; SABiosciences, Frederick, MD
Human immortalized and non-transformed cell lines have served as model biological systems for studying
drug function, metabolism, toxicity and signal transduction mechanisms. Although cell lines have been
shown to acquire additional genetic abnormalities during culture, little is known about epigenetic
differences between human tissue and cultured cell lines. The methylation status of homeobox genes are
known to play important roles in tissue specificity and therefore likely hot spots for such epigenetic
changes. In this study, the methylation status of 96 CpG islands near homeobox genes was measured in
human liver, kidney and lung tissue samples, along with samples from transformed cell lines derived from
human liver (HepG2), kidney (HEK293H) and a non-transformed cell line derived from human lung tissue
(IMR-90). A restriction enzyme-based quantitative PCR array was used to profile the DNA methylation
status. The results showed significant differences in the DNA methylation status of homeobox genes
between human tissues and transformed cell lines. In all three human tissues the homeobox gene
associated CpG islands were mostly unmethylated, with only 1-5% being hypermethylated. This also held
true for the non-transformed cell line, IMR-90, with similar levels of methylation as in lung tissue. In
contrast, 24-40% of the homeobox gene associated CpG islands were hypermethylated in the
transformed cell lines (HepG2 and HEK293H). Furthermore, the gene expression profile of the
hypermethylated genes in HepG2 cells was investigated using a SYBR Green real-time RT-PCR. Most of
the hypermethylated genes (23 out of 24) showed undetectable or very low levels of expression,
suggesting that the hypermethylation is associated with the expression silencing of these genes. Taken
together, our data suggest that the epigenetic signatures of transformed cancer cell lines are significantly
different from that of corresponding human tissues and should be considered when developing gene
expression models from those culture systems.
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3045/L020
Cyclic Mechanical Stretch Induces Secretion of VEGF through an NF-κB Pathway in Adult Rat
Cardiac Myocytes.
1
2
1 1
A. Leychenko , E. Konorev , M. Matter ; Cell & Molecular Biology, University of Hawaii-Manoa, John A
2
Burns School of Medicine, Honolulu, HI, Pharmaceutical Sciences, University of Hawaii-Hilo College of
Pharmacy, Hilo, HI
Cardiovascular diseases such as hypertension and myocardial infarction are associated with the onset of
hypertrophy. Hypertrophy occurs as a compensatory response mechanism to an increase in mechanical
load due to pressure or volume overload. It is characterized by extracellular matrix remodeling and
hypertrophic growth of cardiomyocytes. Production of Vascular Endothelial Growth Factor (VEGF), a key
signaling molecule which acts as an angiogenic factor and a modulator of cardiomyocyte function, is
regulated by mechanical stress. We sought to determine whether cyclic mechanical stretch promotes
VEGF secretion in primary adult rat cardiac myocytes (ARCMs). Using an in vitro model of cultured
ARCMs, our study investigated the process of mechanotransduction and its effects on VEGF secretion.
Cardiomyocytes were isolated from adult (6-week old) Sprague-Dawley rats and allowed to attach to
laminin-coated flexible-bottomed plates in serum-free conditions for 24h prior to initiation of experiments.
Cells were subjected to cyclic mechanical stretch at an extension level of 10% at 30 cycles/min that
induces hypertrophic responses. Secreted VEGF levels were determined from conditioned media by
ELISA. Cyclic mechanical stretch of ARCMs induced VEGF secretion approximately by 2.2-fold at 24h
and 3-fold at 48h compared to non-stretched controls. This increase in stretch-induced VEGF secretion
was paralleled by an increase in FAK phosphorylation at Y861 but not Y397 and the activation of the NFκB signal transduction pathway. The observed stretch-induced VEGF secretion was blocked by an NF-κB
inhibitor, but not by inhibitors of the MAPK or PI3K pathways. These findings indicate that NF-κB
activation plays a central role in mediating VEGF secretion upon cyclic mechanical stretch in adult cardiac
myocytes. These findings also provide a mechanistic insight in that signaling by FAK-NF-κB initiated in
response to stretch coordinates the hypertrophic response in ARCMs.
3046/L021
Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) Induced Mitochondrial
Pathway to Apoptosis and Caspase Activation Is Potentiated by Phospholipid Scramblase-3.
K. Ndebele, R. Khosravi; Pathology, Harvard Medical School, Boston, MA
Abstract Death receptors initiate pathways of cell death in which caspase activation is mediated either
directly (without mitochondrial amplification), or indirectly via the release of apoptogenic factors from
mitochondria. Phospholipid scramblases (PLS) are enzymes that play a key role in cellular function by
inducing bidirectional movement of membrane lipids. Changes in mitochondrial membrane lipids,
cardiolipin, are critical for mediating apoptotic response in many cell-types. PLS3 is a phospholipid
scramblase that is localized to mitochondria and is thought to be involved in the regulation of apoptotic
signals. Here we report that over-expression of PLS3 significantly enhances apoptotic death induced by
the Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) by potentiating the
mitochondrial arm of the extrinsic death pathway. Thereby, PLS3 expression facilitates changes in
mitochondrial membrane lipids that promote the release of apoptogenic factors and consequent full
activation and processing of the caspase cascade. Finally, we demonstrate that TRAIL-induced activation
of PKC-delta mediates regulation of the PLS3-induced changes in cardiolipin.
3047/L022
Enhanced Oxidative Stress and Incidence of Pathological Lesions in the Ventral Prostate of Obese
Rats.
1,2
2
2
2
2
2 1
D. L. Ribeiro , M. G. Gobbo , M. E. Pinto , S. R. Taboga , E. A. Almeida , R. M. Góes ; ICBIM2
Histologia, Federal Univeristy of Uberlandia, Uberlandia, Brazil, IBILCE, São Paulo State University, São
José do Rio Preto, Brazil
The objective of this investigation was to evaluate cell proliferation, oxidative stress and incidence of
pathological lesions in the prostate of obese rats, seeking for subsides to clarify the relation between
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obesity and prostate cancer. Obesity was induced by high-fat diet (20% fat) feeding while control animals
received balanced diet (4% fat). After 17 weeks, rats were killed and ventral prostate was removed,
weight and processed for light miscroscopy to detection of pathologycal lesions, for
immunohistochemistry (cell proliferation- PCNA, androgen receptor-AR and estrogen receptors- ER) for
western blotting (ER, Phosphatidylinositol-3 kinase - PI3K, Fibroblast growth factor-FGF) and also for
quantification of oxidative stress-related enzimes (glutathione S-transferase- GST and catalase) and end
products (malondialdehyde). Also, blood sample was collected for analysis of testosterone, estradiol and
insulin plasma levels. Histopathological analysis of prostate showed greater incidence of acinar atrophy,
prostatic intraepithelial neoplasia (PIN) and inflammation in obese when compared to control rats. Obesity
caused a significant increase in insulin and a decrease in testoterone plasma levels. Also, cell
proliferation increased 150% in the prostate of obese group. Regarding sexual steroidal receptors, AR
reduced 50% while ERα and β enhanced 150% and 121% respectively in this group. PI3-K and FGF also
had a significant increase in obese rats in relation to control. The catalase activity in prostate increased
significantly while GST and MDA did not change. This data clearly demostrated that in the early stages of
obesity the control of oxidative stress is impaired in prostate, which in association to increased cell
proliferation could cause PIN and inflammation. This cell proliferation is probably not triggered by AR, but
by ERs signaling in association to PI3K, a survival pathway. Moreover, FGF is considered a potent
angiogenic factor and can be associated to tumor formation. Thus, this experimental investigation
discriminate initial morphological and molecular alterations caused by obesity that could trigger malignant
development in prostate. Financial Support- FAPESP and FAPEMIG.
3048/L023
Bortezomib Induces Nuclear Translocation of Iκbα Resulting in Gene Specific Suppression of
NFκB-Dependent Transcription and Induction of Apoptosis in Human Cutaneous T-Cell
Lymphoma.
A. Juvekar, S. Manna, S. Ramaswami, T. Chang, I. Vancurova; Biology, St. John's University, Queens,
NY
Cutaneous T cell lymphoma (CTCL) is characterized by constitutive activation of NFκB, which plays a
crucial role in the survival of CTCL cells and their resistance to apoptosis. NFκB activity in CTCL is
inhibited by the proteasome inhibitor bortezomib (Velcade); however, the mechanisms remained
unknown. In this study, we investigated mechanisms by which bortezomib suppresses NFκB activity in
CTCL Hut-78 cells. We demonstrate that bortezomib suppresses NFκB activity in Hut-78 cells by a novel
mechanism that consists of inducing nuclear translocation and accumulation of IκBα, which then
associates with NFκB p65 and p50 in the nucleus and inhibits NFκB DNA binding activity. Surprisingly,
however, while expression of NFκB-dependent anti-apoptotic genes cIAP1 and cIAP2 is inhibited by
bortezomib, expression of Bcl-2 is not suppressed. Chromatin immunoprecipitation indicated that cIAP1
and cIAP2 promoters are occupied by NFκB p65/50 heterodimers, while Bcl-2 promoter is occupied
predominantly by p50/50 homodimers. Collectively, our data reveal a novel mechanism of bortezomib
function in CTCL and suggest that the inhibition of NFκB-dependent gene expression by bortezomib is
gene specific and depends on the subunit composition of NFκB dimers recruited to NFκB-responsive
promoters. Funded by: NIH grants GM079581 and AI085497 to I.V.
3049/L024
Vaccinia A25 and A26 Proteins Are Fusion Suppressors for Mature Virions and Determine StrainSpecific Virus Entry Pathways into Hela, CHO-K1 and L Cells.
W. Chang; IMB, Academia Sinica, Taipei, Taiwan
Shu-Jung Chang1,2, Yu-Xun Chang1, Roza Izmailyan1, Yin-Liang Tang1,3 and Wen Chang1* 1Institute
of Molecular Biology, Academia Sinica, 2Institute of Genomics Sciences, National Yang-Ming University,
3Graduate Institute of Life Sciences, National Defense Medical Center, Taiwan, R.O.C. Objective:
Vaccinia mature virus enters cells through either fluid-phase endocytosis/ macropinocytosis or plasma
membrane fusion. This may explain the wide range of host cell susceptibility for vaccinia virus entry;
however, it is not known how vaccinia virus chooses between these two pathways and which viral
envelope proteins determine such processes. Methods: By screening several recombinant viruses and
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different strains we found that mature virions containing vaccinia A25 and A26 proteins entered HeLa
cells preferentially through a bafilomycin-sensitive entry pathway, whereas virions lacking these two
proteins entered through a bafilomycin-resistant pathway. To investigate whether A25 and A26 proteins
contribute to entry pathway specificity, two mutant vaccinia viruses, WRΔA25L and WRΔA26L, were
subsequently generated from wild-type WR strain. In contrast to the WR strain, both WRΔA25L and
WRΔA26L became resistant to bafilomycin suggesting that removal of A25 and A26 proteins bypassed
the low pH endosomal requirement for mature virions entry. Indeed, WRΔA25L and WRΔA26L infections
of HeLa, CHO-K1 and L cells immediately triggered cell-to-cell fusion at neutral pH at 1-2h p.i., providing
direct evidence that viral fusion machinery is readily activated after removal of A25 and A26 proteins to
allow virus entry through plasma membrane. Conclusion: In summary, our data support a model that, on
vaccinia mature virions, viral A25 and A26 proteins are low pH-sensitive fusion suppressors whose
inactivation during endocytic route resulting in viral and cell membrane fusion. Our results also suggest
that during virion morphogenesis, incorporation of A25 and A26 proteins into mature virions may help
restrain viral fusion activity until the time of infections.
3050/L025
The Role of Micro-RNA in Mesenchymal to Stem-Like Transition.
1,2
1
1
1,2
1
1,2 1
H. Xia , J. Li , G. Lu , H. Yao , W. Poon , M. Lin ; Brain Tumour Centre and Division of
Neurosurgery, Department of Surgery, The Chinese University of Hong Kong, Hong Kong, Hong Kong,
2
Department of Chemistry, The University of Hong Kong, Hong Kong, Hong Kong
The microRNAs (miRNAs) are known to regulate tumor progression by suppressing target gene
expressions. Ample studies have implicated the important role of miRNAs in regulating Epithelialmesenchymal transition (EMT). EMT is a crucial developmental program in which immotile epithelial cells
acquire mesenchymal traits. Activation of EMT triggers tumor cell invasion and dissemination, and is thus
considered as the initiating step of cancer metastasis. Recently, several studies have demonstrated that
EMT is associated with cells with stem cell properties. We report here that miR-200 family members
regulate mesenchymal to stem-like transition in nasopharyngeal carcinoma (NPC) cells. Among the five
members, the expressions of miR-200b~200a~429 and miR-200c~141 clusters, presumably encoded in
form of polycistronic transcripts, are reciprocally repressed by their downstream targets ZEB1 and ZEB2.
We showed that suppressiong miR-200, induced C666-1 into expressing several stem cell-like traits
including CD133+ side population, sphere formation capacity, in vivo tumorigenicity in nude mice and
stem cell markers expression. Consistently, stable overexpression of miR-200 switched a significant
reduction of stem-like cell features. The in vitro differentiation of C666-1 tumorsphere resulted in
diminished stem-like cell population and miR-200 induction. Our findings reveal for the first time the
function of miR-200 family members in shifting NPC cell states via a reversible process coined as
epithelial-mesenchymal to stem-like transition (EMST) through differential and specific mechanisms.
3051/L026
The Influence of Hepatocyte Growth Factor during Outer Segment Phagocytosis by Retinal
Pigment Epithelium.
1,2,3
1,2,3
1,3 1
J. F. Blaize , J. Tachjadi , W. J. L'Amoreaux ; Biology, College of Staten Island, Staten Island,
2
3
NY, Neuroscience, CUNY Graduate Center, New York, NY, Advanced Imaging Facility, College of
Staten Island (CUNY), Staten Island, NY
Inhibition of outer segment (OS) processing by retinal pigment epithelium (RPE) has been linked to
photoreceptor injury; as such, complete understanding of the cellular miscues that precede RPE failure is
crucial for the maintenance of visual health. Sub-retinal clearance by RPE is facilitated by specialized
phagocytosis featuring both RPE specific and traditional FCγR mediated signaling cascades. This
integration renders RPE capable of internalizing both specific and non-specific targets. The discovery that
lack of c-Met signaling results in impairment of phagocytosis in alveolar and hepatocyte macrophages by
Huh et al. suggests c-Met’s role as modulator of this process. Since PI3K has been shown to activate
Rac, a key regulator of phagosome formation during FCγR mediated phagocytosis, we hypothesize that
PI3K phosphorylation by c-Met is capable of mediating OS clearance by RPE and that secretion of HGF
is involved in regulation of this behavior. To test our hypotheses, ARPE-19 were cultured until 70%
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confluent and then serum deprived for 24hrs. Cells were then exposed to various concentrations of HGF
prior to chemical fixation. Established immunocytochemical techniques were employed to prepare cells
for florescence microscopy where receptor expression was evaluated. Intensity values suggest that
ARPE-19 respond maximally to concentrations of 25 ng/ml of HGF when compared to controls (0ng/ml
HGF). Similar studies evaluating expression of phosphorylated c-Met, downstream targets of c-Met and
binding of non-specific targets were conducted with cells prepared as before. Our findings suggest that
RPE respond to increases of exogenous HGF concentrations by up-regulating its receptor and certain
downstream targets. Additionally, our data show a significant increase of fluorescently labeled E.coli and
fluorescently labeled latex bead binding in treated groups. Taken together, these findings suggest that
RTK cross-talk initiated by c-Met activation can partially mediate uptake of non-specific debris and may
influence OS recognition by RPE.
3052/L027
The Hereditary Spastic Paraplegia Protein Strumpellin Binds Members of the WASH and Retromer
Complexes.
1,2
2,3
2,3
1,2 1
C. L. Freeman , M. Harbour , M. Seaman , E. Reid ; Dept of Medical Genetics, University of
2
Cambridge, Cambridge, United Kingdom, Cambridge Institute for Medical Research, University of
3
Cambridge, Cambridge, United Kingdom, Dept of Clinical Biochemistry, University of Cambridge,
Cambridge, United Kingdom
The WASH complex comprises a number of proteins that are recruited by retromer to endosomal
compartments, where they act at the interface of endosome dynamics and cytoskeletal regulation.
Knockdown of any of several WASH complex members induces aberrant endosomal tubulation. One of
these proteins, strumpellin, is mutated in a rare form of hereditary spastic paraplegia, a group of
neurodegenerative disorders where progressive distal axonopathy leads eventually to lower limb
paralysis. Three point mutations in the strumpellin protein have been identified in patients thus far, but
their precise effects upon protein function remain unknown. We aim to elucidate the implications of
strumpellin mutation upon the protein’s behaviour in both neuronal and non-neuronal cell types. We have
demonstrated, by co-immunoprecipitation, an interaction between strumpellin, the actin regulating protein
WASH1 and the retromer component VPS26. We have further shown that these proteins interact in
rodent brain tissue. Using similar techniques, we have demonstrated that strumpellin mutation does not
ablate its interaction with members of either the WASH complex or the retromer complex, indicating that
mutation is likely to affect functional rather than binding domains. We have examined the localisation of
these complex components in rat primary neurons using co-immunofluorescence techniques, and have
shown that their presence at endosomal compartments, previously reported for non-neuronal cell types, is
recapitulated in neuronal cells. We therefore suggest that an interaction between the WASH and retromer
complexes is maintained in neuronal cells, and that disruption of their role in vesicle trafficking has
relevance to the phenotype of progressive axonopathy
3053/L028
Effect of Glycine on the Cyclooxigenase Pathway of the Kidney Arachidonic Acid Metabolism in a
Rat Model of Metabolic Syndrome.
1
1
1
2 1
E. Soria , I. Perez , B. Ibarra , G. Baños ; Pathology, Instituto Nacional de Cardiologia"Ignacio Chavez",
2
Mexico, Mexico, Cardiovascular Biomedicine, Instituto Nacional de Cardiología "Ignacio Chavez",
Mexico, Mexico
The kidneys are organs that may be severely impaired in the metabolic syndrome (MS). MS is
characterized by the association of various pathologies such as; hypertension, dyslipidemia,
hyperinsulinemia and type 2 diabetes. Glycine, a non-essential amino acid, is known to possess various
protective effects in kidney such as: decrease in deterioration of renal function, the inhibition of proximal
sodium reabsorption and the damage caused by hypoxia. In a rat model of MS, the effect of glycine on
the cyclooxygenase pathway of the arachidonic acid (AA) metabolism was studied in isolated perfused
kidney. MS was induced in Wistar rats by feeding them a 30% sucrose solution for 16 weeks. The
addition of 1% glycine to their drinking water containing 30% sucrose, for 8 weeks, reduced high blood
pressure, triglycerides, insulin concentration, HOMA index, albuminuria, free AA concentration, renal
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perfusion pressure, prostaglandin levels, PLA2 expression and COX isoform expression, compared with
MS rats which did not receive this additional glycine. GlyR β receptor expression decreased significantly
in MS, but glycine treatment increased it. The results suggest that 1% glycine treatment improves the
renal function when acting as anti-inflammatory on the COX pathway of AA metabolism in kidney,
reversing some of the damage provoked by the high sucrose consumption in the MS model.
3054/L029
MUC1 Mucin Inhibits Flagellin-Stimulated TLR5 Signaling through MyD88.
1
2
1 1
K. Kato , H. Kai , K. Kim ; Department of Physiology & Lung Center, Temple University School of
2
Medicine, Philadelphia, PA, Department of Molecular Medicine, Faculty of Pharmaceutical Sciences,
Kumamoto University, Kumamoto, Japan
Background: MUC1 is a membrane-tethered mucin glycoprotein expressed on the surface of mucosal
epithelial cells as well as on hematopoietic cells. Recently, we demonstrated that MUC1 suppressed
flagellin-induced Toll-like receptor 5 (TLR5) signaling in vivo and in vitro. Objective: To further elucidate
the mechanism through which MUC1 suppresses flagellin-TLR5 signaling. Methods: HEK293 cells were
transfected with expression plasmids encoding MUC1 and TLR5, or empty vector as a negative control,
and the levels of phosphorylated MAPK and IkB-a were measured by Western blot analysis after
treatment with flagellin (TLR5 stimulus) or TNF-a (non-TLR5 stimulus). In addition, NF-kB activation was
measured using the ELAM-1 promoter-luciferase reporter assay. Finally, the cells were cotransfected with
plasmids encoding TLR5 downstream signaling proteins (MyD88, IRAK1, or TRAF6) and abrogation of
the inhibitory effect of MUC1 expression on flagellin-induced NF-kB activation was determined by
comparison with cells transfected with empty vector Results: (a) MUC1 overexpression attenuated TLR5dependent flagellin-induced phosphorylation of MAPK and IkB-a as well as activation of NF-kB, compared
with MUC1 non-expressing cells. In contrast, TNF-a-induced activation of MAPK and NF-kB was
unaffected by MUC1 expression. (b) Overexpression of MyD88 abrogated MUC1-dependent inhibition of
flagellin-TLR5-dependent NF-kB activation compared with cells expressing endogenous levels of MyD88,
whereas overexpression of IRAK1 or TRAF6 had no effect. (c) Flagellin-induced association of TLR5 and
MyD88 was inhibited by the presence of MUC1. Conclusions: These results suggest that MUC1
expression inhibits MyD88 recruitment, thereby suppressing MAPK and NF-kB activation downstream of
the TLR5 signaling pathway. We believe that MUC1 plays a crucial role in the resolution of inflammation
during airway infection.
3055/L030
Evidence for Functional TAP in the Mycobacterium Tuberculosis Phagosome.
1,2
3
1,2 1
M. Harriff , D. A. Lewinsohn , D. Lewinsohn ; Research, Portland VA Medical Center, Portland, OR,
2
Department of Pulmonary and Critical Care Medicine, Oregon Health & Sciences University, Portland,
3
OR, Pediatrics, Oregon Health & Sciences University, Portland, OR
Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that can reside in both myeloid and nonmyeloid cells in the lung. In myeloid cells such as dendritc cells (DC), Mtb survives and replicates in
phagosomes that do not fuse with the lysosome. Recognition of those cells infected with Mtb is essential
to containing infection. Recent studies have shown that the Mtb phagosome is a competent antigen
presenting organelle, as it contains HLA-I and the transporter associated with antigen processing (TAP),
and has the ability to directly elicit an IFN-γ response from HLA-E-restricted CD8+ T cells. Furthermore, it
has been shown that RD1 is not required for efficient processing and presentation of Class I antigens. In
this study, we looked at the role of phagosomal TAP in the transport of peptides into the phagosome for
subsequent loading on Class I molecules. Highly pure Mtb phagosomes contained TAP and Class I, as
well as other molecules involved in peptide loading in the endoplasmic reticulum (ER), such as ERp57.
To show that TAP in the phagosome was functional, we synthesized a 40 amino acid peptide
representing the lumenal domain of the bovine herpes virus TAP inhibitor, UL49.5. We showed that in the
presence of this peptide, we could inhibit presentation of OVA and Mtb antigen to T cell clones, as
measured by IL-2 or IFN-γ. TAP-dependent peptide transport across the phagosome membrane was
observed in highly pure Mtb phagosomes isolated from DC. Furthermore, coupling of the UL49.5 peptide
directly to Mtb inhibited presentation of Mtb antigens to the HLA-E-restricted T-cell clone, D160 1-23. Our
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data are consistent with the hypothesis that the Mtb phagosome is involved in presentation of Mtb
antigens, and show that phagosomal TAP is involved in the loading of these antigens.
3056/L031
Unfolded Protein Response and Autophagy Behave as Complement Pathways to Dispose ZAAT in
Hepatocytes.
1,2
1
1
1
1,2 1
K. Xiao , L. Wang , E. J. McAndrew , F. N. Rouhani , M. L. Brantly ; Department of Medicine,
2
University of Florida, Gainesville, FL, Genetics & Genomics Program, University of Florida, Gainesville,
FL
Alpha-1 antitrypsin (AAT) is a 52 kDa serine protease inhibitor (PI) that is predominantly synthesized and
secreted by hepatocytes. One of its major functions is to inhibit neutrophil elastase in the lung. The most
common disease associated mutation is the Z mutation (Glu342Lys). ZAAT polymerizes in the
endoplasmic reticulum (ER) of liver cells and accumulation may be associated with liver disease.
Accumulation of ZAAT leads to a series cellular responses including the Unfolded Protein Response
(UPR), Autophagy and ER associated degradation (ERAD). The objective of this study is to determine the
relationship between UPR and Auotphagy in disposing of ZAAT in human hepatocytes. We hypothesized
that UPR and Autophagy are co-responsible for disposing accumulated ZAAT. To assess our hypothesis,
human hepatocytes for a PI*ZZ individuals were transfected with a plasmid expressing ZAAT for 6, 12, 24
and 48 hours. And cells were harvested for total RNA and protein at each time point. UPR gene
expression was determined by qPCR and autophagy related status was determined by Western blot
using LC3 antibody. The results show that UPR gene expression level and LC3 II/LC3 I ratio are
significantly increased after transfection. The time course experiment also revealed that UPR activation is
a time and cargo-amount dependent response. Overall, understanding the relationship between these
stress responses and disease progression is critical to identify potential therapeutic targets for liver
disease in AAT deficiency individuals.
3057/L032
Mechanism of Toll-Like Receptor 7 (TLR7) Agonist-Driven Increase in Cytopathic Effect in
Coronavirus (Cov)-Infected Macrophages.
1,2
2
2 1
L. Mazaleuskaya , J. Martin-Garcia , S. Navas-Martin ; Pharmacology and Physiology Graduate
2
Program, Drexel University College of Medicine, Philadelphia, PA, Department of Microbiology and
Immunology, Center for Molecular Virology and Translational Neuroscience, Institute for Molecular
Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA
Coronaviruses (CoVs) are enveloped, single-stranded, positive-sense RNA viruses that cause a variety of
diseases in different animal species, including humans. A hallmark of all CoVs is their ability to infect
macrophages. However, the precise role of macrophages in CoV-induced pathogenesis and the effects of
CoV infection in macrophage function remain poorly understood. Infections of mice with Mouse Hepatitis
Virus (MHV), the prototype of Group II CoVs, provide models of viral infection of the central nervous
system (CNS). MHV-A59 strain is used to study virus-induced chronic demyelination in the mouse model.
MHV-induced demyelination is associated with infiltrating macrophages and resident microglia in the CNS
white matter. Our understanding of CoV interaction with the host innate immune system is quite limited.
Toll-Like Receptors (TLRs) are pattern recognition receptors that recognize conserved microbial motifs.
TLR2 and TLR4 may sense viral glycoproteins, while TLR3 and TLR7 detect viral dsRNA and ssRNA,
respectively. Activation of TLRs leads to the production of type I interferon and pro-inflammatory
cytokines and ultimately triggers adaptive immunity. It is still unclear whether CoVs induce inflammation
through activation of TLRs and if so, which TLRs activate innate immune response during viral infection.
During our studies, we have found that TLR7 agonist Imiquimod induces increased cytopathic effect in
MHV-infected macrophages. Here, we aim at elucidating the mechanism behind this phenomenon. Our
data suggest that this effect is mediated by TLR7 and depends on MHV spike glycoprotein. In contrast,
Imiquimod has no effect on the virus production in macrophages. Overall, our studies highlight a novel
effect of Imiquimod on virus-induced cell-to cell fusion and have implications towards the understanding
of TLR7 signaling in macrophages.
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3058/L033
Saffron Protects against Liver Cancer in Animal Model: Molecular and Imaging Insights.
1
2
3
4
1 1
A. Amin , D. Mahmoud-Ghoneim , P. Corr , A. Hamza , S. Daoud ; Biology, UAE University, Al-Ain,
2
3
United Arab Emirates, Physics, UAE University, Al-Ain, United Arab Emirates, Radiology, UAE
4
University, Al-Ain, United Arab Emirates, Pathology, Twaam Hospital, Al-Ain, United Arab Emirates
Saffron is a natural product that is a promising chemo-preventive agent against different cancer. The aim
of the present work was to investigate the chemo-protective effect of saffron ethanol extract on
chemically-induced liver cancer in rats. Hepatocarcinogenesis was induced by a single intraperitoneal
injection of diethyl nitrosamine (DEN) (200 mg/kg body weight) and 2 weeks later, the carcinogenic effect
was promoted by 2-acetylaminofluorene (2-AAF) (0.05%). 42 Wister rats were divided into seven groups:
negative control, positive control (DEN+2-AAF), saffron-alone group (saffron for 22 weeks, no DEN or 2AAF) and 3 preventive groups (where saffron was administered at 300, 150 and 75 mg/kg for 2 weeks
before and 20 weeks after inducing cancer). The incidence and multiplicity of liver tumors as well as the
expression of cancer marker, glutathione S-transferase placental form, were significantly reduced in
preventive groups. This hepatopreventive effect was confirmed by the decreasing in cancer marker, α feto
protein in plasma. Immunohistochemical analyses showed a decrease in cell proliferation marker (Ki67)
and an increase of TUNEL-and caspase 3-positive apoptotic cells along with strong expressions of p53 in
liver of preventive groups. These protective effects are mediated by anti-inflammatory and antioxidant
effects. The anti-inflammatory activity was confirmed by saffron-based inhibition of inducible nitric oxide
synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression. The effect of saffron on the
expression of nuclear factor nuclear factor-Kappa B (NF-kappa B) was also assessed. The expression of
NF-kappa B in the preventive groups was significantly low compared to positive control. The in vitro
antioxidant capacity of saffron (FRAP Assay, DPPH Scavenging activity and ABST+ Assay) was
significantly high in ethanol-based extract. In addition, NF-kappa B transcription factor seems to regulate
the saffron chemo-protective effects. Finally, texture analysis was applied to analyze in vivo MRI images
and results were consistent with that of histopathology.
3059/L034
The Two Human [2Fe-2S] Ferredoxins Perform Distint Functions in Steroidogenesis, Heme, and
Iron-Sulfur Biosynthesis.
1
2
2
2
2
3
3
A. Sheftel , O. Stehling , H. Elsässer , U. Mühlenhoff , H. Webert , A. Hobler , F. Hannemann , R.
3
2 1
2
Bernhardt , R. Lill ; OHI / University of Ottawa, Ottawa, ON, Canada, Institut für Zytobiologie, Philipps3
Univesität-Marburg, Marburg, Germany, FR 8.3 - Biologie, Institut für Biochimie, Universität des
Saarlandes, Saarbrücken, Germany
Iron sulfur clusters (Fe/S) are essential cofactors utilized by numerous, indispensable proteins in virtually
every organism. The majority of these metallocofactors are generated and used by proteins within the
mitochondria. Because of the generally oxidizing conditions to which most organisms are subject, as a
consequence of the atmospheric oxygen concentration, the formation of Fe/S is a process that relies on
the orchestrated cooperation of numerous, dedicated proteins. Among these proteins is a [2Fe-2S]
ferredoxin. In yeast, the [2Fe-2S] protein Yah1 (yeast adrenodoxin homolog 1) has been shown to be a
core component of the mitochondrial Fe/S biogenesis machinery. In addition, this protein has been
implicated in the formation of heme a, an essential cofactor of cytochrome oxidase. The human genome
contains two [2Fe-2S] ferredoxins that exhibit appreciable sequence homology to Yah1. The first of these
proteins to be characterized, FDX1 (a.k.a., adrenodoxin), reduces mitochondrial cytochrome P450
enzymes in the synthesis of, e.g., pregnenolone, aldosterone, and cortisol. It has long been assumed that
FDX1 also performs essential roles in human Fe/S and heme biosynthesis, owing to is sequence
homology to Yah1. Our results show this assertion to be false. By RNAi-mediated depletion of FDX1 or
FDX2 (a.k.a., FDX1L; the second human homolog to Yah1), we demonstrate that only cells deficient in
FDX2 have compromised Fe/S protein and cytochrome oxidase function. Furthermore, in contrast to
FDX1, FDX2 was unable to reduce a mitochondrial cytochrome P450 enzyme in vitro. Taken together,
these data demonstrate that the two human [2Fe-2S] ferredoxins perform exclusive roles in distinct
biochemical pathways. The downstream consequences of Fe/S protein deficiency on iron metabolism
were also investigated. These latter experiments show concurrent, aberrant increases in iron uptake and
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decreases in storage, with excess iron accumulating in mitochondria. Although mitochondrial iron
accumulation has been observed in experimental animals and in patients with diseases of Fe/S
biogenesis (e.g., Friedreich ataxia,), this is the first acute model of mitochondrial iron overload in human
cells with compromised Fe/S biogenesis.
3060/L035
Amyloid-β Internalization Dynamics and Redistribution of Myosin-Vb.
1
1
2
3
4
1
L. T. Oliveira , P. A. Matos , D. Provance , L. R. Andrade , F. G. de Mello , M. M. Sorenson , V. P.
1,5 1
2
Salerno ; Instituto de Bioquímica Médica, UFRJ, Rio de Janeiro, Brazil, Instituto Osvaldo Cruz, Rio de
3
4
Janeiro, Brazil, Departamento de Histologia e Embriologia, UFRJ, Rio de Janeiro, Brazil, Instituto de
5
Biofísica Carlos Chagas Filho, UFRJ, Rio de Janeiro, Brazil, Departamento de Biociências da Atividade
Física-EEFD, UFRJ, Rio de Janeiro, Brazil
The 42 amino-acid β-amyloid peptide (Aβ42) is the main suspect underlying the pathogenesis observed
in Alzheimer’s disease (AD). Aβ42 peptide is one of several potential peptides produced from the
proteolytic cleavage of amyloid precursor protein. It is unique from the others by its ability to selfassemble into a variety of oligomers, including the insoluble fibrils observed in the senile plaques that are
postmortem hallmarks of AD. Recently, Aβ42 peptide has been demonstrated to accumulate within
neurons and correlate with alterations in axonal trafficking, morphological abnormalities, synaptic
dysfunction and cognitive difficulties. Intracellular accumulation may result from one or a combination of
processes that include decreased degradation, increased production and increased uptake of external
Aβ42. To examine the uptake of Aβ42 peptide, chick retinal neurons were challenged with low
concentrations of small oligomers of fluorescently labeled Aβ42 peptide. The pattern of internalization
was directly observed by fluorescent confocal microscopy over a 60 minute time course. We observed a
transition from a diffuse pattern to punctate within 30 minutes. Furthermore, the distribution of Aβ42
peptide converges with myosin-Vb and both have a change in distribution from uniform to predominately
localized to the cell body. Perturbation of the cytoskeleton suggests that the internalization of Aβ42
peptide is an actin-dependent process and the distribution is microtubule-dependent. These observations
are consistent the hypothesis that internalized Aβ42 peptide disrupts intracellular trafficking, which could
influence the balance between Aβ42 production and clearance.
3061/L036
Differential Regulation of ICAM1 and VCAM1 during Spontaneous Differentiation of Caco 2 Cells.
E. Astarci, S. Banerjee; Middle East Technical University, Ankara, Turkey
The intestinal mucosa has a dynamic cell population in the crypt-villus axis whereby the crypt cells in their
undifferentiated proliferative state are transformed (differentiated) into mature epithelial villi. This
differentiation process is accompanied by functional and morphological changes. The inflammatory NFkappaB pathway has been shown to be abrogated during epithelial differentiation. The Caco-2 colon
carcinoma cell ceases proliferation and undergoes spontaneous differentiation when it is reaches
confluency. Using this cell line as a model system, we wanted to examine whether the loss of NF-kappaB
activity during differentiation was associated with a loss in expression of the inflammatory NF-kappaB
target genes intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1
(VCAM-1). Surprisingly, expression analysis of these genes using semi-quantitative RT PCR and real
time PCR have indicated that while the mRNA expression of VCAM-1 is lost over 21 days of post
confluent Caco-2 culture, the expression of ICAM-1 remained steady. Interestingly, the ICAM-1 (but not
VCAM-1) promoter contains a CAAT box enhancer binding protein beta (C/EBPbeta) consensus site.
This transcription factor is known to be expressed more during epithelial differentiation. Western blot,
reporter gene assays and EMSA indicated a loss of NF-kappaB activity during differentiation, correlating
with the loss in VCAM-1 expression. On the other hand, C/EBPbeta activity was found to increase during
differentiation, thereby resulting in a recovery of ICAM-1 expression. Our results indicate that although
ICAM-1 and VCAM-1 have very similar functions, their transcriptional regulation during the process of
differentiation appears to be very different. Future studies will enlighten us on functional significance of
this differential regulation of ICAM-1 and VCAM-1 during epithelial differentiation.
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3062/L037
Acetylation Modulates Prolactin Receptor Dimerization.
L. Ma, J. Gao, Y. Guan, Z. Zhang, Y. E. Chin; Department of Surgery, Brown University School of
Medicine-Rhode Island Hospital, Providence, RI
Prolactin (PRL) and PRL receptor (PRLR) are essential in mammary gland development during
pregnancy and lactation. Overexpression of PRLR has been widely detected in breast cancers. Upon
ligand PRL binding, PRLR undergoes extracellular domain dimerization and the intracellular membraneproximal regions of the two dimerized PRLR monomers are brought into defined proximity to ensure
activation of the protein kinase JAK2, which docks within the membrane-proximal proline box. JAK2
phosphorylates PRLR cytoplasmic loop (C-loop) on multiple tyrosine sites and the C-loop-associated
STAT5 on C-terminal tyrosine site Y699. Y699-phosphorylated STAT5 dissociates from PRLR C-loop,
forms a transcriptional active dimer, and translocates into the nucleus where it regulates gene expression
associated with the functions of the ligand prolactin (PRL). However, in breast cancer cells, whether
PRLR acetylation is responsible for its dimerization is unknown. Here we have recently found that in
T47D and MCF7 cells, PRL treatment induces CREB-binding protein (CBP) nuclear exportation and CBPPRLR association. Mass spectrometry analysis showed that PRLR associated CBP acetylates PRLR on
multiple lysine sites along the C-loop. Fluorescence resonance energy transfer (FRET) gave the evidence
that PRLR also undergoes C-loop dimerization which is tightly regulated by acetylation. PRLR C-loop
undergoes acetylation-dependent dimerization for SIRT specific inhibitor nicotinamide (NAM) treatment
enhanced PRLR full length and PRLR C-loop protein-protein interaction. Such C-loop dimerization relies
on the interaction in multiple regions from the membrane-proximal region to the membrane-distal region,
because the C-loop dimerization is disrupted only when all acetylated lysines (K) are substituted with
arginines (R). C-loop associated STAT5 is only activated by the dimerized PRLR, suggesting a receptor
transactivation mechanism. PRLR activated STAT5 is also acetylated by CBP and undergoes acetylationdependent dimerization and gene regulation. Overexpression of SIRT2 or HDAC6 blocks PRLR and
STAT5 activation. Our results indicate that reversible acetylation provides the rheostat-like regulation for
the PRLR-STAT5 route for activation.
3063/L038
Cell-Cell Interactions in Breast Cancer.
N. E. Georgoulia; Systems Biology / SEAS, Harvard, Boston, MA
In this study, a cell culture approach is employed to identify patterns of pair-wise interaction between
cancer cells over a panel of 20 established breast cancer cell lines. It is our hypothesis that different
clones within a tumor, as well as different cell lines in cultured conditions, interact in one of two ways: a)
by competitive growth or, b) by synergistic growth. We investigate this by culturing cell lines in serum-free
conditioned medium (CM), i.e. medium which has already been used on other cell lines. Cell population
growth is monitored by incubating cultures with 5-Ethyl-2’-deoxyuridine (EdU). Preliminary results from a
10x10 interaction matrix indicate that CM has in some cases an enhancing, in other cases a suppressing
effect on cell growth when compared with the serum-free control growth. This provides evidence that
indeed different cancer cell lines interact by secreted factor cross-talk. Moreover, results indicate that
medium which had been pre-conditioned by a specific cell line, has a non-trivial effect on the growth of
that same cell line. Ultimately we desire this research to lead to the purification of tumor suppressor
factors from the various CMs. Such factors - secreted by cancer cells and addressed to cancer cells make attractive low-toxicity drug candidates, since they might specifically target malignancy while leaving
healthy tissue intact.
3064/L039
Eicosanoid Synthesis in Estrogen Receptor Positive (ER+) and Negative (ER-) Breast Cancer Cells
Are Modulated Differentially by Arachidonic Acid.
D. Roy; Biological Sciences, University of Texas at El Paso, El Paso, TX
Breast cancer is the most frequently diagnosed cancer among women, worldwide. Epidemiological and
molecular studies suggest there is an association between lipid-rich diet containing arachidonic acid (AA)
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and the occurrence of breast cancer. Rapid metabolism of AA produces prostaglandins (PGs),
leukotrienes (LT), and hydroxyeicosatetraenoic (HETE) that cause proliferation, multiplication, and
invasion of breast cells during malignancy. Estrogen receptors (ERs) are also known to play critical roles
in inducing breast tumorigenesis and therefore are considered as potential therapeutic targets. In
contrast, the rapid growth of ER- invasive breast cancer cells is not regulated by estrogen and therefore
cannot be treated with tamoxifen or related estrogen antagonist. Because cycloxygenase-2 (COX-2)
overexpression and hyper-PG synthesis have been reported in invasive breast carcinoma, we asked if AA
metabolism and eicosanoid synthesis in ER+ and ER- breast cancer cells are differentially regulated. We
used MDA-MB-231 (ER-) and MCF7 (ER+) breast cancer cells and evaluated eicosanoid synthesis. We
found that while ER- cells produced high levels of PGE2 and PGD2, ER+ cells synthesized mostly HETE
compounds (HETE 5 and HETE 8). Arachidonic acid (AA, 100μM) treatment exhibited differential effects
on eicosanoid synthesis in ER- and ER+ breast cancer cells, respectively. In ER- cells, AA stimulated the
syntheses of HETE8 and LTB4 by ~3 fold but lowered PGE2 and PGD2 productions by ~2 fold.
Interestingly, AA treatment reduced the syntheses of all eicosanoids that were produced in ER+ cells. Our
results demonstrate for the first time that ER- and ER+ breast cancer cells respond differentially to AA
and that ER- cells have the machinery to metabolize AA to produce HETE and LTB4 that could be directly
or indirectly involved in cell proliferation, inflammation, and invasion.
3065/L040
A New Synthetic Isoflavonoid, TCH2972, Enhances Osteogenesis in Cultured Human Adipose
Derived Stem Cells.
Y. Chang; Orthopaedic Research Center, Kaohsiung Medical University, Kaohsiung, Taiwan
Nature products and their components have been used as the food factor for improving osteoporosis
[1,2]. Isoflavones, such as daidzin, daidzein, genistin and genistein, are nature products and useful for
preventing and treating osteoporosis [3]. Ipriflavone (7-isopropoxyisoflavone), one kind of isoflavone
which synthesized from soy isoflavone daidzein, has been approved for treating involutional osteoporosis
in European and in Japan [4,5]. We synthesized several new isoflavone derivatives and screened their
potential for promoting osteogenesis. We found that one potent compound, TCH2972, significantly
enhanced the osteogenesis of osteogenic cells. Here, we investigated the effect of TCH2972, compared
with ipriflavone and raloxifene, on cell viability, cytotoxicity, and osteogenesis in human adipose derived
stem cells (hADSC) .
3066/L041
Cellular Micrornas Mediate the Effects of Human Papillomavirus 16 E5 Expression in Human
Epithelial Cells.
1
2,3
4
4
2,3 1
D. Greco , N. Kivi , K. Qian , P. Auvinen , E. Auvinen ; Women's Clinic, Helsinki University Hospital,
2
Helsinki, Finland, Haartman Institute Department of Virology, University of Helsinki, Helsinki, Finland,
3
4
HUSLAB, Department of Virology, Helsinki University Hospital, Helsinki, Finland, Institute of
Biotechnology, University of Helsinki, Helsinki, Finland
We have earlier studied the effects of the human papillomavirus (HPV) type 16 E5 oncogene on the
expression of cellular genes in a genome-wide cDNA microarray screen (Kivi et al., Oncogene 27:253241, 2008). Genes involved in cell motility, cell adhesion, cell proliferation, mitogenic signaling,
differentiation, and antigen presentation were significantly altered due to E5 expression. Cell motility was
shown to be enhanced in vivo. In order to better understand the role of E5 in cervical carcinogenesis, we
have proceeded to perform a large time-scale analysis of E5 effects on cellular gene expression as well
as on cellular microRNA expression. MicroRNAs have been implicated in many chronic diseases,
particularly in a number of cancers. Many viruses alter the expression of cellular microRNA expression,
and several DNA viruses, excluding papillomaviruses, have been shown to express their own microRNAs.
In this work, E5 expression was induced in HaCaT cells by dexamethasone and total RNA was at 0, 2, 4,
24, 48, 72 and 96 h after induction. Control cells without the E5 gene were treated in a similar manner.
RNA was labeled and hybridized to Agilent human expression microarrays and miRNA microarrays in
triplicates. The expression of a set of cellular microRNAs was found significantly altered due to E5
expression. Microarray results for 13 microRNAs were validated by TaqMan quantitative PCR assays at
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each time point in quadruplicates, and miR-146a, miR-203 and miR-324-5p were studied further. Using
eight prediction algorithms we searched for putative target genes of the selected micro RNAs. The
predicted microRNA target genes found to be counterexpressed among the genes that were significantly
altered in expression array analysis were identified. Gene expression findings were validated by SYBR
Green qPCR. Further, gene expression was studied at protein level in immunoblotting and
immunohistochemical staining of three-dimensional collagen raft cultures and human cervical
intraepithelial neoplasia tissue. Altogether, alterations in the expression of cellular miRNAs and their
target genes due to HPV 16 E5 oncogene seem to to repress epithelial differentiation and favor increased
cell proliferation and tumorigenesis.
3067/L042
Quantitative Phosphoproteomics of CXCL12 (SDF-1) Signaling.
1
2
2
1 1
J. A. Wojcechowskyj , J. Y. Lee , S. H. Seeholzer , R. W. Doms ; Microbiology, School of Medicine,
2
University of Pennsylvania, Philadelphia, PA, Protein and Proteomics Core, Children's Hospital of
Philadelphia Research Institute, Philadelphia, PA
A growing body of evidence suggests that viruses modulate host signal transduction pathways to facilitate
infection of cells. Understanding host pathways activated by viruses and that are required for replication
may provide novel targets for antiviral therapy. Human immunodeficiency virus 1(HIV-1) enters target
cells by specifically engaging a primary receptor, CD4, and then a chemokine coreceptor, either CCR5 or
CXCR4. We employed quantitative phosphoproteomics technology to examine host signaling pathways
modulated through engagement of CXCR4 with its natural ligand, CXCL12 as a prelude to studies with
HIV-1. CEM cells, a lymphoblastic leukemia cell line, were stable isotope labeled by amino acids in cell
culture (SILAC) and treated with CXCL12 for 5 mins. Phosphopeptides were enriched through hydrophilic
interaction chromatography (HILIC) and immobilized metal affinity chromatography (IMAC) before being
analyzed on an LTQ Orbitrap XL mass spectrometer. Over 4,000 unique SILAC pairs were sequenced
and quantified representing about 1,500 proteins. 87 phosphopeptides significantly changed in
abundance, the majority of which have not been previously implicated in CXCL12/CXCR4 signaling.
Several of these significantly regulated phosphoproteins are known to impact the cell cycle, T cell
activation and HIV-1 infection. Current studies are underway to independently validate the regulation of
these phosphoproteins, which will serve as a proof of concept for utilizing quantitative phosphoproteomics
in screening for host factors important for virus infection.
3068/L043
Novel Roles for ARNO/Arf6 Signaling Axis in Glucose-Stimulated Insulin Secretion in Pancreatic
β-Cells.
1,2
1,2
1,2
3
1,2 1
B. Jayaram , I. Syed , C. N. Kyathanahalli , C. J. Rhodes , A. Kowluru ; Pharmaceutical sciences,
2
3
Wayne State University, DETROIT, MI, John D. Dingell VA Medical Center, DETROIT, MI, Section of
Endocrinology, Diabetes and Metabolism, Kovler Diabetes Center, University of Chicago, CHICAGO, IL
ARNO, an Arf-guanine nucleotide exchange factor, regulates insulin secretion via vesicular trafficking and
membrane dynamics. Herein, we tested the hypothesis that signaling events leading to glucosestimulated insulin secretion [GSIS] underlie activation of the small G-protein Arf6 by ARNO. We also
hypothesized that such a signaling axis, in turn, controls the activation of Cdc42 and Rac1, which have
been implicated in cytoskeletal remodeling and exocytosis of insulin. We employed molecular biological
and pharmacological tools to assess the roles for ARNO/Arf6 in insulin secretion in normal rat islets and
INS 832/13 cells. Arf6 and Cdc42/Rac1 activation were quantitated by GST-GGA3 and PAK-1 kinase
pull-down assays, respectively. Insulinotropic concentrations of secretagogues like glucose or KCl
significantly stimulated the activation of Arf6 [i.e., Arf6.GTP] in a time-dependent manner [0-5 min].
Overexpression of inactive mutants of Arf6 [Arf6-T27N] or ARNO [ARNO-E156K] markedly reduced GSIS
in these cells. Silencing of endogenous ARNO [siRNA-ARNO] also attenuated GSIS and KCl-stimulated
insulin secretion [KSIS]], further confirming a role for ARNO in insulin secretion. SecinH3, a potent
inhibitor of ARNO/Arf6 signaling pathway, blocked glucose-induced Arf6 activation and GSIS.
Furthermore, secinH3 inhibited glucose-induced Cdc42/Rac1 activation suggesting a plausible role for
ARNO in regulating glucose-induced Cdc42/Rac1 activation. Immunofluorescence studies revealed an
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increased association of ARNO with Arf6 in glucose-stimulated β-cells. Together, our findings provide
evidence for the involvement of ARNO, in the sequential activation of Arf6, Cdc42 and Rac1 leading to
insulin secretion from the pancreatic β-cell.
3069/L044
Isoprenylcysteine Carboxyl Methyltransferase [ICMT] Facilitates Glucose-Induced Rac1
Activation, ROS Generation and Insulin Secretion in Pancreatic β-Cells.
1,2
1,2
1,2
1,2 1
A. Singh , I. Syed , C. W. Subasinghe , A. Kowluru ; Department of Pharmaceutical Sciences,
2
Wayne State University, Detroit, MI, John Dingell VA Medical Center, Detroit, MI
Isoprenylcysteine carboxyl methyltransferase [ICMT] catalyzes the post-translational methylation of Cterminal cysteines of isoprenylated proteins, including small G-proteins and the γ-subunits of
heterotrimeric G-proteins. It is widely felt that carboxylmethylation [CML] promotes efficient membrane
association of the methylated proteins and specific protein-protein interactions. In the current study, we
tested the hypothesis that protein CML plays a regulatory role in glucose-stimulated insulin secretion
[GSIS]. Western blot data indicated that lCMT is expressed in INS 832/13 β-cells. siRNA-mediated
knockdown of endogenous expression of ICMT markedly attenuated glucose, but not KCl-induced insulin
secretion. These findings were further supported by pharmacological observations, which suggested a
marked reduction in GSIS by acetyl farnesyl cysteine [AFC], a selective inhibitor of ICMT. In addition,
glucose-induced Rac1 activation, a hallmark signaling step involved in GSIS, was markedly inhibited
following pharmacological [AFC] or molecular biological [siRNA-ICMT] inhibition of ICMT. Last, we also
noticed a marked reduction in glucose-induced acute increase in the generation of reactive oxygen
species [ROS] in INS 832/13 cells pre-treated with AFC or transfected with siRNA-ICMT. Together, these
data suggest that ICMT facilitates glucose-induced Rac1 activation, ROS generation and insulin secretion
in pancreatic β-cells.
3070/L045
Is the α-Subunit of Prenyltransferase a Substrate for Proapoptotic Caspase-3 in Pancreatic βCells?
1,2
1,2
1,2
1,2 1
2
C. W. Subasinghe , I. Syed , A. Singh , A. Kowluru ; Wayne State University, Detroit, MI, John
Dingell VA Medical Center, Detroit, MI
Post-translational prenylation is a prerequisite for functional competence of candidate proteins involved in
insulin secretion from pancreatic β-cells. Prenyltransferases catalyze incorporation of either farnesyl
(C15) or geranylgeranyl (C20) derivatives of mevalonic acid into C-terminal cysteines of G-proteins. Both
farnesyltransferase [FTase] and geranylgeranyl-transferase [GGTase] share the same α-subunit
[FTase/GGTase-α], but distinct β-subunits. Published evidence, albeit limited, appears to suggest that
prenyltransferases α subunit and Tiam1, a guanine nucleotide exchange factor for Rac1, are potent
substrates for proapoptotic caspase-3 [CSP-3] cleavage. Herein, we investigated potential degradation of
FTase/GGTase-α and Tiam1 in INS 832/13 cells under conditions of increased CSP-3 activation. INS
832/13 cells were treated with etoposide [60 µM; 6 h], a known inducer of CSP-3 mediated apoptosis, or
the recombinant, biologically active CSP-3, and the degradation product of FTase/GGTase-α or Tiam1
were quantified by Western blotting followed by densitometry. Both etoposide and recombinant CSP-3
treatment conditions promoted a significant degradation of FTase/GGTase-α or Tiam1, which was
reversed by Z-DEVD-FMK (25 µM), a known CSP-3 inhibitor, suggesting that the later is a bonafide
substrate for CSP-3. Along these lines, even though CSP-3 activation was noticeable, a negligible
FTase/GGTase-α and Tiam1 degradation was evident in cells exposed to a variety of conditions known to
cause β-cell demise, including exposure to a mixture of inflammatory cytokines [IL-1β, IF-γ, TNF-α;
10ng/mL; 48 h], glucose [30 mM; 48 h] or palmitate [0.2 mM; 48 h]. Our findings suggest that CSP-3
mediated degradation of FTase/GGTase-α or Tiam1 may not be occurring under the duress of prolonged
exposure of isolated β-cells to cytokines, glucotoxic or lipotoxic conditions. Based on these findings we
conclude that while FTase/GGTase-α and Tiam1 is degraded by CSP-3, it is unlikely that such a signaling
step underlies cell death induced by chronic exposure of isolated β-cells to elevated glucose, palmitate or
cytokines.
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3071/L046
Xanthohumol Uptake and Intracellular Kinetics in Liver Cells.
1
2
3
2
2 1
H. Wolff , M. Motyl , C. Hellerbrand , J. Heilmann , B. Kraus ; Institute of Virology, Helmholtz Ctr
2
Munich, Neuherberg, Germany, Institute of Pharmacy, University of Regensburg, Regensburg, Germany,
3
Department of Internal Medicine, University Hospital Regensburg, Regensburg, Germany
Xanthohumol (XN) is the major prenylated flavonoid in the female inflorescences of hops (Humulus
lupulus L.) and has long been used in traditional medicine, with sleep disturbances being the main
indication. Over the past years, scientific evidence has accumulated pointing to the cancer preventive
potential of XN and numerous effects of XN on transcription factor activation states and cellular signalling
cascades have been reported, indicating potential mechanisms of action. One largely unresolved issue is
the low bioavailability of XN in the human organism and there is considerable lack of data about actual
concentrations and pharmacokinetic of the compound and its metabolites in liver and intestinal cells.
Therefore, we aimed at gathering data concerning uptake and intracellular kinetics of XN in these cells.
We employed fluorescence imaging to study the cellular uptake of XN into various cell lines, like
hepatocellular carcinoma cells (HuH-7), hepatic stellate cells (HSC) and colorectal adenocarcinoma cells
(Caco-2), as well as into primary hepatocytes. Additionally, the intracellular kinetics of XN was assessed
by fluorescence recovery after photobleaching (FRAP) experiments. We observed a rapid accumulation
of XN in cells reaching a maximum already after 30-60 min, with HSC and Caco-2 cells showing a higher
final XN concentration as well as a higher uptake rate than HuH-7 cells and primary hepatocytes. We
found that the half time of recovery of XN in the cells ranged between 0.4 s (HuH-7) and 0.9 s (HSC), with
mobile fractions of approx. 90% in all cell types. Our data provides evidence that XN binds to cellular
proteins with a mean molecular size of approximately 15 kDa. Timelapse microscopy and high-resolution
imaging further revealed that there is constant nucleo-cytoplasmic exchange of XN but that no other
prominent cellular compartment participates significantly in the intracellular life cycle of XN. Our data
provide deeper insights into XN biology on the cell level and provides possible explanations for the poor
bioavailability of XN, and could enable further progress in dissecting the biological properties of XN, such
as transcription factor suppression, from the pharmacokinetic properties.
3072/L047
Vps35 Mediates Vesicle Transport between the Mitochondria and Peroxisomes.
E. Braschi, V. Goyon, R. Zunino, A. Mohanty, L. Xu, H. McBride; pathology and laboratory medicine, univ
of ottawa heart institute, Ottawa, ON, Canada
Mitochondria-derived vesicles (MDVs) have been shown to transport cargo from the mitochondria to the
peroxisomes. Mitochondria and peroxisomes share common functions in the oxidation of fatty acids and
the reduction of damaging peroxides. Their biogenesis is also linked through both the activation of master
transcription factors such as PGC-1α and the common use of fission machinery, including DRP1, Mff, and
hFis1. We have previously shown that MDVs are formed independently of the known mitochondrial fission
GTPase Drp1 and are enriched for a mitochondrial small ubiquitin-like modifier (SUMO) E3 ligase called
MAPL (mitochondrial-anchored protein ligase). Here, we demonstrate that the retromer complex, a known
component of vesicle transport from the endosome to the Golgi apparatus, regulates the transport of
MAPL from mitochondria to peroxisomes. An unbiased screen shows that Vps35 and Vps26 are found in
complex with MAPL, and confocal imaging reveals Vps35 recruitment to mitochondrial vesicles. Silencing
of Vps35 or Vps26A leads to a significant reduction in the delivery of MAPL to peroxisomes, placing the
retromer within a novel intracellular trafficking route and providing insight into the formation of
MAPLpositive MDVs.
3073/L048
Development of an Adenovirus Vector-Mediated Assay System for Hepatitis C Virus Replication.
1
1
1
2
1 1
T. Yoshida , F. Satoh , M. Kondoh , H. Mizuguchi , K. Yagi ; Laboratory of Bio-functional Molecular
2
Chemistry, Graduate School of Pharmaceutical Sciences, Osaka Univ, Osaka, Japan, Department of
Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka Univ, Osaka,
Japan
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Objective: 170 million people are infected with Hepatitis C virus (HCV), and chronic infection with HCV is
a major cause of hepatocellular carcinoma. Interferon therapy is the only effective therapy for HCV
patients, and novel therapeutic strategies for HCV are needed. Transfer of HCV RNA full or subgenome
into hepatic cells is useful for basic and pharmaceutical studies. The adenovirus (Ad) vector is a
convenient and efficient tool for the transduction of foreign genes into cells. However, an Ad vector
expressing the HCV genome has never been reported. We previously tried to prepare an RNA
polymerase (pol) I-driven Ad vector expressing HCV sub-genome, but we made no success on
preparation of the Ad vector particles coding the HCV sub-genome because of influence of HCV subgenome. In the present study, to overcome the influence we developed a tetracycline-regulated RNA pol
I-driven Ad vector system. Methods: A hybrid promoter consisting of the tetracycline-responsive element
and the RNA pol I promoter was constructed. An HCV replicon fragment, containing a luciferase gene
and HCV non-structural genes essential for replication, was inserted into the chimeric RNA pol Idependent expression cassette. Ad vectors were prepared by an improved in vitro ligation method
(Mizuguchi and Kay, 1998, 1999). Huh7 cells were transfected with the Ad vectors, and HCV replication
was monitored by expression of luciferase and HCV non-structural components. Results: Ad vector
particles coding the tetracycline-controllable RNA pol I driven HCV sub-genome could be amplified, and
HCV replication was observed in cells transduced with the Ad vectors. Moreover, interferon, an inhibitor
of HCV replication, prevented HCV replication in the cells. Conclusion: We developed an Ad vectormediated HCV replicon system which could be used to evaluate inhibitors of HCV replication. This system
will contribute to understanding the mechanism of HCV replication or development of anti-HCV drug.
3074/L049
High Glucose Down-Regulates Soluble Epoxide Hydrolase Expression in Insulin-Producing Cells
and in Hepatocytes.
1
1
2
2
1 1
J. Irminger , S. Dupuis , J. Coulaud , F. Homo-Delarche , P. Halban ; Genetic Medecine and
2
Development, University of Geneva, Geneva, Switzerland, Unité BFA, University Paris-Diderot, Paris,
France
Soluble Epoxide Hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) into their less active
form, dihydroxyeicosatrienoic acids (DHETs). EETs have numerous protective autocrine and paracrine
effects on a variety of cells and organs. Lowering sEH expression increases cellular concentration of
EETs potentiating their beneficial effects. In particular, EETs improve glucose homeostasis and insulin
secretion and reduce beta-cell apoptosis. Recently, inhibiting sEH activity has been shown to prevent
hyperglycemia in streptozotocin-induced diabetic mice, a model of Type 1 Diabetes. We have now
analyzed sEH expression in the Goto-Kakizaki (GK) rat, a spontaneous non-obese model of type 2
diabetes (T2D). sEH mRNA levels are drastically (up to 97%) decreased in liver and pancreas of mildly
hyperglycemic E21 GK fetuses and at the maternal-fetal interface (placenta, blood cord). Such underexpression of sEH would increase EET levels in various crucial organs during development and might
reflect an attempt to protect the fetus from adverse effects of maternal hyperglycemia. We also analyzed
various parameters (kinetics, dose-response) of sEH mRNA modulation by glucose in a human
hepatocyte cell line (hUH), insulinoma (INS1) cells, isolated rat islets and purified rat beta cells
(mean±SE, n=3-4). In hUH cells treated for 24 h with 11.2 mM glucose, sEH mRNA expression was 62 ±
8 % of cells treated with 1.4 mM glucose. In insulin-producing INS1 cells treated for 24 h with 25 mM
glucose, sEH mRNA expression was 45 ± 4 % of cells treated with 2.8 mM glucose. In isolated rat islets
and purified rat beta cells treated for 24 h with 16.7 mM glucose, sEH mRNA expression was 35 ± 7 and
29 ± 2 %, respectively, as compared to their corresponding controls at 2.8 mM glucose. Soluble EH is
involved in inflammation, cardiovascular diseases, lipid metabolism and has been suggested to play a
role in T2D. Here we present further evidence for potential involvement of sEH in vivo in T2D and in vitro
in response to elevated glucose, suggesting that enhancing sEH activity might be a potential promising
therapeutic approach in this disease.
3075/L050
Akt Signaling Facilitates Herpes Simplex Virus Entry and Required for Cell-To-Cell Spread.
1
1
2
1,2 1
N. V. Cheshenko , J. Trepanier , E. A. Eugenin , B. C. Herold ; Pediatrics, Albert Einstein College of
2
Medicine, Bronx, NY, Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY
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Herpes Simplex virus (HSV) entry is a complex process that requires multiple interactions at the cell
surface as well as activation of signaling pathways that promote viral entry and transport of viral capsids
to the nuclear pores. Calcium signaling plays a central role in this process and release of inositol
triphosphate receptor (IP3R) -regulated intracellular calcium stores is required for fusion of the viral
envelope with the cell membrane and HSV entry into human epithelial cells. We hypothesize that integrin
and Akt contribute to the requisite signaling pathways. Silencing of integrin αvβ3 with small interfering
RNA (siRNA) impedes viral entry and prevents activation of focal adhesion kinase, which is required for
transport of viral capsids to the nuclear pores. Silencing of Akt does not prevent HSV binding to the cell
surface but blocks penetration of viral capsids from bound virions into cytoplasm and nuclear transport of
viral capsids to the nuclear pores. Surprisingly, silencing of AKT prevents both the initial plasma
membrane HSV induced calcium response as well as release of Ca2+ from intracellular stores, whereas
silencing of integrin αvβ3 only prevents intracellular calcium release. These results suggest that Akt may
act both upstream and downstream of integrin signaling and are consistent with the observation that HSV
induced Akt phosphorylation with multiphasic kinetic. Moreover, Akt and integrin signaling were required
for cell-to-cell spread of HSV. Silencing of Akt or integrin αvβ3 did not trigger any interferon response.
Together, these findings implicate Akt and integrin signaling in HSV entry and cell-to-cell spread. The
observation that Akt acts both upstream and downstream of integrin and calcium signaling may reflect
interactions between the pleckstrin homology domain of Akt and phosphinositides in the process of
infection and cell to cell spread.
3076/L051
Turnover of Amyloid Precursor Protein (APP): Lysosomal Degradation vs. Proteolytic Processing.
1,2
2
2
2
1,2
2 1
P. Burgos , Y. Prabhu , Y. Lin , K. Tifft , G. A. Mardones , J. S. Bonifacino ; Physiology, Universidad
2
Austral de Chile, Valdivia, Chile, NICHD/CBMP, National Institutes of Health, Bethesda, MD
Introduction: APP is a transmembrane protein that exists for a very short time as an intact protein due to
its efficient proteolytic processing and turnover. The endo-lysosomal system (ELS) has been implicated in
pathogenic A-beta peptide production; however, the exact contribution of the ELS to this process has
been poorly defined. The main goal of this study is to characterize the role of lysosomes in the turnover of
APP. Materials and Methods: H4 neuroglioma cells were transfected with constructs encoding HAAPP695-EGFP. We compared the trafficking and proteolytic processing of APP-WT with APP variants
containing amino acid substitutions within its cytosolic tail. To study lysosomal turnover we generated a
mutant version of APP (APP-R) that is resistant to cleavage by secretases. The assays used include
Western blotting, pulse-chase analyses, immunoprecipitation, cell surface biotinylation and confocal
microscopy. Results: The APP cytosolic tail contains critical residues for its delivery to the ELS. Mutation
of these residues disrupts targeting to the ELS without preventing APP proteolytic processing by alpha-,
beta- and gamma-secretases. Discussion: Our results show that proteolytic processing of APP can occur
in compartments other than the ELS, suggesting that targeting to lysosomes mediates complete
degradation rather than A-beta peptide production. FONDECYT 1100027
3077/L052
Identification of a Conserved Domain in Myc, MB0, Which Is Essential for Transcriptional
Activation and Transformation.
K. West-Osterfield, Q. Zhang, Z. Li, A. Panaccione, S. Hann; Cell & Developmental Biology, Vanderbilt
University, Nashville, TN
Numerous studies demonstrate a critical role for the transcription factor c-Myc in the control of cell
proliferation and apoptosis. c-Myc expression is deregulated in many different types of human cancers
and overexpression of c-Myc causes transformation of cells in culture and tumorigenesis in animals.
Apoptosis induced by oncogenic c-Myc activation prevents the unrestricted proliferation of a cell leading
to tumorigenesis. Although c-Myc regulates hundreds of target genes involved in numerous cellular
processes that influence proliferation and transformation, the transcriptional mechanism by which c-Myc
controls the transcription of these target genes is unclear. In the N-terminal transcriptional regulatory
domain there are several conserved domains of c-Myc, termed Myc Boxes, which are necessary for the
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transcriptional activity and biological functions of c-Myc. These different Myc Boxes appear to influence
different c-Myc activities to varying degrees and the specific domains that are involved in the ability of cMyc to transactivate target gene promoters are not well defined. In this report we have identified a novel
conserved domain, designated MB0, which is essential for c-Myc transactivation and target gene
induction. MB0 is also necessary for c-Myc transformation of primary fibroblasts in cooperation with
activated Ras and efficient c-Myc-induced apoptosis of p53-independent fibroblasts.
3078/L053
NPM Controls c-Myc Nucleolar Localization and c-Myc-Mediated rDNA Trancription.
Z. Li, S. Hann; Cell and Developmental Biology, Vanderbilt University, Nashville, TN
The transcription factor c-Myc has a critical role in proliferation and cell growth and the control of
ribosome biogenesis by c-Myc through the regulation of all three RNA polymerases plays an integral part
in this role. Specifically, in the nucleolus, c-Myc has been shown to bind to ribosomal DNA and activate
Pol I-mediated transcription of rRNA genes. c-Myc accumulates in nucleoli upon inhibition of the
proteasome, suggesting nucleolar localization influences c-Myc degradation. Nucleophosmin, a
predominantly nucleolar protein, is also critical in ribosome biogenesis and, like c-Myc, is found
overexpressed in many types of tumors. Here, we show that transient NPM overexpression can cause cMyc to relocalize from the nucleoplasm to nucleoli, which leads to enhanced c-Myc proteolysis. NPM is
essential for both exogenous and endogenous c-Myc nucleolar accumulation. In contrast, c-Myc nucleolar
accumulation is independent of p53, Mdm2 and ARF. In addition, NPM overexpression stimulates c-Mycmediated upregulation of rDNA transcription. Conversely, reduction of endogenous NPM by shRNA
inhibits the ability of c-Myc to induce rDNA transcription, suggesting that NPM is necessary for c-Myc to
activate rDNA transcription. Taken together, these results suggest that NPM controls c-Myc nucleolar
localization and rDNA transcription.
3079/L054
Vitamin E and Membrane Repair.
A. C. Howard, P. L. McNeil; Medical College of Georgia, Augusta, GA
The cellular function of vitamin E (VE) remains unknown almost 100 years after its discovery as a dietary
deficiency that results in a lethal muscular dystrophy. At least one form of muscular dystrophy is caused
by defective membrane repair. We hypothesized that one important biological role of VE is to promote
muscle membrane repair. Cultured muscle cell models, C2C12, were loaded with VE in a 24 hr preincubation, washed free of exogenous vitamin and their plasma membrane disrupted with a microscope
laser in the presence of a membrane impermeant fluorescent dye. Cellular uptake of the dye was then
monitored quantitatively over time: repair prevents further dye entry and attenuates the rate of increase in
the fluorescence intensity measured from the wounded cell. VE loading significantly decreased cellular
dye influx, indicating that repair, normally robust in the C2C12 cell, had been promoted. Strikingly, the
HeLa cell, a non-muscle cell normally not capable of repair of the laser disruption, was converted by the
VE treatment into a repair competent cell. Lastly, VE loading significantly improved the defective repair
exhibited by cells exposed to high glucose. Vitamin C, another anti-oxidant that can be loaded into cells,
also significantly decreased dye influx after laser injury. However, glutathione, an anti-oxidant that cannot
be transported across the plasma membrane, was ineffective. Cells injured in the presence of H2O2
displayed significantly more dye influx than controls injured in physiological saline lacking this oxidant. If
however cells were first loaded with VE, then H2O2 was without effect on repair. Skeletal muscle myocyte
repair in intact muscle was similarly impaired by H2O2, and VE protective. We conclude that VE promotes
skeletal muscle plasma membrane repair, and that its capacity as an anti-oxidant is crucial in this role.
TUESDAY
3080/L055
The Role of IL-1beta in Reduced IL-7 Production by Stromal and Epithelial Cells: A Model for
Impaired T-Cell Numbers in the Gut during HIV-1 Infection.
1,2
1
3
1
2
2
4
1
T. H. Pham , N. Ruffin , D. Brodin , B. Rethi , C. P. Dac , H. T. Nguyen , L. Lopalco , N. Vivar , F.
1 1
Chiodi ; Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden,
2
3
Department of HIV/AIDS, National Institute of Hygiene and Epidemiology, Hanoi, Viet Nam, Department
4
of Biosciences and Nutrition, Karolinska Institute, Stockholm, Sweden, Division of Immunology,
Transplantation anfd Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy
Objective: Interleukin (IL)-7 is a key cytokine in T-cell homeostasis. Stromal cells, intestinal epithelial cells
and keratinocytes are known to produce this cytokine. The mechanisms and cellular factors regulating IL7 production are still unclear. We assessed whether IL-1beta and interferon (IFN)-gamma, cytokines
produced during inflammatory conditions, may impact on IL-7 production. Methods: We used human
intestinal epithelial cells (DLD-1 cell line) and bone marrow stromal cells (HS27 cell line), known to
produce IL-7; IL-7 production was evaluated at the mRNA and protein levels. To assess whether
treatment of HS27 cells with IL-1beta and/or IFN-gamma leads to changes in the gene expression of
cytokines, Toll-like receptors (TLRs) and chemokines, we analysed gene expression profiles using the
whole-genome microarray Human Gene 1.0 ST. Results: We found that IFN-gamma enhanced the
expression of IL-7 mRNA (P < 0.001) in both cell lines. IL-1beta treatment led to a significant downregulation (P < 0.001) of IL-7 mRNA expression in both cell lines. The IL-7 concentration in supernatants
collected from treated DLD-1 and HS27 cell cultures reflected the trend of IL-7 mRNA levels. The gene
profiles revealed dramatic changes in expression of cytokines and their receptors (IL-7/IL-7R alpha; IL1alpha, IL-1beta/IL-1R1; IFN-gamma/IFN-gammaR1), of IFN regulatory factors (IRF-1 and 2), of TLRs
and of important chemo-attractants for T cells. The microarray results were verified by additional
methods. Conclusions: Our results are discussed in the setting of inflammation and T-cell survival in the
gut compartment during HIV-1 infection where stromal and epithelial cells may produce factors that
contribute to impaired IL-7 homeostasis and homing of T cells.
3081/L056
Characterization of Mgrn1 and Mgrn2, a Novel Family of E3 Ligases Implicated in Spongiform
Neurodegeneration.
D. Fallaize, P. Reynolds, L. Li, L. Chin; Pharmacology, Emory University, Atlanta, GA
Spongiform neurodegeneration is the pathological hallmark of prion diseases and is also a feature of
human immunodeficiency virus (HIV) encephalitis, Alzheimer’s disease, and diffuse Lewy Body disease.
The pathogenic mechanisms that trigger spongiform neurodegeneration remain elusive. Interestingly, a
null mutation in the novel E3 ligase Mahogunin RING finger 1 (Mgrn1) causes spongiform
neurodegeneration in mice and depletion of Mgrn1 by the pathogenic forms of prion protein has recently
been implicated in prion disease pathogenesis. In mammals, there is an Mgrn1 homologue, Mahogunin
RING finger 2 (Mgrn2), which shares 65% amino acid identity and 72% similarity to the sequence of
Mgrn1. The tissue distribution and subcellular localization of the Mgrn family of proteins remain mostly
uncharacterized. We have generated several distinct rabbit polyclonal antibodies against the unique or
overlapped regions of Mgrn1 and Mgrn2 and characterized the antibody specificity. Western blot analysis
with these antibodies revealed that Mgrn1 and Mgrn2 are expressed in cells as a 64-kDa and a 78-kDa
protein, respectively. Using these antibodies, we found that Mgrn1 and Mgrn2 exhibit overlapping yet
distinct tissue distribution and expression patterns. We have shown that Mgrn1 binds and colocalizes with
ESCRT-I component TSG101 on early endosomes. Although Mgrn2 lacks the TSG101-binding P(S/T)AP
tetrapeptide motif, our immunofluorescence microscopic analysis reveals that Mgrn2 is also localized to a
vesicular compartment(s) in mammalian cells. Further characterization of the Mgrn family of E3 ligases
will provide a better understanding of the pathogenic mechanisms underlying spongiform
neurodegeneration.
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3082/L057
Kaposi Sarcoma Virus: Virus-Host Protein Interactions.
2,1
1
1,2
2
2
2
1,2
S. Wanamaker , D. Meine , A. Fronduto , T. Hao , D. Hill , M. Vidal , J. Roecklein-Canfield ;
1
2
Chemistry, Simmons College, Boston, MA, Center for Cancer Systems Biology, Dana Farber Cancer
Institute, Boston, MA
Mapping virus-host protein-protein interactions uncovers global properties of cellular networks, clarifies
gene-disease associations, and shows how viruses manipulate cellular networks to their advantage. We
present a network of protein-protein interactions between Kaposi Sarcoma virus (KSHV), a gamma
herpesvirus, and human. Using yeast two-hybrid (Y2H) we systematically screened KSHV baits against
the Human ORFeome v5.1 collection, which contains approximately 21,000 human cDNA clones. We
present here a preliminary virus-host interactome. Mapping of KSHV protein interactions with the human
host generates greater knowledge of KSHV pathogenesis and benefits further understanding of other
herpesviruses. Comparative analysis of this interactome with other existing viral networks will provide a
foundation to study common mechanistic methods for infection.
3083/L058
Purinergic Signalling Is Involved in the Malaria Parasite Plasmodium Falciparum Invasion to Red
Blood Cells.
J. C. Levano Garcia, R. P. Markus, C. R. Garcia; Physiology Department, Biosciences institute - São
Paulo University, São Paulo, Brazil
Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly
complex cell cycle that is essential for its successful replication within the host. A number of evidence
2+
suggests that changes in parasite Ca levels occur during the intracellular cycle of the parasites and play
a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium
2+
receptors linked with calcium signaling and causal relationship between Ca increases and parasite
2+
functions are still largely mysterious. We here describe that increases in P. falciparum Ca levels,
induced by extracellular ATP modulate parasite invasion. In particular, we show that addition of ATP
2+
leads to an increase of cytosolic Ca in trophozoites and segmented schizonts. Addition of the
2+
compounds KN62, Ip5I on parasites blocked the ATP-induced rise in cytosolic Ca concentration.
Besides, the compounds or hydrolysis of ATP with Apyrase added in culture drastically reduce RBC
infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of
purinoceptor antagonists Ip5I and KN62 in this study suggest the presence of putative purinoceptor in P.
2+
falciparum. In conclusion, we have demonstrated that increases in cytosolic Ca concentration in the
malarial parasite Plasmodium falciparum, by ATP leads to modulation of its invasion of red blood cells.
3084/L059
Visualization and Quantification of Influenza a Virus Infection in Primary Human Dendritic Cell
Subsets.
1
2
1
1 1
C. M. Chalouni , A. Smed-Sorensen , L. Komuves , I. Mellman ; Genentech, South San Francisco, CA,
2
Karolinska institutet, Stockholm, Sweden
Influenza A virus (IAV) infection is a threat to human health normally controlled by generation of
pathogen-specific adaptive immune responses that limit and eradicate infection in the infected individual.
The impact of Influenza is augmented as IAV infection often predisposes individuals to a secondary
infection suggesting that IAV infection affects the ability of the host to manage a second pathogen.
Dendritic cells (DCs) are professional antigen-presenting cells that initiate and shape these immune
responses. We assessed the susceptibility to IAV infection in the two major human DC subsets found in
blood by confocal microscopy, followed by analysis using Volocity software. Both plasmacytoid DCs
(PDCs) and myeloid DCs (MDCs) captured IAV particles; 61% of immature PDCs and 89% of immature
MDCs were positive for the virus, and the vast majority of the positive cells had internalized the virus.
Prior maturation of DCs with TLR7/8 agonist did not affect IAV capture by PDCs, but significantly reduced
the ability of MDC to internalize IAV to 38%. We next examined the capacity of DCs to support IAV
replication, and found that only immature MDCs were able to replicate the virus showing abundant viral
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structures below the cell membranes 6h after infection. To assess the functional consequences of IAV
infection of MDCs, we compared the ability of IAV infected and uninfected MDCs to endocytose
inactivated cytomegalovirus (CMV). Similar percentages of uninfected and IAV infected MDCs captured
CMV. However, the uninfected immature MDC contained only half the number of CMV particles per cell
compared to IAV infected MDCs. In summary, these data show that both immature and mature MDCs
and PDCs can capture and internalize IAV, but only immature MDC can replicate IAV.
3085/L060
Novel Evidence that p53 Regulates TGF-β1-Induced Plasminogen Activator Inhibitor-1 Expression.
J. M. Overstreet, R. Samarakoon, P. J. Higgins; Albany Medical College, Albany, NY
Plasminogen activator inhibitor-1 (PAI-1) is a direct downstream target of p53 in the induction of
replicative senescence and mediates transforming growth factor-β1 (TGF-β1)-induced growth arrest.
Typically, p53-responsive genes, including PAI-1, contain one or more p53 consensus binding sites in
their promoter regions. Our preliminary studies indicated that genetic deletion of p53 in mouse embryonic
fibroblasts (MEFs) virtually eliminated TGF-β1-induced PAI-1 expression. PAI-1 expression “rescue” was
evident in p53-/- MEFs engineered to re-express human p53. Similarly, transient siRNA knockdown of
p53 drastically inhibited the PAI-1 induction in response to TGF-β1 in HaCaT human keratinocytes. The
objective of this study is to investigate the molecular mechanisms underlying p53 participation in TGF-β1induced PAI-1 expression in HaCaT keratinocytes. TGF-β1-stimulated PAI-1 expression requires
cooperation of the SMAD pathway and the non-canonical (e.g. ERK1/2, p38 MAPK) signaling elements
as inhibition of SMAD3 as well as MAPK suppressed PAI-1 induction. Consistent with findings that posttranslational modifications (e.g. phosphorylation) regulate the transcriptional function of p53, Ser 9, 15
(but not Ser 20, 37, 46, 392) were rapidly phosphorylated (30 mins) upon TGF-β1 stimulation, while total
p53 levels remain relatively unchanged. The pattern of phospho-p53Ser9,15 correlated with stimulation of
ERK1/2 and p38 MAPK phosphorylation suggesting that p53 is phosphorylated as a downstream target of
MAP kinases. Interestingly, p53 co-immunoprecipitated with activated pSMAD2/3 in HaCaT following
TGF-β1 stimulation (1-2 hours). Transient complex formation, with maximal p53-pSMAD2/3 interaction at
1 hour post TGF-β1 treatment, preceded PAI-1 induction. These data suggest that p53 is a downstream
target of non-canonical TGF-β1 signaling and is a required element for TGF-β1-induced PAI-1
transcription integrating the two distinct, yet cooperating pathways (canonical and non-canonical) by
interacting with SMAD proteins. Our future studies will characterize the upstream signaling control
elements of PAI-1 induction via cooperative p53 and SMAD transcriptional networks in keratinocytes.
(Supported by NIH grant GM057242)
3086/L061
Novel Alternative Transcripts in the CTNNA3 Gene Are Overrepresented in Late-Onset Alzheimers
Disease.
G. V. De Ferrari, M. Alarcon, E. Perez, B. Bustos, M. Avila, C. F. Burgos; Center for Biomedical
Research, Universidad Andres Bello, Santiago, Chile
Copy Number Variants (CNVs), briefly defined as a polymorphic portions of genomic DNA between 1kb
and 3Mb in size behaving as deletions and/or multiple gains, have been proposed as one the most
prevalent type of genetic variation in the human genome. Considering that the CTNNA3 gene (also
known as α-T-catenin or VR22) is located in the long arm of chromosome 10, within a genomic region
showing genome-wide significant evidence of linkage to late-onset Alzheimer’s disease (LOAD) and that
contains one of the highest frequency CNV deletion hotspot in the human population, here we examined
whether these putative CNVs events may affect the integrity of the CTNNA3 message and thus its
biological function as a component of the Wnt/β-catenin signaling pathway. Based on the annotated
CTNNA3 cDNA sequence, we designed 4 sets of overlapping primers to sequentially amplify the full
CTNNA3 transcript in 47 human brain cortical mRNA samples, including 22 individuals diagnosed with
LOAD, 11 controls and 14 individuals with other neurological disorders. Sequencing of the RT-PCR
amplification products revealed that two novel alternative transcripts exist: CTNNA3Δ8-12, which is
overrepresented in LOAD individuals (5 out of 22 LOAD; 0 out of 11 controls), lacking 685 bp
corresponding to skipping of exons 8 to 12 and introducing a premature stop codon; and CTNNA3Δ8-17,
TUESDAY
lacking 1353 bp corresponding to skipping of exons 8 to 17, without affecting the normal ORF.
Subsequent cloning and assessment of the biological function of CTNNA3Δ8-12 and CTNNA3Δ8-17
constructs indicated a differential pattern of activity in Wnt/β-catenin reporter assays. Our results thus
reveal novel CTNNA3 alternative transcripts supporting previously described CNV events within the
genomic region and which may affect the function of the gene in LOAD individuals. Supported
FONDECYT 1100942.
3087/L062
Monoclonal Antibody for LRP6 Inhibits the Wnt Pathway and Inflammation in Diabetic
Retinopathy.
K. Lee, Y. Chen, Y. Hu, J. Ma; ouhsc, 941 stanton L young blvd, OK
It has been shown that the canonical wnt signaling pathway plays a major pathogenic role in diabetic
retinopathy through up-regulation of angiogenic and inflammatory factors, suggesting that the wnt
signaling represents a new therapeutic target for this ocular disease. The purpose of this study is to
evaluate the effect of a blocking antibody for LRP6, a co-receptor of wnt signaling, on wnt signaling in the
retina and diabetic retinopathy. A monoclonal antibody (mAb) specific for the wnt-binding domains of
LRP6 was generated using a recombinant peptide. The mAb showed a high specificity and affinity to
LRP6 ectodomain. Wnt signaling was induced by wnt3a conditioned media or high-glucose in cultured
retinal cell lines. The mAb inhibited LRP6 phosphorylation at Ser1490 and beta-catenin accumulation in
cytosol and down-regulated the expression of VEGF, ICAM-1, and TNF-alpha induced by wnt3a and by
high-glucose. However, the mAb did not inhibit LiCl-induced wnt signaling, confirming that the mAb
functions via blocking the receptor-ligand interactions on the plasma membrane. In vivo, the mAb was
injected into the vitreous of streptozotocin (STZ)-induced diabetic rats, and its effects were evaluated by
Western blotting, leukostasis, and retinal vascular permeability assay. In STZ rats, intravitreal injection of
the mAb resulted in significant down-regulation of inflammatory cytokine expression including ICAM-1 and
TNF-alpha, and subsequently, decreased vascular leukostatsis. Moreover, injection of the mAb also
significantly reduced retinal vascular permeability in the diabetic model. In conclusion, the mAb specific
for the LRP6 ectodomain suppresses wnt signaling activation, inflammation and vascular leakage in
diabetes, demonstrating therapeutic potential for diabetic retinopathy.
3088/L063
MTM Phosphoinositide Phosphatase and Pi3-Kinases Regulate Integrin Trafficking Required for
Muscle Attachment and Maintenance.
1
2
2
3
1 1
2
I. Ribeiro , L. Yuan , G. Tanentzapf , J. Dowling , A. Kiger ; UCSD, La Jolla, CA, University of British
3
Columbia, Vancouver, BC, Canada, University of Michigan Medical Center, Ann Arbor, MI
Muscles must maintain cell compartmentalization when remodeled during development and use. Identity
of membrane trafficking compartments is in part conveyed by phosphoinositide phosphates (PIPs), which
are spatially-temporally regulated by dedicated kinases and phosphatases. Mutations in human
phosphoinositide 3-phosphate phosphatase, Myotubularin1 (MTM1), underlie X-linked myotubular
myopathy (XLMTM), suggesting tight regulation of PIP homeostasis is essential in muscle. We show that
the Drosophila MTM1-homologue, myotubularin (mtm), is required for integrin- mediated myofiber
attachments, and that mtm-depleted myofibers exhibit hallmarks of human XLMTM myopathy. Depletion
of mtm leads to increased turnover of sarcolemmal integrin and mislocalization of integrin with PI(3)P on
endosomal-related membrane inclusions. The depletion of Class II, but not Class III, PI3-kinase rescued
mtm-dependent integrin defects, identifying an important pathway that regulates integrin recycling.
Surprisingly, depletion of Class III Pi3-kinase mimicked mtm muscle detachment defects, which could be
rescued by co-depletion with Class II Pi3-kinase. Moreover our results on Rab21 and Rab6 localization
and functions implicate their roles in regulation of integrin trafficking in myofibers revealing an intricate
pathway to maintain muscle integrin adhesions. Importantly, similar integrin localization defects were
found in human XLMTM myofibers signifying conserved MTM1 function in muscle membrane trafficking.
Our results indicate that regulation of distinct phosphoinositide pools plays a central role in maintaining
cell compartmentalization and attachments during muscle remodeling, and suggest involvement of Class
II PI3-kinase in XLMTM disease mechanism.
TUESDAY
3089/L064
A Mutation in an Autosomal Dominant Retinitis Pigmentosa Causative Gene Affects Its Cullin
Complex Formation.
Y. Kigoshi, F. Tsuruta, T. Chiba; Graduate School of Life and Environmental Sciences, University of
Tsukuba, Tsukuba, Japan
Retinitis pigmentosa (RP) are a genetically heterogeneous group of progressive retinal dystrophies,
resulting in degeneration of rod and cone photoreceptors. Patients experience night blindness and visual
field loss, often leading to complete blindness. RP can be inherited in autosomal dominant (ad),
autosomal recessive, and X-linked manner. It has been recently reported that mutations in a BTB-Kelch
protein, cause adRP. BTB-Kelch proteins are the adaptor/substrate recognition proteins, which target
their specific substrate to Cullin3 (Cul3)-based ubiquitin ligases for degradation. Here, we investigated
whether the BTB-Kelch protein function in the ubiquitin proteasome system and how the reported
mutations affect the characteristics. Co-immunoprecipitation experiments showed that the BTB-Kelch
protein does indeed interact with Cul3 via the BTB and BACK domain, and the disease causing mutation
decreased those binding. Interestingly, immunofluorescence analyses revealed that wild-type protein colocalized with Cul3 in relatively large punctate structures in the cytoplasm, while its mutants did not colocalize with Cul3 and dispersed as relatively small dot-like structures. The wild-type protein also showed
positive interaction and co-localization with poly-ubiquitin chain suggesting that Cul-3 mediated
ubiquitination is linked to the formation of a large puncta. We also found that the mutant BTB-Kelch
protein formed heterooligomer with the wild-type protein. Furthermore, co-expression of wild-type and
mutant proteins inhibited the binding of wild-type protein with Cul-3. These results suggest that the BTBKelch protein is a bona fide component of Cul3-based E3 complexes and that the autosomal dominant
phenotype of the disease causing mutation is due to its dominant-negative effect.
3090/L065
Development of a Cell-Free Based Screening Method to Identify Cancer Specific Autoantigen
Proteins.
K. Nishimori; University, Matsuyama, Ehime, Japan
Autoantigen protein is known as a causal factor for autoimmunity diseases. Recently many autoantigen
proteins have also been found in cancer patients. In addition, certain autoantigen proteins have
abundantly expressed in cancer cells. These evidences suggest that the autoantige proteins may
implicate in cancer formation. Identification of cancer-related autoantigen proteins, therefore, is a key
issue for understanding of its biological roles in cancer development. We developed a cell-free based
protein production system, biotin labeling technology and highly specific/sensitive detection for
autoantigen-autoantibody reaction by luminescent assay. Using these system, our recent work
demonstrates two key issues: 1) many autoantibodies can recognize a conformational epitope in
autoantigen proteins, and 2) autoimmune susceptibility loci, and membrane and extracellular proteins are
good substrates for screening of autoantigen proteins . In this study, we challenged to identify prostate
cancer specific autoantigen proteins using our screening method, and to analyze its biological function in
cancer formation. According to these results, we constructed a human protein library for screening of
cancer specific autoantigen proteins. Based on annotation date from human autoimmune susceptibility
loci, and membrane and extracellular genes, 1,994 genes were selected from Mammalian Gene
Collection clones, and were used as templates. A biotin ligation site (bls) and histigine tag (HisT) were
fused to N-terminal region of the gene by “spilt-primer PCR” we made. DNA templates for translation,
SP6-E02-bls-HisT-gene were constructed, and then used for a wheat cell-free protein production . The
protein productivity was determined by AlphaScreen system using anti-His antibody. Finally, human
protein library consisting of 1,688 proteins were generated. They were mixed with sera of prostate cancer
patients, and subsequently autoantigen proteins were detected by AlphaScreen system. At least 52
proteins were prostate specific autoantigen proteins. Certain proteins were highly abundant in prostate
cell lines. Now, we are trying to investigate biological function of these proteins in prostate cancer
development.
TUESDAY
3091/L066
Screening of Cancer-Related E3 Ubiquitin Ligase by Wheat Cell-Free System.
S. Yasuoka; University, Matsuyama, Japan
Posttranslational modification of proteins is a key issue for cancer formation, e.g. phosphorylation of
proteins is known as a main player of cancer transformation. Recently, as a new modification, a protein
ubiquitination has been reported to be remarkable type of cancer transformation. Because the protein is
ubiquitinated by a specific E3 ligase, it is crucial to identify the specific E3 ligase recognizing a cancerrelated protein. However, conventional methods using the living cells have limitations on production of
recombinant E3 ligase protein. Since eukaryotic cells have proteasomal degradation system, recombinant
E3 ligase proteins induce internal protein degradation. Fortunately it is not found the proteasome system
in a wheat cell-free system (Takahashi, et al. 2009). In general, E3 ligases is classified by three families:
HECT, RING and U-box. RING-type E3 ligase family is involving in functional regulation of protein by
ubiquitination, rather than degradation. From their situation, we challenged to construct RING-type E3
ligase library by the wheat cell-free system, and to screen the E3 ligase binding to cancer-related protein.
The 256 of full-length RING-type E3 ligase genes were collected from Mammalian Gene Consortium
(human) and FANTOM (mouse), and then were used for construction of DNA templates for the cell-free
system by GenDecoder 1000 with biotinylation on the N terminal. As a model case, MDM2, well-known
RING-type E3 ligase for ubiqutination of p53, was used. Binding and ubiqutination between biotinylated
MDM2 and Flag-p53 were detected by the luminescent method using AlphaScreen. Using this system,
we found the novel E3 ligases binding to p53 and LKB1 as cancer-related proteins.
3092/L067
Structural and Biophysical Analysis of BST-2/Tetherin Ectodomains Reveal an Evolutionary
Conserved Design to Inhibit Virus Release.
1,3,4
1
2
2 1
T. J. Brett , S. M. Scheaffer , M. Swiecki , M. Colonna ; Internal Medicine, Washington University
2
School of Medicine, Saint Louis, MO, Pathology and Immunology, Washington University School of
3
Medicine, Saint Louis, MO, Cell Biology and Physiology, Washington University School of Medicine,
4
Saint Louis, MO, Biochemistry and Molecular Biophysics, Washington University School of Medicine,
Saint Louis, MO
BST-2 is a recently discovered host antiviral factor that potently blocks viral replication by trapping
progeny enveloped viruses on the surface of infected cells, eventually leading to internalization and
degradation, thereby preventing their spread.In return, viruses have evolved antagonists to this activity.
BST-2 traps budding virions by using two separate membrane-anchoring regions that simultaneously
incorporate into the host and viral membrane. Here we detail the structural and biophysical properties of
the full BST-2 ectodomain, which spans the two membrane anchors. The 1.6 Å crystal structure of the
complete mouse BST-2 ectodomain reveals a ~145 Å parallel dimer in an extendedα -helix conformation
that predominantly forms a coiled-coil bridged by three intermolecular disulfides that are required for
stability. Sequence analysis in the context of the structure reveals an evolutionarily conserved design that
destabilizes the coiled-coil, resulting in a labile superstructure, as evidenced by solution X-ray scattering
displaying bent conformations spanning 150 Å and 180 Å for mouse and human BST-2 ectodomains,
respectively. Additionally, crystal packing analysis reveals possible curvature-sensing tetrameric
structures that may aid in proper placement of BST-2 during the genesis of viral progeny. Overall, this
extended coiled-coil structure with inherent plasticity is undoubtedly necessary to accommodate the
dynamics of virus budding while ensuring separation of the anchors.
3093/L068
The Effects of Onion Extract and Quercetin on Blood-Brain Barrier (BBB) Dysfunction during
Ischemia/Hypoxia.
1,2
1,3
5
5
1,2
1,2,4
1,2
5
1,3,4
S. Hyun , S. Park , E. Ko , M. Jang , C. Moon , S. Lee , E. Baik , S. Park , Y. Jung ;
1
2
Physiology, Ajou University School of Medicine, Suwon, Republic of Korea, Brain Korea 21 for Medical
3
Sciences, Suwon, Republic of Korea, Brain Korea 21 for Molecular Sciences and Technology, Suwon,
4
Republic of Korea, Research Institute of Pharmaceutical Sciences and Technology, Ajou University,
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5
Suwon, Republic of Korea, Molecular Biotechnology, College of life and Environmental Sciences,
Konkuk University, Seoul, Republic of Korea
Blood-Brain Barrier (BBB) dysfunction is a critical event during cerebral ischemia, and is followed by an
influx of water, leading to edema and secondary brain injury. Onion and its major component, quercetin,
has protective effects against brain damage from cerebral ischemia. However, the effects of onion extract
and quercetin on dysfunction of BBB are still not clearly known. In this study, we examined the effects of
onion extract and quercetin on ischemia/hypoxia-induced BBB dysfunction and edema using both in vivo
and in vitro model. We induced focal cerebral ischemia by occlusion of left middle cerebral artery (MCA)
using an intraluminal filament technique in vivo and hypoxic condition through incubation in anaerobic
chamber in vitro. Onion extract and quercetin reduced brain water content in the ischemic hemisphere in
vivo. They also significantly decreased BBB permeability and inhibited the disruption of tight junction
protein zonula occludens-1 (ZO-1) in vivo and in vitro. In addition, onion extract and quercetin also
attenuated ROS accumulation. Taken together, our results suggested that onion extract and quercetin
may have protective effects on brain edema through decreasing BBB dysfunction in focal cerebral
ischemia via, at least in part, an antioxidant effect. This work was carried out with the support of
"Cooperative Research Program for Agriculture Science & Technology Development (Project No.
PJ005453)" Rural Development Administration, Republic of Korea.
3094/L069
Activation of the Anti-Oxidative Transcription Factor Nrf2 in Renal Ischemia Reperfusion Injury.
M. Leonard, D. Crean; Conway Institute, University College Dublin, Dublin, Ireland
Ischemic Reperfusion injury (IRI) is associated with a diverse array of disease states where blood flow is
disrupted and subsequently reintroduced. One aspect of adaptation to injury is to up-regulate an array of
cytoprotective genes responsible for buffering the cells’ antioxidant capacity and has recently been
suggested to involve the transcription factor Nrf2. Both in vivo and in vitro models were used to define the
activation and role for nrf2 in renal IRI. Microarray analysis of renal tissue from a murine in vivo IRI model
revealed seven of the twenty most highly upregulated genes as Nrf2 dependent with
antioxidant/detoxification function. Activation of Nrf2 within these tissues was also observed by western
blot analysis and immunohistochemistry. Microarray analysis of renal epithelial (HK-2) cells demonstrated
a reoxygenation specific activation of Nrf2. This was paralleled by a rapid nuclear accumulation of Nrf2
protein and the induction of the Nrf2 dependent gene NQO1 promoter reporter activity. Oxygen/glucose
deprivation (OGD) and subsequent reintroduction (R) also demonstrated an activation of Nrf2 and NQO1
expression. The functional consequence of Nrf2 activation in this model was assessed using siRNA
targeted against Nrf2. Knockdown of Nrf2 did not alter toxicity of OGD/R in HK-2 cells assessed using
lactate dehydrogenase release. However the activation of NQO1 gene expression was diminished by
Nrf2 siRNA. In summary we have demonstrated the activation of Nrf2 in models of IRI. Future work will
investigate precise signalling roles for Nrf2 in protection against acute and chronic injury in this important
ischemic disease condition.
3095/L070
Host Cell Entry of Influenza a Virus Requires Early Ubiquitination Events and the ESCRT
Machinery.
J. Huotari, A. Gabryjonczyk, A. Helenius; Biochemistry, ETH Zurich, Zurich, Switzerland
Influenza A virus infects cells by hijacking the host cell endocytic machinery. Uptake of the virus results in
trafficking to acidic late endosomes, the site of penetration by viral and endosomal membrane fusion.
Most membrane-associated cargo destined for the degradative pathway requires the attachment of
ubiquitin to its cytosolic domain. Ubiquitin serves as a signal for inclusion into intralumenal vesicles and is
recognized at the level of early endosomes by the ESCRT machinery. Previous studies have suggested a
role for ubiquitination in the trafficking of the virus. We show here that inhibition of ubiquitination using an
E1-ligase inhibitor, UBEI-41, results in a block of influenza virus infection. Ubiquitination is required early,
as addition of UBEI-41 15 min after the virus no longer has an inhibitory effect. The block occurs during
endosomal trafficking and results in accumulation of the virus in acidic endosomal compartments. In
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addition, siRNA-mediated depletion of ESCRT-0 and ESCRT-I components resulted in a similar
phenotype. We propose that a cellular receptor is activated and ubiquitinated upon influenza infection,
and that this is required for ESCRT-mediated sorting of the virus into the late endosomal pathway or for
correct endosome maturation.
3096/L071
Activation of Sirt1 Results in the Induction of the Nrf2/ARE Pathway.
1,2
2
2
2 1
P. M. Irusta , R. Paul , A. Martin-Motalvo , R. de Cabo ; Georgetown University, Washington, DC,
2
National Institute on Aging, Baltimore, MD
SirT1 is a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase that regulates
diverse stress responses. In this study we show that SirT1 interacts with NF-E2-related factor 2 (Nrf2), a
transcription factor that regulates the expression of numerous detoxifying and antioxidant genes via the
antioxidant response element (ARE). Furthermore, pharmacological activation of Sirt1 either in wild type
mouse embryonic fibroblasts (MEFs) or in C57BL/6J mice results in increased Nrf2 protein levels and
activation of various Nrf2 downstream targets, including NAD(P)H quinone oxidoreductase1 (NQO1),
glutathione S-transferase Ya subunit (GST Ya subunit) and Ubiquitin-conjugating enzyme E2 R1
(UB2R1). Taken together, these results reveal a novel physical and functional interaction between SirT1
and Nrf2, and suggest that these proteins may act in concert to execute the oxidative stress response.
The interplay between SirT1 and Nrf2 represents a potential therapeutic target for treating diseases
resulting from oxidative damage.
3097/L072
Modulation of Yeast [PIN+] Prion Variants by Hsp90-Related Chaperones.
D. L. Lancaster, R. A. Rachubinski; David Lancaster, University of Alberta, Edmonton, AB, Canada
Prion-related diseases, such as Creutzfeldt-Jakob disease, vary in severity depending upon the unique
folded state adopted by the prion protein. These states, called variants, are also a feature of prions in the
yeast, Saccharomyces cerevisiae. [PSI+] is the prion form of the yeast translation termination factor,
Sup35p. [psi-] cells can adopt the [PSI+] phenotype if Sup35p misfolds into a prion. For this to occur
efficiently, the cell must contain the [PIN+] prion, the prion state of the Rnq1 protein. Rnq1p can adopt
three [PIN+] variants: low, medium, and high, named for their [PSI+] induction potentials. These variants
can also be distinguished in vivo by the morphology of Rnq1p aggregates. Rnq1p forms predominantly a
single aggregate per cell in low and medium [PIN+] backgrounds and multiple aggregates in high [PIN+]
backgrounds. While [PIN+] variants serve as a powerful means by which the cell can regulate the rate of
[PSI+] induction, no mechanism has yet been identified for regulating which [PIN+] variant the cell carries.
Here we provide evidence for a role for several Hsp90-related chaperones in modulating [PIN+] variants.
Deletion of the HSC82, AHA1, CPR6, CPR7, TAH1, SSE1, or YDJ1 gene in low and medium variant
backgrounds increased the [PSI+] induction efficiency to that of the high variant. Deletion of the SBA1
gene in medium and high variant backgrounds decreased the [PSI+] induction efficiency to that of the low
variant. That this shift in induction efficiency is due to a bona fide change in the [PIN+] variant is
supported by concurrent shifts in the morphology of Rnq1p. Additionally, treatment of low and medium
variants with chemical inhibitors of Hsc82p and Cpr6/7p led to similar phenotypic shifts to the high variant.
Tah1p and Cpr7p were also shown to interact with Rnq1p by yeast two-hybrid analysis. Taken together,
our results implicate Hsp90-related chaperones in maintaining specific [PIN+] variants and suggest that
disruption of their activity through genetic deletion or chemical inhibition leads to a shift from one stable
[PIN+] variant to another. By understanding the factors that affect prion variants, we can better devise
variant-specific therapeutic strategies for prion diseases.
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3098/L073
miR-138 Deregulation Promotes Epithelial-Mesenchymal Transition in Head and Neck Squamous
Cell Carcinoma.
1,2
1,2
1,2
1,2
1,2 1
C. Wang , X. Liu , Z. Chen , Y. Jin , X. Zhou ; Center for Molecular Biology of Oral Diseases,
2
College of Dentisty, University of Illinois at Chicago, Chicago, IL, Graduate College, UIC Cancer Center,
University of Illinois at Chicago, Chicago, IL
Cancer cells can dedifferentiate through activation of specific biological pathways associated with
epithelial-mesenchymal transition (EMT), thereby gaining the ability to migrate and invade. Recent
studies suggested that deregulation of microRNAs (miR) contributes to EMT, and is associated with
disease progression in a number of cancer types, including head and neck squamous cell carcinoma
(HNSCC). Our previous data suggest that miR-138 plays a critical role in tumor invasion by targeting
RhoC and ROCK2, concurrently. Here, we shown that miR-138 was markedly down-regulated in patients
with metastatic HNSCC and cell lines with a mesenchymal phenotype. Knockdown of miR-138 in HNSCC
cells with high endogenous miR-138 expression induced a partial EMT. Conversely, ectopic expression of
miR-138 in mesenchymal HNSCC cells with low endogenous miR-138 level reversed the EMT
phenotype. Several EMT regulators (including Vim, Zeb2, and EZH2) were predicted to be direct targets
of miR-138 based on bioinformatics analysis. The direct interactions of miR-138 and the specific targeting
sequences in the mRNAs of Vim, Zeb2 and EZH2 genes were confirmed by luciferase reporter gene
assays. Taken together, our results suggest that miR-138 deregulation may involve in EMT dynamics,
thereby contributing to migration and invasion during HNSCC progression.
3099/L074
Shiga Toxins Induce Autophagy Leading to Different Signaling Pathways in Toxin-Sensitive and
Toxin-Resistant Cells.
M. Lee, C. Rama, V. L. Tesh; Microbial and Molecular pathogenesis, Texas A & M University Health
Science Center, College station, TX
The bacterial virulence factors Shiga toxins are expressed by Shigella dysenteriae serotype 1 and certain
Escherichia coli strains. Shiga toxins are potent protein synthesis inhibitors and induce apoptosis, or
programmed cell death, in many cell types including epithelial, endothelial and neuronal cells. Previously,
we showed that Shiga toxins rapidly induced apoptosis through ER stress in monocytic (undifferentiated)
THP-1 cells, while a slower onset of apoptosis was observed in macrophage-like (differentiated) THP-1
cells. Both extrinsic and intrinsic apoptotic signaling cascades were activated by the toxins. Numerous
studies have shown that autophagy, a highly conserved lysosome-dependent catabolic process,
characterized by the formation of double-membrane vesicles to engulf damaged organelles, is associated
with activation of death processes, although autophagy may also activate pro-survival signals. It is
currently not known if autophagy contributes to apoptosis induction by Shiga toxins. To study the role of
autophagy in apoptosis induction, we intoxicated human macrophage-like cells (THP-1), renal proximal
tubule epithelial cells (HK-2), and primary human monocyte-derived macrophages transfected with GFPLC3 to detect autophagosome formation. Confocal microscopy revealed the punctate green fluorescence
which is characteristic of GFP-LC3+ autophagosome formation. Autophagy induction was also confirmed
from the observation that Shiga toxin treatment of THP-1 and HK2 cells rapidly triggered LC3-I → LC3-II
conversion (lipidation), a well known marker of autophagy. Proteolytic cleavage of Beclin-1 and Atg5
plays pivotal roles in switching death signaling from non-toxic autophagy to apoptosis. We observed that
cleaved forms of Atg5 and Beclin-1 were detected in Shiga toxin-treated THP1 and HK2 cells, which are
Shiga toxin-sensitive cells. In contrast, cleaved Atg5 and Beclin-1 were not detected in toxin-resistant
primary human monocytes and macrophages following toxin treatment. These findings suggest that there
is a molecular link between autophagy and apoptosis induced by Shiga toxins.
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3100/L075
Proteasome Activation Delays Stress-Induced Senescence and Reduces Oxidative Damage in
Human Astrocytes.
1
1,2
1
1
1 1
E. P. Crowe , S. Hartley , A. Bitto , C. Sell , C. Torres ; Pathology and Laboratory Medicine, Drexel
2
University College of Medicine, Philadelphia, PA, Gettysburg College, Gettysburg, PA
A decline in proteasome activity has been established in models of aging and Alzheimer Disease. This
decline is associated with increases in oxidative stress and accumulation of oxidized proteins. Human
astrocytes demonstrate increased sensitivity to oxidative stress and a propensity to undergo stressinduced senescence compared to human fibroblasts. Senescent astrocytes exhibit an increase in reactive
oxygen species production and a decline in the proteasomal protein degradation pathway; alternatively,
proteasome inhibition induces a senescent phenotype in astrocytes. Astrocytes play a critical role in the
maintenance of neuronal homeostasis and accumulation of oxidative damage may contribute to the
dysfunction associated with aging and neurodegenerative disease. Previously, we demonstrated
proteasome stimulation by insulin-like growth factor (IGF-I) enhances the clearance of oxidized proteins in
the brain. This study elucidates the role of the proteasome in the response to oxidative damage and
stress-induced senescence in human astrocytes. Compared to young astrocytes, senescent astrocytes
exhibit a decline in proteasome activity and a concomitant increase in protein carbonyl content and
reactive oxygen species production. Treatment with compounds that induce nuclear erythroid factor 2
(Nrf-2), a transcription factor that directs the cellular antioxidant response pathway, results in upregulation
of catalytic proteasomal subunits and proteasome activation in astrocytes. Protein carbonyl levels in
response to oxidative damage are diminished by pretreatment with Nrf2-inducing compounds.
Proteasome activation via Nrf-2 delays stress-induced senescence and may affect astrocyte lifespan in
vitro. Taken together, these data suggest the proteasome plays a major role in the regulation and
alleviation of oxidative damage in human astrocytes.
3101/L076
Isolation of Cells with Characteristic of Stem/Progenitor Cells from Epithelial Ovarian Cancer.
1
1
1
1
2
3
1
1
S. Miotti , I. Bortolomai , A. Zacchetti , A. Vecchi , F. Raspagliesi , B. Valeri , P. Perego , G. Cossa , S.
1 1
Canevari ; Experimental Oncology, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy,
2
3
Gynecological Surgery, Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy, Anatomic Pathology,
Fondazione IRCCS Istituto Nazionale Tumori, Milan, Italy
Several studies support the presence of putative cancer stem cells (CSC) in epithelial ovarian cancer
(EOC). The chemokine receptor CXCR4, expressed by CSCs of different solid tumors, is frequently
present on a subset of ovarian tumor cells, but not yet described as CSC marker in EOC. The present
study aims to investigate whether, like CD133, also CXCR4 could identify a subpopulation of cells with
characteristics of tumour initiating cells in human primary EOC and ascites. By FACs analyses CD133
and CXCR4 expression was found in a panel of epithelial tumour cells obtained from primary ovarian
cancer (range 0.1%-19% and 4.9%-54% respectively) and from ascite (range 0.3%-35% and 1%-64%
respectively). Serial in vivo passages of ascitic cells derived from a poorly differentiated EOC selected for
the expression of both CD133 and CXCR4, showed in vivo selection of CXCR4 bright tumor cells,
whereas CD133 expression decreased. CXCR4 or CD133 sorted tumor cells from two patient xenografts
were evaluated by Real Time PCR for stem markers. Higher mRNA levels of stem genes were found to
be reproducibly associated with CXCR4 positive tumour cells. Variable results were obtained with CD133
sorted cells. To verify if CXCR4 identifies a tumour subpopulation, which expresses markers linked to
chemoresistance, CXCR4 sorted fractions were analyzed by real time PCR for the expression of a wide
panel of drug transporters. The obtained results pointed out an increment in the expression of several
drug transporters in CXCR4 positive cells. In contrast with the literature, the injection of sorted CXCR4
positive and negative cells in SCID mice gave rise to ascites containing similar levels (9%) of CXCR4
positive cells. These results suggest that CXCR4 could be an unstable stem cell marker which expression
could be inducible in negative cells by the host microenvironment. Partially supported by a grant of
Cariplo Foundation.
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3102/L077
A Toxic Oligomer Acts as the Direct Precursor for the Formation of Mature Amyloid Fibrils by the
Mouse Prion Protein.
S. Jain, G. Kulasegaran, J. B. Udgaonkar; National Centre for Biological Sciences, Bangalore, India
In order to characterize the intermediate aggregates formed during amyloid fibril formation by the mouse
prion protein and to elucidate their exact kinetic role, the reaction was studied at the physiological pH by
multiple structural probes, and over a range of protein concentration. Atomic force microscopy (AFM)
images suggest that the reaction proceeds with transient formation of spherical oligomers, and culminates
into the formation of mature amyloid fibrils. The observed rate of fibrillation is the same, whether
measured by changes in thioflavin T fluorescence upon binding, or total scattering intensity. This
suggests that β-sheet conformational change occurs concurrently with aggregate assembly. The
observed rate increases with an increase in protein concentration, and saturates at protein concentrations
above 25 μM. This suggests that aggregate growth is rate-limiting at low protein concentrations, and that
a conformational change, that would be independent of protein concentration, becomes rate-limiting at
higher protein concentrations. Hence, fibril formation by moPrP occurs in at least two separate steps. A
detailed kinetic analysis suggests that the reaction occurs via a nucleated conformation conversion, which
has been described earlier in the case of a yeast prion protein, Sup35. Furthermore, spherical oligomers
are seen to populate during the initial lag phase of the kinetics of amyloid fibril formation by the mouse
prion protein. These spherical oligomers are found to be toxic to cell lines, as the mature amyloid fibrils.
Interestingly, they are capable of seeding the aggregation reaction, which suggests that they are on the
direct pathway to the mature fibrils. The data supports the notion that prion-mediated toxicity originates
from the aggregation reactions of the prion proteins.
3103/L078
Bacterial Cyclic AMP-Elevating Toxins Can Inhibit Autophagy in Host Cells.
1,2
1
1,4
1,2,3 1
S. Shahnazari , A. Namolovan , P. K. Kim , J. H. Brumell ; Hospital for Sick Children, Cell Biology,
2
Toronto, ON, Canada, Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada,
3
4
Institute of Medical Science, University of Toronto, Toronto, ON, Canada, Department of Biochemistry,
University of Toronto, Toronto, ON, Canada
Autophagy is the process by which long-lived proteins and organelles residing in the cytoplasm are
recycled. It can be triggered by starvation and stress but is also thought to play a protective role against
the progression of some human diseases including cancer, muscular disorders and neurodegenerative
diseases. Importantly, it has also been found to play a role in adaptive and innate immune responses.
Second messengers have been implicated in the regulation of autophagy but their mechanism of action
has yet to be characterized. Cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) have
been found to play a negative role in the regulation of autophagy in yeast and most recently in
mammalian cells through phosphorylation of different autophagy components including Atg1, Atg13 and
LC3. Some bacteria including Bacillus anthracis (B. anthracis) and Vibrio cholerae (V. cholerae) produce
toxins that are capable of elevating intracellular cAMP levels. B. anthracis produces edema factor which
is a calmodulin-activated adenylate cylcase whereas V. cholerae produces cholera toxin which can
activate host adenylate cyclases. Both toxins have been previously found to suppress immune
responses. We were interested in testing whether these bacterial toxins are also capable of inhibiting
autophagy through production of cAMP. We tested a role for these toxins and cAMP-elevating
pharmacological agents and observed a significant inhibition in multiple types of autophagy including
autophagy of bacteria (xenophagy). Using a PKA specific inhibitor we were able to determine that these
toxins and drugs were functioning through PKA to inhibit this process. These confirm a role for
cAMP/PKA in the negative regulation of autophagy and demonstrate for the first time that bacterial
virulence factors are capable of repressing autophagy.
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3104/L079
Hydration and Transparency of the Rabbit Cornea Irradiated with UVB-Doses Equivalent to 2.5 and
5.0 Hour Exposure Time of UVB Rays Reaching the Human Cornea from Sunlight.
C. Cejka, J. Cejkova, T. Ardan; Department of Eye Histochemistry and Pharmacology, Institute of
Experimental Medicine, Prague 4, Czech Republic
Purpose: Exposure of the cornea with UVB rays evokes the damage of the corneal epithelium and the
development of intracorneal inflammation, photokeratitis. The mechanism by which the cornea maintains
the normal hydration and transparency is disturbed and the cornea swells, light scattering appears and
the cornea becomes opalescent. The aim of this study was to examine changes of corneal hydration and
transparency (evaluated by corneal light absorption) in the dependence of repeated irradiation of the
rabbit cornea with two different UVB doses corresponding to 2.5 hours and 5 hours of UVB exposure of
the human cornea from sunlight. Methods: The rabbit corneas were irradiated with the daily UVB dose of
0.25 J/cm2 or 0.5 J/cm2 for four days. Every day before the irradiation and the last day before the
sacrificing the animals corneal hydration was examined using an ultrasonic Pachymeter. One day after
the end of irradiations the rabbits were sacrificed and corneal light absorption measured using our
spectrophotometric method. Some rabbits were sacrificed on day four after the end of irradiations to
examine the development of maximal corneal hydration. Results: Changes of corneal hydration and
transparency appeared after the UVB dose of 0.25 J/cm2 and massively increased after the UVB dose of
0.5 J/cm2. Maximal corneal hydration developed on day two following the end of irradiations after the
lower UVB exposure and on day three after the higher UVB exposure. Conclusions: Repeated exposure
of the rabbit cornea to the daily dose of 0.25 J/cm2 equivalent to 2.5 hours of solar UVB radiation
reaching the human cornea from sunlight evoked significant changes of corneal hydration and
transparency. Thus even a short repeated daily stay in sunshine without UV protection is potentially
harmful for the human eye.
3105/L080
The Role of FXR in the Regulation of GpBAR-1 and Megalin in Gallbladder.
1
1
2
1 1
F. Cabezas , J. Lagos , M. Arrese , M. Marzolo ; Departamento de Biologia Celular y Molecular, Fac.
2
Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Santiago, Chile, Depto. Gastroenterologia,
Fac. Medicina, Pontificia Universidad Catolica de Chile, Santiago, Chile
The gallbladder expresses Megalin, an endocytic receptor for several relevant ligands, and also GpBAR1, a GPCR activated by bile acids. The mice KO for GpBAR-1 are resistant to develop gallstones. We are
interested to evaluate the role and regulation of GpBAR-1 in gallbladder, as well as its functional
relationship with megalin. Previously we have shown that megalin expression is regulated by bile acids,
which are also ligands for the nuclear receptor FXR. Interestingly and contrarily to GpBAR-1, FXR KO
mice are more sensitive to develop gallstones, however it is not known how gallbladder itself is affected
under this condition. In this work, we evaluated the expression of megalin and GpBAR in gallbladder
epithelia mediated by FXR, both in vitro and in vivo. Methods: Using wt and FXR KO mice, fed with
control and lithogenic diets, we evaluated the role of FXR ligands (that does not activate GpBAR-1) in the
gallbladder expression of megalin, GpBAR-1 and the FXR-target gene SHP, measured by qPCR. In vitro,
using gallbladder epithelial cells we determined the expression of megalin and GpBAR by incubation of
the cells with a FXR specific agonist. Results: Our data indicate the both megalin and GpBAR expression
were significantly different in FXR KO mice compared to the controls. In addition the expression of
megalin was significantly decreased in wild type mice treated with the FXR agonists under lithogenic diet
in contrast to the expression of GpBAR-1 that was significantly increased. In vitro, we found that megalin
expression was also decreased at protein and mRNA level, by treatment with an FXR agonist in human
gallbladder epithelial cells in culture. Finally, we also found significant differences in the expression of
megalin and GpBAR-1 in patients having gallstones compared to the controls. Conclusions: Complex
regulatory mechanisms are involved in the regulation of Megalin and GpBAR-1, including the effects of
bile acids, FXR and GpBAR-1 ligands, which composition can be different under gallstone disease.
Supported by: Fondecyt 1070373
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3106/L081
Celecoxib Increases Activation-Induced Cell Death of CD4 T Cells through COX-2-Independent
Pathway.
M. Li, Y. Zhang, K. Becker, W. Wood, E. Lehrmann, F. Bosetti, R. Sen; National Institute on Aging/NIH,
Baltimore, MD
Activation-induced cell death (AICD), a Fas ligand (FasL)-dependent pathway, is important for
maintaining T-cell homeostasis. FasL cross-links Fas on the CD4+ T cell surface, promoting a series of
intracellular signaling events leading to the release of active caspase-8 and initiating apoptosis. CD4 T
cell blasts activated with CD3 and CD28 showed significantly reduced viability in 12 and 24 hours after
activation. To confirm that cell death under these conditions was medicated by the death receptor
pathway, we used a Fas-deficient LPR mice. LPR CD4 T cell blasts did not die in response to CD3 and
CD 28. Activation of T cells with CD3 and CD28 rapidly induced nuclear p65, which peaked at 4 hours
and then reduced after 8 hours. Cyclooxygenase (COX) is one of NF-kB target genes, it a key enzyme
mediating the inflammatory process, catalyzes the rate-limiting step involved in the synthesis of a series
of prostaglandins (PGs) and leukotrienes from arachidonic acid. There are two isoforms of COX, COX-1
and COX-2. COX-2, an inducible isozyme were induced in CD4 T cell blasts stimulated by TCR
activation. Celecoxib, COX-2 specific inhibitor, exacerbated CD3 and CD28-induced cytotoxicity
concentration and time dependent manner. However viability were not significant different between COX2-deficient and wild-type CD4 T cells, indicating that celecoxib inhibited cell survival in a COX-2
independent manner. In celecoxib-treated CD4 T cells, the expression of TNF superfamily members, such
as CD70, TNFSF9, TNFSF11, TNFSF4 and TNFSF14 were significantly decreased. Our study provides
novel molecular mechanism into the role of celecoxib in TCR induced AICD.
3107/L082
Pseudorabies Virus Infection Disrupts Neuronal Transport of Mitochondria In Vitro.
T. Kramer, L. Enquist; Department of Molecular Biology, Princeton University, Princeton, NJ
Mitochondria are highly dynamic organelles that are important for energy production and metabolism,
regulation of calcium homeostasis, and apoptosis. Mitochondria are transported throughout neurons by
microtubule-dependent molecular motors and accumulate in regions of the cell that require increased
2+
ATP concentrations such as synapses. Specifically, regulation of mitochondrial dynamics is Ca dependent such that motility is inhibited when synaptic activity and action potential (AP) firing are
increased. Pseudorabies virus (PRV) is a neuroinvasive alphaherpesvirus with a broad host range that is
closely related to the human pathogens herpes simplex virus type 1 and 2 (HSV-1 and HSV-2), and
varicella-zoster virus (VZV). Previous studies have identified PRV proteins that localize to mitochondria
and affect mitochondrial function. However, little is known about mitochondrial motility and dynamics in
infected neurons. Our lab has shown that PRV infected neurons become electrically coupled resulting in
propagation of spontaneous electrical activity with elevated AP firing rates. Additionally, we determined
that glycoprotein B (gB), an essential member of the viral membrane fusion complex, is required for
electrical coupling and increased AP firing rates. Here we show that mitochondrial motility and
morphology is disrupted in PRV infected superior cervical ganglion (SCG) neurons cultured in vitro using
time-lapse microscopy. In neurons infected with a gB-null mutant, disruption of mitochondrial trafficking
and morphology is reduced compared to neurons infected with wild type PRV. In the presence of
tetrodotoxin (TTX), a potent inhibitor of AP firing, motility is partially restored in infected neurons. Finally,
2+
live-cell imaging of neurons loaded with the Ca -sensitive dye Oregon Green 488 BAPTA-1, revealed
2+
that Ca increases dramatically during PRV infection. We postulate that gB-dependent membrane fusion
results in increased electrical activity that may affect mitochondrial dynamics. Furthermore, disruption of
mitochondrial trafficking may be a previously uncharacterized pathogenic effect of PRV infection in
neurons cultured in vitro.
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3108/L083
The Hepatitis B Virus X Protein Modulates Intracelluar Calcium Signaling.
1
2 1
B. Yang , M. Bouchard ; Molecular and Cell Biology and Genetics Program, Drexel University College of
2
Medicine, Philadelphia, PA, Biochemistry Department, Drexel University Colldege of Medicine,
Philadelphia, PA
Chronic hepatitis B virus (HBV) infection is related to the development of hepatocellular carcinoma (HCC).
The hepatitis B virus X protein (HBx) is thought to play an important role in the development of HBVmediated HCC. Multiple functions of HBx have been reported including regulation of HBV replication,
cellular signal transduction, transcription, cell cycle, and apoptosis pathways. One essential HBx activity
is the modulation of intracellular calcium (Ca2+) signaling; this HBx activity may be responsible for many
HBx activities. We have shown that HBx increases basal cytosolic calcium ([Ca2+]c), that this increased
[Ca2+]c is indispensable for HBV replication, and that HBx is localized to mitochondria outer membrane.
Exactly how HBx modulates intracellular calcium signaling is not clear. In the studies described here, we
show that in HBx-expressing cells, the plateau level of IP3-linked Ca2+ spike is higher than in control
cells and can be inhibited by blocking mitochondrial Ca2+ uptake and storage-operated channel (SOC)
Ca2+ entry. In HBx-expressing cells, mitochondria have increased Ca2+ uptake; however, there is no
difference in thapsgargin-induced storage-operated Ca2+ entry in HBx-expressing and control cells.
These studies suggest that HBx may increase the extent of the opening of SOC by increasing
mitochondrial Ca2+ uptake, which results in an increased plateau level in IP3-linked Ca2+ signaling. In
addition, our studies suggest that HBx does not impact the inactivation of SOC. Overall, understanding
how HBx modulates cellular Ca2+ will enhance our understanding of the role of HBx during HBV
replication and in the development of HBV-associated HCC.
3109/L084
Signature Microrna Profile Distinguishes the Stem Cell Enriched Region of the Colon Crypt from
Colon Carcinoma.
3,1
3,1
1,3
3
2
1,3,2
V. Viswanathan , S. S. Haghighat , L. Opdenaker , G. Gonye , T. Zhang , B. Boman , N. J.
1 1
2
Petrelli ; Helen F. Graham Cancer Center, Newark, DE, Thomas Jefferson University, Philadelphia, PA,
3
University of Delaware, Newark, DE
The initiation of Colon Cancer is attributed by the dysruption of crypt dynamics. The base of the crypts
harbors stem cells which are responsible for the renewal of the lumen of the colon every five days. The
cancer stem cell model hypothesizes that disturbance in the self renewal properties of the stem cells
could lead to colon tumorigenesis. One of the factors which lead to the deregulation of crypt cell
proliferation and differentiation is MicroRNA. MicroRNAs are small non-coding RNA molecules (14-24
nucleotides long) which downregulate gene expression through post-transcriptional regulation. Aberrant
expression of various microRNAs has already been implicated mainly in cancer of the lung, breast,
prostate and colon. We performed microarray using a 368 gene chip to identify miRNAs which are
differentially expressed in the crypt bottom (stem cell enriched region). Total RNA was isolated using
standard TrizolTM method. RNA was transcribed into complementary DNA (cDNA) through reverse
transcription (RT) which was then amplified through QPCR. Statistical analysis and plots of expression
data for miRNAs were done to find miRNA genes that are differentially expressed. Profiling of purified
colonic crypt subregions showed that a distinct signature involving 16 differentially expressed miRNAs
characterizes the crypt bottom. Out of the sixteen, three miRNAs (has-mir-206, hsa-mir-7, and has-mir23b) were independently capable to distinguish colorectal tissue from normal colonic epithelia (p<0.01).
To investigate whether these miRNAs play role in colon stem cell function, we used the rna22 tool to
predict their common targets. The results indicated these 3 miRNAs together targeted 30 genes including
important genes like MeCP2, SOX11 and LRAT. MicroRNA 23b by itself is predictive to target ALDH1 and
LGR5 which are considered as important stem cell markers of colon. Further studies using stable or
transient expression or knockdown of the genes targeted by these miRNAs in colon cancer cell lines will
help us understand the mechanisms involved in initiation of colon cancer.
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3110/L085
C-Jun-N Terminal Kinase (JNK) Inhibitor as Potential Drug Candidate for Pancreatic Cancer
Treatment.
B. Bandyopadhyay, A. Mohr, T. Caffrey, N. Chaika, S. Pankaj, M. Hollingsworth, M. Wheelock, K.
Johnson; University of Nebraska Medical Center, Omaha, NE
Objective: Pancreatic cancer, the fourth leading cause of cancer-related deaths in the United States, is
one of the most lethal types of solid tumor. Almost 95% of cases are detected in their later stages, and
the median survival period is between 6 months to a year. Besides the present regime of treating
pancreatic cancer patients with gemcitabine, new therapies and methods of early disease screening are
required to increase the survival rate and improve life expectancies. Pancreatic cancer cell lines exhibit
Epithelial to Mesenchymal Transition (EMT) and show up-regulation of mesenchymal markers. We have
shown in these lines that EMT occurs when cells are plated upon collagen, which was accompanied by
up-regulation of N-cadherin (N-cad) expression. We identified JNK1 as one of the components in this
signaling pathway. Hence for our studies, JNK1 has been selected as a drug target. Methods and
Results: SP600125, an inhibitor of JNKs, has been used in an orthotopic mouse model of pancreatic
cancer generated by injecting the pancreatic human cancer cell line CFPAC1 into nude mice. SP600125
at 30 mg/kg body weight caused a 2.5 fold reduction in primary tumor volume and reduced tumor burden
in the treated group compared to the vehicle control. Hematoxylin and eosin stained sections obtained
from these studies have shown reduction in metastasis to distant organs like liver and lung in the treated
group. The expression patterns of N-cad and markers of angiogenesis, cell growth and EMT by
immunohistochemistry have shown a reduced signal for these markers in the treated versus the untreated
samples. The N-cad signal was confined to vesicle-like structures in the treated samples. Conclusion: The
results from our studies, both in vitro and in vivo, suggested JNKs are viable targets in pancreatic cancer.
In ongoing studies, we are using SP600125 together with gemcitabine to determine if these agents are
additive or synergistic in their effects. If successful, our data will support the development of more specific
JNK inhibitors for use in combination with gemcitabine for human subjects in the treatment of pancreatic
cancer.
3111/L086
Tau Over-Expression Suppresses TNFα-Induced Apoptosis in the Breast Cancer Cell Line MCF7.
1,2
2
2
1,2
1,2 1
S. Debnath , P. Hannon , K. Toropova , A. Alonso , J. Fata ; Biochemistry Program, Graduate
2
Center,City University of New York (CUNY), New York, NY, Department of Biology, The College of
Staten Island, New York, NY
The multifunctional cytokine, TNFα induces a number of important biologic responses, such as
inflammation and apoptosis. While systemic administration of TNFα as an anticancer therapy is not
feasible because of serious side effects, specific targeting of TNFα to cancer cells has been shown to
markedly increase chemotherapy-induced apoptosis, including apoptosis induced by the breast cancer
drug Paclitaxel. Tau is a neuronal Microtubule Associated Protein (MAP) and its most recognized
biological function is to promote tubulin assembly into microtubules and to stabilize the preformed
microtubules. In cancer cells, often Tau is abnormally expressed. Recent evidence indicates that high
levels of Tau expression have been associated with breast cancer cell resistance to Paclitaxel; however
the mechanism behind this resistance has yet to be fully elucidated. Here we present evidence that Tau
over-expression in the MCF7 breast cancer cell line suppresses TNFα-induced apoptosis. MCF-7 cells,
when treated with TNFα do not show signs of apoptosis unless the translational inhibitor cyclohexamide is
co-administered. Under these conditions MCF-7 cells show onset of TNFα-induced apoptosis as early as
6 hours post-treatment, with characteristic cell shrinking, membrane blebbing, disruption of tubulin
architecture, and caspase-3 activation. Over-expression of a GFP-Tau in MCF-7 cells completely
abrogates all TNFα-induced apoptosis associated phenotypes. We are investigating whether the
mechanism behind this suppression is related to our finding that GFP-Tau stabilizes tubulin microtubules
in MCF7 cells. These findings are the first to associate Tau and TNFα signaling as it relates to apoptosis
and they may become an important consideration when designing TNFα-directed therapies.
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3112/L087
Role of miR-143 and -145 for Human Glioblastoma Cell Invasion.
1
1
1,2 1
S. Koo , G. S. Martin , L. Toussaint ; Texas A&M University System Health Sceince Center, College
2
Station, TX, The Texas Brain and Spine Institute, College Station, TX
Glioblastoma multiforme (GBM) is the most common and most malignant type of primary brain tumor. The
median survival of only 12-18 months is at least partially due to its invasive phenotype - rendering
complete surgical resection nearly impossible. In this study, we hypothesize that specific microRNA
molecules are key regulators of the invasive phenotype of glioblastoma. To test this assumption, we used
a modified Boyden chamber assay to select, in a serial fashion, subpopulations of glioblastoma cell lines
with enhanced invasion. This phenotype was stable over multiple passages and allowed for comparative
expression analysis of microRNA species. miR-145 and miR-143, two microRNA species in close genetic
proximity and likely sharing a single transcript, were over-expressed in the invasive subpopulations of
U87, U373, and U251 human GBM lines. Expression was confirmed in human tumor samples, especially
at the tumor-vascular interface. Antagomir treatment against these molecules abrogated invasion, and the
effect was most pronounced when expression of miR-143 and miR-145 were targeted simultaneously.
Herein, we present a straightforward method for creating invasive subpopulations of glioblastoma cells,
and we use those cells to generate data supporting a role for miR-143 and miR-145 in regulating
invasion. Currently, the phenotypic consequences of miR-143 and miR-145 expression are being defined
in multiple neoplastic and vascular diseases. Previous reports suggest a tumor-suppressive effect for
these molecules, focusing on cell-cycle regulation. Taken together, these data could be helpful in
understanding the dichotomy between glioma proliferation and invasion, and may illustrate the unique
biology of the most invasive GBM cells.
3113/L088
Investigating the Role of the Cell Cycle Protein E2F1 in HIV-Induced Neurotoxicity.
J. Zyskind, Y. Wang, J. Ting, C. Akay, D. Kolson, K. Jordan-Sciutto; University of Pennsylvania,
Philadelphia, PA
HIV-associated neurocognitive disorder (HAND) results from release of inflammatory factors into the
extracellular milieu by HIV-infected macrophages, provoking neuronal damage, synaptic loss, and
ultimately, neuronal death. Our previous data indicate that cell cycle regulatory machinery, notably
transcription factor E2F1, which is known to activate genes required for G1 to S phase progression of the
cell cycle, are elevated in neurons from post-mortem brain tissue of patients with HAND, as well as in
neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Despite its increased
expression, E2F1 target genes are unaltered by HIV-induced neuronal damage in vitro. Furthermore,
E2F1 displays a predominantly cytoplasmic localization, a site inconsistent with its role as a transcription
factor. In addition to its ability to induce apoptosis via transcriptional regulation, E2F1 can also induce
apoptosis by a less well-defined transcription-independent mechanisms. At least one of these
mechanisms has been shown to include activation of the calcium-sensitive cysteine protease calpain,
which leads to proteolytic degradation of the pro-survival factor MDMx. Using an in vitro model of HIVinduced neuronal loss, we have demonstrated that neuronal E2F1 is upregulated following insult and that
it is primarily cytoplasmic. Additionally, E2F1 is cleaved to a lower molecular weight product by calpain
following HIV-mediated neurotoxic insult. Our preliminary evidence indicates that calpain-dependent
cleavage of E2F1 occurs at the N-terminus of the protein. Further, our data suggest that the increased
levels of E2F1 are due to changes in protein stability of the lower molecular weight E2F1. As
pharmacological inhibition of calpain activation or genetic deletion of E2F1 attenuates neuronal death
from HIV insult, our data suggest that calpain cleavage of E2F1 contributes to HIV-induced neurotoxicity.
Interestingly, we have found that E2F1interacts with MDMx in neurons and that this interaction is
disrupted following HIV-mediated insult. As MDMx has been implicated as a pro-survival factor in several
models of neurodegeneration, we propose that E2F1 contributes to neuronal death by blocking MDMx
activity in neurons.
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3114/L089
Identification and Analysis of Retinoic Acid Signaling Molecules Regulating Stem Cells in Colon
Cancer Progression.
1,2
1,2
2
1,2,3 1
A. Gupta , S. Modarai , N. J. Petrelli , B. Boman ; Biological Sciences, University of Delaware,
2
3
Newark, DE, Helen F. Graham Cancer Ctr, Newark, DE, Thomas Jefferson University, Philadelphia, PA
Introduction-Colonic stem cells are critical in embryonic and fetal gut development and are thought to be
regulated by retinoids. But retinoic acid pathway is compromised in different types of cancer. To better
understand this pathway in relation to stem cells in colorectal cancer, different members of this pathway
were studied in two colorectal cancer cell lines SW480 and HT-29 and a fetal human colon (FHC) cell
line. Aldehyde dehydrogenase (ALDH) was used to identify the stem cells using aldeflour assay and
expression of other members of retinoic acid pathway validated by immunocytochemistry and western
blot analysis. To see an effect of exogenous retinoic acid on cell lines, SW40 cells were treated with
exogenous all-trans retinoic acid (ATRA) (10^-3 to 10^-10) and 9-cis retinoic acid (10^-5 to 10^-10).
Result- High percentages of cells were positive for ALDH and RXR (Retinoid X Receptor) alpha in FHC.
In SW480, 14% of cells were positive for ALDH, 41% for RXR alpha, 34% for CTBP1(C-terminal binding
protein 1) which is a retinol dehydrogenase (RDH) enzyme inhibitor. In HT-29 cells, 2% of ALDH
expression, 11% of RXR alpha expression and 18% of CTBP1 expression were seen. CYP26A1
(Cytochrome P450 26A1) which metabolizes retinoic acid was present in both colorectal cancer cell lines.
With ATRA treatment, I-C50 for SW480 is 10^-6 and with 9-cis retinoic acid treatment, I-C50 for SW480 is
10^-6. Conclusion- High expression of RXR alpha and ALDH in FHC confirms a relation between retinoic
acid and stem cells. RXR alpha is present in both cell lines but probably not active due to no or minimal
amount of cellular retinoic acid present to bind as ligand. This decrease in cellular retinoic acid can be
due to the presence of CYP26A1 and Ctbp1 in high percentage in these two colorectal cancer cell lines.
Treatment with exogenous ATRA and 9-cis retinoic acid in SW480 cells showed to have an effect on their
proliferation.
3115/L090
Assessing the Contribution of HAART to Neuronal Damage and Death as a Mediator of Cognitive
Decline in HIV-Associated Neurocognitive Disorder.
1
2
2
2
2
2
2
P. J. Gannon , A. Odeleye , M. Cooper , D. Cook , M. White , C. Akay , K. Jordan-Sciutto ;
1
2
Pharmacology, University of Pennsylvania, Philadelphia, PA, Pathology Department, University of
Pennsylvania, Philadelphia, PA
The use of highly active anti-retroviral therapy (HAART) for HIV treatment has led to a significant
reduction in the incidence of HIV-associated dementia (HAD), a severe motor/cognitive disorder afflicting
AIDS patients. However, the prevalence of more subtle CNS dysfunctions, termed HIV-associated
neurocognitive disorders (HAND), has continued to escalate in the post-HAART era. While increased
longevity of HIV-infected populations is thought to contribute to alterations in HAND pathology, other risk
factors, such as peripheral and potential central toxicities of remain unexplored. Previous work has shown
that HAART is toxic to both the peripheral nervous system in vivo as well as dorsal root ganglion neurons
in vitro. Thus, we hypothesized that chronic HAART exposure leads to neuronal unfolded protein
response (UPR) activation, observed in HAND. We treated primary rat cortical neuroglial cultures
individually with commonly prescribed HAART compounds from two classes, nucleoside reverse
transcriptase inhibitors (NRTI), and HIV protease inhibitors (PI). Our preliminary studies suggest that PIs
indinavir, ritonavir and saquinavir lead to a statistically significant MAP2 loss over the course of 8 days of
treatment, whereas NRTIs zidovudine, stavudine and zalcitabine did not induce MAP2 loss. Moreover,
PIs ritonavir and saquinavir lead to an early and persistent mitochondrial membrane depolarization, while
NRTIs did not induce mitochondria membrane depolarization. We also observed an early increase in
cleaved caspase 3 and followed by increased calpain cleaved spectrin in ritonavir and saquinavir treated
cultures by immunoblotting. Ritonavir and saquinavir lead to increases in BiP and peIF2alpha, indicators
of UPR activation. However, we did not detect activation of autophagy. Overall, our results suggest that
neuronal responses to anti-retrovirals are class specific, and PIs, not NRTIs induce mitochondrial
membrane depolarization, caspase and calpain activation, and neuronal death. Further studies will
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examine the effect of clinically relevant combination drug treatments on neuronal survival and stress
activation pathways using in vitro and in vivo experimental models.
3116/L091
PKCγ Induces Neuronal Differentiation in Multipotential Neural Precursor Cells and Gliomas Stem
Cells.
1
1
2
1,2 1
R. Steinhart , G. Kazimirsky , S. Finniss , C. Brodie ; Bar-Ilan University, Ramat Gan, Israel,
2
Neurosurgery, Henry Ford Hospital, Detroit, MI
Multipotential neural precursor cells (MNPC) exhibit self-renewal and are capable of differentiating along
the different lineages of the CNS. In this study we explored the role of the neural-related PKC isoform,
PKCγ in the differentiation of MNPC. We found that the expression of PKCγ increased upon
differentiation of the MNP spheres and it was mainly expressed in the neuronal lineage cells. To examine
the role of PKCγ in the differentiation of the MNPC, we overexpressed it in these cells using adenovirus
vectors. Overexpression of PKCγ selectively increased the number of betaIII tubulin positive cells and
significantly enhanced their migration. PKCγ also mediated the effect of BDNF on neuronal differentiation.
Thus, BDNF increased the differentiation and migration of neurons from MNPC and this effect was
inhibited by a PKCγ-KD mutant. To delineate the mechanisms involved in PKCγ effect on neuronal
differentiation, we examined the expression of the proneural basic helix-loop-helix (bHLH) transcription
factor Mash1 and that of ATF5 in neurospheres overexpressing PKCγ. We found that overexpression of
PKCγ increased the expression of Mash1 after 3 days, whereas it significantly decreased the expression
of ATF5. Similarly, silencing of ATF5 increased the neuronal differentiation of the MNPC, whereas
silencing of Mash1 significantly abolished the effect of PKCγ. In addition to its role in neuronal
differentiation, ATF5 has been also shown to be highly expressed in gliomas and glioma stem cells
(GSCs). We therefore examined the role of PKCγ and ATF5 in the differentiation of these cells. We found
that GSCs did not express PKCγ and that overexpression of this isoform decreased their self-renewal
ability and induced a significant neuronal differentiation of the cells. In addition, overexpression of PKCγ
decreased the expression of ATF5. Similarly, silencing of ATF5 decreased the self-renewal of the GSCs
and induced neuronal differentiation of these cells. Our data suggest an important role of PKCγ in the
neuronal differentiation of both MNPC and GSCS and implicate Mash1 and ATF5 as potential mediators
of this effect.
3117/L092
Effect of GPR3 Single Nucleotide Polymorphisms on Gamma Secretase Assembly.
J. L. Hallgren, Z. Qiao, Z. Song; Pharmacology and Toxicology, University of Louisville School of
Medicine, Louisville, KY
It was recently discovered that G protein coupled receptor 3 (GPR3), a constitutively active orphan Gprotein coupled receptor, is able to regulate γ-secretase assembly and localization in a manner which
enhances Amyloid Precursor Protein processing but has no effect on Notch cleavage. We hypothesize
that polymorphic variations of GPR3 may have functional consequences ultimately affecting the
production of Amyloid Beta (Aβ). Three nonsynonymous single nucleotide polymorphisms (SNPs) of
GPR3 are listed in the NIHs dbSNP database build #131; A42fs, T79G, and G222A. These three
mutations were created in recombinant human GPR3 via site directed mutagenesis and stably
transfected, along with wild type (WT) GPR3 into HEK293 cells. Constitutive cAMP production was
evaluated by radioimmunoassay. The level of gamma secretase complex assembly was evaluated by
blue native polyacrylamide gel electrophoresis (BN-PAGE). γ-secretase activity was quantified using a
fluorescent enzymatic assay. Cells containing WT, T79G and G222A receptors had increased cAMP
activity compared to control cells containing empty vector. This effect was not seen with the A42fs
receptor. However, cells expressing the G222A receptor had reduced levels of cAMP compared to cells
expressing WT and T79G receptors. An increase in the molecular weight of the γ-secretase complex was
apparent in cells transfected with WT, G222A, and T79G receptors. BN-PAGE band intensity for γsecretase was decreased for cells expressing both A42fs and T79G receptors compared to WT receptor
expressing cells. WT cells had increased γ-secretase activity compared to control. The enhanced
enzymatic activity was retained in cells transfected with T79G and G222A receptors, but not in cells
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expressing A42fs receptor. In addition, the γ-secretase activity was significantly reduced in cells
expressing T79G receptor compared to cells expressing G222A receptor. These findings demonstrate
that polymorphisms of GPR3 may lead to different levels of cAMP production, different amounts of fully
assembled γ-secretase and different activity of γ-secretase. These could ultimately result in variations in
individual susceptibility to Alzheimer’s disease.
3118/L093
From Inflammation to Cancer: Deconstructing Mechanism of TNFa-Induced Stabilization of Hif1a
Transcription Factor.
1
2 1
2
Y. Fedorov , A. Schoolmeesters ; Thermo Fisher Scientific, Pittsburgh, PA, Thermo Fisher Scientific,
Lafayette, CO
Hypoxia-inducible factor-1 (Hif-1) and its regulatory subunit Hif1a plays a central role in tumor progression
by modulating expression of genes involved in cancer cell survival, proliferation and metastasis. Hif-1
activity is regulated by several mechanisms including modulation of protein stability and degradation.
Among recent advances are the discoveries that inflammation-induced cytokines affect protein
accumulation of Hif1a under normoxia conditions. TNFa is a major pro-inflammatory chemokine that is
known to induce Hif1a activity and promote tumorigenesis. To improve our understanding of TNFamediated regulation of Hif1a accumulation we screened a kinome-wide pre-defined ON-TARGETplus®
siRNA library using a cell imaging -based reporter assay, Redistribution® U2OS-Hif1a. Redistribution
technology monitors the cellular translocation of GFP-tagged proteins in response to drug compounds or
other stimuli and allows easy acquisition of multiple readouts from the same cell in a single assay run. In
addition to the primary readout, high content assays provide supplementary information about cell
morphology, compound fluorescence, and cellular toxicity. Interestingly, our screen determined that
depletion of kinases involved into conventional TNFa signaling - IKK/NFkB and JNK pathways, has no
detrimental effect on Hif1a accumulation. On the other hand, depletion of several other kinases or related
genes significantly decreases effect of TNFa on Hif1a stability more then 1.5 fold: PRKAR2B, ADCK2,
TRPM7, RIOK2, TRIO and TRIB2. Furthermore, we found that expression of ADCK2, TRPM7, RIOK2
and TRIB2 is modulated by TNFa. Taken together, our findings indicate that TNFa-mediated stabilization
of Hif1a is regulated by a non-conventional TNFa signaling pathway comprised of TNFR1 receptor and
downstream effectors. In conclusion, we demonstrated the utility of RNAi-based screening combined with
high content cell imaging to assess the molecular factors involved in TNFa-driven regulation of HIF-1
transcription factor.
3119/L094
The Characterization of Laulimalide as an Antimitotic Agent.
1
2
2
1
1 1
M. J. Bennett , G. Chan , L. Vos , J. B. Rattner , D. C. Schriemer ; Biochemistry and Molecular Biology,
2
University of Calgary, Calgary, AB, Canada, Department of Oncology, University of Edmonton,
Edmonton, AB, Canada
Laulimalide is a polyketide macrolide derived from the marine sponge Cacospongia mycofijiensis. It has
been recently discovered that this class binds to a region removed from the taxol site on the exterior of βtubulin. It is our objective to characterize the effects of laulimalide treatment in comparison with the well
studied taxane class of antimitotics. Asynchronous HeLa cells were treated for 2.5 hours with low dose
laulimalide or taxotere (0-50nM). The cells were stained with a β-tubulin antibody, M4491, a marker for
kinetochore and centrosomal proteins, and DAPI and analyzed by indirect immunofluorescence. Cells
accumulated in mitosis, with abnormal spindle morphologies consisting primarily of multipolar structures
and surrounded by circular chromosomal arrays. EM analysis confirmed these results. Time-lapse
imaging was then conducted on HeLa cells treated with laulimalide and taxotere, synchronized at G1/S
using a thymidine block. Results confirmed the immunofluorescence data, and revealed that laulimalidetreated cells enter mitosis but do not proceed beyond metaphase. We then wanted to determine whether
decongression of the chromosomes occurred dependently or independently of multiple pole formation.
Time-lapse imaging was performed on HeLa cells synchronized in metaphase using a MG132 block. The
cells were treated with 30nM laulimalide, or 0.5μM taxol for 2 hours. There was a significant reduction in
multipole formation in laulimalide-treated cells, but chromosomal decongression remained. We also noted
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a complete loss of K-fibres and nonkinetochore microtubules (MTs). These MTs were very persistent in
the taxol-treated cells, and retained kinetochore-MT interactions with a well defined metaphase plate.
Although laulimalide and taxol are both microtubule stabilizers, laulimalide appears to destabilize
kinetochore-MT interactions whereas taxol stabilizes them. Interphase cells also have more MT bundling
in the presence of taxol than with laulimalide and taxotere. In conclusion, preliminary data suggests that
laulimalide has similar characteristics to taxotere, however differs from taxol in both interphase and mitotic
cells.
3120/L095
Role of Myosin IIA Domains in Natural Killer Cell Cytotoxicity: Insights from Human MYH9-Related
Disease.
1
2
3
1,4 1
K. B. Sanborn , R. Favier , J. B. Bussel , J. S. Orange ; Immunology, University of Pennsylvania,
2
Philadelphia, PA, Assistance Publique-Hopitaux de Paris, CRPP, Armand Trousseau Children's Hospital,
3
4
Paris, France, Weill Cornell Medical College, Cornell University, New York, NY, Children's Hospital
Research Institute, Children's Hospital of Philadelphia, Philadelphia, PA
Natural killer (NK) cells kill transformed or infected target cells via the formation of an actin-rich
immunological synapse (IS) and the directed secretion of granzyme- and perforin-containing secretory
lysosomes (SL). We have recently defined a critical role for myosin IIA in directly enabling the interaction
of SL with actin and their transit through the actin-rich IS to the synaptic membrane. In order to determine
which portions of the myosin IIA protein are required for this function, we analyzed the effect of naturally
occurring human myosin mutations on NK cell cytotoxicity using 51Cr-release cytotoxicity assays.
Mutations in the head and neck region, as well as the nonhelical tailpiece, of the myosin IIA molecule
reduced NK cell cytotoxicity, while most mutations in the rod domain do not adversely affect NK cell
killing. Biochemical analysis of SL-associated myosin IIA revealed phosphorylation of residue Ser1943 in
the nonhelical tailpiece, suggesting that the myosin tailpiece may be required for proper regulation of its
function on SL. These results point to a requirement for myosin IIA to bind to cargo and move along Factin in order to help SL reach the synaptic membrane for secretion, and highlight tailpiece
phosphorylation of SL-associated myosin IIA as an important regulatory mechanism for NK cell
cytotoxicity.
3121/L096
Trypanosoma Cruzi Subverts the Sphingomyelinase-Mediated Plasma Membrane Repair Pathway
for Cell Invasion.
1
1
1
1
2
1 1
M. C. Fernandes , M. Cortez , A. Flannery , C. Tam , R. A. Mortara , N. Andrews ; Department of Cell
2
Biology and Molecular Genetics, University of Maryland, College Park, MD, Department of Microbiology,
Immunology and Parasitology, Federal University of Sao Paulo, Sao Paulo, Brazil
In order to invade host cells, the protozoan parasite Trypanosoma cruzi triggers cytosolic free Ca2+
transients that stimulate exocytosis of lysosomes. However, the exact mechanism by which lysosomal
exocytosis promotes T. cruzi internalization remains unclear. Here we show that host cell entry by T. cruzi
mimics the process of plasma membrane injury and repair. This process involves calcium triggered
exocytosis of lysosomes, delivery of the lysosomal enzyme acid sphingomyelinase (ASM) to the outer
leaflet of the plasma membrane, followed by a rapid form of endocytosis which mediates internalization of
membrane lesions. Our results demonstrate that T. cruzi trypomastigotes transiently wound host cells and
promote increased endocytosis levels. In addition, host cells become more susceptible to infection when
injured with pore forming toxins. T. cruzi invasion is significantly impaired after chemical inhibition or
genetic silencing of lysosomal ASM, treatments that also block plasma membrane repair. Extracellular
addition of sphingomyelinase stimulates host cell endocytosis, facilitates T. cruzi invasion, and restores
normal invasion levels in ASM-depleted cells. Control cells contain newly formed parasitophorous
vacuoles enriched in ceramide, the product of sphingomyelin hydrolysis, while the few parasite-containing
vacuoles formed in ASM-depleted cells are ceramide deficient. Thus, T. cruzi subverts ASM-dependent,
ceramide-enriched endosomes that function in plasma membrane repair to infect host cells.
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3122/L097
Novel Pathways of Combination Antiretroviral Therapy (cART) -Mediated Toxicity in the Central
Nervous System.
1
1
1
2
1
1
1 1
A. Odeleye , M. Cooper , M. G. White , D. Cook , G. Ritson , C. Akay , K. Jordan-Sciutto ; Pathology,
2
School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, Neurology, School of Medicine,
University of Pennsylvania, Philadelphia, PA
Combination antiretroviral therapy (cART) efficiently controls levels of HIV; however, the prevalence of
HIV-associated Dementia remains high. Antiretroviral drugs have been demonstrated to cause
neurotoxicity to the peripheral nervous system via alterations in lipid and protein metabolism in addition to
mitochondrial damage-induced production of reactive oxygen species (ROS). We hypothesize that cART
compounds induce mitochondrial and endoplasmic reticulum (ER) stress, leading to neuronal damage in
the central nervous system, contributing to the changing clinical and pathological picture seen in HIV
positive patients. To test this hypothesis, we treated primary rat cortical neurons with commonly
prescribed antiretroviral compounds from two classes, nucleoside reverse transcriptase inhibitors (NRTI)
and HIV protease inhibitors (PI) in therapeutically relevant doses and assessed mitochondrial, oxidative,
ER stress and neuronal damage. Our data suggest PIs (indinavir, ritonavir and saquinavir) are neurotoxic
alone or in combination with NRTIs (zidovudine, stavudine and zalcitabine), as detected by loss of MAP2
and activation of calpain. Zidovudine, ritonavir, and saquinavir, when applied in combination, lead to early
induction of ER stress as indicated by increases in BiP, p-eIF2α and ATF6-β expression, followed by
mitochondrial membrane depolarization. Interestingly, early increases in HO-1 and NQO-1 protein levels
suggest the activation of the endogenous antioxidant response element (ARE) in response to the
compounds as well. Interestingly, compounds that alter the ARE attenuated neuronal mitochondrial
dysfunction, ER stress and death. Overall, our results suggest antiretrovirals at therapeutically relevant
combinations induce a multi-pronged stress response, resulting in neuronal damage, which can be
attenuated by altered endogenous antioxidant response. Understanding the underlying mechanisms of
the toxicity will provide new strategies to develop adjunctive therapies to complement cART, as well as
potential diagnostic means for testing the neurotoxicity of new cART treatments.
3123/L098
Specific Phosphorylation of pRb Alters Neuronal Viability in HIV-Induced Neuronal Damage.
1
1
2
2
1
1
1
C. Akay , A. Yeager , E. Moscado , J. Isaacman-Beck , K. A. Lindl , Y. Wang , M. G. White , K. Jordan1 1
2
Sciutto ; School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, School of Medicine,
University of Pennsylvania, Philadelphia, PA
HIV Associated Dementia (HAD) is characterized by microgliosis, astrogliosis, neuronal loss and dendritic
damage. While the mechanisms of neuronal death in HAD remain only partially defined, neuronal
damage has been linked to soluble factors released by HIV infected, and non-infected, activated
macrophages/microglia in the brain. The inflammatory infiltrate secretes both neurotoxic and
neuroprotective factors. Phosphorylation of pRb has been shown to be required in in vitro models of
neurodegeneration including trophic factor withdrawal, beta-amyloid treatment and oxidative stress, as
well as in conditions where neuroprotective factors were present. To define the differential effects of
neurotoxic and neuroprotective factors on pRb phosphorylation in HAD, we used an in vitro model where
we treated primary rat neuroglial cultures with supernatants from human monocyte-derived-macrophages
infected with a neurotropic strain of HIV-1 (HIV-M/M). We assessed pRb phosphorylation on
characterized sites and the interaction of these phospho-pRb isoforms with cell cycle proteins. Using
dilutions of HIV-M/M that produced 25% neuronal loss by 4 hours or 50% neuronal loss by 20 hours, we
observed an early increase in p-pRbSer795 by western blotting (WB) and immunofluorescence analysis
(IFA), whereas pro-survival factors, CCL5 and BDNF lead to an early accumulation of p-pRb S608 and
ppRb S807/811 in the cytoplasm of neurons, as detected by WB and IFA. We observed similar increases
in ppRb S795 by WB and IFA in the mid-frontal cortices of autopsied brain tissue of HIV(+) individuals.
We did not observe an increase in ppRb Ser608 or ppRb S807/811 by WB in vivo. Interestingly,
overexpression of a mutant of ppRb nonphosphorylatable at S795 protected against neuronal damage
induced by HIV-M/M, suggesting an alternative role for the specific phosphorylation of pRb at S795 in
neuronal damage / death.
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3124/L099
Comprehensive Analyses of Gene Expression Pattern Based on Tumor Differentiation in
Hepatocellular Carcinoma. A Relationship with Hepatic Stellate Cells.
1
1,2
1
1
3
3
2
3
S. Nagaie , K. Terashima , K. Mogushi , Y. Mahmut , N. Nakamura , S. Tanaka , M. Kitagawa , S. Arii ,
1 1
2
H. Tanaka ; Bioinformatics, Tokyo Medical and Dental University, Tokyo, Japan, Pathology, Tokyo
3
Medical and Dental University, Tokyo, Japan, Hepato-Biliary-Pancreatic Surgery, Tokyo Medical and
Dental University, Tokyo, Japan
Purpose:Gene expression pattern of hepatocellular carcinoma (HCC) was analyzed based on tumor
differentiation. Materials and Methods:Among 168 cases of HCC microarray analyzed for full scaled-gene
expression, 63 cases were selected for the present microarray analyses (well differentiated (WD) = 28
cases and poorly differentiated (PD) = 35 cases). Wilcoxon rank sum tests are applied to estimate the
significance levels of gene expression and KEGG pathway are used to investigate the molecular
pathways. Immunostaining was performed with frozen sections following acetone-fixation and HRP-LSAB
method. Result:Gene expression of LDB2 (p < 0.001), NOTCH1 (p = 0.003), NGF (p = 0.004), NGFR (p =
0.013), CD34 (p = 0.005) was up-regulated in WD HCC. From the molecular pathway analyses,
metabolism pathway (especially retinol metabolism), pathway of complement and coagulation cascades,
PPAR signaling pathway showed the significant difference in WD. In addition, immunostaining showed
RBP7 and RBP1 were positive in normal hepatic stellate cells and WD-tumor cells, but negative for
normal hepatocytes and PD-tumor cells. Conclusion:Up-regulation of LDB2 gene which is related to
mesenchymal stem cell, retinol metabolism pathway as well as NGF and NGFR suggested that there is a
relationship between WD HCC and hepatic stellate cells.
3125/L100
The Combination Antiretroviral Drug Treatment Induces an Integrated Stress Response in Primary
Macrophages Directly, Leading to Mitochondrial Dysfunction and Decreased Viability in Primary
Neurons Indirectly.
1
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1
2
3
2
1
M. Cooper , A. Odeleye , M. G. White , D. Cook , M. Cushman , D. Kolson , C. Akay , K. Jordan1 1
Sciutto ; Pathology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA,
2
3
Neurology, School of Medicine, University of Pennsylvania, Philadelphia, PA, Neuroscience Graduate
Group, School of Medicine, University of Pennsylvania, Philadelphia, PA
The introduction of the combination antiretroviral therapy (cART) has lead to a significant decrease in the
incidence of HIV-associated Dementia, the most severe form of HIV-Associated Neurocognitive Disorders
(HAND), due to efficient systemic viral control and reduced mortality and morbidity rates. However, the
frequencies of less severe forms of HAND have been increasing in cART-treated, HIV-positive patients. In
HAND, cells of monocyte/macrophage (M/M) lineage constitute the major source of viral presence as well
as neuroinflammatory stress in the CNS. The effects of cART on M/Ms and their contribution to
neurodegeneration in HIV positive patients remains a critically unexplored explanation for the increased
prevalence of HAND. Antiretrovirals are known to induce cellular toxicity in the periphery by a number of
mechanisms, including alteration of protein metabolism, increased endoplasmic reticulum stress and
mitochondrial damage-induced generation of reactive oxygen species (ROS). We hypothesized that
antiretroviral compounds activate stress pathways in M/M, altering function of these cells, leading to
indirect neurotoxicity. To test this hypothesis, we treated primary human monocyte-derived macrophages
(MDMs) with commonly prescribed antiretroviral compounds from two classes, nucleoside reverse
transcriptase inhibitors (NRTIs) and HIV protease inhibitors (PIs) in therapeutically relevant doses and
assessed ER and oxidative stress in MDMs and the effect of antiretroviral-treated MDM supernatants
(ART-MDM) on neuronal mitochondrial function and viability. We observed that antiretroviral-treated
MDMs displayed increases in the markers of ER and oxidative stress, including BiP and p-eIF2a.
Furthermore, ART-MDMs induced loss of mitochondrial membrane potential and loss of MAP2, indicative
of cellular damage in neurons. Overall, our results suggest antiretrovirals as activators of multiple stress
pathways that can alter macrophage function leading to indirect neurotoxicity. These studies underscore
the need to develop adjunctive therapies to alleviate macrophage dysfunction to support neuronal health
in the post-cART era.
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3126/L101
AGR2 Induces Enterocyte Differentiation of Rat Primary Intestinal Epithelial Cells.
A. Gupta, A. Dong, M. T. Tun, A. W. Lowe; Medicine, Stanford University, Stanford, CA
Background: Agr2 is a 19 kD protein with a thioredoxin like domain, a signal peptide, and an ER retention
signal (KTEL). AGR2's specific function is unknown, but has been implicated in Xenopus glandular organ
development, newt limb regeneration, and human adenocarcinoma growth. Normal mouse intestine
express AGR2 in secretory cells (i.e. Paneth, goblet, and enteroendocrine cells); including replicating
cells within the intestinal crypt. Using a primary intestinal cell line, we explored potential role of agr2 in cell
differentiation Methods: IEC-6 cells originally isolated from rat intestinal crypts do not express AGR2.
AGR2's effect on intestinal cells was studied by stably transfecting AGR2 cDNA into IEC-6 cells. AGR2’s
impact was evaluated using qPCR, immunoblotting and immunofluorescence. We also studied the
importance of AGR2's carboxy-terminal KTEL sequence by introducing a stop before KTEL or conversion
to a known ER retention signal, KDEL. Results: CDX2 expression is not detected in wild-type IEC-6 cells.
qPCR revealed that AGR2 expression in IEC-6 cells induced CDX2 expression. CDX2 expression was
confirmed with immunoblotting and immunofluorescence. CDX2 is a transcription factor that promotes
intestinal development, including proliferation and differentiation. IEC-6 cells also exhibited enhanced cell
proliferation with AGR2 expression. The CDX2 positive cells are also positive for sucrase isomaltase, a
protein expressed by differentiated enterocytes. In vitro labeling for cells for a nucleoside analog shows
that the cells positive for CDX2 expression are negative for proliferation. Further, confirming that AGR2
can induce terminal differentiation of enterocytes that cannot divide. The alteration of the ER retention
signal from KTEL to KDEL or KTEL to putting a STOP before KTEL, results in loss of Cdx2 induction and
function of AGR2. Conclusion: AGR2 induces CDX2 expression in IEC-6 cells; consistent with their
known expression in proliferating and differentiating intestinal cells. Consistent with AGR2's established
role in Xenopus glandular development, it may serve a similar role in mammalian intestinal development.
AGR2 activity requires a KTEL ER retention signal for its function.
3127/L102
Slc35d3 Regulates Biogenesis of Platelet Dense Granules from an Early Endosomal Compartment.
1
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2
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2
2
2
2
R. Meng , D. Harper , Y. Wang , A. Sitaram , Y. Yao , J. Opalinska , M. Weiss , M. Poncz , M. S.
1 1
2
Marks ; Pathology and laboratroy medicine, University of Pennsylvania, Philadelphia, PA, Children's
hospital of Philadelphia, Philadelphia, PA
Slc35d3 is a recently defined member of a family of sugar-nucleotide transporters. A mutation in the gene
underlies the bleeding disorder in the ashen-Roswell mouse, correlating with a depletion of platelet dense
granules. This finding suggests that Slc35d3 regulates the formation of dense granules, but the
distribution of Slc35d3 in platelets or megakaryocytes is not yet known. Dense granules are members of a
lysosome-related organelle (LRO) that includes melanosomes in melanocytes. Studies have shown that
melanosomal cargoes are delivered from distinct early endosomal domains to melanosomes. However,
how the different sorting pathways are coordinated to drive the maturation of melanosomes is unknown.
Here, we hypothesize that Slc35d3 is a component of this regulatory system in platelets/
megakaryocytes. To test this hypothesis, we have expressed HA-tagged Slc35d3 in a megakaryocytoid
G1ME cell line. Immunofluorescence microscopy (IFM) analysis shows that HA-Slc35d3 co-localize with
tubular early and recycling endosomes marked by syntaxin 13 (STX13), but not with dense granules.
Consistently, immunoelectron microscopy analysis shows HA-Slc35d3 is mostly on tubulovesicular
structures of the endosomes. Subcellular fractionation shows that Slc35d3 is co-fractioned with STX13 in
G1ME cells. Further IFM analysis indicates that endogenous slc35d3 is on a tubular structure underneath
the plasma membrane in platelets, which is similar to the STX13 positive endosomal structure. Upon
activation, slc35d3 and STX13 rearrange in a similar pattern and both move to the interior of the platelet.
Our results provide evidence that Slc35d3 seems not a cargo of dense granule, rather a regulatory
protein reside on the tubulovesicular domains of early endosomes. Our studies suggest that the
endosomal network in megakaryocytes are not only critical for the formation of dense granules, also likely
involved in the function of platelets. Further characterization of Slc35d3 is necessary for delineation of
how early endosomal system is involved in the biogenesis of dense granules.
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3128/L103
NIGMS PSI: Biology Initiative - Enabling High-Throughput Structural Biology and Structural
Genomics.
W. Smith, R. Basavappa, J. Chin, C. Edmonds, P. Flicker, P. Preusch, J. Wehrle, C. Lewis, J. Berg;
NIH/NIGMS, Bethesda, MD
The National Institute of General Medical Sciences has announced PSI:Biology to continue the
development of high-throughput structural biology methods and apply them to important biological
problems. This will be accomplished by establishing a network of collaborations between centers for
structure determination and biologists with interests in problems involving particular proteins or collections
of proteins that would benefit from structural information. Collaborations will be established through
separate awards to investigators outside the structure determination centers. These awards are named
Consortia for High-Throughput-Enabled Structural Biology Partnerships and successful applicants will
help to define targets for structure determination by the centers and will receive funds for functional
studies in the applicants’ laboratories. This mechanism provides an on-going opportunity for the wider
biomedical research community to obtain funding to participate in the PSI through collaboration with the
high-throughput structure determination centers and with the centers for membrane structure
determination. The PSI:Biology Network will include Centers for High-Throughput Structure
Determination, Centers for Membrane Protein Structure Determination, the PSI:Materials Repository and
the PSI:Biology Knowledgebase and the Consortia investigators. In addition, R01 and P01 applications
are solicited from individual investigators or groups of investigators to participate in the PSI:Biology
Network. The applicants may propose to further develop high-throughput structural biology methods,
novel methods for comparative molecular modeling or additional biological partnerships as member of the
PSI:Biology Network. Individual researchers may also suggest proteins for structure determination by the
PSI Structure Determination Centers on the PSI Knowledgebase Community Nomination site.
3129/L104
Quantitative Phosphoproteomics of Tau and Synaptic Proteins in Alzheimers Disease.
P. Rudrabhatla, H. Jaffe, H. C. Pant; NIH, NINDS, Bethesda, MD
Tau protein in human brain exists as a series of six isoforms generated by alternative splicing from a
single gene. The neurodegenerative diseases including Alzheimer’s disease (AD), Pick’s disease (PiD),
progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and fronto-temporal dementia
with Parkinsonism linked to chromosome 17 (FTDP-17) have been collectively termed the tauopathies
due to the presence of pathological deposits of tau in affected brain regions. AD is the best characterized
of the tauopathies, the paired helical filaments (PHF) that form characteristic neurofibrillary tangles are
comprised of insoluble tau (PHF-tau). We used quantitative phosphoproteomics method, iTRAQ (isobaric
tag for relative and absolute quantitation) and compared the phosphorylation abundance of individual
phosphorylation sites of insoluble Tau from normal and AD brain. We report here that the proline-directed
phosphorylation of Tau is increased in AD brain compared to normal brain at Thr205 (4-fold), Ser396,
Ser404 (10-fold) and Thr231 (7-fold). We also report the higher abundance of non-proline directed
phosporylation sites in AD brain compared to normal brain at Ser191 (2-fold), S198, S199 (3-fold),
Ser208 (3-fold) Thr403 (10-fold). We have observed dramatic increase of Tyr phosphorylation of Tau at
Tyr394 in AD brain compared to normal brain, suggesting the involvement of Tyr phosphorylaiton of Tau
in AD. AD is also pathologically characterized by synaptic loss in the affected brain areas. We
demonstrate that Ser14 and Thr5, CKII phosphorylation sites of syntaxin 1A are down regulated in AD
brain. Furthermore, the phosphorylation of other synaptic proteins, syntaxin 1B (Ser 14), septin 5 (Ser
226, Y218), septin 2 (Ser 248) and vesicle transport related protein (VTRP, Thr403) are down regulated
in AD brain. This study forms the first comprehensive iTRAQ analysis of Tau and synaptic proteins in AD
brain. Unraveling the phosphorylation sites of Tau and synaptic proteins would lead to the successful
kinase and/or phosphatase-based therapeutics in AD.
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3130/L105
Characterization of Single Chain Antibody Targets Using the Yeast Two-Hybrid Technology.
3
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1
1
1
2
3
3
M. Nunez , O. Vielemeyer , C. Nizak , A. Echard , B. Goud , J. Camonis , P. Tafelmeyer , J. Rain , F.
1 1
Perez ; Molecular Mechanisms of Intracellular Transport, UMR CNRS 144, Institut Curie, Paris, France,
2
3
Transduction Networks Analysis Group (ART), INSERM U528, Institut Curie, Paris, France, Hybrigenics
Services SAS, Paris, France
Because of their ability to bind antigens with extremely high specificity, antibodies are widely used in
fundamental and biomedical research, are indispensable in innumerable clinical diagnostic tests and
represent one of the most promising therapeutics. Detailed knowledge about the domain(s) recognized by
a given antibody is crucial especially before any clinical application. Such data include the epitope
mapping and the identification of secondary targets due to identical or analogous sequences present in
any other proteins. However, such data is frequently difficult to obtain. Here we describe a novel
approach, which allows target identification and epitope characterization of already selected monoclonal
antibodies using the yeast two-hybrid technology. By using single chain Ab (scFv) as bait and a highly
complex random-primed cDNA prey library, coupled with a rigorous scoring system to identify interacting
clones while eliminating non-specific binders, decisive additional information about several scFv targets
was obtained. This included data about conformational specificity, domain/epitope mapping, and
secondary targets identification. For example, using this approach, we precisely mapped the epitope
recognized by the AA2(1) and ROF7 antibodies, two conformation-specific sensors that specifically bind
to the activated forms of the small GTPase Rab6 and Rab1, respectively. For two other scFv, TA10 and
SF9, which recognize the giantin and non-muscle myosin IIA(2), the epitope sequence was defined.
Finally, for some antibodies, secondary targets within and across species could be revealed. All together,
we show that the yeast two-hybrid technology can be a powerful tool to characterize monoclonal antibody
target(s). Our novel approach may become an invaluable tool for investigators who wish to characterize
their selected antibody prior to using them in research or clinical medicine.
3131/L106
The Mitochondria Dynamics in Isolated Senescent Yeast Cells.
C. Chang; The Institute of Biotechnology, National Tsing Hua University, Hsin Chu City, Taiwan
Mitochondria are critical organelles in living cells. Unlike static organelles, they are continuously fusing
and dividing. Many researches have provides evidences that mitochondria dynamics is critical for
maintaining their function. Mitochondria dysfunction has long been correlated to cell senescent. In
addition, many age-related neurodegenerative diseases such as Parkinson disease and Huntington
disease are associated with abnormal mitochondria morphology. However, little is understood about the
aging effects on mitochondria dynamics and function. In order to address this issue, we use budding
yeast Saccharomyces cerevisiae as a model to investigate. Senescent yeast cells were isolated from the
culture medium. These cells are used to compare with younger cell population on the aspects of
mitochondria dynamics and functions.
3132/L107
The Use of Rigid and Non-Rigid Lipids in Enhancing Nucleic Acid Delivery with a Novel Polyene
Cationic Nucleic Acid Delivery Vector.
1
1
2
2
2
3
1 1
R. Jones , M. Mirza , C. Opstad , R. Sliwka , V. Partali , P. Leopold , M. Pungente ; Weill Cornell
2
Medical College in Qatar, Doha, Qatar, Norwegian Univeristy of Science and Technology, Trondheim,
3
Norway, Stevens Institute of Technology, Hoboken, NJ
The success of therapeutic nucleic acid delivery requires the development of novel delivery vectors and
formulations that overcome cellular barriers for effective transport. Herein, a novel cationic phospholipid,
C14, constructed of one rigid, highly unsaturated chain based on a carotenoid structure and a second
saturated hydrocarbon chain with a length of 14 carbons is shown to effectively deliver pDNA into CHOK1 cells and siRNA into HR5CL11 cells. Lipid-DNA particles were generated with C14 employing two
distinct co-lipids, namely 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol, and
the performance of these formulations were compared to two established non-viral delivery vectors, 3β-
TUESDAY
[N-(N’,N’-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and 1,2-dimyristoyl-sn-glycero-3phosphoethanolamine (EPC). Particle size analysis demonstrated that the C14 carotenoid lipid-DNA
complex at a N/P (+/-) ratio of 1.5 formed smaller particles when mixed with DOPE as compared to with
cholesterol as co-lipid, with sizes of 199.8 and 238.9 nm, respectively. DNase I degradation assays
revealed that complexes with DC-Chol offered the best plasmid protection with DOPE as co-lipid. In gene
transfer experiments using a beta-galactosidase expression plasmid, the C14 vector out performed both
DC-Chol and EPC mediated expression when complexed with cholesterol. In knockdown experiments
using luciferase-expressing HR5CL11’s, C14 complexed with siLuciferase and colipid cholesterol
outperformed DC-Chol and EPC. These data establish this novel C14 rigid lipid as a promising nucleic
acid transfer reagent.
3133/L108
Extending the Range of Applications of Transmission-through-Dye Microscopy.
M. A. Model, M. Pelts, C. Oh; Kent State University, Kent, OH
INTRODUCTION. Transmission-through-dye microscopy (TTD) provides quantitative information about
the 3D shape and volume of transparent objects, including biological cells (Gregg et al, Pflüger’s Arch,
2010). It is based on imaging cells in transmitted light in the presence of a nontoxic external dye with
strong absorption, so that image contrast represents cell thickness. The vertical dimensions of a cell can
be measured with precision exceeding the diffraction limit, and the volume and thickness can be
continuously monitored for long time periods. OBJECTIVE. The method is most straightforward when
using objective lenses with moderate NA and when applied to living cells spread on glass. The goal of the
present study was to investigate whether other type of specimens and high power lenses can be used for
this type of imaging. RESULTS. (1) TTD images obtained with high-NA objectives can be contaminated
with contrast resulting from intracellular compartments with a different refractive index. This effect can be
mitigated by growing cells on a slide (as opposed to a coverslip) and imaging them from their apical side.
(2) In glutaraldehyde and formaldehyde-fixed samples, many cells preserve the integrity of the plasma
membrane. Since quantitative interpretation of TTD images assumes that the dye is excluded from the
cell interior, such cells can be imaged and analyzed without any modification of the method. (3) TTD
images of rounded cells may be affected by refraction on steep cell boundaries and, if a low-NA objective
is used, by limited spatial resolution. A good estimate of the volume of average-sized cells can be
obtained by using an objective with medium resolution (such as x20/0.7) and calculating the volume from
the lateral and vertical diameters. CONCLUSION. The TTD microscopy is a simple technique that
provides direct information about both volume and thickness of living and aldehyde-fixed spread cells at
moderate xy resolution. The membrane morphology (but not the volume) can also be obtained at high
spatial resolution, and the volume (but not morphology) can be measured for round cells.
3134/L109
Investigation of Optimum Temperature to Thaw Frozen Human Tissue Samples.
D. McGarvey, T. Kannanayakal, L. Lovecchio, C. Arana, E. Yoffee, V. A. LiVolsi; Pathology, University of
Pennsylvania, Philadelphia, PA
Endogenous enzyme activity can cause degradation of proteins in tissue during collection and
processing. To reduce this degradation, biosamples are frozen then stored in liquid nitrogen (LN2) or 80°C environments. Depending on the research protocol, the tissues are thawed at various temperatures
(37°C, or 4 °C) prior to use. Little information exists regarding the optimum thaw process or temperature
for proteomic research. In an earlier study, we investigated the effects of different thaw temperatures in
human thyroid tissues by comparing routine thaw temperatures along with a new method of preserving
human tissue at a temperature of 95°C. We observed a 67.8% decrease in tyrosine phosphatase activity
in thyroid tissues that were thawed at 95°C (using the Denator AB tissue stabilizing system) compared to
traditional thaw methods. In the current study, we extended the investigation to other types of human
tissues to determine if the optimum thaw temperature is tissue specific. Materials and Methods: Small
bowel and uterus (endometrium and myometrium) tissue samples were rapidly frozen in LN2 at collection.
The tissues were thawed either on ice (4°C), or in the oven (37°C) or at 95°C. Non-thawed, frozen tissues
were used as controls. The tissues were then homogenized without using protease or phosphatase
TUESDAY
inhibitors. The collected supernatant was stored at -80°C until protein yield was estimated using Pierce
BCA Assay. Phosphatase activity was calorimetrically assayed using the pNPP Protein Phosphatase
Assay kit. Results: In comparison to traditional thaw methods, the tissues thawed at 95°C had a
significant reduction in Tyrosine phosphatase activity in both small bowel (63.2%) and uterus (95.2%)
tissues. These results are consistent with our earlier study of thyroid tissues and indicate that thaw
temperature of 95°C seems best for several human tissue types. Whether this holds for other human
tissues requires further study. Conclusion: The optimum thawing temperature for inactivating tyrosine
phosphatase activity in frozen human tissues is high temperature stabilization at 95°C which might better
preserve the proteome.
3135/L110
A Novel Method for Temporal Assessment of Wound Healing Using Parallel Laminar Flow for
Controlled Epithelial Cell Wounding.
M. Schwartz, C. G. Conant, J. Nevill, C. Ionescu-Zanetti; Fluxion, S. San Francisco, CA
The migratory or proliferative responses mounted by wounded cell monolayers are important to drug
discovery and drug safety testing, as well as to basic research across a number of disciplines including
stem cell biology, cell biology, ophthalmology, endocrinology, microbiology, oncology and developmental
biology. Scratch wounding by mechanical means is the gold standard to achieve an appropriate model
system in which to study these cellular reactions. The scratch wounding technique is plagued by wound
size irregularity and release of cytosolic contents along the wound edge. To address these issues, we
developed a method to produce wounds enzymatically using tightly controlled laminar parallel flow
streams. Resulting wound size was highly uniform in comparison to reported wound size variability of
manual scratch wounding. Wound healing in the presence of epidermal growth factor (EGF), cytochalasin
D and/ or starvation conditions was investigated. Treatment of cells with EGF led to an early increase in
migration followed by a prolonged period of cell proliferation, with full wound healing occurring within
24hours. As expected, cytochalasin D abrogated this effect. Under starvation conditions, the period of cell
migration occurred for a period of 5 hours followed by a significant decrease in cell viability.
3136/L111
Novel Disposable Bioreactor Supports Maintenance of Mixed Cell Populations in 3D.
1
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1,2 1
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W. P. Pfund , L. A. Noll , K. M. McPherson ; RealBio Technology, Inc., Kalamazoo, MI, Department of
Chemistry, Western Michigan University, Kalamazoo, MI
The objective was to demonstrate the ability of a novel 3D culture system to maintain the varied cell
populations of a model tissue in the proper proportions such that the culture represents a biorelevant in
vitro model of the native tissue. Methods: Human Bone Marrow Mononuclear Cells (BMMCs) were
4
7
seeded onto the open 3-dimensional scaffolds of RealBio D ™ Culture Systems (3x10 cells/system).
Growth medium (IMDM w/ serum and growth factors) was perfused above and below the culture scaffolds
in a tangential fashion using low shear pulses. Metabolic gas exchange was accomplished by passive
diffusion from dual gassing chambers through the culture medium flow chambers above and below the
culture scaffolds, effectively decoupling the delivery of gasses from nutrient flow. Metabolic activity of the
cultures was monitored by measuring glucose consumption and lactate production three times/week, and
any cells migrating from the culture scaffolds into the lower medium flow chambers were collected and
characterized with respect to total cell number, viability, and cell surface marker expression. Results:
Robust mixed-cell cultures of human BMMCs were successfully established in each culture system. The
cultures functioned similar to natural bone marrow tissue in that each continually released a mixture of
cell types into the circulating medium just as natural bone marrow releases cells into the circulating blood
6
in vivo. The yield of cells migrating from the culture scaffolds was on the order of 10 cells/day/system
after 2 weeks in culture with viabilities in excess of 80%. Cell surface marker analysis by flow cytometry
showed that the distribution of different cell types migrating from the culture systems reflected the
distribution of the same cell types in the BMMC suspension used to seed the cultures. Conclusions: The
results show that the novel bioreactor described is capable of maintaining mixed cell populations of
BMMCs that reflect the composition of natural human bone marrow. Also, the continuous migration of
TUESDAY
viable cells (representing the make-up of natural bone marrow) from the cultures indicates that BMMC
cultures in the novel bioreactors represent a model of functional hematopoietic tissue in vitro.
3137/L112
Microfluidic Devices for Eukaryotic Chemotaxis, Mammalian Cell Culture, and C. elegans
Embryogenesis Studies.
1
1
2
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3
3 1
A. Groisman , E. Gutierrez , M. Skoge , W. Loomis , A. Carvalho , K. Oegema ; Department of Physics,
2
3
UCSD, La Jolla, CA, Division of Biological Sciences, UCSD, La Jolla, CA, Department of Cellular &
Molecular Medicine, UCSD, La Jolla, CA
We present several new microfluidic devices for cell biology and report the results of preliminary studies
performed with these devices. Quantitative studies of cellular chemotaxis require application of well
defined gradients of chemo-attractants to cells. We built and tested a microfluidic device that generates a
ladder of linear concentration profiles in an array of shallow gradient chambers by molecular diffusion
between two deep flow-through channels that are continuously perfused with a solution of attractant and
plain buffer. The device has two novel features. First, a special compensation mechanism makes the
linear profiles in the gradient chambers very stable and robust, allowing chemotaxis assays to be
successfully performed by a minimally experienced operator. Second, the small depth of the gradient
chambers (~2 um) constrains the cells in the vertical dimension, facilitating confocal imaging, such that
sub-cellular localization of fluorescently tagged proteins can be followed for up to 30 min without
noticeable phototoxicity. We also built and tested a microfluidic device where adherent mammalian cells
are cultured in a shallow (~300 um) chamber for many days without medium exchange or drying, but with
precisely controlled gas content in the medium. We used the device to measure the motility of endothelial
cells as a function of oxygen concentration and found cells to be most motile at low oxygen levels. Finally,
we built a novel microdevice to allow imaging of permeabilized C. elegans embryos during addition of
drugs to the medium. Permeabilized embryos are extracted from an adult worm inside the device and
immediately moved to nearby microwells. The microwells protect the embryos from fluid flow during
medium exchange, holding them motionless close to the coverslip to allow continuous high-resolution
imaging.
3138/L113
Total Internal Reflection Fluorescence Microscopy, Cell Adhesion, and Cell Traction Force
Measurements on Soft Silicone Gels.
1
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2
2
1 1
E. Gutierrez , E. Tkachenko , S. Lee , M. H. Ginsberg , A. Groisman ; Physics, University of California,
2
San Diego, La Jolla, CA, Medicine, University of California, San Diego, La Jolla, CA
Substrate rigidity impacts cellular behaviors such as migration, gene expression, and cell fate. Total
Internal Reflection Fluorescence (TIRF) microscopy enables selective visualization of the dynamics of
substrate adhesions, vesicle trafficking, and biochemical signaling at the cell-substrate interface. Here we
apply high-refractive-index silicone gels to perform TIRF microscopy on substrates with a wide range of
physiological elastic moduli, measure traction forces exerted by cells on the substrate, and perform
analysis of the cell spreading and the biochemical signaling that drives this process. We successfully
implemented TIRF on mouse endothelial cells plated on ~30 micron thick layers of silicone gels with a
refractive index of 1.49 and obtained high quality images. We also functionalized gel surfaces and
covalently bound to them 40nm far-red fluorescent beads that were distributed nearly evenly with a high
surface density. Measurements of displacements of the beads under cell traction forces made it possible
to obtain high-resolution substrate deformation maps that were converted to traction force maps. We
show mouse embryonic fibroblasts (MEFs) spreading over a significantly larger area on silicone gels of
moderate physiological stiffness, 30 kPa, versus those grown on softer substrates of 0.5 and 3.5 kPa, in
agreement with cells grown on polyacrylamide substrates of similar stiffness. We developed a novel
microfluidic technique to measure the elastic moduli of silicone gels and used it to prepare gel substrates
with elastic moduli covering nearly the entire physiological range in different wells of a 6-well plate. Cells
grown in different wells were harvested and analyzed for biochemical changes. Western-blot analysis
revealed varying levels of phosphorylated Focal Adhesion Kinase (FAK) in MEFs grown on silicone gels
with different elastic moduli.
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3139/L114
Kinase Profiling of TrkA and TrkB Expressing Neuroblastoma Cell Lines Using Desthiobiotin
Nucleotide Probes.
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M. M. Rosenblatt , R. Bomgarden , E. Cichon , C. Etienne , M. Patricelli , J. Rogers ; Thermo Fisher
2
Scientific, Rockford, IL, ActivX, La Jolla, CA
Neuroblastoma is the most common and deadly tumor in children. Clinical behavior of this disease is
variable in that some cases show spontaneous regression, whereas others are untreatable despite
maximally tolerable chemotherapy. Although much genetic data has been collected on these tumors, the
biology responsible for this disease is not well understood. Current evidence suggests that the
tropomyosin-receptor-kinase (TRK) family of neurotrophin receptors plays a critical role in neuroblastoma
phenotypes and prognosis. Tumors expressing TrkA are biologically favorable and prone to spontaneous
regression, whereas tumors expressing TrkB have poor prognosis and are associated with MYCN
amplification. To gain more insight into this pathology, we examined the relative expression of kinases in
parental SH-SY5Y cells or cells stably expressing TrkA or TrkB. We employed a proteomic approach
using desthiobiotin nucleotide probes to specifically capture and profile the kinome of each cell line using
mass spectrometry to identify labeled kinase active-site peptides. Differential expression of protein
kinases between cell lines was validated using a parallel Western blot workflow. Overall, we identified and
quantified over 50 human kinases using this approach including many which had different expression
profiles in Trk receptor expressing cell lines. These studies were further complemented via analysis of the
phosphoproteome of the cell lines in response to ligand specific stimulation.
3140/L115
Confocal Fluorescence Anisotropy for the Detection of Conformational States of Proteins in C.
elegans In Vivo.
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4
1 1
V. C. Henschel , A. Esposito , E. Butkevich , C. F. Schmidt , F. S. Wouters , D. R. Klopfenstein ; Drittes
2
Physikalisches Institut - Biophysics, Georg-August University Göttingen, Göttingen, Germany, MRC
3
Cancer Cell Unit, Hutchison/MRC Research Centre, Cambridge, United Kingdom, Biochemistry II,
Center for Molecular Physiology of the Brain (CMPB), Georg-August University Göttingen, Göttingen,
4
Germany, Laboratory of Cellular and Molecular Systems, Department of Neuro- and Sensory Physiology,
Georg-August University Göttingen, Göttingen, Germany
The study of protein-protein interactions in vivo is often hindered by the limited acquisition speed of
typical instrumentation used, for instance, for lifetime imaging microscopy. Anisotropy polarization is
altered by the occurrence of Foerster Resonance Energy Transfer (FRET) and anisotropy imaging was
shown to be comparatively fast and simple to implement. Here, we present the adaptation of a spinning
disc confocal microscope for fluorescence anisotropy imaging that allowed to achieve in vivo imaging at
high spatial and temporal resolution. We demonstrate the capabilities of this system and in-house
developed analysis software by imaging living Caenorhabditis elegans expressing constitutive dimeric
and monomeric proteins that were tagged with GFP. Studies were funded by Deutsche
Forschungsgesellschaft (DFG) and Center for Molecular Physiology of the Brain (CMPB).
3141/L116
Ethidium Bromide/Acridine Orange Viability Staining Method for Counting Cell Populations of
Tetrahymena pyriformis.
A. E. Ogle, J. Swann; Biology, Maryville College, Maryville, TN
Fast, inexpensive, reliable techniques to quantify cell populations in culture are crucial for laboratory
experiments. Currently, standardized methods for cell counting of culture populations include hand
counting with a hemacytometer, digital measurement with a Coulter Counter, or spectrophotometric
absorbance. In this study, the objective was to investigate a new method for enumeration of Tetrahymena
pyriformis (ciliated protozoa) using an ethidium bromide/acridine orange (EB/AO) viability stain. Initial
subcultures from dense cultures were diluted to a concentration of 5,000 cells/mL. For the three days of
linear population growth, daily subsamples of T. pyriformis cultures (n=16) were analyzed via standard
hand counting with a hemacytometer and with EB/AO staining. The stained cultures were plated in
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triplicate and absorbances at 495nm were read by a microplate reader. The equation for hand counting
2
by hemacytometer was y = 4,312.5x + 916.67, r =0.9916. The equation for absorbance at 495nm was y =
2
1,709.4x + 976.83, r =0.9983. The absorbance data from the microplate reader were multiplied by
100,000 to compare the hemacytometer counts on a similar scale. We conclude that the ethidium
bromide/acridine orange cell counting method is recommended for further validation studies.
3142/L117
Innovative Vaccine Complex for More Effective Cancer Therapy.
1,2
2 1
2
R. C. Ennis , P. A. Jose ; Biochemistry, Georgetown University, Washington, DC, Physiology,
Children's National Medical Center, Washington DC, VA
Fraught with chronic side effects, the current paradigm of cancer therapy includes chemotherapy and
radiation. Advancements in nanomedical techniques have lead to the emergence of a new field of
research, cancer immunotherapy, which is the study of stimulating the human immune system to elicit an
anti-tumor response through the proliferation of cytotoxic T lymphocytes (CTL) and, the more recently
discovered killer dendritic cells. The major problem with current cancer vaccines is immunological
overstimulation due to indiscriminant activation of a variety of signal cascades. The present research
proposes an innovative vaccine delivery system of tumor associated immunomodulatory agent CpG
oligodeoxyribonucleotide (CpG ODN) conjugated to poly(lactic-co-glycolic-acid) nanoparticles all
encapsulated within a human-derived mannose glycoprotein. Together, this vaccine complex promotes
CTL clonal expansion through the innate immunological pathways of dendritic cells. Experimental studies
on the vaccine in vivo showed the localization of immune cells to the area of injection with 100% efficacy
in organisms with a closed vasculature, while in vitro experiments showed the direct phagocytosis of the
vaccine complex and morphogenesis of 92% of post-vaccine treatment dendritic cells. RT-PCR of
vaccine-treated dendritic cell mRNA output showed a 4-fold and 17-fold increase in mRNA expression
levels of IL-12 and IFN-γ, which are specific interleukins associated with the anti-tumor immune response.
Pre-clinical mouse studies proved that even in extremely metastatic Balb/c-4T1 mouse models, tumor
growth was halted by the immunol response elicited by the vaccine. Taken together, the proposed
vaccine complex elicits a tumor-restricted immune response without concomitant immunological
overstimulation seen in current cancer modalities.
3143/L118
Detecting and Characterizing Cytosolic Phosphatidylinositol 4-Kinase II Using Fluorescence
Fluctuation Spectroscopy.
1
2
2
1
1 1
J. Li , B. Barylko , J. Albanesi , J. Mueller , Y. Chen ; Physics and Astronomy, University of Minnesota,
2
Minneapolis, MN, Departments of Pharmacology, University of Texas Southwestern Medical Center,
Dallas, TX
Mammalian cells express two classes of phosphatidylinositol 4-kinase (PI4K), designated Types II and III.
These enzymes phosphorylate phosphatidylinositol to generate PI4P. A number of studies indicate that
these enzymes are important for Golgi trafficking and early as well as late stages of endocytosis. There
are two Type II kinases : PI4KIIalpha, an integral membrane protein involved in membrane trafficking; and
PI4KIIbeta, distributed evenly between membranes and cytosol and responsive to growth factor signaling.
In this study, we focus on PI4KIIbeta. Using advanced fluorescence spectroscopic methods such as
fluorescence fluctuation spectroscopic, we identified that PI4KIIbeta exists as two distinctive species in
the cytosol. One species exists as a soluble monomer, while the other species is associated with cytosolic
vesicles. We further determined that PI4KIIbeta on vesicles moves together with transferrin receptor and
epidermal growth factor receptor using advanced dual-color fluorescence fluctuation spectroscopy with
heterospecies partition analysis. The heterospecies partition analysis allows us to isolate the co-moving
species in the presence of strong background contributions from the monomeric PI4K. Our results
demonstrate that brightness analysis is a powerful tool to characterize cellular cytosolic vesicles that are
otherwise difficult to characterize by other techniques. This work is supported by the National Institutes of
Health (R01 GM64589 and GM75401)
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3144/L119
Direct Protein Deposition for Single Cell Analysis.
J. M. Collins, A. B. Smetana, R. Lam, S. Nettikadan; NanoInk, Inc., Skokie, IL
The objective of this study is to pattern cells onto surfaces using direct deposition of extracellular matrix
(ECM) proteins and deliver multiple compounds to individual cells. First, actin polymerization and stress
fiber formation is followed over a 2 hour time period. Second, multiple ECM proteins are patterned on the
same substrate and side-by-side analysis of single cells is done to characterize differential responses.
Finally, polyethylene glycol (PEG) with or without Cytochalasin-D is delivered to individual cells. A method
is established for single cell analysis with multiple compounds on the same substrate. The NLP 2000
(NanoInk, Inc., Skokie, IL) fabrication system is used for patterning of ECM proteins and hydrogel
composites. After approximately 4 hours, substrates are rinsed and non-specific cell binding is blocked
with a solution of bovine serum albumin. NIH 3T3 fibroblasts (ATCC) are added at high density for 30
minutes, at which point non-adherent cells are washed and removed. Complete media is then added for
0.5 or 1.5 hours before paraformaldehyde fixation, staining and analysis. Cells attach to approximately
75% of the patterns deposited onto glass surfaces. Actin polymerization is more developed and stress
fibers are more elongated at 2 hours versus earlier time points. Cell attachment to laminin or fibronectin
ECM proteins is demonstrated and allows side-by-side analysis of cell function. Finally, delivery of
Cytochalasin-D in PEG and the corresponding decline in actin polymerization demonstrates the ability to
address single cells with multiple compounds. Combinatorial experimentation is increasingly important
with regard to the cellular microenvironment. Here, cell patterning with two common ECM proteins and
PEG hydrogel, for delivery of Cytochalasin-D, demonstrates a simple, flexible and fast method of
targeting single to few cells with multiple factors for analysis on a single substrate.
3145/L120
Dissecting Interactions between Coactivator and Nuclear Receptors by Fluorescence Fluctuation
Spectroscopy in Living Cells.
J. Li, Y. Chen, J. Mueller; Physics and Astronomy, University of Minnesota, Minneapolis, MN
Coactivators play an essential role in gene transcription of nuclear receptors (NR) by binding to ligand
activated NRs. We previously applied fluorescence fluctuation spectroscopy to characterize the
interaction between the steroid receptor coactivator (SRC-1) and retinoid X receptor (RXR) inside living
cells. RXR forms a homodimer upon ligand activation, and we identified that the RXR dimer interacts with
SRC-1 to form a heterotrimeric complex with a stoichiometry of two RXR and one SRC-1. This
observation confirms the expected model of dimeric NR interacting with a coactivator. In this study, we
examine RXR’s heterodimer partner, retinoid acid receptor (RAR) and investigate its interactions with the
coactivator SRC-1 in cells. Unlike RXR, RAR cannot form a homodimer and therefore remains monomeric
in the absence of RXR. The interaction between monomeric RAR and the coactivator has been measured
with dual-color time-integrated fluorescence cumulant analysis. To our surprise, we found that two RAR
bind to SRC-1 in the presence of RAR-specific agonist. This result implies that the coactivator may act as
a scaffold for assembling two RAR that potentially interact with their DNA response element. NRs bind to
the coactivator via the LXXLL motif. SRC-1 has three of these signature motifs that collectively determine
the stoichiometry of RAR upon ligand activation. In this study, we will explore the function of each SRC-1
NR interaction motif. This work is supported by the National Institutes of Health (R01 GM64589).
3146/L121
Prokaryotic Annotation: A Tool for Teaching Undergraduates about Biochemistry, Microbiology,
Molecular Biology, Evolution.
1
2
3 1
2
A. Rosenwald , R. Madupu , J. Russell ; Biology, Georgetown University, Washington, DC, Prokaryotic
3
Annotation Group, J. Craig Venter Institute, Rockville, MD, Center for New Designs in Learning and
Scholarship, Georgetown University, Washington, DC
With improvements in technology and sharp decreases in costs of genome sequencing, the number of
sequences currently available far exceeds the capacity of professional manual curators to annotate these
genomes. As bioinformatics researchers have therefore increasingly turned to so-called “community
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annotation”, it seems reasonable to include undergraduate students in the community of annotators.
Indeed, recent educational research has shown that authentic scientific research experiences in the
classroom are as valuable in terms of encouraging students to pursue careers in science as summer
research experiences. In our pilot experience, seven undergraduate students at Georgetown University
engaged in a project to examine the genome structure of a Salmonella enterica serovar using annotation
tools developed at the J. Craig Venter Institute as well as other tools available on the internet. We found
this platform to be a useful way to reinforce previously acquired knowledge from formal course-work in
biology. In addition, using the Salmonella serovar under study the students investigated several
hypotheses based on their annotations, including sensitivity to heavy metals and antibiotics. We are
currently looking for additional undergraduate faculty to join in development of curricular tools and in an
online community consisting of a network of faculty and students.
3147/L122
Development of an Inquiry-Based Cell Biology Laboratory with Emphasis on Scientific
Communication Skills.
1
2
1
1
3
2
1
T. F. Bruce , E. M. Hughes , P. A. Vigueira , K. S. Paul , S. B. Katz , D. M. Switzer , L. A. Temesvari ;
1
2
Biological Sciences, Clemson University, Clemson, SC, Eugene T. Moore School of Education,
3
Clemson University, Clemson, SC, English, Clemson University, Clemson, SC
We have developed an inquiry based senior-level undergraduate Cell Biology lab designed to emphasize
and improve critical thinking, communication and ethical reasoning. Students participated in 2 projects
during which they were required to design and conduct experiments, collect data, analyze data, and
present their findings in poster or manuscript styles. Students were also engaged in discussions of
science communication ethics. Pre- and post-tests of critical thinking skills, attitudes towards careers in
science, and attitudes towards science communication ethical standards were administered. Critical
thinking skills were initially monitored using TIPSI and TIPSII testing which assesses the ability to identify
variables, define operationally, state hypotheses, interpret graphical data, and design experiments. No
statistically significant differences were observed in percent change in TIPS test scores (pre-test vs posttest) between semesters (non-inquiry-based vs inquiry-based). However, interpreting graphical data and
designing experiments improved more dramatically in the inquiry-base course. The data suggested a
ceiling effect. Therefore, an alternative test, the CAT test, was adopted for one semester. CAT testing
indicated that students significantly improved their ability to think critically (P<0.01). To assess ethics,
students responded to an ethics statement explaining why they agreed or disagreed. Responses were
scored using a rubric to assess change in responses over time. Responses were qualitatively analyzed
using a style analysis technique. To determine changes in responses to writing in the disciplines, a
General Linear Model for repeated measures was conducted to determine change over time for 4
statements. Results indicate significant changes regarding beliefs about writing laboratory reports to
assist with understanding (a) key concepts and (b) key steps in hands-on practice for advanced cellbiology. Science-related attitudes were assessed relating to four constructs: value of goal, value of
behavior, expectancy, and instrumentality. Results indicated a statistical growth across certain constructs,
notably value of goal.
3148/L123
Bioengineering of Nanoscale DNA Baskets for Delivery of siRNA.
A. C. Zirzow, A. Baranova; Molecular and Microbiology, George Mason University, Fairfax, VA
Silencing of gene expression by small interfering RNA (siRNA) is promising for drug target discovery and
as a therapy. However, a major impediment to the use and development of this technology is the inherent
instability of siRNA in the bloodstream, partly due to susceptibility to nucleases. To address this
restriction, this research evaluates a novel, DNA/albumin-based delivery vehicle for siRNA. The role of
the delivery vehicle is to form a “basket” surrounding the siRNA, which provides both steric separation of
siRNA from nucleases and local excess of the substrate for nuclease action; thus, slowing the rate of the
degradation of siRNA. The DNA component of this vehicle is a non-viral heterologous polynucleotide
approved for use for humans as an immunomodulator. The albumin is a large protein used to stabilize
and interact with the nucleic acids while providing further spatial separation of siRNA from nucleases.
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This investigation finds that variation of the albumin concentration in basket construction can significantly
decrease the mean size distribution of the basket. Smaller siRNA-containing DNA baskets may increase
cellular uptake. Previous studies have shown definitive nanoparticle sizes that are ideal for cellular uptake
and nanoparticle uptake is greatest where a large number of proteins are associated with the particle.
Dynamic light scattering is used to quantify the mean size distribution of DNA baskets. The siRNA
designed for this study is a 21nt double stranded RNA custom ordered to include a fluorophore on one
strand and a quencher on the other strand. Degradation dependent separation of quencher and
fluorophore results in increased fluorescence intensity allowing siRNA degradation to be measured via
fluorescence spectroscopy. This investigation finds that the degradation of siRNA is delayed when siRNA
is prepared with this delivery vehicle, implying that DNA baskets are a promising technology for further
research as a siRNA delivery vehicle.
3149/L124
Targeted Cancer Cell Detection and Destruction Based on RNAi Mediated In Vivo Expression
Profiling.
L. Wroblewska, H. Chung, R. Weiss; Biological Engineering, MIT, Cambridge, MA
Modern breast cancer therapies utilize non-specific approaches to kill or remove cancerous cells, inflicting
significant collateral damage to healthy cells. In response to the need for highly targeted detection and
destruction of cancerous cells, we propose to implement an RNAi-enhanced logic circuit capable of
identifying multiple markers of breast cancer and selectively destroying only cancerous cells, leaving
other cells to grow normally. In this project we focus on the MCF-7 breast adenocarcinoma cell line, a
well-characterized cell line derived from a common form of breast cancer. MCF-7 cells overexpress
Gata3, NPY1R and TFF1 mRNA relative to healthy cells. Based on our bioinformatics analysis, taking into
account the three biomarkers allows for dramatically improved specificity in comparison to targeting single
genes. We therefore design a three-input RNAi-based AND gate that enables specific mRNA biomarker
analysis and targeted destruction of MCF-7 cells. We have designed, tested and successfully
implemented each of the necessary circuit components: very efficient siRNA and microRNA gene
knockdown, hBax dependent apoptosis, expression of short, regulatory mRNA sequences, and nonintegrating lentivirus as a potential delivery mechanism. Our current efforts are focused on
characterization of the modules as a preliminary step for final implementation and fine-tuning of the full
circuit. Ultimately, we anticipate that our RNAi-based logic circuits will be able to reliably and selectively
differentiate between cancerous and noncancerous cells in vivo, killing the cells deemed cancerous, while
producing no significant deleterious effects in healthy cells. Using this project as a foundation, our longterm goals are to create a highly selective system capable of detecting and destroying many cancer cell
types or cells in various cancer progression stages and to create a general-purpose mechanism for
sophisticated analysis of cell state.
3150/L125
Effect of Controlled Local Delivery of Simvastatin/PLGA Carrier on Bone Healing.
L. Tai; Orthopaedic Research Center, KAOHSIUNG MEDICAL UNIVERSITY, Kaohsiung, Taiwan
Statins, a hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, is known to inhibit
cholesterol biosynthesis. Currently, many studies indicated that simvastatin stimulate bone formation in
vitro by stimulate BMP-2 expression, but the in vivo studies and clinical applications are still controversial.
It is because that statins will undergo first pass metabolism by oral administered and do not likely reach
sufficient blood concentrations to reliably cause substantial increases in bone formation. In order to avoid
first pass metabolism, many repot use local delivery to stimulate bone formation. Accordingly, we
hypothesized that local administration of simvastatin carried by biomaterial with control release property
may enhance bone healing. The drug can locally releasing continued and maintain the therapeutic
concentration in tibia fracture model will improve bone healing quality. Simvastatin encapsulated PLGA
microspheres were produce by double emulsion (w/o/w) method.The bioactivity of released simvastatin
on bone mesenchymal stem cells (BMSCs) was tested by Alizarin Red S Staining. The bone healing
efficiency of simvastatin/PLGA was tested on a tibia fracture animal model (osteonucrosis defect). The
osteonucrosis defect was performed in mouse tibia by following the standard animal center protocol. We
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used Hematoxylin and eosin staining, immunochemistry to evaluate the effect of bone healing. The
results show that the the simvastatin released from PLGA microspheres were potentially enhanced the
cell mineralization on BMSCs, which confirmed the bioactivity of the released simvastatin. The H&E stains
quantification further confirmed that the simvastatin/PLGA microsphere group was significantly increased
than control group. The simvastatin/PLGA mircrospheres treated groups showed the higher number of
endothelial cell per mm2 in the graft bone in comparison with control group.We suggest that simvastatin
released from PLGA microsphere can enhance cell mineralization and local delivery of control released
simvastatin/PLGA can improve bone healing in tibia fracture.
3151/L126
Development of a New Gelatin-Fibronectin Cryogel as a Supporting Material for Stem Cells.
1
2
3
4
4,5
1
1,5
K. Kita , W. M. Cox , K. J. Smith , R. A. Cox , H. K. Hawkins , C. C. Finnerty , D. N. Herndon ;
1
Department of Surgery and Shriners Hospitals for Children, The University of Texas Medical Branch,
2
Galveston, TX, Medical Student Summer Research Program and School of Medicine, The University of
3
Texas Medical Branch, Galveston, TX, School of Medicine, The University of Texas Medical Branch,
4
Galveston, TX, Department of Pathology and Shriners Hospitals for Children, The University of Texas
5
Medical Branch, Galveston, TX, Department of Pediatrics, The University of Texas Medical Branch,
Galveston, TX
We recently reported the isolation and characterization of mesenchymal stem cells (MSCs) from human
umbilical cord (K. Kita et al. (2010) Stem Cells Dev., 19, 491-502). In order to use MSCs in regenerative
medicine, the development of appropriate supporting materials is necessary. Here we describe a biodegradable, easily prepared, and inexpensive sponge-like gel that supports the growth of human cells.
This material was developed using gelatin and fibronectin (FN) in order to take advantage of FN’s
function in promoting wound healing. Gelatin (4.3%) was mixed with a minimum amount of FN (0.03%) on
ice. Cross-linking of the matrix was achieved by adding varying concentrations of glutaraldehyde. This
mixture was then frozen at -20°C overnight. The pore size of the sponge-like structure was approximately
80-200μm. Both light and electron microscopic images indicate that higher concentrations (0.3-0.5%) of
glutaraldehyde enabled the formation of denser mesh structures. Materials cross liked with lower
concentrations of glutaraldehyde were softer than highly cross linked materials. Nuclear staining showed
that both MSCs and human foreskin fibroblasts migrated into the gels and survived for at least 5-7 days
with no apparent cytotoxicity. Although the softer gels were sensitive to trypsin digestion when gels were
incubated in a 0.005% trypsin solution, highly cross linked gels took longer to degrade (16 hours). In
addition, the gelatin-FN gels were less sensitive against trypsin digestion in comparison to gelatinfibrinogen gels. The gelatin-FN gels are stable at least for a week in cell culture, implying slow
degradation/absorption upon in vivo applications. We also prepared a sheet-like prototype of this gel for
future animal trials and clinical applications. In conclusion, we successfully developed gelatin-FN cryogels
for use with MSCs. The healthy growth of cells in the materials suggests negligible cytotoxicity by this
new material, and our material may be useful as a carrier to build up artificial tissues/organs. Supported
by Anderson Foundation for Research and the Shriners Hospitals for Children Morphology Special
Shared Facility grant.
3152/L127
Towards Patterning Complex Multiple Tissues.
1,3
7
4
2,3
5
6
6
4
D. Ker , A. Nain , L. L. Schultz , B. Chu , J. A. Phillippi , B. Gharaibeh , J. Huard , L. Weiss , P.
1,2,3 1
2
Campbell ; Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, Biomedical Engineering,
3
Carnegie Mellon University, Pittsburgh, PA, Institute for Complex Engineered Systems, Carnegie Mellon
4
5
University, Pittsburgh, PA, Robotics Institute, Carnegie Mellon University, Pittsburgh, PA, Surgery,
6
University of Pittsburgh Medical Center, Pittsburgh, PA, Stem Cell Research Centre of Children’s
7
Hospital, University of Pittsburgh Medical Center, Pittsburgh, PA, Mechanical Engineering, Virginia Tech,
Blacksburgh, VA
A fundamental knowledge of how the cellular microenvironment imparts cues to direct stem cell behavior
and function is crucial for understanding the development of complex multi-tissue units such as
orthopedic interfaces since stem cells must be directed toward multiple cell types that are spatially
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organized to achieve structure and function. Towards this goal, growth factor (GF) inkjet printing was
used to create printed patterns of GFs on 2D and 3D substrates to direct stem cell behavior. This
approach mimics some aspects of biological patterning of GFs, which normally occur during development,
homeostasis and wound healing, by providing biochemical and structural cues via the immobilization of
GFs with native ECM. Initially, immunofluorescence screening identified fibroblast growth factor-2 (FGF-2)
as being capable of inducing C3H10T1/2 mesenchymal stem cells, C2C12 myoblasts and primary
muscle-derived stem cells towards a tendon lineage through upregulation of the tendon marker Scleraxis
(Scx). Quantitative PCR studies subsequently suggested that FGF-2 directs stem cells towards a tendon
lineage via members of the Ets family of transcription factors such as pea3 and erm. Following this,
immobilized, printed patterns of FGF-2 on fibrin-coated coverslips were shown to upregulate Scx in a
dose-dependent fashion. Furthermore, printed patterns of bone morphogenetic protein-2 (BMP-2) and
FGF-2 on either fibrin-coated coverslips (2D) or a novel sub-micron fibrous scaffold (3D; STEP scaffolds)
directed C2C12 myoblasts towards osteoblast (bone) and tenocyte (tendon) fates, respectively with
spontaneous fusion of myotubes outside the printed regions. Additionally, STEP scaffolds induced the
alignment of myotubes along the fiber length. Together, these results demonstrate spatial control of cell
differentiation into 3 different cell lineages as well as control of cell alignment, simultaneously. This
approach has applications for studying the interactions between neighboring cell regions (co-culture
studies) and may translate into creating complex tissues requiring precise spatial arrangement such as
muscle-tendon-bone relevant for related orthopaedic repair.
3153/L128
A Novel Mini-Liver Model for Studying the Impact of HBV Infection or Toxic Agents on Liver
Physiology.
1
2
2
1 1
S. Rawat , D. Snyder , H. Noh , M. Bouchard ; Biochemistry and Molecular Biology, Drexel University
2
College of Medicine, Philadelphia, PA, Mechanical Engineering, Drexel University, Philadelphia, PA
The liver is responsible for regulating many metabolic processes and detoxifying chemicals. It is also a
target of viral infections such as those with the hepatitis B virus (HBV). Chronic HBV infections are
associated with the development of liver cancer. Studies involving HBV infections and the consequential
effects on hepatocytes are currently conducted in established cell lines or cultured primary hepatocytes.
Unfortunately, many cell lines do not mimic authentic hepatocytes, and its difficult to maintain
differentiated primary hepatocytes in traditional cell culture systems. We are developing a novel liver
model that recreates the microarchitecture of a basic functional unit of the liver, the liver sinusoidal
functional unit. This model will facilitate studying the impact of HBV infections or other toxic agents on
hepatocyte physiology. A liver sinusoidal functional unit is a specialized capillary lined with liver sinusoid
endothelial cells, Kuppfer cells, and stellate cells; hepatocytes line the outer edge of this unit. We have
fabricated microchannels that approximate the dimensions of a liver sinusoid. Here we show that primary
rat and human hepatocytes can be cultured in these microchannels and remain differentiated. The
hepatocytes can be infected with recombinant adenoviruses, allowing expression of transduced genes.
We can also perform RT-PCR and immuno-fluoroscence analysis in microchannels. In addition, even
though the number of cells is small, we can detect endogenous proteins by western analysis. In future
studies, we will expand this system to include other resident liver cells and establish a dynamic flow
system to mimic fluid circulation in the sinusoid.
3154/L129
Characterizaion of GAF-Only Domains from Cyanobacteriochromes of Thermosynechococcus
elongatus BP-1.
1
1
1
1
1
1
1
D. B. Salantes , R. F. Meza-Acevedo , M. Schwartz , K. Beck , S. Kwong , C. Kwong , S. Zarifis , C.
1
1
1
1
1
1
1
1
Lopez , A. Breen , L. M. Dunn , S. Dwojak , N. Szoka , A. Yeaton-Massey , B. Vaccaro , J. Darnton , L.
1
1
1
2
2
1 1
Roberts , E. Charvonneau , P. Delgado , N. Rockwell , J. C. Lagarias , S. C. Spiller ; Biology, Mills
2
College, Oakland, CA, CBST, UC Davis, Sacramento, CA
Phytochromes are widely occurring photoreceptors that mainly respond to red/far-red light in the visible
spectrum. The phytochrome contains a linear tetrapyrrole (bilin) chromophore which is covalently bound
within a knotted PAS-GAF domain pair. A mutation of the GAF domain results in a change in the binding
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pocket, restructuring the space where the chromophore is nested. Inhibition of chromophore movement
within the binding pocket causes the emission of fluorescent energy. Thermophilic cyanobacteria such as
Thermosynechococcus elongatus BP-1 contain more distant relatives of phytochromes that lack the PASGAF knot. These cyanobacteriochromes function as blue/green switchable photoreceptors. In this study
we characterized isolated GAF domains from class II cyanobacteriochromes of T. elongatus, tlr0924,
tll0569, tll0899. The project explores form and function of the GAF only domain of these proteins with and
without a mutation, which modifies cyanobacteriochromes into a short, bright red fluorescent biliprotein.
These proteins are: 179-168,174,and 152 amino acids long. These truncated cyanobacteriochromes are
much shorter than previously characterized Infrared-Fluorescent Proteins (IFPs). When our proteins are
expressed in E.coli, they yield yellow, blue, or green colored pellets as a representation of the successful
expression of the gene, and binding to the PCB chromophore. We have been able to successfully
express all of these proteins. Our ultimate goal is to purify and characterize them. In addition, our plan is
engineer a plasmid that can transfect them into mammalian cells using DsRed-Monomer-Actin vector as a
model. Transfected lymphocyte cells expressing the red-tagged actin cytoskeleton will be imaged during
infection with GFP-labeled HIV in collaboration with partners at the Center for Biophotonics Science and
Technology (CBST-UC Davis.)
3155/L130
Three-Dimensional Measurement of Bacterial Colony Growth on Semi-Solid Agar Substrates.
1,2
2 1
J. K. Kim , E. J. Park ; School of Mechanical Engineering, Kookmin University, Seoul, Republic of
2
Korea, Department of Mechanical Engineering, Graduate School, Kookmin University, Seoul, Republic of
Korea
Bacteria have responses to substrates by detecting environmental signal and lead changes in physiology
and morphology, which can affect their proliferations and growths. We conducted a systematic study to
decipher the interplays among bacteria motility, nutrient and concentration of agar when bacteria colonize
on semi-solid substrate. We observed the growth kinetics of three kinds of bacteria, swarming Escherichia
coli (E.coli), non-motile E.coli, and gliding Myxococcus Xanthus (M.xanthus), grown on semi-solid agar
substrates with different amount of agar and nutrients. At the beginning of the colony growth, the changes
in viable cell numbers of both motile E.coli and M.xanthus followed the same trend that the numbers are
higher on the substrates of lower agar concentration. In course of time, we observed that motile E.coli
changed the tendency of colony growth, while non-motile E.coli kept the cell numbers independent of
agar concentration. This indicates that agar concentration of substrates could affect bacterial mobility
because non-motile E.coli would not react to agar concentration. After passing a threshold of time, motile
E.coli showed an opposed growth inclination depending on agar concentration. Especially, when highdensity bacterial solution was inoculated on the agar substrate, the threshold time moved up, meaning
that the power of influence by agar concentration increased after quorum sensing started in our system.
In conclusion, the colony of motile bacteria showed more sensitive response to the agar concentration of
gel than that of non-motile ones. And bacteria having different modes of motility had different growth
patterns controlled by agar concentration of gel. Generally, the rate of bacterial proliferation on agar
substrates was proportional to the concentration of nutrients. The collective behavior of bacteria cultured
on the agar substrate is affected by both biochemical and biophysical properties of their
microenvironment. Although other kinds of bacterial strains should be studied further to verify our results,
we believe that our findings can be applied to designing surfaces for preventing bacterial adhesion and
growth.
3156/L131
Reversible Cell Labeling Exploiting Fluorescent Aptamer and Nuclease Digestion.
1
1
1
1,2 1
H. Terazono , M. Hayashi , H. Kim , K. Yasuda ; Yasuda"On-chip Molecular Cell" Project, Kanagawa
2
Academy of Science and Technology, Kawasaki, Japan, Department of Biomedical Information, Institute
of Biomaterials and Bioengineering, Chiyoda-ku, Japan
1. Objective Separation of uniform phenotype cells whilst maintaining their native states is important both
for post genome cell based studies and practical applications of those cells for regenerative medicine. A
number of cell separation methods have been reported to date, such as density-gradient centrifugation,
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antibiotic screening and fluorescence-activated cell sorting (FACS). The latter gained significant
popularity because of its simplicity and throughput. One of the problems of the FACS and other antibodybased labelling and separation techniques is that strong, often poorly reversible conjugation between
antibody and antigen molecules on cell surface may affect the function of the targeted cells and their
interaction efficacy with other cells. As an alternative to antibodies, we developed nucleotide based
probes capable of binding target cell antigens. Aptamers are single-strand DNAs (ssDNA), RNAs or
modified nucleic acids. The development of ssDNA aptamers for specific binding to live cells (CellSELEX) expanded their potential for use in cell separation and labelling. Here we describe a method for
reversible labelling of the cells with fluorescently tagged aptamers. 2. Methods Our method uses high
affinity single strand ssDNA aptamers against surface exposed target molecules on cells. The aptamers
are removed from the cell surface using DNase nuclease. Binding of the fluorescent-aptamers to the cells
was evaluated by measuring fluorescence intensity and was further confirmed using flow cytometry. 3.
Results The target-specific aptamer was attached to the cells, and the removal of Qdot-aptamer by
DNase nuclease treatment allowed recovery of the initial distribution of intact cells. Non-specific negative
control DNA aptamer showed lack of interaction and absence of any noticeable effects on the treated
cells. The growth rates of treated cells were almost the same as that of the non-treated intact cells. 4.
Discussion Our labelling procedure produces no noticeable damage to the labelled cells. Our method is
advantageous over traditional antibody labelling because it allows non-invasive cell separation and
reversible cell labelling.