Table Of Content

Abstracts The American Society for Cell Biology th 45 Annual Meeting December 10-14, 2005 The Moscone Center San Francisco CONTENTS SATURDAY Coordination of Cytoskeletal Networks.....................................207 Nuclear Compartments ..............................................................208 Big Science, Little Science........................................................ 4 Pathogens Co-opting Host Cell Functions.................................210 Regulating Intercellular Junctions .............................................211 Signaling in 3D Environments...................................................213 SUNDAY Trafficking Proteins & Complexes ............................................215 Quantitative Studies of Cell Signaling Networks...................... 5 Signal Transduction II ...............................................................216 Bruce Alberts Award Presentation ............................................ 5 Apoptosis I.................................................................................224 Prokaryotic Origins of the Cytoskeleton ................................... 5 Mitosis & Meiosis II..................................................................231 E. E. Just Lecture....................................................................... 6 G2-M .........................................................................................237 Cytokinesis I ..............................................................................240 Cargo Sorting & Vesicular Transport........................................ 7 Cell Biology of the Synapses..................................................... 8 Actin-Associated Proteins II......................................................245 Actin Dynamics & Assembly I ..................................................250 Differentiation & Cancer........................................................... 10 Extracellular Matrix & Signaling .............................................. 11 Conventional Myosin.................................................................255 Formins & Arp2/3: Regulators of Actin.................................... 13 Tubulin ......................................................................................260 Dynein II....................................................................................262 Nuclear Envelope Functions...................................................... 15 Regulation of the Cell Cycle ..................................................... 16 Microtubule Dynamics & Assembly I .......................................269 Signaling in the Immune System............................................... 18 Cilia & Flagella I .......................................................................275 Cell Motility II ...........................................................................282 EB Wilson Medal Presentation & Lecture ................................ 20 Growth Factors & Receptors ..................................................... 20 Cytoskeletal Organization I .......................................................290 Signal Transduction I ................................................................ 26 Cytoskeleton-Membrane Interactions I......................................296 Cell Attachment to the Extracellular Matrix..............................300 Mitosis & Meiosis I................................................................... 32 G1-S/DNA Replication ............................................................. 39 Extracellular Matrix & Cell Signaling II ...................................306 Actin.......................................................................................... 42 Cell-Cell Interactions II .............................................................312 Organization and Regulation of the Extracellular Matrix ..........316 Actin-Associated Proteins I....................................................... 44 Unconventional Myosins........................................................... 50 Membrane Receptors.................................................................318 Dynein I..................................................................................... 57 ER to Golgi Transport ...............................................................326 Microtubule-Associated Proteins............................................... 60 Endocytosis II ............................................................................330 Cell Motility I............................................................................ 67 Protein Targeting to the Cell Surface.........................................335 Centrosomes I............................................................................ 75 Protein Folding & Assembly in the Endoplasmic Reticulum I ..............................................................................339 Intracellular Movement............................................................. 79 Intermediate Filaments I............................................................ 85 Mechanisms of Nuclear Transcription .......................................342 Chromatin & Chromosomes I....................................................347 Focal Adhesions........................................................................ 91 Cell-Cell Adherens Junctions.................................................... 96 Nuclear Import and Export Signals............................................352 Extracellular Matrix & Cell Signaling I .................................... 100 Organogenesis ...........................................................................355 Cell Polarity II ...........................................................................358 Cell-Cell Interactions I .............................................................. 106 Membrane Channels & Transporters I....................................... 110 Development & Carcinogenesis ................................................362 Membrane Fusion...................................................................... 117 Synapse Formation & Function I...............................................364 Golgi Complex ..........................................................................368 Endocytosis I............................................................................. 119 Protein Targeting....................................................................... 124 Epithelia II .................................................................................375 Gene Structure and Expression.................................................. 132 Cancer II ....................................................................................379 Metabolic Diseases ....................................................................383 Chromatin Remodeling ............................................................. 137 Nuclear Matrix and Nuclear Architecture ................................. 139 Other Diseases I.........................................................................391 Germ Cells & Fertilization ........................................................ 142 Cell Polarity I ............................................................................ 148 TUESDAY Neurotransmitters, Peptides & Receptors.................................. 152 Chloroplasts & Mitochondria .................................................... 157 Reprogramming Cell Fate..........................................................399 Endoplasmic Reticulum............................................................. 163 Community Building to Promote Careers in Biomedical Epithelia I .................................................................................. 169 Science.....................................................................................399 Parasitology............................................................................... 173 Host-Pathogen Interactions........................................................400 Cancer I ..................................................................................... 180 Building Sensory Networks .......................................................401 Imaging Technology.................................................................. 186 Coordinating Adhesion & Signaling..........................................402 Molecular Biology..................................................................... 193 Cytoskeletal Molecular Motors..................................................404 Blood Vessels............................................................................ 198 Intermediate Filaments ..............................................................405 Intersection of Signaling & Trafficking: Small GTPases ..........407 Mitosis & Meiosis .....................................................................409 MONDAY Organelle Dynamics ..................................................................410 Wiring the Nervous System....................................................... 202 Protein Misfolding & Disease....................................................412 Keith R. Porter Lecture..............................................................413 The Nature of Life: An Orientation Course that Introduces Freshmen to the Disciplines of Biology, Builds Community, Oncogenes & Tumor Suppressors .............................................414 and Teaches Strategies for Success in College ........................ 202 Cell Cycle Controls I .................................................................419 Apoptosis II ...............................................................................425 Adapting to Stress: Spotlight on Organelles.............................. 203 Cell Migration/Motility............................................................. 203 Mitosis & Meiosis III.................................................................431 Chromatin Dynamics................................................................. 205 Kinetochores ..............................................................................440 CONTENTS Pre-College and College Science Education.............................. 447 Caveolae ....................................................................................718 Actin-Associated Proteins III .................................................... 455 Trafficking in Polarized Cells....................................................721 Actin Dynamics & Assembly II ................................................ 461 Developmental Control of Gene Expression..............................726 Muscle: Biochemistry & Cell Biology I.................................... 466 Ribonucleoproteins ....................................................................728 Kinesin I.................................................................................... 470 Structure of Nuclear Envelope...................................................732 Microtubule Dynamics & Assembly II...................................... 474 Invertebrate Development..........................................................737 Cell Motility III ......................................................................... 479 Signal Transduction in Development.........................................740 Cytoskeleton-Membrane Interactions II .................................... 486 Stem Cells II ..............................................................................747 Extracellular Matrix & Cell Behavior I ..................................... 490 Endosomes & Lysosomes II ......................................................753 Extracellular Matrix & Morphogenesis..................................... 494 Leukocytes.................................................................................757 Cadherins................................................................................... 499 Cell Culture ...............................................................................760 Gap Junctions............................................................................ 504 Cancer IV...................................................................................765 Structure & Function of Membrane Proteins I .......................... 511 Neuronal Diseases II..................................................................771 Golgi to Cell Surface Transport................................................. 517 Other Diseases III ......................................................................778 Endocytic Machinery: Structure, Function & Regulation.......... 522 Bioinformatics/Biological Computing .......................................785 Protein Targeting to the Endocytic Pathway ............................. 529 RNAi Technology......................................................................787 Protein Folding & Assembly in the Endoplasmic Reticulum II............................................................................. 534 Tissue-Specific Gene Expression .............................................. 538 Chromatin & Chromosomes II .................................................. 543 Mammalian Development ......................................................... 548 Growth Factors in Development................................................ 555 Stem Cells I............................................................................... 558 Synapse Formation & Function II ............................................. 564 Endosomes & Lysosomes I ....................................................... 568 Endothelial Cells ....................................................................... 571 Cancer III .................................................................................. 575 Neuronal Diseases I................................................................... 581 Other Diseases II ....................................................................... 588 WEDNESDAY Cell Growth and Division.......................................................... 594 Yeast Genomics in the Classroom: Using the Yeast Deletion Collection to Study Environmental Toxins and Food Additives ................................................................................. 594 Cytoskeletal Dynamics in Living Cells ..................................... 595 Epithelial Morphogenesis & Polarity ........................................ 596 Lipid-Mediated Signals ............................................................. 598 The Membrane Cytoskeleton..................................................... 599 Neuronal Polarity & Axo-Dendritic Growth ............................. 601 Protein Folding & Quality Control............................................ 603 RNA Silencing Mechanisms ..................................................... 604 Stem Cell Niches ....................................................................... 606 Signal Transduction III.............................................................. 607 Cell Cycle Controls II................................................................ 614 Mitosis & Meiosis IV ................................................................ 621 Cytokinesis II ............................................................................ 629 Actin Dynamics & Assembly III............................................... 633 Muscle: Biochemistry & Cell Biology II................................... 638 Kinesin II................................................................................... 643 Cilia & Flagella II...................................................................... 648 Cell Motility IV......................................................................... 655 Cytoskeletal Organization II...................................................... 662 Centrosomes II .......................................................................... 669 Nerve Cell Cytoskeleton............................................................ 673 Intermediate Filaments II .......................................................... 679 Extracellular Matrix & Cell Behavior II.................................... 684 Integrins..................................................................................... 689 Metalloproteases........................................................................ 695 Tight Junctions .......................................................................... 699 Structure & Function of Membrane Proteins II ......................... 705 Membrane Domains .................................................................. 708 Exocytosis: Regulated Secretion............................................... 713 Saturday Big Science, Little Science (1-2) 1 Unraveling Smell L. Buck; Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, WA Odorants are detected by ~1000 different odorant receptors (ORs), which are located on olfactory sensory neurons in the nose. Our studies show that ORs are used combinatorially to detect different odorants and encode their unique identities. To explore how the nervous system translates odorous chemicals into perceptions, we asked how inputs derived from different mouse ORs are organized as signals travel from the nose to the olfactory bulb and then the olfactory cortex. In the nose, each sensory neuron expresses a single OR gene. Neurons with the same OR are dispersed in the nose, but their axons converge in a few glomeruli at two fixed locations in the bulb. The result is a stereotyped sensory map in which inputs from different ORs are segregated in different glomeruli and relay neurons. In the olfactory cortex, inputs from one OR are targeted to clusters of neurons at specific sites, creating a stereotyped map unrelated to that in the bulb. In contrast to the segregation of different OR inputs seen in both the nose and bulb, it appears that different OR inputs overlap extensively in the cortex and single neurons receive combinatorial inputs from multiple different ORs. Using c-Fos as an indicator of neuronal activator, we found that different odorants elicit different, but partially overlapping, activation patterns in the cortex. The representation of each odorant is composed of a small subset of sparsely distributed neurons. Quantitative analysis of the odor representations suggests that cortical neurons may function as coincidence detectors that are activated only by correlated inputs from different ORs. 2 Discovery-Driven Research: A New Frontier in the Biological Sciences C. Fraser; The Institute for Genomic Research, Rockville, MD The application of large-scale approaches to the study of biological questions has produced a fundamental change in the way that we approach scientific discovery. In this new era, high-throughput technologies are providing enormous amounts of new data and computational biology is allowing us to make links between genome sequence and biological processes and function. The ultimate goal of such big science is to achieve a predictive understanding of biology. 4a Sunday Quantitative Studies of Cell Signaling Networks (3-4) 3 The Deeper Correlations: Single Cell Measures of Kinase Signaling for Mechanistic and Clinical Analyses G. Nolan; Microbiology/Immunology, Stanford University, Stanford, CA Intracellular assays of signaling systems has been limited by an inability to correlate functional subsets of cells in complex populations based on active kinase states or other nodal signaling junctions. Such correlations could be important to distinguish changes in signaling status that arise in rare cell subsets during functional activation or in disease manifestation. Simultaneous detection of activated kinases and phosphoproteins in simultaneous pathways in subpopulations of complex cell populations by multi-parameter flow cytometric analysis allows identification of signaling cascades for disease states by ordering of kinase activation and phosphoprotein status in signaling hierarchies. Importantly, we demonstrate that ordering of these activations requires multiple interrogations of cells, and that the networks discovered are reflective of deeper correlations. Using Bayesian Network analysis (a form of machine learning) one can infer pathway connectivity in an automated fashion, allowing for high throughput derivations of signaling system networks graphs in PRIMARY CELLS. The approach has powerful applications in mechanistic understanding, drug screening, and patient stratification for prediction of disease outcome in cancer, autoimmunity, infection, based on signaling network status. (1) Irish J.M., Hovland R., Krutzik P.O., Perez O.D., Bruserud O., Gjertsen B.T., Nolan G.P. (2004) Single Cell Profiling of Potentiated Phospho-Protein Networks in Cancer Cells. Cell. 118:217-228. (2) Sachs K., Perez O., Pe'er D., Lauffenburger D.A and Nolan G.P. 2005. Causal protein-signaling networks derived from multiparameter single-cell data. Science. 308:523-9. 4 Systems Biology of Cytokine Signaling in Human Cells P. Sorger, D. Lauffenburger, K. Janes, S. Gaudet, J. Albeck, B. Schoeberl; Department of Biology and Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA Cytokines and their receptors activate complex signaling cascades to regulate cell proliferation, death and differentiation. We seek to develop quantitative, mechanistic models that describe cytokine-induced signaling with an eye to understanding cell-type variation and the differences between healthy and diseased states. We focus on decisions controlled by pro-apoptotic cytokines such as Tumor Necrosis Factor (TNF) and TRAIL and pro-survival cytokines such as EGF and the insulin-like growth factors (IGF). By mining a compendium comprising ~10,000 measurements of signal protein activities elicited by cytokines individually and in combination we can construct both statistical and physicochemical models. Classifier-based regression has been particularly helpful in establishing that cells respond to TNF directly, via activated TNF receptor, and indirectly via autocrine circuits involving transforming growth factor alpha (TGF- α), interleukin-1α(IL-1α) and IL-1 receptor antagonist (IL1-ra). These cytokines participate in a three-part autocrine loop that plays out over at least 24 hr and adds sequential layers of pro and anti-apoptotic signaling that sets cell death at a self-limiting level. Experimental work to date has been in human tumor cells. However, it seems highly likely that TNF-triggered autocrine cascades will differ from one cell type to the next. We are currently attempting to compare diseased and normal primary cells from breast and connective tissue. One interesting initial finding is that the logic of the TNF-TGF-IL-1α- IL-1ra cascade can be re-wired in some cell types by inflammatory cytokines such as interferons. There is much interest in the role of intracellular crosstalk among signaling circuits. We propose that that time-dependent crosstalk among synergistic and antagonistic autrocrine circuits may equally important. Morevoer, it should be easier to modulate the activity of autocrine than intracellular loops thanks to the increasing range of protein-based therapeutics available to target cytokines and their receptors. Bruce Alberts Award Presentation (5) 5 Columbia University’s Summer Research Program for Secondary School Science Teachers S. C. Silverstein; Dept Physiology/Cell Biophys, Columbia Univ Coll Phys & Surg, New York, NY Concerns about the quality of secondary science education stimulated me in 1990 to found Columbia’s Summer Research Program (www.scienceteacherprogram.org). The program’s purpose is to increase student interest and achievement in science by improving the quality of science instruction. To this end, Columbia’s program provides secondary school science teachers with paid fellowships that support their participation in life and physical science research laboratories for two consecutive summers under the guidance of Columbia faculty. To date, 202 teachers have participated in the program. At ASCB’s 2004 Education Forum, I reported that 8.4% more students in classes of participating teachers pass a NY State Regents exam in science than students studying the same subject in classes of non-participating teachers in the same school. This is objective evidence that teacher participation in Columbia’s program has a significant positive impact on student achievement in science. The present value of this increase in Regents science exam pass rate is $11,782 per teacher in school costs saved annually, and $32,885 per teacher in additional tax revenues generated annually, yielding total annual economic benefits of each teacher’s participation in Columbia’s program that are 3.4-fold greater than the program’s annual cost per teacher. Policy implications of these findings: A national investment of $75 million annually could support similar programs at 250 U.S. medical schools and research universities, while returning over $220 million annually in school costs saved and tax revenues generated. If each of these 250 programs enrolled 10 new science teachers annually, over 10 years they could provide science work experiences for 25,000 science teachers, approximately half the current membership of the National Science Teachers Association. Prokaryotic Origins of the Cytoskeleton (6-8) 6 Bacterial tubulin homolog FtsZ H. P. Erickson; Department of Cell Biology, Duke University Medical Center, Durham, NC 5a Sunday FtsZ is the major cytoskeletal protein in bacterial cytokinesis. When viewed by light microscopy it appears as a “Z ring” in the center of the cell. The Z ring constricts to divide the cell, disassembles during the constriction and then reassembles in the daughter cells. The substructure of the Z ring has not been visualized by EM, but we have turned to in vitro studies to deduce it. FtsZ assembles into short, single-stranded protofilaments (pfs), which are structural homologs of the tubulin protofilaments that make the microtubule wall. We have developed fluorescence techniques to study the kinetics of initial assembly and subunit turnover at steady state. FtsZ assembly is cooperative, showing a weak dimer nucleus and a critical concentration. (It is an enigma how a single-stranded pf can assemble cooperatively.) The assembly is very dynamic - pfs are turning over with a half time of 8 sec at steady state. The subunit turnover is regulated by GTP hydrolysis, and may involve a mechanism like microtubule dynamic instability. We believe that the Z ring in vivo is constructed from these dynamic pfs. This must involve a lateral association (the mechanism for this is unknown) and attachment to the membrane. FtsZ is tethered to the membrane by FtsA, a bacterial actin homolog. We used FRAP to determine the assembly dynamics of the Z ring in vivo. It is turning over with a half time of 8 sec, just as we determined in vitro. This is the most rapid cytoskeletal dynamics known. An important question is what generates the force of constriction? We have found in vitro that FtsZ can form curved pfs, which are equivalent to tubulin rings. The straight-to-curved pf conformational change is powered by GTP hydrolysis, and may generate the force for constriction. 7 Dynamics of a DNA-Segregating Cytoskeletal System in Prokaryotes D. Mullins, C. Campbell, E. Garner; Cellular & Molecular Pharmacology, University of California, San Francisco, San Francisco, CA The mechanisms that provide force to segregate bacterial chromosomes are still mysterious. We do, however, understand one important example of bacterial DNA segregation in molecular detail - segregation of the R1 and R100 drug-resistance plasmids. These large (100kb), low-copy plasmids encode genes for antibiotic and heavy-metal resistance and have been isolated from many pathogens. To ensure inheritance by both daughter cells during division, the R1 par operon constructs a simple DNA-segregating machine from three components. One of these components, ParM, is related to eukaryotic actins and assembly of ParM into actin-like filaments appears to drive plasmid segregation directly. We find that this simple prokaryotic cytoskeleton exhibits a remarkable collection of activities usually associated with eukaryotic cytoskeletons, including: dynamic instability, processive capping, insertional polymerization, and the ability to generate force. We also find that R1 plasmid segregation is a dynamic process in which assembly of unstable ParM filaments induces plasmids to oscillate rapidly from pole to pole of the cell producing a dynamic rather than static bipolar distribution. Our results indicate that the assembly dynamics of prokaryotic cytoskeletal systems are important to their cellular function and that the prokaryotic systems also make use of mechanisms similar to those of the eukaryotic cytoskeleton to establish long- range order and to move intracellular cargo. 8 The Bacterial Cytoskeleton and Cell Shape 1 2 1 1 1 C. Jacobs-Wagner, N. Ausmees, G. Charbon, M. Cabeen ; Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT, 2 Uppsala University, Uppsala, Sweden Similarly to eukaryotic cells, prokaryotic cells come in a variety of shapes. In eukaryotes, the cytoskeleton, which is made of microtubules, microfilaments and intermediate filaments, constitutes an internal framework that is essential for the maintenance of cell shape. For decades, it was thought that the external cell wall was the sole determinant of cell shape in bacteria. It is now apparent that bacteria also possess an actin-like cytoskeleton made of MreB and that this cytoskeleton is involved in determining rod cell morphology. Our laboratory has recently discovered a prokaryotic counterpart of intermediate filament (IF) proteins, termed crescentin. Crescentin is a fibrous protein with a tripartite domain architecture similar to that of metazoan IF proteins. Purified crescentin self-assembles spontaneously into ~10 nm wide filaments in vitro without exogenous energy sources, which is a distinct biochemical property of IF proteins. The function of crescentin is required for the characteristic crescent shape of Caulobacter crescentus as cells lacking crescentin lose their curvature and adopt a straight-rod cell morphology. Consistent with its role in cell curvature, crescentin forms a filamentous structure along the inner cell curvature of wild-type cells. Localization and proper organization of the crescentin cytoskeleton is dependent on the bacterial homolog of actin, MreB, indicating that MreB also plays an active role in cell curvature. E. E. Just Lecture (9) 9 Still Waters Run Deep: Investigations into the Quiescent State in Yeast M. Werner-Washburne; Department of Biology, University of New Mexico, Albuquerque, NM My laboratory has studied entrance into, survival during, and exit from stationary phase in yeast for the past 17 years. Previously, yeast and other microbes were thought to lack a true G0 phase, because budded cells were always present in stationary-phase cultures. We have recently isolated two distinct cell populations from yeast stationary-phase cultures that contain very different cell types. We have concluded from our analysis that one fraction contains quiescent (G0) cells and the other non-quiescent cells. The quiescent cells are generally the last cells formed after yeast cells exhaust glucose (the diauxic shift). They are refractile by phase contrast microscopy, unbudded, thermotolerant, and synchronous during exit from quiescence. Strangely, in the quiescent cells only nuclei and vacuoles are visible by EM. Non-quiescent cells contain many more membrane-bound organelles, including ER, Golgi, and mitochondria and lipid bodies and lack glycogen. Both cell types are metabolically active, but non-quiescent cells show a reduced ability to form colonies. We are continuing to characterize these cell types and their formation. The identification and characterization of these cell types provides the basis for ongoing, novel analyses of the quiescent state, asymmetric cell division, and the processes of cell differentiation and aging in yeast. 6a Sunday Cargo Sorting & Vesicular Transport (10-15) 10 An Intramolecular t-SNARE Complex Functions in vivo without the Syntaxin N-terminal Regulatory Domain J. S. Van Komen, X. Bai, B. L. Scott, J. A. McNew; Biochemistry and Cell Biology, Rice University, Houston, TX Membrane fusion in the secretory pathway is mediated by SNAREs (located on the vesicle membrane (v-SNARE) and the target membrane (t- SNARE). In all cases examined, t-SNARE function is provided as a three-helix bundle complex containing three ~70 amino acid ‘SNARE-motifs’. One SNARE motif is provided by a syntaxin family member (the t-SNARE heavy chain) and the other two helices are contributed by additional t- SNARE light chains. The syntaxin family is the most conformationally dynamic group of SNAREs and appears to be the major focus of SNARE regulation. An N-terminal region of plasma membrane syntaxins has been assigned as a negative regulatory element in vitro. This region is absolutely required for syntaxin function in vivo. We now show that the required function of the N-terminal regulatory domain of the yeast plasma membrane syntaxin, Sso1p, can be circumvented when t-SNARE complex formation is made intramolecular. Our results suggest the N-terminal regulatory domain is required for efficient t-SNARE complex formation and does not recruit necessary scaffolding factors. 11 Arf1p, Chs5p, and the ChAPs are Required for Export of Specialized Cargo from the Golgi M. Trautwein, C. Schindler, R. Gauss, A. Spang; Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Tübingen, Germany In Saccharomyces cerevisiae, the synthesis of chitin is temporally and spatially regulated through the transport of Chs3p (Chitin synthase III) to the plasma membrane in the bud neck region. Traffic of Chs3p from the TGN/early endosome to the plasma membrane requires the function of Chs5p and Chs6p. Chs6p belongs to a family of four proteins that we have named ChAPs for Chs5p-Arf1p-binding Proteins. This novel protein family is conserved throughout fungi and seemed to have arisen by three gene duplication events. We show that all ChAPs physically interact not only with Chs5p but also with the small GTPase Arf1p. A short sequence at the C-terminus of the ChAPs is required for protein function and the ability to bind to Chs5p. Disruption of two members, Δbud7 and Δbch1, phenocopies a Δchs6 or Δchs5 deletion with respect to Chs3p transport. Moreover, the ChAPs interact with each other and form higher molecular weight complexes. In addition, they are all at least partially localized to the TGN in a Chs5p-dependent manner. Most importantly, the ChAPs interact physically with Chs3p. We propose that the ChAPs facilitate export of cargo out of the Golgi. 12 The Role of ARF4 and ARF-GAPs in Rhodopsin Trafficking 1 1 1 2 1 D. Deretic, L. Astuto-Gribble, N. Ransom, P. A. Randazzo ; Surgery/Ophthalmology, University of New Mexico, Albuquerque, NM, 2 Laboratory of Cellular Oncology, National Cancer Institute, Bethesda, MD The small GTP-binding protein ARF4, a class II ARF, specifically recognizes and binds to the VXPX-COOH sorting motif of the light receptor rhodopsin. Using a retinal cell- free system, which reconstitutes rhodopsin trafficking in vitro, we have established that the rhodopsin-ARF4 interaction regulates the budding from the trans-Golgi network (TGN) and the incorporation of rhodopsin into the transport carriers (RTCs). RTCs are targeted to the rod outer segment (ROS), a highly specialized subcellular domain of retinal photoreceptors. To test if rhodopsin controls the ARF4-GTPase reaction by regulating the access of an ARF-GAP to ARF4, in this study we sought to identify the ARF-GAP that interacts with ARF4 in retinal photoreceptors. ARF-GAPs containing ankyrin repeats and plekstrin homology domains (AZAPs) were particularly attractive candidates given their known roles in the regulation of membrane trafficking and actin cytoskeleton. By western blotting with specific antibodies, we determined that photoreceptors express high levels of ASAP1, an AZAP that also contains the Src homology domain 3 (SH3), and has a preference for ARF1, a class I ARF, and ARF5, a class II ARF. ARAP1, an AZAP that contains a rho-GAP domain, was detected at lower levels. By confocal microscopy ASAP1 was localized to punctate structures distributed along the photoreceptor microfilaments and in the vicinity of the Golgi/TGN. ARAP1 was particularly concentrated at the outer limiting membrane where the actin cables are anchored. We are currently testing the GAP activity of ASAP1 on ARF4. Since the BAR domain of ASAP1 participates in membrane bending and fission, we are exploring if ARF4- dependent recruitment of ASAP1 to the sites of RTC budding may be the underlying mechanism for the regulation of rhodopsin trafficking by ARF4. Supported by NIH grant EY 12421. 13 Functional Involvement of Annexin II in cAMP-Induced AQP2 Exocytosis in Renal Cells G. Tamma, A. Strafino, F. Addabbo, M. Svelto, G. Valenti; Department of General and Environmental Physiology, University of Bari, Bari, Italy The membrane associated protein annexin II is known to be required for the apical transport in epithelial cells. In this study we investigated the involvement of annexin II in cAMP-induced AQP2 translocation to the apical membrane. RT-PCR performed using degenerated primers revealed the presence of annexin II mRNA transcript in AQP2 expressing renal CD8 cells. Interestingly, annexin II was found in AQP2-containing vesicles immunoisolated from CD8 cells. Consistent with this observation, cell fractionation followed by Western blotting analysis showed that stimulation of CD8 cells with the cAMP-elevating agent forskolin, caused a significant increase of annexin II in the particulate fraction paralleled with a decrease in the soluble fraction. To investigate the functional involvement of annexin II in AQP2 exocytosis the fusion process between highly purified AQP2 bearing vesicles and plasma membrane was in vitro reconstructed and monitored by a fluorescence assay based on the dequenching of the lipophilic fluorescent probe octadecylrhodamine B-chloride (R18). We designed a peptide reproducing the N-terminal 14 aminoacids of annexin II and including binding site of the the calcium binding protein p11, a protein required for the formation of a complex with annexin necessary for tightly anchoring the protein to the cortical cytoskeleton. A control peptide having 1000 times reduced affinity for p11 was used as a control. We found that preincubation of cellular fractions with annexin II peptide strongly inhibited the fusion induced by the addition of cytosol. In contrast, the control peptide had no effect on fusion. Together these data demonstrate the association of annexin II with AQP2 containing vesicles and point to a possible functional involvement of annexin II in cAMP-induced AQP2 exocytosis in renal cells. 7a Sunday 14 Rab Coupling Protein (RCP) Regulates the Sorting of Membrane Proteins in Recycling Endosomes 1 2 2 2 1 1 J. J. Burden, E. Schonteich, G. Wilson, R. Prekeris, C. R. Hopkins ; Department of Biological Sciences, Imperial College, London, United 2 Kingdom, University of Colorado Health Sciences Center, Aurora, CO Rab coupling protein (RCP) is a class I member of the Rab11 family of interacting proteins (FIPs), a family characterised by their ability to bind Rab11 via a Rab11/25 binding domain (RBD), and to bind phospholipids via a C2 domain. Through its association with Rab11, RCP has been proposed to play a role in protein recycling. Whilst many proteins, like the transferrin receptor (TfR), have been extensively characterised and shown to faithfully follow this recycling pathway, little is known about the molecular machinery involved in regulating this trafficking route. Using RNA interference and a combination of biochemical techniques and electron microscopy, we have investigated the role that RCP plays in the intracellular trafficking of recycling proteins. By immuno-electron microscopy we have localised RCP to a tubular-vesicular compartment, that can be loaded with endocytosed transferrin-HRP. Depletion of RCP in Hela cells, was found to significantly reduce the amount of internalised transferrin in comparison to control cells, whilst the rate of internalisation of the TfR was unaffected. The reduction in transferrin uptake was accompanied by a reduction in the levels of TfR, suggesting mis-sorting of the TfR away from the recycling pathway towards the degradation pathway. To investigate this further, we used RNA interference to deplete cells of another class I FIP member, Rip11, and found that TfR recycling was not inhibited (see Wilson et al. poster). Interestingly, the co-down-regulation of RCP with Rip11 was able to rescue the effect of RCP down- regulation on the TfR, implicating Rip11 in sorting membrane proteins towards the degradative pathway. Thus, we propose that RCP plays a role in the regulation of TfR trafficking, specifically sorting the receptor from the endosomal compartment towards the plasma membrane and away from the degradative pathway. 15 Ubiquitin Binding Proteins and Lysosomal Sorting R. C. Piper, S. Winistorfer, J. McDermott; Physiology and Biophysics, University of Iowa, Iowa City, IA One of the sorting signals that directs membrane proteins to lysosomes is their post-translational attachment to ubiquitin. Ubiquitin acts as a self- contained sorting signal, which acts at may intracellular locales to ultimately send membrane proteins to the lysosome. Ubiquitin works as a signal for internalization, as a TGN sorting signal and as a signal for incorporating proteins into lumenal vesicles of multivesiclular bodies. In order to understand how ubiquitin sorts proteins to the lysosome, we have focused on identifying ubiquitin-sorting receptors at the TGN and endosomes that bind cargo proteins and guide them to lysosomes. At the TGN, we find that the GGA family of clathrin binding proteins bind ubiquitin via two equivalent motifs within their GAT domains. This binding is required to direct ubiquitinated membrane proteins from the TGN directly to endosomes. We also find that several ubiquitin-binding proteins at the endosome are required for sorting into multivesicular bodies. Among these are the Vps27-Hse1 complex, the ESCRT-I complex and ESCRT-II complex. We show that physical interaction of the Vps27-Hse1 complex and ESCRT-I complex triggers a hand-off mechanism whereby ubiquitinated cargo could be transferred from one complex to the next. Interestingly, however, we find that the ubiquitin binding capacity of the ESCRT-I and ESCRT-II complexes is not required for proper sorting of ubiquitinated cargo into the MVB. Thus, we propose that the ubiquitin-binding function of these complexes is used to help Vps27-Hse1 release from cargo binding and help prevent the Vps27-Hse1 complex from being incorporated into the MVB lumen. Cell Biology of the Synapses (16-21) 16 ProN-cadherin Inhibits Synapse Formation A. G. Reines, W. Shan, A. W. Koch, D. R. Colman; BTRC, Montreal Neurological Institute, McGill University, Montreal, PQ, Canada N-cadherin participates in the regulation of synaptic strength and stabilization. This protein is synthesized as a precursor molecule (ProN-cadherin) which has anti-adhesive properties. The Pro domain is thought to be cleaved off in the late Golgi by a furin protease, after which adhesively activated N-cadherin is directed to the cell membrane. We raised an antibody recognizing the ProN sequence, and studied the expression of ProN- cadherin in developing hippocampal cultures. We demonstrated the presence of this immature N-cadherin throughout neuronal differentiation, from 5h to 14 days in vitro. Biotinylation of the proteins on the neuronal surface revealed that a proportion of ProN-cadherin is sorted to the plasma membrane and that the mature/immature N-cadherin ratio on the surface increases as neuronal differentiation progresses. Interestingly, we found that the Pro piece is released into the culture media coincident with its decrease on the plasma membrane. To study the function of ProN-cadherin when expressed on the neuronal surface, we employed a construct in which the endogenous cleavage site was replaced by a factor Xa cleavage site. ProN-cadherin overexpression had a large impact on synapse number, measured as a decrease in the number of synaptophysin and PSD-95 puncta. The same effect was observed when the functional labeling of presynaptic boutons with the FM4-64 dye was analyzed. This effect was partially overcome when factor Xa was applied to the cultures. Our results demonstrate that anti-adhesive ProN-cadherin is expressed in neurons and sorted to the plasma membrane where it may act as a negative regulator of synapse formation. We propose that changes in the ratio of mature/immature N-cadherin on the neuronal surface might be a novel mechanism by which neurons regulate synaptic junction formation. 17 Immaculate Connections, a Kinesin Motor, is Necessary for Presynaptic Differentiation and for the Transport of Synaptic Components E. Pack-Chung, D. K. Dickman, T. L. Schwarz; Children's Hospital, Harvard Medical School, Boston, MA The building blocks of synapses are present within growing axons and are recruited to sites of target cell contact. Thus, the transport of synaptic components, including the proteins of synaptic vesicles and active zones, is necessary for synaptogenesis. The identity and regulatory mechanisms of the molecular motors that move synaptic cargoes, however, remain elusive. We have identified a Drosophila gene (named immaculate connections) that is required for synaptogenesis. immaculate connections (imac) encodes a member of the kinesin 3 family of motors. Mutations in imac do not prevent axon outgrowth but prevent growth cones from transforming into synapses. At the embryonic neuromuscular junction, growth cones reach their target muscles but do not mature into synaptic boutons. Thus the development of nmj appears to be blocked just at synaptogenesis. Examination of intracellular transport revealed that the trafficking of synaptic vesicle and active zone components are blocked in 8a Sunday imac. These proteins were lacking in imac axons and accumulated in the cell bodies. However, post-Golgi vesicles, mitochondria, and cytoskeletal components were properly trafficked in imac axons. Interestingly, the absence of presynaptic differentiation in imac did not prevent the assembly of postsynaptic components; clustering of postsynaptic receptors and postsynaptic density elements were detected. We thus conclude that Imac is selectively required for the transport of materials for synaptogenesis and that in its absence, presynaptic differentiation fails to transpire. Our data also proves that it is distinct from the motor or motors necessary for axon outgrowth. By bringing synaptic materials into the growing axon, imac permits the rapid formation of functional synaptic connections. The coupling and uncoupling of the motor and its cargo are likely to be important in regulating the formation of synapses. 18 HIP1 Expression is Required for Normal NMDAR Function: Implications for a Role of HIP1 in Huntington’s Disease 1 1 1 2 2 2 1 2 1 1 M. Metzler, L. Gan, J. Helm, T. P. Wong, L. Liu, Y. Wang, L. Liu, Y. T. Wang, M. R. Hayden ; CMMT, Dept. of Medical Genetics, UBC, 2 Vancouver, BC, Canada, The Brain Research Centre, UBC, Vancouver, BC, Canada The initial identification of the endocytic protein HIP1 (huntingtin interacting protein 1) resulted from its interaction with the polyglutamine- containing protein huntingtin that, in its polyglutamine-expanded form, causes Huntington's Disease (HD). The interaction between HIP1 and huntingtin is significantly altered following polyQ-expansion in huntingtin suggesting that HIP1 is a possible component of the pathogenic mechanism in HD. In previous studies we have shown that AMPA-induced AMPA receptor (AMPAR) trafficking is blocked in cortical neurons from HIP1 knock-out mice (Metzler et al., 2003). Here, we demonstrate a similar block in NMDA-induced AMPAR endocytosis, and hence the reduction of cell-surface AMPAR expression, in cultured hippocampal neurons from HIP1 knock-out mice. Moreover, the NMDA-induced long term potentiation of AMPAR-mediated synaptic transmission at the CA1 synapses in hippocampal brain slices from HIP1 knock-out mice is significantly reduced compared to wild-type littermates. Results from coimmunoprecipitation and GST-pulldown experiments revealed direct interaction between HIP1 and the NR2 subunit of NMDARs. Furthermore, colocalization between HIP1 and NR2-cointaining NMDARs is observed in primary hippocampal neurons. Most important for our understanding of HD, NMDA-induced excitotoxicity is blocked in neurons from HIP1 knock-out mice. Together, these data provide strong evidence that HIP1 regulates NMDAR function and that this function of HIP1 may be contributing to enhanced excitotoxicity in HD. 19 Association of an AKAP Signaling Scaffold with Cadherin Adhesion Molecules in Neurons and Epithelial Cells 1 1 2 1 1 J. A. Gorski, L. L. Gomez, J. D. Scott, M. L. Dell'Acqua ; Pharmacology, University of Colorado Denver Health Sciences Center, Aurora, CO, 2 Vollum Institute, Howard Hughes Medical Institute, Oregon Health Sciences University, Portland, OR A-kinase anchoring protein (AKAP) 79/150 organizes a scaffold of PKA, PKC and protein phosphatase 2B/calcineurin that is localized to epithelial adherens junctions and the postsynaptic density of neuronal synapses. Targeting of the AKAP to these subcellular locations requires three N-terminal basic domains that bind F-actin and acidic phospholipids. Here we report a novel interaction of this targeting domain with cadherin adhesion molecules that are linked to actin through β-catenin (β-cat). Mapping the AKAP binding site in cadherins identified overlap with β-cat binding; however, no competition between AKAP and β-cat binding to cadherins was detected in vitro. Accordingly, AKAP79/150 exhibited polarized localization with β-cat and cadherins in epithelial cell lateral membranes, and β-cat was present in AKAP-cadherin complexes isolated from epithelial cells, cultured neurons, and rat brain synaptic membranes. Inhibition of epithelial cell cadherin adhesion induced by extracellular calcium switch and inhibition of actin polymerization by treatment with LatrunculinA redistributed intact AKAP-cadherin complexes from lateral membranes to intracellular compartments. In contrast, stimulation of neuronal pathways implicated in long term depression that depolymerize postsynaptic F-actin disrupted AKAP-cadherin interactions and resulted in loss of the AKAP, but not cadherins, from synapses. This neuronal regulation of AKAP79/150 targeting to cadherins may be important in functional and structural synaptic modifications underlying plasticity. 20 Structural Plasticity with Preserved Topology in a Postsynaptic Protein Network 1 2 1 2 T. A. Blanpied, M. D. Ehlers ; Physiology, University of Maryland School of Medicine, Baltimore, MD, Neurobiology, Cell Biology, Pharmacology and Cancer Biology, Howard Hughes Medical Institute and Duke University Medical Center, Durham, NC Multiprotein complexes form structural networks to mediate diverse cellular events including adhesion, signaling, nuclear transport, and intercellular communication. The function of such interconnected protein machines is determined by the spatial positioning and dynamic rearrangements of individual components within the complex. The postsynaptic density (PSD) is a prominent example of such networks. The PSD positions neurotransmitter receptors across from presynaptic sites of neurotransmitter release and links postsynaptic receptors with intracellular signaling cascades. Nearly all molecular theories of learning postulate morphological and molecular alteration of the PSD during plasticity at excitatory synapses. However, despite recent documentation of rapid mobility of receptors near the PSD, there is little known about dynamic behavior of core PSD constituents in the complex. To probe the structure of living PSDs, we have measured their internal dynamics using high- resolution imaging analyses. These experiments indicate that the PSD is assembled as a flexible yet topologically stable matrix on which enduring changes in function can be rapidly encoded. Exchange, addition, or removal of PSD elements can occur at independent matrix coordinates, providing a molecular map for organizing synaptic nanoarchitecture. 21 Two Types of Endocytic Intermediates at the Periactive Zone of a Central Synapse A. Sundborger, N. Tomilin, O. Shupliakov; Neuroscience, Karolinska Institutet, Stockholm, Sweden Dynamin is the GTPase implicated in fission of vesicles from the plasma membrane. In synapses it is an important component of the protein complex responsible for detaching clathrin vesicles from the presynaptic membrane. It has been shown in in vitro studies with isolated membranes and with synaptosomes that GTPγS blocks fission resulting in the accumulation of clathrin-coated pits with elongated necks decorated with dynamin-containing spirals. To visualize dynamin-dependent endocytic intermediates in an intact synapse we microinjected GTPγS into living giant axons in lamprey and studied them using electron microscopy. GTPγS did not alter morphology of synapses at rest. Stimulation of 9a Sunday microinjected axons with action potentials at 5 Hz induced a reduction in the number of synaptic vesicles at active zones and the appearance of numerous endocytic intermediates at periactive zones. Two different classes of intermedients were found at periactive zones: clathrin-coated pits with elongated necks, and membrane invaginations. Coated pits were present only on some of these membrane invaginations. Both were connected to the presynaptic membrane. Spiral-like structures were found at sites of these membrane connections. To investigate if these spirals contain dynamin, axons microinjected with GTPγS we cut along the longitudinal axis and stained with anti-dynamin (DG-1) antibodies using a pre- embedding immunogold technique. An accumulation of gold particles occurred at necks of clathrin-coated pits and at sites of connection of membrane invaginations to the presynaptic membrane. Our results show that two endocytic intermediates, clathrin-dependent and clathrin- independent, are formed at periactive zones during synaptic activity. Thus, in addition to the clathrin mechanism, bulk retrieval of large membrane compartments may occur in intact synapses during neurotransmitter release. Differentiation & Cancer (22-27) 22 Understanding Wnt Signaling in Development and Disease 1,2 1 2 X. He ; Children's Hospital, Boston, MA, Harvard Medical School, Boston, MA Wnt signaling is essential for development and tissue homeostasis. Disruption of Wnt signal transduction causes abnormal embryogenesis and cancers. Using a combination of molecular, biochemical and embryological techniques, we have focused on the mechanism of Wnt signaling in Xenopus embryo development and human cancer. We are particularly interested in how the Wnt receptor complex transduces Wnt signal across the plasma membrane, how the Wnt receptor complex specifies distinct transduction pathways to govern different aspects of embryogenesis, and the molecular composition and logic of these transduction pathways. Protein phosphorylation is pivotal for Wnt signaling. We have characterized two key phosphorylation events in the canonical Wnt/beta-catenin pathway. One is phosphorylation of the Wnt coreceptor, LDL receptor related protein 6 (LRP6). This phosphorylation leads to LRP6 activation and the initiation of Wnt signal transduction. The other is beta-catenin phosphorylation, which results in beta-catenin degradation and is inhibited upon Wnt signaling. 1). The mechanism of phosphorylation and activation of the Wnt coreceptor LRP6. We have shown that Wnt induces LRP6 phosphorylation at PPPS/TP motifs, and this phosphorylation is necessary and sufficient to trigger Wnt signaling. We have generated antibodies that specifically recognize phosphorylated LRP6. These antibodies are useful tools for detection of Wnt signaling activation in vivo and for identification of kinases involved in LRP6 phosphorylation. I will discuss our recent progress in studying Wnt-induced LRP6 phosphorylation and identifying LRP6 kinases. 2). The mechanism of phosphorylation and degradation of beta-catenin. We have demonstrated that two kinases, casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3), sequentially phosphorylate beta-catenin in a protein complex assembled by the scaffolding protein Axin. I will discuss how Wnt receptor activation may lead to inhibition of beta-catenin phosphorylation, and our attempt to identify other key molecules via in vitro reconstitution. 23 p21WAF1/Cip1 Mediates Notch1-dependent Suppression of Wnt Expression and Signaling 1 2 3 4 2 1 V. Devgan, C. Mammucari, S. E. Millar, C. Brisken, G. P. Dotto ; Cutaneous Biology Research Center, Massachusetts General Hospital, 2 3 Charlestown, MA, Department of Biochemistry, Lausanne University, Epalinges, Switzerland, Department of Dermatology, University of 4 Pennsylvania Medical School, Philadelphia, PA, Swiss Cancer Research Institute, Epalinges, Switzerland WAF1/Cip1 Besides controlling cyclin/CDK and PCNA activities, p21 can directly bind transcription factors and co-activators, modulating their functions. However, the biological significance of these findings has not been established. In keratinocytes, p21 is a direct downstream target of Notch1 activation, and loss of either p21 or Notch1 expands keratinocyte stem cell populations and facilitates tumor development. The tumor suppressor function of Notch1 has been associated with negative regulation of β-catenin signaling, through an unknown mechanism. We show here that Notch1 activation down-regulates β-catenin signaling through suppression of Wnts gene expression, and that p21 is a key mediator of this down-modulation. p21 suppresses Wnts expression independently of the cell cycle, while lack of p21 prevents Notch-dependent suppression of Wnt gene expression and results, both in cultured keratinocytes and in the intact skin in vivo, in increased Wnt 4 expression. More specifically, p21 associates with the E2F-1 transcription factor at the Wnt4 promoter and causes curtailed recruitment of c-Myc and p300, and histone hypoacetylation at this promoter. Thus, p21 functions as a mediator of the negative effect of Notch1 activation on Wnt signaling, by specific down- modulation of Wnt gene expression at the transcription-chromatin level and independently of cell cycle. 24 Gata-3 is a Critical Regulator of Differentiation in the Mammary Gland and Breast Cancer H. Kouros-Mehr, Z. Werb; Anatomy, UCSF, San Francisco, CA A series of breast cancer microarray studies have shown that the transcription factor GATA-3 is strongly correlated with Estrogen Receptor (ER) status, tumor grade and survival. We show here that GATA-3 plays a fundamental role in maintaining the luminal epithelial cell fate in the mammary gland and in breast cancer. We initially identified GATA-3 through a microarray screen as the most highly expressed transcription factor in the mammary gland. Immunostaining revealed that GATA-3 is expressed exclusively in all luminal progenitors and differentiated luminal cells. To determine the function of GATA-3, we crossed floxed GATA-3 mice with MMTV-Cre and WAP-rtTA-Cre lines. Homozygous floxed GATA-3 mice carrying MMTV-Cre displayed runting, progressive alopecia, and a highly defective mammary gland. The mammary glands displayed a near lack of epithelium due to an inability to form terminal end buds. To more closely analyze the function of GATA-3, we crossed the floxed mice with the Tet-inducible Cre line WAP-rtTA-Cre. After short-term (72 hr) administration of doxycycline to adult mice, GATA-3 null luminal cells displayed loss of basal polarity and died within the ductal lumen as single cells. To determine how GATA-3 affects breast cancer progression, we used the PyMT mouse model of breast cancer. We transplanted GFP+ hyperplasias into syngeneic mice to determine when malignant conversion occurred. We found that loss of GATA-3 strongly correlated with loss of tumor differentiation, the progression from adenoma to early carcinoma, and the onset of tumor dissemination into distant sites. Furthermore, overexpression of GATA-3 in primary PyMT tumors was sufficient to induce elements of tumor differentiation. This work suggests that GATA-3 specifies and maintains luminal cell differentiation and suggests that loss of cell fate is a critical event in the malignant progression of breast cancer. 10a