HM Medical Clinic

 

Abstract

Nankai University May 21, 2010
7:00-8:00
Tom Rapoport "How the ER gets into shape" May 22, 2010
Proteins in generating membrane vehicles I
8:00-8:40
Harvey McMahon "Sculpting Cell Membranes: Understanding pathways of endocytosis and exocytosis" 8:40-9:20
Pietro De Camilli "Bending membranes at sites of endocytosis" 9:20-9:40
Coffee and photo
9:40-10:20
Yeguang Chen "Endocytotic regulation of TGFbeta signaling" 10:20-11:00 Thomas Pucadyil "Dynamic remodeling of membranes
catalyzed by dynamin" 11:00-11:30 Baoliang Song "NPC1L1-mediated vesicular transport of
cholesterol during cholesterol absorption" Proteins in generating membrane vehicles II
4:30-5:10
Jenny Hinshaw "Dynamins role in membrane remodeling" 5:10-5:50
Winfried Weissenhorn "ESCRT-III structure and regulation" 5:50-6:20
Michael Kozlov "Theory of membrane shaping and remodeling 6:30-9:00
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University May 23, 2010
Proteins in membrane remodeling: fusion and fission
8:00-8:40
Reinhard Jahn "Membrane fusion mediated by SNARE proteins" 8:40-9:20
Leonid Chernomordik "Membrane fusion mediated by viral and developmental fusogens" 9:20-9:40
9:40-10:20
Senfang Sui "Functional oligomerization of DegP on membranes" 10:20-11:00 Joshua Zimmerberg "Membrane remodeling in exocytosis and
malaria parasite discharge" 11:00-11:40 Patricia Bassereau "Sensing curvature and curving membrane:
in vitro studies of dynamin and amphiphysin 1" 11:40-11:55 Junjie Hu "A class of dynamin-like GTPases involved in the
generation of the tubular ER network" Proteins in shaping cellular membranes
2:00-2:40
Pekka Lappalainen "Mechanism and physiological role of I- BAR domain induced membrane deformation" 2:40-3:20
Pingsheng Liu "Interaction between lipid droplets and other cellular organelles" 3:20-3:30
3:30-4:10
Ruth Collins "Prenylated small GTPases and their membrane partners: defining membrane shape and compartment identity in the eukaryotic microbe S. cerevisiae" 4:10-4:50
Quan Chen "Molecular regulation of mitochondrial dynamics" 4:50-5:20
Yanzhuang Wang "Cell cycle-regulated Golgi stack assembly and function" 7:00-9:00
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University HOW THE ER GETS INTO SHAPE
Tom A. Rapoport, Ph.D.
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115 Many organelles have characteristic shapes. For example, the Golgi consists of a stack of flat membrane sheets and the inner membrane of mitochondria has invaginations, called cristae. A particularly striking example is the endoplasmic reticulum (ER), which consists of both sheets, found in the inner and outer nuclear membrane as well as in the peripheral ER, and of tubules that are interconnected by three-way junctions to form a network. The entire ER is a continuous membrane system with a common luminal space. How the different morphologies are generated and maintained and how these morphologies correlate with different functions are important questions that have not yet been addressed.
We have started to investigate how the tubular ER is generated and maintained. Previous work has demonstrated that tubules can be generated by being pulled out of membrane reservoirs by molecular motors that move along microtubule or actin filaments, or by the tips of filaments as these grow by polymerization. However, the alignment of membrane tubules with the filaments of the cytoskeleton is not perfect, the ER network does not retract upon deplomerization of actin filaments in yeast and retracts only slowly upon depolymerization of microtubules in mammals, and ER tubules can also be generated from vesicles. Thus, other mechanisms are likely responsible for stabilizing the tubules.
We have used an in vitro assay to address the mechanism of ER tubule formation. Xenopus egg membranes, consisting of small vesicles, served as starting material. These vesicles can fuse to generate an elaborate ER network, a process that can be followed visually with hydrophobic fluorescent dyes or by the efflux of Ca2+ ions from the ER lumen. Based on the inhibitory effect of sulfhydryl reagents and 2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University antibodies, we found that ER network formation requires the integral membrane protein reticulon 4a (Rtn4a)/NogoA. This protein belongs to the ubiquitous reticulon family, whose members contain the reticulon domain, a segment of 200 amino acids that contains two relatively long hydrophobic segments (30-35 residues). In agreement with the postulated role for the reticulons in ER tubule formation, they are largely restricted to the tubular ER both in mammalian cells and in yeast and are excluded from the continuous sheets of the nuclear envelope and peripheral ER. Upon overexpression, the reticulons form tubular membrane structures. The reticulons interact with DP1/Yop1, a conserved integral membrane protein that also localizes to the tubular ER. DP1/Yop1 also has two relatively long hydrophobic segments. The simultaneous absence of the reticulons (Rtn1 and Rtn2) and Yop1 in yeast converts the tubular cortical ER into sheets, indicating that these proteins are essential for ER tubule formation. The absence of the abundant Rtn1 and Yop1 proteins has the same effect, and the overexpression of the less abundant Rtn2 protein can restore ER tubules, indicating that abundance of these proteins is important for ER tubule formation. Purified yeast Rtn1 or Yop1, when reconstituted with pure lipids, can form tubules in vitro. The tubules have a very narrow diameter, likely caused by the high membrane concentration of the proteins. Together, these results indicate that the reticulons and DP/Yop1 are both necessary and sufficient for the formation of membrane tubules. These data thus give first insight into how the shape of an organelle is generated and maintained.
The two hydrophobic segments of the reticulons and DP/Yop1 sit in the membrane as hairpins, thus exposing all hydrophilic segments to the cytoplasm. We postulate that these proteins have a wedge-like shape in the membrane that would allow them to induce the high curvature that is characteristic for tubules in cross section. In addition, these proteins form oligomers and could thus form a scaffold around the Recent experiments show that the formation of the tubular network requires a class of 2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University dynamin-like GTPases. The mammalian atlastins are dynamin-like, integral membrane GTPases that interact with the tubule-shaping proteins. The atlastins localize to the tubular ER and are required for proper network formation in vivo and in vitro. Depletion of the atlastins or overexpression of dominant-negative forms inhibits tubule interconnections. The Sey1p GTPase in S. cerevisiae is likely a functional ortholog of the atlastins; it shares the same signature motifs and membrane topology and interacts genetically and physically with the tubule-shaping proteins. Cells simultaneously lacking Sey1p and a tubule-shaping protein have ER morphology defects. These results indicate that formation of the tubular ER network depends on conserved dynamin-like GTPases. Since atlastin-1 mutations cause a common form of hereditary spastic paraplegia, we suggest ER shaping defects as a novel neuropathogenic mechanism.
Du, Y., Ferro-Novick, S., and Novick, P. (2004) J. Cell Sci. 117, 2871-78. Dynamics
and inheritance of the endoplasmic reticulum.
Baumann, O., and Walz, B. (2001) Int. Rev. Cytol. 205, 149-214. Endoplasmic
reticulum of animal cells and its organization into structural and functional domains.
Voeltz, G.K., Rolls, M.M., and Rapoport, T.A. (2002) EMBO Reports 3(10),
944-950. Structural organization of the endoplasmic reticulum.
Voeltz, G.K., Prinz, W.A., Shibata, Y., Rist, J.M., and Rapoport, T.A. (2006) Cell 124,
573-86. A class of membrane proteins shaping the tubular endoplasmic reticulum.
Shibata, Y., Voeltz, G.K., and Rapoport, T.A. (2006) Cell 126, 435-439. Rough sheets
and smooth tubules.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Voeltz, G.K., and Prinz, W.A. (2007) Nat. Rev. Mol. Cell Biol. 8, 258-64. Sheets, ribbons and tubules - how organelles get their shape.
Hu, J., Shibata, Y., Voss, C., Shemesh, T., Li, Z., Coughlin, M., Kozlov, M.M., Rapoport, T.A., and Prinz, W.A. (2008) Science 319, 1247-1250. Membrane proteins of the endoplasmic reticulum induce high-curvature tubules.
Shibata, Y., Voss, C., Rist, J.M., Hu, J., Rapoport, T.A., Prinz, W.A., and Voeltz, G.K. (2008) J. Biol. Chem. 283, 18892-904. The reticulon and DP1/Yop1p proteins form
immobile oligomers in the tubular endoplasmic reticulum.
Hu, J., Shibata, Y., Zhu, P.-P., Voss, C., Rismanchi, N., Prinz, W.A., Rapoport, T.A., and Blackstone, C. (2009) Cell 138, 549-561. A class of dynamin-like GTPases
involved in the generation of the tubular ER network.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Sculpting Cell Membranes: Understanding pathways
of endocytosis and exocytosis
Harvey T. McMahon
MRC, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
Cell shape is adapted to function. Organelle shape and local membrane architectures are likewise optimised for the processes that take place on and within these microenvironments. We focus on the dynamic regulation of membrane shape, which can occur by the interplay between the transient and regulated insertion of membrane bending motifs and the detection and stabilisation of membrane shape. This approach has allowed us not only to describe the biophysics of membrane shape changes but also to take a fresh look at membrane dynamics in physiological processes like exocytosis and endocytosis. In doing so we have noted that proteins with amphipathic helices or hydrophobic membrane-inserting loops are likely to effect or respond to curvature and that the membrane interaction surfaces of proteins can sense shape (like proteins of the BAR Superfamily). This molecular view has allowed us to ascribe novel cell-biological functions to proteins (e.g. the mechanistic affect of synaptotagmin in membrane fusion) and to give a more insightful view of how these processes work. Thus we can now go from the biophysics of a molecule, to better understanding of known pathways and to the molecular characterisation of novel cellular trafficking pathways both of endocytosis and exocytosis. See: Richard Lundmark, Gary J. Doherty, Mark T. Howes, Katia Cortese, Yvonne Vallis, Robert G. Parton and Harvey T. McMahon (2008) The GTPase-activating protein GRAF1 regulates the CLIC/GEEC endocytic pathway. Current Biology, 18,
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Sascha Martens, Michael M. Kozlov and Harvey T. McMahon (2007) How Synaptotagmin Promotes Membrane Fusion. Science, 316, 1205-1208.
Eva M. Schmid and Harvey T. McMahon (2007) Integrating molecular and network biology to decode endocytosis. Nature, 448, 883-888.
William Mike Henne, Helen M. Kent, … Philip R. Evans, and Harvey T. McMahon (2007) Structure and Analysis of FCHo2 F-BAR Domain: a Dimerising and Membrane Recruitment Module that Effects Membrane Curvature. Structure, 15,
Oli Daumke, Richard Lundmark, Yvonne Vallis, Sascha Martens, Jo Butler and Harvey T. McMahon (2007) Architectural and mechanistic insights into an EHD ATPase involved in membrane remodelling. Nature, 449, 923-927.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Endocytotic regulation of TGF-β signaling
State Key Laboratory of Biomembrane and Membrane Biotechnology, School of Life Sciences, Tsinghua University, Beijing 100084, China Phone: 86-10-62795184 TGF-β signaling plays pivotal roles in embryogenesis and tissue homeostasis, and deregulation of this signaling pathway is associated with a variety of diseases including cancer. The signal transduction is mediated by transmembrane TGF-β type I and type II receptors and intracellular Smad proteins. We and others found that the TGF-β type I ALK5 constitutively internalized into the cell mostly via the clathrin- dependent pathway. Endofin, a member of the FYVE domain protein family, has been suggested to regulate endosome trafficking. We found that endofin functions as a scaffold protein to facilitate TGF-β signaling in early endosomes. Knockdown of endogenous endofin expression specifically led to reduction of the transcriptional responses of TGF-β. Endofin interacts with Smad4 and TGF-β type I receptors. Therefore, endofin can facilitate TGF-β signaling as a scaffold protein in early endosome to promote the Smad heterocomplex formation by bringing Smad4 to the proximity of the receptor complex. Once ALK5 and Nodal type I receptor ALK4 reach to late endosomes, we found that they interacts with Dapper2, which targets the internalized receptors to lysosome for degradation, thereby negatively regulating TGF-β/Nodal signaling and fine-tuning signal output. Finally, our recent data show that lipid rafts are required for TGF-β to activate MAPK, suggesting that signal specificity can be orchestrated by receptor distribution on the plasma membrane. 2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Dynamic remodeling of membranes catalyzed by
Thomas J. Pucadyil
The Scripps Research Institute, La Jolla, CA 92037, U.S.A.
Membrane compartments in the cell are under a constant state of flux, both in shape and composition. The creation and consumption of membrane compartments are attributed to the function of protein machinery that manages to remodel membrane shape, often by the consumption of energy. These reactions occur under non- equilibrium conditions inside the cell. Time-resolved approaches are therefore necessary to unravel its mechanistic details. Dynamin 1, a prototypical member of the large GTPase superfamily, is involved in the scission and release of vesicles in clathrin-dependent and -independent transport pathways. We have recently recreated a dynamin-catalyzed membrane fission reaction under conditions of constant GTP turnover on a novel membrane system of supported lipid bilayers with excess membrane reservoir (SUPER). Insights gained from monitoring dynamics of this extreme membrane remodeling processes that makes vesicles from a planar bilayer will be described in the talk. 2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University NPC1L1-mediated vesicular transport of
cholesterol during cholesterol absorption
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. 320 Yue-Yang Rd, New Biochem Building #1101, Shanghai 200031, China.
Cholesterol is an essential component of the phospholipid bilayer to maintain proper functions of the membrane. However, high level of cholesterol causes severe problems including cardiovascular disease. Excessive cholesterol uptake from diet is a major risk factor for cardiovascular disease. Niemann-Pick C1 Like 1 (NPC1L1) is an essential protein for cholesterol absorption. Ezetimibe, an inhibitor of NPC1L1, decreases cholesterol absorption and is clinically used to lower plasma cholesterol level. However, the molecular mechanism of NPC1L1-mediated cholesterol uptake and how ezetimibe inhibits this process are poorly defined. Here we find that cholesterol specifically promotes the internalization of NPC1L1 and this process requires microfilaments and clathrin/AP2 complex. Blocking NPC1L1 endocytosis dramatically decreases cholesterol internalization, indicating NPC1L1 mediates cholesterol uptake via its vesicular endocytosis. Ezetimibe prevents NPC1L1 from incorporating into clathrin-coated vesicles and thus inhibits cholesterol uptake. Together, our data suggest a model wherein cholesterol is internalized into cells with NPC1L1 through clathrin/AP2-mediated endocytosis and ezetimibe inhibits cholesterol absorption by blocking the internalization of NPC1L1. In addition, I will talk about our latest findings about cholesterol absorption. 2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Dynamins role in membrane remodeling
Jenny E. Hinshaw
LCBB, NIDDK, NIH. Building 8, Room 1A03, 8 Center Dr. Bethesda, MD 20892 Dynamin, a 100 kDa GTPase, is involved in the final stages of fission during endocytosis and vesiculation from the Golgi. Other dynamin family members are involved in organelle division, such as the dynamin-related protein, Drp1, that is required for mitochondrial fission. During membrane fission, dynamin family members are believed to self-assemble into short helices around sites of constriction and drive fission upon GTP hydrolysis. In support of this model, both purified dynamin and the yeast Drp1 (Dnm1) readily form protein-lipid tubes, which further constrict the membrane upon GTP addition. However, the dimensions of the protein assemblies and the extent of constriction is tailored to the proteins function. Dynamin forms helices with a diameter of 50 nm , which is ideal for wrapping around the necks of budding vesicles, and constricts the membrane by 10 nm (outer diameter of 50 nm to 40 nm). Dnm1 assembles into significantly larger helices with an outer diameter of 110 nm, which exactly matches mitochondrial constricted sites observed in vivo, and constricts the membrane by 60 nm (110 nm to 50 nm). The 3D structure of both proteins, solved by cryo-electron microscopy methods, reveal differences in their architecture that would lead to slight variations in the mechanism of constriction. The 3D maps of dynamin in the constricted and non-constricted states revealed a twisting motion between subunits that suggests a corkscrew model for dynamin constriction. The 3D map of Dnm1-lipid tubes reveals a 2-start helix, instead of a 1-start helix observed for dynamin, and lacks a direct interaction with the lipid bilayer, which correlates with the absence of a pleckstrin homology domain in Dnm1. Both of these features allows for a more flexible helix, a characteristic that may be necessary for the large conformational change required for organelle division. Overall, these results suggest that although dynamin family members share common mechanochemical properties, the structure of each member may vary to fit their unique function. 2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University ESCRT-III structure and regulation
Bettina Hartlieb1, Suman Lata1, Julianna Solomons1, Gur Fabrikant2, Guy
Schoehn1,3 John Briggs4, Heinrich G. Göttlinger5, Michael Kozlov2 and Winfried
1UVHCI, UMI 3265 UJF-EMBL-CNRS, Grenoble, France 2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 3IBS UMR 5075 CEA-CNRS-UJF, Grenoble, France 4EMBL, Heidelberg, Germany 5University of Massachusetts Medical School, Worcester, MA, USA The ESCRT-III complex, formed by CHMP family member proteins, is recruited to membranes and functions at budding steps during multivesicular body biogenesis, cytokinesis and release of some enveloped viruses including HIV-1. ESCRT-III polymers (composed of CHMP2, 3, 4 and 6) and the ATPase VPS4 are essential for all three processes and most likely constitute the minimal budding and membrane scission machinery. We present structural studies on ESCRT-III regulators ALIX, CC2D1 and AMSH that regulate ESCRT-III polymerization. A structural model of ALIX dimerization reveals its mode of ESCRT-III CHMP4 filament interaction and role during HIV-1 budding. In contrast, the CHMP4 effector CC2D1 targets only CHMP4 monomers but not polymers. AMSH, an ubiquitin hydrolase, interacts with a C-terminal helical motif of ESCRT-III CHMP3, which permits interaction with ESCRT-III CHMP2A/CHMP3 helical polymers. The CHMP2A/CHMP3 helical polymers have an outer diameter of 40 nm and associate with cellular membranes via their outer surface while VPS4 binds on the inside to catalyze their disassembly. Some of the ESCRT-III polymers associate into "dome-like" structures which appear closed and capped by a smaller CHMP2A ring-like structure, indicating that such structures could bend membranes and catalyze spontaneous membrane fission. An integrated model of ESCRT-III regulation and assembly will be discussed.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Theory of Membrane Shaping and Remodeling by
Adi Pick1, Felix Campelo1,2, Gur Fabrikant1, Suman Lata3, Winfried
Weissenhorn3, Harvey T. McMahon4 , Michael M. Kozlov1
1Dept. Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, 2Department of Cell and Developmental Biology, CRG-Centre de Regulació Genòmica, Barcelona, Spain 3Université Joseph Fourier-EMBL-CNRS, Grenoble, France, 4MRC Laboratory of Molecular Biology, Cellular membranes are highly dynamic, undergoing both persistent and dynamic shape changes driven by specialized proteins. The observed membrane shaping can be simple deformations of existing shapes or membrane remodeling involving fission or fusion. I will describe the major mechanistic principles by which membrane shaping proteins act and address, specifically, the models for membrane fission by ESCRT-III proteins and N-BAR domains.
The N-BAR domain containing proteins, endophilin and amphiphysin, have been suggested to induce large membrane curvatures during endocytosis. We propose and substantiate by modeling that, in addition to curvature generation, the N-BAR domains are able to drive membrane fission and, hence, accomplish the whole transformation of flat membranes into separate transport intermediates. The model is based on the interplay and mutual frustration of two modes by which N-BAR domains can bend lipid membranes: the shallow insertion of amphipathic helices into the lipid bilayer matrix and the membrane scaffolding by the concave faces of BAR dimers. We use the elastic models of lipid bilayers and BAR dimers to show that increasing amounts of the membrane-bound N-BARs resulting in membrane bending also generate mechanical stresses which drive transformation of cylindrical or flat membranes into spherical vesicles, i.e., membrane fission. We analyze quantitatively the conditions of this transformation, demonstrate its biological feasibility and predict a difference in the abilities of endophilin and amphiphysin to induce membrane 2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University fission. We propose that the suggested principle is not limited to N-BAR domains but may underlie membrane remodeling by numerous proteins known to have a potential to bend membranes by both the hydrophobic insertion and scaffolding mechanisms.
ESCRT-III proteins catalyze membrane fission during multi vesicular body biogenesis, budding of some enveloped viruses and cell division. We propose that the ESCRT-III subunits, CHMP2 and CHMP3, self-assemble into hemi-spherical dome- like structures within the necks of the initial membrane buds generated by CHMP4 filaments. The dome formation is accompanied by the membrane attachment to the dome surface, which drives narrowing of the membrane neck and accumulation of the elastic stresses leading, ultimately, to the neck fission. Based on the bending elastic model of lipid bilayers, we determine the degree of the membrane attachment to the dome enabling the neck fission and compute the required values of the protein- membrane binding energy. We estimate the feasible values of this energy and predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission. We support the computational model by electron tomography imaging of CHMP2- CHMP3 assemblies in vitro.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Membrane fusion mediated by SNARE proteins
Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen/ The secretory pathway in eukaryotic cells connects the biosynthetic endoplasmic reticulum with the plasma membrane and the lysosome via intermediate sorting compartments such as the Golgi apparatus and the endosomal system. Communication is mediated by vesicular traffic where transport vesicles form at the donor compartment and then fuse with the acceptor compartment. Membrane recognition, docking and fusion is mediated by supramolecular machines consisting of conserved proteins that act in concert, with the final step being bilayer fusion that is executed by SNARE proteins. SNAREs constitute a superfamily of conserved, small and mostlyl membrane-anchored proteins. SNARE proteins spontaneously assemble into tight helical complexes that bridge the membranes and initiate fusion. After fusion, dissociation and thus regeneration is brought about by the AAA+- ATPase NSF, in conjunction with cofactors.
In our work we have concentrated on understanding the precise mechanism by which SNAREs fuse membranes. SNARE complexes consist of structurally highly conserved, elongated four-helix bundles in which each position is occupied by a SNARE motif of a different subfamily, termed Qa-, Qb-, Qc-, and R-SNARE. Assembly follows an ordered sequence that may vary between different SNARE complexes and that is highly regulated: An acceptor complex is formed on one of the membranes, with the donor SNARE then binding to the N-terminal end of the SNARE motif, followed by rapid progression ("zippering") towards the C-terminal membrane anchors. Reconstitution experiments with both native and artificial membranes have shown that assembly is directly linked to fusion, with the helical domains extending into the membrane and thus initiating membrane merger. Both large liposomes with reduced curvature and native synaptic vesicles effectively fuse 2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University in a SNARE-dependent manner, documenting that reconstitution of SNARE- dependent fusion is a valid approach to reproduce biological fusion reactions. Fusion results in both lipid and content mixing. Furthermore, a single SNARE complex suffices to fuse membranes, and there is no cooperativity between SNARE 2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Membrane fusion mediated by viral and
Leonid V. Chernomordik
Section on Membrane Biology, Program in Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health Cell entry by enveloped viruses, syncytia formation in development, and intracellular protein trafficking, all these processes share a common stage of membrane fusion. In this talk I will discuss our work on fusion mediated by dengue virus E protein and fusion among myoblasts mediated by yet-unidentified proteins. An important human pathogen dengue virus as many other viruses invade cells by fusion between viral envelope and membrane of acidified endosome. We found that effective fusion between this virus and plasma- and intracellular- membranes, as well as virus fusion to protein-free liposomes requires the target membrane to contain anionic lipids such as bis(monoacylglycero)phosphate, a specific lipid of late endosome. Our data indicate that lipid-dependence of dengue fusion machinery protects it against premature irreversible restructuring and ensures that the transition from the earliest hemifusion intermediates to the latter fusion stages takes place in late endosomes, where virus encounters anionic lipids for the first time during entry.
Myoblast fusion in formation of multinucleated myotubes and muscle fibers is a highly regulated process that involves a series of complex steps. One of the important challenges in exploring mechanisms of the myoblast fusion and other developmental cell fusions is related to the fact that these fusion reactions, in contrast to low pH- triggered viral fusion reactions and calcium-triggered exocytosis, advance rather slowly in what appears to be a relatively unsynchronized manner. In this study, we isolated fusion stage in myotube formation by murine myoblasts (C2C12 cells) labeled with different membrane and cytosolic probes by blocking myotube formation immediately prior to fusion with reversible hemifusion-inhibitor 2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University lysophosphatidylcholine. This approach accumulates cells at a ready-to-fuse stage and, thus, synchronizes fusion upon lifting the inhibitor. Isolation of the fusion stage allowed us to explore myoblast fusion pathway and identify several membrane- shaping proteins that play an important role in opening and expansion of fusion pores. We hope that better understanding of protein-lipid interactions underlying different stages of viral and developmental fusion will bring new ways of controlling membrane fusion in pathophysiology.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Functional oligomerization of DegP on membranes
Qing-tao Shen, Xiao-chen Bai, and Sen-Fang Sui
State Key Lab of Biomembrane and Membrane Biotechnology, Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China The life phenomena depends much on the function of an extraordinarily large number of proteins, most of which have their structures marginally stable and are thus subjected to continuous quality control to keep them in a functional structural state, and the failure of this leads to severe diseases. Molecular chaperones and proteases, both bind to unfolded substrate proteins in functioning, are the two families of proteins that cells generally employ to implement such quality control processes. DegP, present in the periplasmic space of E. coli cells, is a protein that functions as both, exhibiting the dual protease-chaperone activity in an ATP-independent manner, making it an unique case for understanding the quality control mechanism of proteins. Homologs of DegP (collectively named as HtrA) have been identified in almost all organisms and believed to function in protecting cells under stress conditions.
DegP is involved in the quality control of several important membrane-related proteins in the narrow E. coli envelope; therefore, its relationship with the cell membrane is of great interest. Previous studies have suggested that DegP has a high affinity for the periplasmic side of the inner membrane. This study was conducted in attempt to clarify the structural status of DegP functioning on membranes. Our combined cryo EM and biochemistry studies revealed that DegP, induced by lipid membranes, forms a range of bowl-shaped oligomeric structures, each with a 4- or 5- fold symmetry and both with a DegP trimer as the structural unit. The symmetry axis of the bowl-shaped structures is always perpendicular to the membrane plane. These bowl-shaped membrane-bound DegP assemblies have the capacity to recruit and process substrates in the bowl chamber, and they exhibit proteolytic and chaperone- like activities. Our findings imply that DegP might regulate its dual roles during protein quality control, depending on its assembly state in the narrow bacterial 2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Sensing curvature and curving membrane:
in vitro studies of dynamin and amphiphysin 1
Benoit Sorre, Gerbrand Koster, Andrew Callan-Jones, Martin Lenz, Jacques
Prost, Aurélien Roux, Patricia Bassereau
Laboratoire PhysicoChimie Curie, Institut Curie, 11 Rue P. et M. Curie, 75231 Paris Cedex 05, France Membrane transport between intracellular compartments, entry or exit out of the cell, imply similar sequential events: membrane deformation and lipid/protein sorting during the formation of the transport intermediate (vesicle or tube), fission from the donor compartment, transport and eventually fusion with the acceptor membrane. Model membrane are convenient systems to investigate mechanisms involved in cell trafficking, because they are composed of a very limited number of components compared to cellular membranes. In particular, membrane nanotubes with a controlled diameter (15-500 nm) pulled out of Giant vesicles (GUV) are particularly suited to study the dependence of protein/membrane interactions with curvature in membrane trafficking. Dynamin is a protein, which assembles in helical structures around the neck of clathrin vesicles during budding and induces fission upon GTP hydrolysis. We will show that, in a biologically relevant concentration range, dynamin assembly can occur only when the neck diameter is below a threshold value (Fig.1). This curvature-dependent polymerization mechanism guaranties a correct timing for carrier budding. This curvature sensitivity for binding disappears at high dynamin concentration. In a second part, we will show that amphiphysin 1, a N-BAR- domain protein also involved in clathrin vesicle genesis, is also a curvature sensor at a similar concentration with a strong coupling coefficient to curvature; in these biologically relevant conditions, amphiphysin 1 binds exclusively to the vesicle neck. Nevertheless, it can impose a spontaneous curvature to the membrane at high protein concentration that can be measured using confocal microscopy and optical tweezers. These experiments will be analyzed within a general theoretical framework of membrane mechanics. All together, our experiments demonstrate that depending on 2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University its concentration, the same protein can behave as either a curvature-inducer or a curvature sensor. However, the next challenge is to understand how the curvature sensitivities of these two proteins influence their mutual regulation during vesicle Fig. 1: Dynamin (green) polymerization along a membrane nanotube (red)
of diameter lower than 40 nm
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Mechanism and physiological role of I-BAR domain
induced membrane deformation
Juha Saarikangas, Hongxia Zhao, Pieta Mattila, Anette Pykäläinen and Pekka
Institute of Biotechnology, P.O. Box 56, 00014 University of Helsinki, Finland Generation of membrane curvature is critical for the formation of plasma membrane protrusions and invaginations, and for shaping intracellular organelles. Among the central regulators of membrane dynamics are the BAR superfamily domains, which can deform membranes into tubular structures. In contrast to the relatively well characterized BAR and F-BAR domains that promote the formation of plasma membrane invaginations, I-BAR domains induce plasma membrane protrusions when expressed in cells. Mammals have four I-BAR domain proteins (MIM, ABBA, IRSp53 and IRTKS), which in addition to the N-terminal I-BAR domain also contain a C-terminal actin monomer binding WH2 domain. By using time-lapse microscopy of giant unilammelar vesicles and cryo-EM, we provide evidence that I-BAR domains bind to the inner surface of membrane tubules, and consequently bend membranes through strong electrostatic interactions to the opposite direction compared to BAR and most F-BAR domains. We also show that I-BAR domains induce PIP2-clustering and can thus promote the formation of PIP2-rich membrane microdomains. We are currently examining the dynamics of PIP2 and I-BAR domains in these membrane microdomains. To elucidate the physiological role of I-BAR domain proteins, we generated MIM knockout mice. These mice displayed a progressive kidney disease characterized by abnormal tubular morphology, severe urine concentration defect, renal electrolyte wasting and bone abnormalities. Cell biological analysis demonstrated that MIM displays dynamic localization to cell-cell contacts in cultured kidney cells through its membrane binding/deforming I-BAR domain and promotes actin filament assembly at these sites. Together, these data suggest that MIM promotes actin and plasma membrane dynamics to contribute to the maintenance of intercellular junctions in epithelial cells.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Prenylated small GTPases and their membrane partners: defining
membrane shape and compartment identity in the eukaryotic
microbe S. cerevisiae
Catherine Z. Chen1,3, Jared Chen2 and Ruth N. Collins1
1Graduate Field of Pharmacology, Cornell University, Ithaca NY 14853 2RABS Program, Cornell University, Ithaca NY 14853 3Department of Molecular Medicine, Cornell University, Ithaca NY 14853 A hallmark of eukaryotic cells is the presence of membrane-enclosed organelles, and this organization necessitates the regulation of transport between compartments and maintenance of organelle integrity. Rab GTPases are well-known regulators of membrane trafficking as well as micro-domain remodeling. The YIP1 family is a group of integral membrane proteins with the ability to bind Rab GTPases in a manner that is specific for intact COOH-terminal di-geranyl prenyl moieties. The YIP1 family is evolutionarily conserved in all eukaryotes, and the S. cerevisiae genome contains four paralogs, two of which are essential (YIP1 and YIF1), while two are nonessential (YIP4 and YIP5). We report on a series of mutations in YIP1 family members to characterize the nature of the relationship between YIP1 family members and Rab GTPases.
This work was supported by NIH Grant no. 5R01GM069596 to R. Collins. Catherine Chen was supported by NIH training grant (5T32GM008210-18).
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Molecular Regulation of Mitochondrial Dynamics
Zhang Juan, Bin Wang and Quan Chen
The Joint Laboratory of Apoptosis and Cancer Biology, College of Life Sciences, Nankai University, Tianjin 300071 and The State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China Mitochondria undergo frequent fission, fusion, and redistribution throughout the cytoplasm in response to the environmental cues. Mitochondrial fusion is mediated by mitofusins at outer membrane and OPA1 at the intermembrane space, while mitochondrial fission is regulated by Drp1. These proteins are mechanoenzymes belonging to the dynamin GTPase family. Dysregulation of these proteins results in abnormal mitochondrial fission or fusion which are closely associated with a number of neuromuscular diseases. We demonstrate that Mfn1 and Mfn2 are polyubiquitinated and degraded primarily by a proteasome-dependent mechanism. We identify that membrane-anchored Ring Finger Protein 5 (RNF5), an ubiquitin E3 ligase, plays an important role in Mfn1 stability. Our results reveal that Mfn1 protein levels are finely tuned by the RNF5 mediated uitination-dependent degradation to regulate mitochondrial dynamics. I will also discuss our findings on a new regulator of mitochondrial fusion.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Cell cycle-regulated Golgi stack assembly and
Department of Molecular, Cellular and Developmental Biology, University of Michigan. 830 North University Ave, 2127A Natural Science, Ann Arbor, MI 48109, United States.
The molecular mechanism of the Golgi disassembly and reassembly process during the cell cycle of animal cells has been revealed by a defined in vitro reconstitution assay. Mitotic Golgi fragmentation involves membrane vesiculation coupled with cisternal unstacking; post-mitotic Golgi reassembly is mediated by membrane fusion to form single cisternae and stack formation. Stack formation directly involves the Golgi stacking protein GRASP65 and GRASP55, which play complementary and essential roles in Golgi cisternal stacking by forming mitotically regulated trans- oligomers. By inhibition of GRASP65/55 oligomerization we are able to manipulate Golgi stack formation and thus determine the biological significance of stacking for the first time. We demonstrate that Golgi cisternal unstacking stimulates COPI vesicle budding and protein transport. Golgi fragmentation, however, impairs glycosylation of cell surface proteins and reduces cell adhesion. Inhibition of Golgi disassembly at the onset of mitosis also affects cell cycle progression. We propose that Golgi stack formation is a flux regulator for protein trafficking and thereby maintain the quality of protein glycosylation. Structural and functional Golgi defects in disease models are explored in this study.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University PInB – a novel BAR domain protein expressed in
specialised epithelial cells
Anette Pykäläinen, Malgorzata Boczkowska, Hongxia Zhao, Grzegorz
Rebowski, Juha Saarikangas, Janne Hakanen, Helena Vihinen, Marjo Salminen,
Eija Jokitalo, Roberto Dominguez and Pekka Lappalainen
Department of Molecular and Cell Biology, Institute of Biotechnology. Viikinkaari 9, Helsinki, Finland, 00014.
The actin cytoskeleton plays a central role in a number of cellular processes involving membrane dynamics. However, the exact mechanisms by which the polymerizing actin filaments interact with cellular membranes remain poorly understood. A central group of proteins functioning at the interface between plasma membrane and the actin cytoskeleton are the I-BAR family proteins. Mammals have five I-BAR family proteins, which all contain an N-terminal membrane binding I-BAR domain and an actin monomer binding WH2 domain at the C-terminus. All I-BAR domains studied so far - MIM, IRSp53, IRTKS and ABBA - induce filopodia formation when expressed in cells and deform PI(4,5)P2-rich membranes to tubular structures in vitro. We show that, in contrast to four other relatively widely expressed I-BAR proteins, the fifth member of the family, PInB, is only expressed in the epithelial cells of the bowel and in the kidney. Interestingly, the biochemical properties of the PInB BAR domain differ from the other I-BAR domains. Electron tomography analysis revealed that PInB BAR domain induces a formation of narrow sheet-like structures and does not deform membranes into tubular structures like other I-BAR domains. We also determined the crystal structure of the BAR domain of PInB. The structure displays significant differences, both in charge distribution and overall bending, compared to the I-BAR domains of MIM and IRSp53. The biochemical data and crystal packing contacts suggest a mechanism of cooperative self-association during membrane binding. Thus, PInB appears to regulate membrane dynamics through at least partially different mechanism compared to other I-BAR domain proteins.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University ADF/cofilin binds phosphoinositides in a multivalent
manner to act as a PIP2-density sensor
Hongxia Zhao, Markku Hakala and Pekka Lappalainen
Institute of Biotechnology, University of Helsinki. Viikinkaari 9, Helsinki, Finland.
Actin-depolymerizing-factor (ADF)/cofilins have emerged as key regulators of cytoskeletal dynamics in cell motility, morphogenesis, endocytosis and cytokinesis. The activities of ADF/cofilins are regulated by membrane phospholipid PI(4,5)P2 in vitro and in cells, but the mechanism of ADF/cofilin - PI(4,5)P2 interaction has remained controversial. Interestingly, recent studies suggested that ADF/cofilins interact with PI(4,5)P2 through a specific binding pocket and that this interaction is dependent on pH. Here, we combined systematic mutagenesis with biochemical and spectroscopic methods to elucidate the phosphoinositide-binding mechanism of ADF/ cofilins. Our analysis revealed that cofilin does not harbor a specific PI(4,5)P2- binding pocket, but instead interacts with PI(4,5)P2 through a large positively charged surface of the molecule. Importantly, cofilin interacts simultaneously with multiple PI(4,5)P2 headgroups in a cooperative manner. Consequently, interactions of cofilin with membranes and actin exhibit sharp sensitivity to PI(4,5)P2-density. Finally, we show that cofilin binding to PI(4,5)P2 is not sensitive to changes in the pH at physiological salt concentration, although the PI(4,5)P2-clustering activity of cofilin is moderately inhibited at elevated pH. Collectively, our data demonstrating that ADF/cofilins bind PI(4,5)P2 headgroups through multivalent, cooperative mechanism, suggest that the actin filament disassembly activity of ADF/cofilin can be accurately regulated by small changes in the PI(4,5)P2-density at cellular membranes.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Function of FAP overexpression and its potent
inhibitor in cancer progression
Hsin-Ying Lee; Hsiang-Yun Tang; Chia-Hui Chien; Xin Chen
Division of Biotechnology and Pharmaceutical Research, National Health Research Institut. 35 Keyan, Zhunan, Miaoli county 350, Taiwan, 35053 Fibroblast activation protein (FAP) is a type II membrane protease, with prolyl- cleaving endopeptidase activity. It is exclusively expressed in embryonic mesenchyme, wounded tissues and activated stromal fibroblasts in more than 90% of the malignant epithelial tumors, but not in benign tumors or normal adult cells. As the results, FAP has been speculated to be a drug target for cancer treatment. However, the role of FAP in cancer remains to be investigated. Other than that, there is no potent and selective FAP chemical inhibitors have been reported. To understand its function, we have developed potent and selective FAP inhibitors. These inhibitors are slow-tight binding inhibitors of FAP. Using these chemical inhibitors, we have evaluated the effects of inhibiting FAP enzymatic activity in colony formation, wound healing, migration/invasion and growth.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Membrane topology of human NPC1L1, a key
protein in enterohepatic cholesterol absorption
Jiang Wang, Bei-Bei Chu, Liang Ge, Bo-Liang Li, Yan Yan, Bao-Liang Song
Institute of Biochemistry and Cell Biology. 320 Yueyang Road, Shanghai, China,200031 The Niemann-Pick C1-like 1 (NPC1L1) is a predicted polytopic membrane protein that is critical for cholesterol absorption. NPC1L1 takes up free cholesterol into cells through vesicular endocytosis. Ezetimibe, a clinically used cholesterol absorption inhibitor, blocks the endocytosis of NPC1L1 thereby inhibiting cholesterol uptake. Human NPC1L1 is a 1332-amino acid protein with a putative sterol-sensing domain (SSD) that shows sequence homology to HMG-CoA reductase (HMGCR), Niemann- Pick C1 (NPC1) and SREBP cleavage-activating protein (SCAP). Here, we use protease protection and immunofluorescence in selectively permeabilized cells to study the topology of NPC1L1. Our data indicate that NPC1L1 contains 13 transmembrane helices. The NH2-terminus of NPC1L1 is in the lumen while the COOH-terminus projects to the cytosol. NPC1L1 contains 7 small cytoplasmic loops, 4 small and 3 large luminal loops, one of which has been reported to bind ezetimibe. Ezetimibe-glucuronide, the major metabolite of ezetimibe in vivo, can block the internalization of NPC1L1 and cholesterol. The membrane topology of NPC1L1 is similar to that of NPC1, and the putative SSD of NPC1L1 is oriented in the same manner as those of HMGCR, SCAP and NPC1. The defined topology of NPC1L1 provides necessary information for further dissecting the functions of different NPC1L1 domains.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Ezetimibe Inhibits Cholesterol Uptake by Blocking
the Sterol-Induced Internalization of NPC1L1
Liang Ge, Jing Wang, Wei Qi, Hong-Hua Miao, Jian Cao, Bo-Liang Li, Yu-Xiu
Qu and Bao-Liang Song
Institute of Biochemistry and Cell Biology. 320 Yueyang Road, Shanghai, China,200031 Niemann-Pick C1 Like 1 (NPC1L1) is a polytopic transmembrane protein that plays a critical role in cholesterol absorption. Ezetimibe, a hypocholesterolemic drug, has been reported to bind NPC1L1 and block cholesterol absorption. However, the molecular mechanism of NPC1L1-mediated cholesterol uptake and how ezetimibe inhibits this process are poorly defined. Here we find that cholesterol specifically promotes the internalization of NPC1L1 and this process requires microfilaments and clathrin/AP2 complex. Blocking NPC1L1 endocytosis dramatically decreases cholesterol internalization, indicating NPC1L1 mediates cholesterol uptake via its vesicular endocytosis. Ezetimibe prevents NPC1L1 from incorporating into clathrin- coated vesicles and thus inhibits cholesterol uptake. Together, our data suggest a model wherein cholesterol is internalized into cells with NPC1L1 through clathrin/ AP2-mediated endocytosis and ezetimibe inhibits cholesterol absorption by blocking the internalization of NPC1L1.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Peng Xu, Juha Okkeri , Susanne Hanisch, Rui-Ying Hu, Qin Xu, Thomas
Günther Pomorski and Xiao-Yan Ding
Institute of Biochemistry and Cell Biology. Yueyang Road 320, Old Biochemistry Building 718, Shanghai, China, 200031 P4-ATPases are transmembrane proteins unique to eukaryotes that play a fundamental role in vesicular transport. They have been proposed to act as phospholipid flippases thereby regulating lipid topology in cellular membranes. We cloned and characterized a novel murine P4-ATPase that is specifically expressed in testis, and named it FetA (flippase expressed in testis splicing form A). When expressed in , FetA localizes partially to the plasma membrane resulting in increased internalization of NBD- labeled phosphatidylethanolamine and phosphatidylcholine, supporting a role for FetA in the inward lipid translocation across cellular membranes. In mouse testis, FetA protein is detected in gamete cells, from pachytene spermatocytes to mature sperms, and its intracellular localization is tightly related with acrosome formation, a process that involves intensive intracellular vesicle formation and fusion. Furthermore, loss-of-function of FetA by RNA interference in mastocytoma P815 cells profoundly perturbs the structural organization of the Golgi complex and causes loss of constitutive secretion at lower temperature. Our findings point to an essential role of FetA in Golgi morphology and secretory function, suggesting a crucial role for this novel murine P4-ATPase in spermatogenesis.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Membrane Deformation by Exo70 In Cell Migration
Yuting Zhao and Wei Guo
Department of Biology, University of Pennsylvania. 433 S University Ave Lynch Lab 305, Philadelphia, United States.
Cell migration requires actin organization and plasma membrane remodeling at the leading edge. Previously, we showed that Exo70, a component of the exocyst complex essential for targeting secretory vesicles to the plasma membrane, regulates cell migration[1]. Exo70 interacts directly with actin nucleator Arp2/3 complex[1] and phospholipids such as PIP(4,5)P2[2]. Overexpression of Exo70 induces extensive actin-based membrane protrusions. Here we report that Exo70 can induce tubular invaginations in PIP2-rich synthetic liposomes resembling those induced by I-BAR proteins. We propose that Exo70 can deform plasma membrane in addition to its role of regulating actin cytoskeleton dynamics during cell migration.
[1] Exo70 interacts with the Arp2/3 complex and regulates cell migration. Zuo X, Zhang J, Zhang Y, Hsu SC, Zhou D, Guo W. Nat Cell Biol. 2006 Dec;8(12):1383-8.
[2] Phosphatidylinositol 4,5-bisphosphate mediates the targeting of the exocyst to the plasma membrane for exocytosis in mammalian cells. Liu J, Zuo X, Yue P, Guo W. Mol Biol Cell. 2007 Nov;18(11):4483-92.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins Nankai University Effects of Fatty Acids on Skeletal Muscle Glucose
Uptake: Two Sides of the Coin
Gong Peng, Jing Pu, Linghai Li, and Pingsheng Liu
Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Plasma fatty acid concentration is significantly elevated during fasting similar as plasma glucose wave after food ingestion. The higher fatty acid level in blood is tightly correlated with insulin resistance in obese and diabetic patients. We examined how palmitic acid (PA), one of the major saturated dietary fatty acids affects skeletal muscle glucose uptake. Using both cell and animal model systems, in a short-term treatment (within 3 h) we found that PA could stimulate GLUT4 translocation and glucose uptake through the activation of Akt. Conversely, in a long-term treatment (over 6 h) PA could reduce insulin-stimulated glucose uptake and Akt phosphorylation by enhancing ER stress. On the other hand, another major dietary fatty acid, a monounsaturated oleic acid (OA) itself did not affect insulin signal but could abolish PA-mediated ER stress and recover Akt phosphorylation. Our data suggest the dual effects of PA on skeletal muscle glucose uptake and the competitive regulation of insulin sensitivity by OA and PA via controlling ER stress.
2010 Nankai Symposium: Membrane Shaping and Remodeling by Proteins

Source: http://sky.nankai.edu.cn/upfiles/201005/Program_Abstract.pdf