DOCSLIB.ORG

SNARE Motif-Mediated Sorting of Synaptobrevin by the Endocytic Adaptors Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) and AP180 at Synapses

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
SNARE Motif-Mediated Sorting of Synaptobrevin by the Endocytic Adaptors Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) and AP180 at Synapses SNARE motif-mediated sorting of synaptobrevin by the endocytic adaptors clathrin assembly lymphoid myeloid leukemia (CALM) and AP180 at synapses Seong Joo Kooa, Stefan Markovicb, Dmytro Puchkova, Carsten C. Mahrenholzc, Figen Beceren-Braund, Tanja Maritzena, Jens Dernedded, Rudolf Volkmerc, Hartmut Oschkinatb, and Volker Hauckea,b,1 aInstitute of Chemistry and Biochemistry, NeuroCure Cluster of Excellence, Freie Universität Berlin, 14195 Berlin, Germany; bLeibniz-Institut für Molekulare Pharmakologie, 13125 Berlin, Germany; cInstitut für Immunologie, Charité Universitätsmedizin Berlin, 10117 Berlin, Germany; and dZentralinstitut für Laboratoriumsmedizin und Pathobiochemie, Charité Universitätsmedizin Berlin, 12200 Berlin, Germany Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved July 7, 2011 (received for review May 3, 2011) Neurotransmission depends on the exo-endocytosis of synaptic endocytic protein AP180. Unc-11/AP180 mutants in Caenorhabditis vesicles at active zones. Synaptobrevin 2 [also known as vesicle- elegans mislocalize synaptobrevin (13, 14), whereas more general associated membrane protein 2 (VAMP2)], the most abundant syn- endocytic defects are seen in Lap/AP180-deficient Drosophila aptic vesicle protein and a major soluble NSF attachment protein melanogaster strains (15, 16). Whether these phenotypes reflect receptor (SNARE) component, is required for fast calcium-triggered a direct association between AP180 family members and syn- synaptic vesicle fusion. In contrast to the extensive knowledge aptobrevin is unknown. In mammals, the ANTH domain family about the mechanism of SNARE-mediated exocytosis, little is known consists of two members, clathrin assembly lymphoid myeloid about the endocytic sorting of synaptobrevin 2. Here we show that leukemia (CALM) and AP180. AP180 is exclusively expressed in synaptobrevin 2 sorting involves determinants within its SNARE mo- neurons, where it accumulates at nerve terminals (17). By con- tif that are recognized by the ANTH domains of the endocytic adap- trast, CALM is a ubiquitous protein found in both neurons and tors AP180 and clathrin assembly lymphoid myeloid leukemia glia and in nonneuronal tissues (18). Though both CALM and (CALM). Depletion of CALM or AP180 causes selective surface accu- AP180 have been implicated in regulating neurite outgrowth mulation of synaptobrevin 2 but not vGLUT1 at the neuronal sur- (19), their relationship to SV cycling has not been explored. face. Endocytic sorting of synaptobrevin 2 is mediated by direct Here we demonstrate that endocytic sorting of synaptobrevin 2 interaction of the ANTH domain of the related endocytic adaptors is achieved by direct interaction of the ANTH domain of the CALM and AP180 with the N-terminal half of the SNARE motif cen- endocytic adaptors CALM and AP180 with the N-terminal half of tered around M46, as evidenced by NMR spectroscopy analysis and the SNARE motif. These data suggest a unique mechanism of site-directed mutagenesis. Our data unravel a unique mechanism of SNARE motif-dependent endocytic sorting and identify the SNARE motif-dependent endocytic sorting and identify the ANTH fi fi ANTH domain proteins AP180 and CALM as cargo-speci c domain proteins AP180 and CALM as cargo-speci c adaptors for adaptors for synaptobrevin endocytosis in the central nervous synaptobrevin endocytosis. Defective SNARE endocytosis may also system. Our work further contributes to the notion that cargo- underlie the association of CALM and AP180 with neurodevelop- selective mechanisms operate at synapses to maintain the high mental and cognitive defects or neurodegenerative disorders. fidelity of SV protein sorting and recycling. clathrin-mediated endocytosis | structure Results Depletion of the ANTH Domain Proteins AP180 or CALM Causes eurotransmission in the brain depends on the calcium-trig- Selective Surface Accumulation of Synaptobrevin 2. To explore the fi Ngered fusion and recycling of neurotransmitter- lled synaptic possible role of the related ANTH domain proteins AP180 and vesicles (SVs) with the presynaptic membrane at active zones (1). CALM in SV recycling, we knocked down either or both proteins Following their exocytic insertion into the presynaptic membrane, by RNA interference. CALM- or AP180-specific siRNA effi- fi SV proteins need to be retrieved at a precisely de ned stoichi- ciently down-regulated expression of their corresponding target ometry by endocytosis, a process involving clathrin, adaptors, and protein in transfected HEK293 cells (Fig. 1A) and in primary other endocytic proteins (2). Fast calcium-triggered SV fusion hippocampal neurons in culture (Fig. S1A) (19). Under the critically depends on the SV arginine (R)-soluble NSF attachment conditions used, depletion of CALM or AP180 did not adversely protein receptor (SNARE) synaptobrevin [or vesicle-associated affect electrical excitability of neurons or stimulation-dependent membrane protein (VAMP)], which by forming a complex with exocytosis as probed by exo-endocytic cycling of the styryl dye the plasma membrane glutamine (Q)-SNAREs syntaxin and FM4-64 (Fig. 1 B and C). synaptosomal-associated protein (SNAP)-25 (3) drives neuro- < To assess the role of CALM and AP180 in synaptobrevin 2 en- exocytosis (4, 5). Synapses lacking synaptobrevin 2 display 1% docytosis, we made use of superecliptic pHluorin-tagged synapto- of wild-type release when stimulated by action potential (AP)- brevin 2 (synaptopHluorin). Fluorescence of synaptopHluorin mediated calcium influx (6). Proteomic studies have shown that synaptobrevin 2 is a highly abundant SV protein (7) that is exo- endocytically sorted with very high precision (8). Similar obser- Author contributions: D.P., H.O., and V.H. designed research; S.J.K., S.M., D.P., C.C.M., vations have been made for other SV proteins, including syn- F.B.-B., and T.M. performed research; C.C.M. and R.V. contributed new reagents/analytic aptotagmin and vesicular glutamate transporters (VGLUTs). tools S.J.K., S.M., D.P., F.B.-B., J.D., R.V., H.O., and V.H. analyzed data; and S.J.K., H.O., and How such precise sorting of synaptobrevin 2 is achieved has V.H. wrote the paper. remained enigmatic. Synaptobrevin lacks recognizable linear The authors declare no conflict of interest. sorting motifs (9), and unlike other SNARE proteins does not This article is a PNAS Direct Submission. contain a folded N-terminal domain that serves as a targeting Freely available online through the PNAS open access option. determinant in other VAMP family members (10–12). 1To whom correspondence should be addressed. E-mail: [email protected]. Genetic data have linked synaptobrevin sorting to the function This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of the AP180 N-terminal homology (ANTH) domain-containing 1073/pnas.1107067108/-/DCSupplemental. 13540–13545 | PNAS | August 16, 2011 | vol. 108 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1107067108 Downloaded by guest on October 1, 2021 pHluorin, indicative of its impaired endocytic retrieval. This effect was augmented in neurons depleted of both ANTH domain pro- teins (Fig. 1D), suggesting that AP180 and CALM serve overlaping functions in synaptobrevin 2 internalization. To test whether loss of CALM or AP180 selectively impairs synaptobrevin 2 endocytosis, or whether other SV proteins are also affected, we assessed the partitioning of pHluorin-tagged vGLUT1, another abundant SV component at excitatory synapses. To our surprise, knockdown of either CALM or AP180 or of both proteins together had no effect on the vesicular-to-surface pool ratio of VGLUT1-pHluorin (Fig. 1E). These effects were corroborated by analyzing the axonal dis- tribution of synaptopHluorin and vGLUT1-pHluorin. A partial dispersion of synaptopHluorin but not of vGLUT1-pHluorin was observed in double-knockdown neurons depleted of both CALM and AP180 (Fig. 1F). Hence, CALM and AP180 exert an over- lapping cargo-specific role in the endocytic retrieval of synapto- brevin 2 from the neuronal surface. Synaptic Vesicle Size Is Altered in AP180-Depleted Neurons. AP180 (termed Lap in flies) mutants in D. melanogaster display enlarged and heterogenously sized SVs (16). To explore possible alter- ations in SV size we turned to ultrastructural analysis by electron microscopy. Synapses from transfected neurons were identified by immunogold labeling using antibodies against eGFP cotrans- fected together with specific siRNA targeting AP180 or CALM. Nerve terminals from AP180- or CALM-depleted neurons were morphologically normal (Fig. S2A) and showed no significant change in SV density compared with those from control neurons (Fig. 1H). However, SVs from AP180 knockdown neurons appeared slightly larger and more heterogenous, as evidenced by Fig. 1. Depletion of AP180 and CALM causes surface accumulation of syn- – aptobrevin 2. (A)Efficient siRNA knockdown of AP180 or CALM expression. the increased variance of SV size (Fig. S2 A C). Similar alter- HEK293 cells transiently expressing rat AP180 were transfected with siRNA ations were observed in neurons depleted of both AP180 and specific for rat AP180 (Left) or CALM (Right) and analyzed by immunoblot- CALM, whereas SV size was unchanged in CALM-depleted ting. Expression levels of AP180 or CALM are efficiently down-regulated. (B neurons (Fig. 1G and Fig. S2 B and C). As alterations in SV size and C) SV exocytosis probed by FM4-64 in AP180-, CALM-, or AP180- and have also been observed in Lap/AP180 mutants in D. mela- CALM-depleted neurons.
Load more

Recommended publications
  • Sorting Nexins in Protein Homeostasis Sara E. Hanley1,And Katrina F
    Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 6 November 2020 doi:10.20944/preprints202011.0241.v1 Sorting nexins in protein homeostasis Sara E. Hanley1,and Katrina F. Cooper2* 1Department of Molecular Biology, Graduate School of Biomedical Sciences, Rowan University, Stratford, NJ, 08084, USA 1 [email protected] 2 [email protected] * [email protected] Tel: +1 (856)-566-2887 1Department of Molecular Biology, Graduate School of Biomedical Sciences, Rowan University, Stratford, NJ, 08084, USA Abstract: Sorting nexins (SNXs) are a highly conserved membrane-associated protein family that plays a role in regulating protein homeostasis. This family of proteins is unified by their characteristic phox (PX) phosphoinositides binding domain. Along with binding to membranes, this family of SNXs also comprises a diverse array of protein-protein interaction motifs that are required for cellular sorting and protein trafficking. SNXs play a role in maintaining the integrity of the proteome which is essential for regulating multiple fundamental processes such as cell cycle progression, transcription, metabolism, and stress response. To tightly regulate these processes proteins must be expressed and degraded in the correct location and at the correct time. The cell employs several proteolysis mechanisms to ensure that proteins are selectively degraded at the appropriate spatiotemporal conditions. SNXs play a role in ubiquitin-mediated protein homeostasis at multiple levels including cargo localization, recycling, degradation, and function. In this review, we will discuss the role of SNXs in three different protein homeostasis systems: endocytosis lysosomal, the ubiquitin-proteasomal, and the autophagy-lysosomal system. The highly conserved nature of this protein family by beginning with the early research on SNXs and protein trafficking in yeast and lead into their important roles in mammalian systems.
    [Show full text]
  • SNARE Proteins and the Timing of Neurotransmitter Release
    Molecular Psychiatry (1998) 3, 293–297 1998 Stockton Press All rights reserved 1359–4184/98 $12.00 NEWS & VIEWS SNARE proteins and the timing of neurotransmitter release The SNARE complex proteins have been implicated in exocytotic neurotransmitter release and other forms of membrane fusion. Recent work shows that NSF, the ATPase of the SNARE complex, regulates the kinetics of neurotransmitter release and can thereby control the inte- grative properties of synapses. Time is one of the most critical parameters in the func- hydrolyzes ATP. Because SNAREs are found on both tioning of the brain. Information transfer on the time- the synaptic vesicle membrane and the plasma mem- scale of milliseconds (10−3 seconds) is typical through- brane, it has been postulated that the various SNARE out the brain and in certain brain regions, such as the complexes mediate the interaction between the two auditory brainstem, time differences on the order of membranes before fusion and thus may be necessary microseconds (10−6 seconds) are used to define the fre- for neurotransmitter release.2 quency and location of perceived sounds. Thus infor- Evidence for a role for SNARE proteins in neuro- mation processing not only depends on a fast underly- transmitter release has come from a variety of sources. ing process but also on the precise timing of synaptic The most compelling indication of the central impor- activity. Such high temporal fidelity must rely upon tance of the three membrane SNARE proteins is that very finely-regulated molecular mechanisms. However, these proteins are remarkably specific targets of tetanus until recently the identity of these mechanisms has and botulinum toxins, a group of potent neurotoxins been remarkably elusive.
    [Show full text]
  • Endoplasmic Reticulum to Golgi Transport Factor Usolp STEPHANIE K
    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 522-526, January 1995 Cell Biology p115 is a general vesicular transport factor related to the yeast endoplasmic reticulum to Golgi transport factor Usolp STEPHANIE K. SAPPERSTEIN*, DAVID M. WALTER*, ALEXANDRA R. GROSVENOR*, JOHN E. HEUSERt, AND M. GERARD WATERS*t *Department of Molecular Biology, Princeton University, Princeton, NJ 08544; and tDepartment of Cell Biology, Washington University School of Medicine, St. Louis, MO 63110 Communicated by Arnold J. Levine, Princeton University, Princeton, NJ, September 7, 1994 (received for review August 4, 1994) ABSTRACT A recently discovered vesicular transport fac- number of proteins involved in vesicle budding-for example, tor, termed p115, is required along with N-ethylmaleimide- coatomer and ARF (ADP-ribosylation factor)-are homolo- sensitive fusion protein (NSF) and soluble NSF attachment gous in yeast and mammals (15-17). In general, it appears that proteins for in vitro Golgi transport. p115 is a peripheral the molecules and mechanisms used by mammals and yeast are membrane protein found predominantly on the Golgi. Biochem- quite similar (18-20). ical and electron microscopic analyses indicate that p115 is an Recently, a set of membrane proteins collectively called elongated homodimer with two globular "heads" and an ex- SNAREs (SNAP receptors) has been shown to form an tended "tail" reminiscent of myosin II. We have cloned and oligomeric complex with NSF and SNAPs (21, 22). The sequenced cDNAs for bovine and rat p115. The predicted trans- SNAREs include VAMP (vesicle-associated membrane pro- lation products are 90%o identical, and each can be divided into tein; also called synaptobrevin), syntaxin, and SNAP-25 (syn- three domains.
    [Show full text]
  • An Arf1 Synthetic Lethal Screen Identifies a New Clathrin Heavy
    Copyright 1998 by the Genetics Society of America An arf1D Synthetic Lethal Screen Identi®es a New Clathrin Heavy Chain Conditional Allele That Perturbs Vacuolar Protein Transport in Saccharomyces cerevisiae Chih-Ying Chen and Todd R. Graham Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235 Manuscript received March 5, 1998 Accepted for publication June 16, 1998 ABSTRACT ADP-ribosylation factor (ARF) is a small GTP-binding protein that is thought to regulate the assembly of coat proteins on transport vesicles. To identify factors that functionally interact with ARF, we have performed a genetic screen in Saccharomyces cerevisiae for mutations that exhibit synthetic lethality with an arf1D allele and de®ned seven genes by complementation tests (SWA1-7 for synthetically lethal with arf1D). Most of the swa mutants exhibit phenotypes comparable to arf1D mutants such as temperature-conditional growth, hypersensitivity to ¯uoride ions, and partial protein transport and glycosylation defects. Here, we report that swa5-1 is a new temperature-sensitive allele of the clathrin heavy chain gene (chc1-5), which carries a frameshift mutation near the 39 end of the CHC1 open reading frame. This genetic interaction between arf1 and chc1 provides in vivo evidence for a role for ARF in clathrin coat assembly. Surprisingly, strains harboring chc1-5 exhibited a signi®cant defect in transport of carboxypeptidase Y or carboxypepti- dase S to the vacuole that was not observed in other chc1 ts mutants. The kinetics of invertase secretion or transport of alkaline phosphatase to the vacuole were not signi®cantly affected in the chc1-5 mutant, further implicating clathrin speci®cally in the Golgi to vacuole transport pathway for carboxypeptidase Y.
    [Show full text]
  • ADP-Ribosylation Factor, a Small GTP-Binding Protein, Is Required for Binding of the Coatomer Protein Fl-COP to Golgi Membranes JULIE G
    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6408-6412, July 1992 Biochemistry ADP-ribosylation factor, a small GTP-binding protein, is required for binding of the coatomer protein fl-COP to Golgi membranes JULIE G. DONALDSON*, DAN CASSEL*t, RICHARD A. KAHN*, AND RICHARD D. KLAUSNER* *Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, and tLaboratory of Biological Chemistry, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Communicated by Marc Kirschner, April 20, 1992 (receivedfor review February 11, 1992) ABSTRACT The coatomer is a cytosolic protein complex localized to the Golgi complex, although their functions have that reversibly associates with Golgi membranes and is Impli- not been defined. Distinct among these proteins is the ADP- cated in modulating Golgi membrane transport. The associa- ribosylation factor (ARF), originally identified as a cofactor tion of 13-COP, a component of coatomer, with Golgi mem- required for in vitro cholera toxin-catalyzed ADP- branes is enhanced by guanosine 5'-[v-thioltriphosphate ribosylation of the a subunit of the trimeric GTP-binding (GTP[yS]), a nonhydrolyzable analogue of GTP, and by a protein G, (G,.) (19). ARF is an abundant cytosolic protein mixture of aluminum and fluoride ions (Al/F). Here we show that reversibly associates with Golgi membranes (20, 21). that the ADP-ribosylation factor (ARF) is required for the ARF has been shown to be present on Golgi coated vesicles binding of (-COP. Thus, 13-COP contained in a coatomer generated in the presence of GTP[yS], but it is not a com- fraction that has been resolved from ARF does not bind to Golgi ponent of the cytosolic coatomer (22).
    [Show full text]
  • Mechanisms of Synaptic Plasticity Mediated by Clathrin Adaptor-Protein Complexes 1 and 2 in Mice
    Mechanisms of synaptic plasticity mediated by Clathrin Adaptor-protein complexes 1 and 2 in mice Dissertation for the award of the degree “Doctor rerum naturalium” at the Georg-August-University Göttingen within the doctoral program “Molecular Biology of Cells” of the Georg-August University School of Science (GAUSS) Submitted by Ratnakar Mishra Born in Birpur, Bihar, India Göttingen, Germany 2019 1 Members of the Thesis Committee Prof. Dr. Peter Schu Institute for Cellular Biochemistry, (Supervisor and first referee) University Medical Center Göttingen, Germany Dr. Hans Dieter Schmitt Neurobiology, Max Planck Institute (Second referee) for Biophysical Chemistry, Göttingen, Germany Prof. Dr. med. Thomas A. Bayer Division of Molecular Psychiatry, University Medical Center, Göttingen, Germany Additional Members of the Examination Board Prof. Dr. Silvio O. Rizzoli Department of Neuro-and Sensory Physiology, University Medical Center Göttingen, Germany Dr. Roland Dosch Institute of Developmental Biochemistry, University Medical Center Göttingen, Germany Prof. Dr. med. Martin Oppermann Institute of Cellular and Molecular Immunology, University Medical Center, Göttingen, Germany Date of oral examination: 14th may 2019 2 Table of Contents List of abbreviations ................................................................................. 5 Abstract ................................................................................................... 7 Chapter 1: Introduction ............................................................................
    [Show full text]
  • SNAP-24, a Novel Drosophila SNARE Protein 4057 Proteins Were Purified on Glutathione Beads and Cleaved from the GST Fig
    Journal of Cell Science 113, 4055-4064 (2000) 4055 Printed in Great Britain © The Company of Biologists Limited 2000 JCS1894 SNAP-24, a Drosophila SNAP-25 homologue on granule membranes, is a putative mediator of secretion and granule-granule fusion in salivary glands Barbara A. Niemeyer*,‡ and Thomas L. Schwarz§ Department of Molecular and Cellular Physiology, Stanford Medical School, Stanford, CA 94305, USA *Present address: Department of Pharmacology and Toxicology, School of Medicine, University of Saarland, D-66421 Homburg, Germany ‡Author for correspondence (e-mail: [email protected]) §Present address: Harvard Medical School, Division of Neuroscience, The Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, USA Accepted 16 September; published on WWW 31 October 2000 SUMMARY Fusion of vesicles with target membranes is dependent is not concentrated in synaptic regions. In vitro studies, on the interaction of target (t) and vesicle (v) SNARE however, show that SNAP-24 can form core complexes with (soluble NSF (N-ethylmaleimide-sensitive fusion protein) syntaxin and both synaptic and non-synaptic v-SNAREs. attachment protein receptor) proteins located on opposing High levels of SNAP-24 are found in larval salivary glands, membranes. For fusion at the plasma membrane, the t- where SNAP-24 localizes mainly to granule membranes SNARE SNAP-25 is essential. In Drosophila, the only rather than the plasma membrane. During glue secretion, known SNAP-25 isoform is specific to neuronal axons and the massive exocytotic event of these glands, SNAP-24 synapses and additional t-SNAREs must exist that mediate containing granules fuse with one another and the apical both non-synaptic fusion in neurons and constitutive and membrane, suggesting that glue secretion utilizes regulated fusion in other cells.
    [Show full text]
  • Is Synaptotagmin the Calcium Sensor? Motojiro Yoshihara, Bill Adolfsen and J Troy Littleton
    315 Is synaptotagmin the calcium sensor? Motojiro Yoshihara, Bill Adolfsen and J Troy Littletonà After much debate, recent progress indicates that the synaptic synaptotagmins, which are transmembrane proteins con- vesicle protein synaptotagmin I probably functions as the taining tandem calcium-binding C2 domains (C2A and calcium sensor for synchronous neurotransmitter release. C2B) (Figure 1a). Synaptotagmin I is an abundant cal- Following calcium influx into presynaptic terminals, cium-binding synaptic vesicle protein [8,9] that has been synaptotagmin I rapidly triggers the fusion of synaptic vesicles demonstrated via genetic studies to be important for with the plasma membrane and underlies the fourth-order efficient synaptic transmission in vivo [10–13]. The C2 calcium cooperativity of release. Biochemical and genetic domains of synaptotagmin I bind negatively-charged studies suggest that lipid and SNARE interactions underlie phospholipids in a calcium-dependent manner [9,14,15, synaptotagmin’s ability to mediate the incredible speed of 16–18]. There is compelling evidence that phospholipid vesicle fusion that is the hallmark of fast synaptic transmission. binding is an effector interaction in vesicle fusion, as the calcium dependence of this process ( 74 mM) and its Addresses rapid kinetics (on a millisecond scale) (Figure 1b) fit Picower Center for Learning and Memory, Department of Biology and reasonably well with the predicted requirements of Department of Brain and Cognitive Sciences, Massachusetts synaptic transmission [15]. In addition to phospholipid Institute of Technology, Cambridge, MA 02139, USA Ãe-mail: [email protected] binding, the calcium-stimulated interaction between synaptotagmin and the t-SNAREs syntaxin and SNAP- 25 [15,19–23] provides a direct link between calcium and Current Opinion in Neurobiology 2003, 13:315–323 the fusion complex.
    [Show full text]
  • Dynamin Action at the Final Stage of Clathrin- Mediated Endocytosis
    Dynamin Action at the Final Stage of Clathrin- Mediated Endocytosis Ning Fang ( [email protected] ) Georgia State University https://orcid.org/0000-0003-4710-0984 Xiaodong Cheng Georgia State University Kuangcai Chen Georgia State University https://orcid.org/0000-0002-9321-9225 Bin Dong Georgia State University https://orcid.org/0000-0002-3196-0712 Meek Yang Georgia State University Seth Filbrun Georgia State University Yong Myoung Georgia State University Teng-Xiang Huang Georgia State University Yan Gu The Bristol-Myers Squibb Company Gufeng Wang Georgia State University Article Keywords: Dynamin, clathrin-mediated endocytosis, scission, vesicle ssion Posted Date: February 5th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-134570/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/25 Abstract Dynamin plays an important role in clathrin-mediated endocytosis by cutting the neck of nascent vesicles from the cell membrane. Gold nanorods were used as imaging probes to observe dynamin action on cargo vesicles during live endocytosis events. Invariant is that at the peak of dynamin accumulation, the cargo-containing vesicle always gives abrupt, right-handed rotations that nishes in a short time (~ 0.28 s). The large and quick twist, herein named the super twist, is the result of the coordinated dynamin helix action upon GTP hydrolysis. After the super twist, the rotational freedom of the vesicle drastically increases, accompanied with simultaneous or delayed translational movement, indicating that it detaches from the cell membrane. These observations suggest that dynamin-mediated scission at the nal stage involves a large torque generated by coordinated actions of multiple dynamins in the helix, which is the main driving force for scission.
    [Show full text]
  • Membrane Remodeling in Clathrin-Mediated Endocytosis Volker Haucke1,2,* and Michael M
    © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs216812. doi:10.1242/jcs.216812 REVIEW Membrane remodeling in clathrin-mediated endocytosis Volker Haucke1,2,* and Michael M. Kozlov3,* ABSTRACT membrane fission and vesicle uncoating to eventually allow fusion Clathrin-mediated endocytosis is an essential cellular mechanism by of the nascent endocytic vesicle with endosomes (Box 1). which all eukaryotic cells regulate their plasma membrane composition In spite of important advances in the characterization of these to control processes ranging from cell signaling to adhesion, migration factors, key questions regarding the endocytic process remain. and morphogenesis. The formation of endocytic vesicles and For example, different models have been proposed regarding the tubules involves extensive protein-mediated remodeling of the initiation of clathrin-coated pit (CCP) formation and the onset of plasma membrane that is organized in space and time by protein– membrane curvature acquisition. Moreover, knowledge regarding protein and protein–phospholipid interactions. Recent studies the nanoscale distribution of endocytic proteins within the bilayer combining high-resolution imaging with genetic manipulations of plane during vesicle formation and the mechanisms that guide the endocytic machinery and with theoretical approaches have led their orchestrated assembly and disassembly is only now beginning to novel multifaceted phenomenological data of the temporal and to emerge. Finally, our understanding of the nature and dynamics spatial organization of the endocytic reaction. This gave rise to of membrane phospholipids (Box 2) and their associations with various – often conflicting – models as to how endocytic proteins endocytic proteins during endocytic membrane remodeling and their association with lipids regulate the endocytic protein remains limited.
    [Show full text]
  • Cryo-Tomography of Coat Complexes ISSN 2059-7983
    research papers Visualizing membrane trafficking through the electron microscope: cryo-tomography of coat complexes ISSN 2059-7983 Evgenia A. Markova and Giulia Zanetti* Institute of Structural and Molecular Biology, Birkbeck College, Malet Street, London WC1E 7HX, England. Received 8 March 2019 *Correspondence e-mail: [email protected] Accepted 12 April 2019 Coat proteins mediate vesicular transport between intracellular compartments, Keywords: cryo-electron tomography; which is essential for the distribution of molecules within the eukaryotic cell. three-dimensional reconstruction; coat proteins; The global arrangement of coat proteins on the membrane is key to their vesicular transport; COPII; subtomogram function, and cryo-electron tomography and subtomogram averaging have been averaging. used to study membrane-bound coat proteins, providing crucial structural insight. This review outlines a workflow for the structural elucidation of coat proteins, incorporating recent developments in the collection and processing of cryo-electron tomography data. Recent work on coat protein I, coat protein II and retromer performed on in vitro reconstitutions or in situ is summarized. These studies have answered long-standing questions regarding the mechanisms of membrane binding, polymerization and assembly regulation of coat proteins. 1. Introduction Recent advances in cryo-electron microscopy (cryo-EM) have enabled numerous high-resolution studies which have addressed long-standing structural biology questions. The large body of cryo-EM work carried out for protein structure characterization has utilized single-particle electron micro- scopy (Cheng, 2018). Recently, developments in hardware, data collection and data processing have placed cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) at the forefront of structural studies of repetitive assemblies, reaching resolutions comparable to those of single-particle EM (Schur et al., 2016; Wan et al., 2017; Hutchings et al., 2018; Dodonova et al., 2017; Himes & Zhang, 2018).
    [Show full text]
  • Molecular Mechanism of Fusion Pore Formation Driven by the Neuronal SNARE Complex
    Molecular mechanism of fusion pore formation driven by the neuronal SNARE complex Satyan Sharmaa,1 and Manfred Lindaua,b aLaboratory for Nanoscale Cell Biology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany and bSchool of Applied and Engineering Physics, Cornell University, Ithaca, NY 14850 Edited by Axel T. Brunger, Stanford University, Stanford, CA, and approved November 1, 2018 (received for review October 2, 2018) Release of neurotransmitters from synaptic vesicles begins with a systems in which various copy numbers of syb2 were incorporated narrow fusion pore, the structure of which remains unresolved. To in an ND while the t-SNAREs were present on a liposome have obtain a structural model of the fusion pore, we performed coarse- been used experimentally to study SNARE-mediated mem- grained molecular dynamics simulations of fusion between a brane fusion (13, 17). The small dimensions of the ND compared nanodisc and a planar bilayer bridged by four partially unzipped with a spherical vesicle makes such systems ideally suited for MD SNARE complexes. The simulations revealed that zipping of SNARE simulations without introducing extreme curvature, which is well complexes pulls the polar C-terminal residues of the synaptobrevin known to strongly influence the propensity of fusion (18–20). 2 and syntaxin 1A transmembrane domains to form a hydrophilic MARTINI-based CGMD simulations have been used in several – core between the two distal leaflets, inducing fusion pore forma- studies of membrane fusion (16, 21 23). To elucidate the fusion tion. The estimated conductances of these fusion pores are in good pore structure and the mechanism of its formation, we performed agreement with experimental values.
    [Show full text]