Exocytotic membrane fusion of synaptic vesicles is driven by the SNAREs syntaxin, SNAP-25, and synaptobrevin/VAMP, which assemble in trans between the membranes in a zipper-like fashion, thus pulling the membranes together and overcoming the energy barrier for fusion. In addition, regulatory proteins including synaptotagmin, Munc18, and complexin interact both with the SNAREs and the participating membranes, thus controlling SNARE reactivity and shaping the specific features of cacium-dependent exocytosis of synaptic vesicles. While the zippering model is intuitively satisfying and confirmed by a large body of evidence, the precise sequence of protein-protein interactions and the site of action of the regulatory proteins along the fusion pathway is still only partially understood and controversially discussed. Here we use in-vitro fusion of native secretory vesicles and liposomes reconstituted with purified proteins to isolate partial reactions of the fusion pathway, to understand the structure, dynamics and stoichiometries of the intermediate states of the participating proteins, and to determine the parameters affecting kinetics of each reaction. In the previous funding period, we solved the ci7stal structure of the neuronal SNARE complex with its linkers and transmembrane domains and characterized the interaction of synaptotagmin with SNARE-containing liposomes. Moreover, we developed refined assays for measuring fusion intermediates including fluorescence cross-correlation and cryo electron microscopy, studied the fusion of synaptic vesicles with SNARE-containing liposomes, and interfered with SNARE assembly at the partially or fully zippered state, thus gaining access to intermediate states of the fusion pathway. We now propose to take advantage of these achievements to characterize the intermediate steps in SNARE-mediated fusion and to determine the precise step at which the regulatory proteins synaptotagmin 1, complexin 1, and Munc18-1 operate on membrane fusion catalyzed by neuronal SNAREs. In the first specific aim, we plan to elucidate how SNARE assembly and zippering is connected to membrane docking, hemifusion, and fusion using native and artificial vesicles. In the second specific aim, we will determine the steps in SNARE nucleation and zippering that are acted upon by the regulatory proteins synaptotagmin, MunclS, and complexin. The program will be carried out in close collaboration with Projects 2 and 3 in which complementary approaches are pursued. We expect to obtain essential mechanistic information about the molecular mechanism of neuronal exocytosis that is not obtainable by other approaches..

Public Health Relevance

; SNARE-mediated exocytosis is fundamental for synaptic transmission at every central and peripheral synapse of our body. Furthermore, mutations within several of the proteins, particularly MunclS and SNAP- 25, are associated with neurological diseases such as alcoholism, schizophrenia, and attention deficit disorder. Understanding how these proteins work will facilitate new therapeutic approaches.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Program Projects (P01)
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Special Emphasis Panel (ZRG1-BCMB-S)
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University of Virginia
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Yavuz, Halenur; Kattan, Iman; Hernandez, Javier M et al. (2018) Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion. J Biol Chem 293:8645-8655
Liang, Binyong; Tamm, Lukas K (2018) Solution NMR of SNAREs, complexin and ?-synuclein in association with membrane-mimetics. Prog Nucl Magn Reson Spectrosc 105:41-53
Hussain, Syed Saad; Harris, Megan T; Kreutzberger, Alex J B et al. (2018) Control of insulin granule formation and function by the ABC transporters ABCG1 and ABCA1 and by oxysterol binding protein OSBP. Mol Biol Cell 29:1238-1257
Blackburn, Matthew R; Hubbard, Caitlin; Kiessling, Volker et al. (2018) Distinct reaction mechanisms for hyaluronan biosynthesis in different kingdoms of life. Glycobiology 28:108-121
Witkowska, Agata; Jablonski, Lukasz; Jahn, Reinhard (2018) A convenient protocol for generating giant unilamellar vesicles containing SNARE proteins using electroformation. Sci Rep 8:9422
Kiessling, Volker; Kreutzberger, Alex J B; Liang, Binyong et al. (2018) A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis. Nat Struct Mol Biol 25:911-917
Nyenhuis, Sarah B; Cafiso, David S (2018) Choice of reconstitution protocol modulates the aggregation state of full-length membrane-reconstituted synaptotagmin-1. Protein Sci 27:1008-1012
Kreutzberger, Alex J B; Kiessling, Volker; Liang, Binyong et al. (2017) Asymmetric Phosphatidylethanolamine Distribution Controls Fusion Pore Lifetime and Probability. Biophys J 113:1912-1915
Tamm, Lukas K (2017) Special Issue on Liposomes, Exosomes, and Virosomes. Biophys J 113:E1
Jakhanwal, Shrutee; Lee, Chung-Tien; Urlaub, Henning et al. (2017) An activated Q-SNARE/SM protein complex as a possible intermediate in SNARE assembly. EMBO J 36:1788-1802

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