The objective of my proposal is to understand the molecular mechanism of calcium-triggered synaptic exocytosis. I will use single-molecule approaches to investigate how Munc18-1, complexin, synaptotagmin, NSF, and ?-SNAP regulate synaptic SNARE assembly and disassembly. SNARE proteins assemble via multiple intermediates to drive fusion of neurotransmitter-containing vesicles with the presynaptic plasma membrane. Key regulatory proteins target these intermediates to activate and/or inhibit SNARE assembly, enabling synaptic exocytosis to be triggered by the calcium influx induced by an action potential. After fusion, NSF and ?-SNAP disassemble the fully assembled SNARE complexes to recycle SNARE proteins, which is required for frequent neuron communication. The precise, rapid SNARE assembly and disassembly is central to calcium-triggered synaptic exocytosis and underlies our thinking, memory, emotions, and actions. Malfunctions in this process cause various neurological disorders and neurodegenerative diseases. Yet, the mechanism underlying the regulated SNARE assembly and disassembly is poorly understood. We have developed a unique single-molecule manipulation approach based on high-resolution optical tweezers to investigate dynamic SNARE assembly and disassembly. We have identified several intermediates of SNARE assembly and characterized the energies, and kinetics associated with these intermediates as well as the fully assembled SNARE complex for the first time, paving a new road to study SNARE regulation. We will expand our single-molecule approach to address several key questions: (1) What is the essential role of Munc18-1 in SNARE assembly and disassembly? (2) Whether and how complexin and synaptotagmin activate and/or clamp SNARE assembly in a calcium-dependent manner? (3) How NSF and ?-SNAP recognize and disassemble only the fully assembled SNARE complexes, but not the partially assembled SNARE intermediates essential for membrane fusion? Our research will provide unique insights into the mechanisms of regulated SNARE assembly and disassembly, calcium-triggered exocytosis, and associated diseases.

Public Health Relevance

Calcium-triggered synaptic exocytosis is mediated by SNARE proteins and regulated by key proteins including Munc18, complexin, synaptotagmin, NSF, and alpha-SNAP with high precision and speed. SNAREs couple their dynamic assembly and disassembly to drive frequent exocytosis. We will characterize how SNARE assembly and disassembly is accurately regulated by the key regulatory proteins to enable calcium-triggered exocytosis.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Flicker, Paula F
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Yale University
Anatomy/Cell Biology
Schools of Medicine
New Haven
United States
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Rebane, Aleksander A; Wang, Bigeng; Ma, Lu et al. (2018) Two Disease-Causing SNAP-25B Mutations Selectively Impair SNARE C-terminal Assembly. J Mol Biol 430:479-490
Zhang, Yongli; Ha, Taekjip; Marqusee, Susan (2018) Editorial Overview: Single-Molecule Approaches up to Difficult Challenges in Folding and Dynamics. J Mol Biol 430:405-408
Zhang, Yongli (2017) Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers. Protein Sci 26:1252-1265
Jiao, Junyi; Rebane, Aleksander A; Ma, Lu et al. (2017) Single-Molecule Protein Folding Experiments Using High-Precision Optical Tweezers. Methods Mol Biol 1486:357-390
Ma, Lu; Cai, Yiying; Li, Yanghui et al. (2017) Single-molecule force spectroscopy of protein-membrane interactions. Elife 6:
Ma, Lu; Kang, Yuhao; Jiao, Junyi et al. (2016) ?-SNAP Enhances SNARE Zippering by Stabilizing the SNARE Four-Helix Bundle. Cell Rep 15:531-539
Zhang, Xinming; Rebane, Aleksander A; Ma, Lu et al. (2016) Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex. Proc Natl Acad Sci U S A 113:E8031-E8040
Rebane, Aleksander A; Ma, Lu; Zhang, Yongli (2016) Structure-Based Derivation of Protein Folding Intermediates and Energies from Optical Tweezers. Biophys J 110:441-454
Zhang, Yongli; Jiao, Junyi; Rebane, Aleksander A (2016) Hidden Markov Modeling with Detailed Balance and Its Application to Single Protein Folding. Biophys J 111:2110-2124
Clapier, Cedric R; Kasten, Margaret M; Parnell, Timothy J et al. (2016) Regulation of DNA Translocation Efficiency within the Chromatin Remodeler RSC/Sth1 Potentiates Nucleosome Sliding and Ejection. Mol Cell 62:453-461

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