Neurotransmitter release is one of the most regulated membrane fusion events. Unlike constitutive vesicle trafficking, synaptic vesicles are recruited to the presynaptic membrane, but do not readily fuse. Instead, an average of ten vesicles are stably docked at a region of the synapse termed the active zone awaiting an action potential. Synaptic vesicle fusion is closely associated with the Ca2+ influx that follows arrival of an action potential. Exocytosis is triggered within approximately 0.2 msec of the Ca2+ arrival. Although many key factors have been found to be essential for Ca2+-dependent neurotransmitter release, such as SNAREs, synaptotagmin, complexin, Munc18, and Munc13, the molecular mechanism of synaptic vesicle membrane fusion and Ca2+- triggering remains unclear. In this renewal application we wish to continue our efforts to decipher the molecular mechanism by using a combination of single molecule and single particle fluorescence imaging microscopy experiments. Driven by our hypothesis that SNAREs and auxiliary proteins do not fully assemble and, thus, do not trigger fusion until a Ca2+ signal arrives, we have designed labeling strategies to monitor distinct conformational states of the protein and protein-complexes involved in the process. We have also developed fluorescent reporters that provide information about content and lipid exchange as the system proceeds from vesicle docking, to membrane contact, and, finally, to fusion. These labeling strategies and the single molecule and single vesicle experimental setups are the cornerstones for the proposed research. Our assays are a breakthrough, since for the first time, we observe fast fusion kinetics upon Ca2+ triggering. Our new assay now enables us to determine the role of the factors involved, their stoichiometry (e.g., how many SNARE complexes and synaptotagmin molecules are required for fast Ca2+ triggered fusion?), to propose and test mutants of these factors that affect their function in the assay, and then to use these mutants as tools for experiments with neuronal cultures to dissect their role in vivo. Furthermore, we have recently demonstrated the ability of super-resolution imaging of synaptic proteins by using labeled antibodies to an unprecedented degree of localization accuracy. This in vivo imaging approach will enable us to image the distribution and correlation of synaptic proteins along axons, and so it will complement our reconstitution and single molecule biophysical studies.
Neurotransmitter release is a key step in neuronal communication. Synaptic vesicle fusion is closely associated with the Ca2+ influx that follows arrival of an action potential. Exocytosis is triggered within approximately 0.2 msec of the Ca2+ arrival. We are utilizing single molecule/particle fluorescence microscopy with a reconstituted system to decipher the mechanism of the molecular machinery that controls the process of Ca2+-triggered neurotransmitter release. We hope that a better understanding of the molecular mechanism of synaptic release can be leveraged for the betterment of human health through the design of specific compounds that modulate the underlying protein-protein interactions.
|Zhao, Minglei; Brunger, Axel T (2016) Recent Advances in Deciphering the Structure and Molecular Mechanism of the AAA+ ATPase N-Ethylmaleimide-Sensitive Factor (NSF). J Mol Biol 428:1912-26|
|Lyubimov, Artem Y; Uervirojnangkoorn, Monarin; Zeldin, Oliver B et al. (2016) Advances in X-ray free electron laser (XFEL) diffraction data processing applied to the crystal structure of the synaptotagmin-1 / SNARE complex. Elife 5:|
|Gong, Jihong; Lai, Ying; Li, Xiaohong et al. (2016) C-terminal domain of mammalian complexin-1 localizes to highly curved membranes. Proc Natl Acad Sci U S A 113:E7590-E7599|
|MalmersjÃ¶, Seth; Di Palma, Serena; Diao, Jiajie et al. (2016) Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion. EMBO J 35:1810-21|
|Martinelli, David C; Chew, Kylie S; Rohlmann, Astrid et al. (2016) Expression of C1ql3 in Discrete Neuronal Populations Controls Efferent Synapse Numbers andÂ Diverse Behaviors. Neuron 91:1034-51|
|Lai, Ying; Choi, Ucheor B; Zhang, Yunxiang et al. (2016) N-terminal domain of complexin independently activates calcium-triggered fusion. Proc Natl Acad Sci U S A 113:E4698-707|
|Choi, Ucheor B; Zhao, Minglei; Zhang, Yunxiang et al. (2016) Complexin induces a conformational change at the membrane-proximal C-terminal end of the SNARE complex. Elife 5:|
|Zhao, Minglei; Wu, Shenping; Zhou, Qiangjun et al. (2015) Mechanistic insights into the recycling machine of the SNARE complex. Nature 518:61-7|
|Zhang, Yunxiang; Diao, Jiajie; Colbert, Karen N et al. (2015) Munc18a does not alter fusion rates mediated by neuronal SNAREs, synaptotagmin, and complexin. J Biol Chem 290:10518-34|
|Diao, Jiajie; Liu, Rong; Rong, Yueguang et al. (2015) ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes. Nature 520:563-6|
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