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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37MH063105-12
Application #
8264742
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Asanuma, Chiiko
Project Start
2000-08-10
Project End
2016-02-29
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
12
Fiscal Year
2012
Total Cost
$395,000
Indirect Cost
$145,000
Name
Stanford University
Department
Biophysics
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Lai, Ying; Diao, Jiajie; Cipriano, Daniel J et al. (2014) Complexin inhibits spontaneous release and synchronizes Ca2+-triggered synaptic vesicle fusion by distinct mechanisms. Elife 3:e03756
Diao, Jiajie; Cipriano, Daniel J; Zhao, Minglei et al. (2013) Complexin-1 enhances the on-rate of vesicle docking via simultaneous SNARE and membrane interactions. J Am Chem Soc 135:15274-7
Diao, Jiajie; Zhao, Minglei; Zhang, Yunxiang et al. (2013) Studying protein-reconstituted proteoliposome fusion with content indicators in vitro. Bioessays 35:658-65
Pertsinidis, Alexandros; Mukherjee, Konark; Sharma, Manu et al. (2013) Ultrahigh-resolution imaging reveals formation of neuronal SNARE/Munc18 complexes in situ. Proc Natl Acad Sci U S A 110:E2812-20
Kyoung, Minjoung; Zhang, Yunxiang; Diao, Jiajie et al. (2013) Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system. Nat Protoc 8:1-16
Cipriano, Daniel J; Jung, Jaemyeong; Vivona, Sandro et al. (2013) Processive ATP-driven substrate disassembly by the N-ethylmaleimide-sensitive factor (NSF) molecular machine. J Biol Chem 288:23436-45
Diao, Jiajie; Burre, Jacqueline; Vivona, Sandro et al. (2013) Native *-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2. Elife 2:e00592
Vivona, Sandro; Cipriano, Daniel J; O'Leary, Sean et al. (2013) Disassembly of all SNARE complexes by N-ethylmaleimide-sensitive factor (NSF) is initiated by a conserved 1:1 interaction between *-soluble NSF attachment protein (SNAP) and SNARE complex. J Biol Chem 288:24984-91
Fischer, Audrey; Sambashivan, Shilpa; Brunger, Axel T et al. (2012) Beltless translocation domain of botulinum neurotoxin A embodies a minimum ion-conductive channel. J Biol Chem 287:1657-61
Vrljic, Marija; Strop, Pavel; Hill, Ryan C et al. (2011) Post-translational modifications and lipid binding profile of insect cell-expressed full-length mammalian synaptotagmin 1. Biochemistry 50:9998-10012

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