# Single molecule studies of SNARE-induced vesicle fusion

> **NIH NIH R37** · STANFORD UNIVERSITY · 2020 · $536,061

## Abstract

Over the last 4.5 years of this Merit award (R37 MH63105) we have achieved a number of milestones: (1) the
development of a single vesicle fusion assay that mimics certain properties of spontaneous and calcium
triggered vesicle fusion observed in neuronal cultures; (2) the atomic resolution structure of the complex
between the calcium sensor synaptotagmin-1 and the neuronal SNARE complex, revealing an unexpected
calcium-independent interface that we believe forms the foundation for the process of calcium-triggered
synaptic vesicle fusion; (3) the near-atomic resolution structure of the complex between NSF, SNAPs, and
SNAREs that has revealed clues how NSF is capable of disassembling the SNARE complex. Moreover, we
have studied the molecular mechanism of complexin-1 and Munc18. We found that complexin-1 inhibits
spontaneous release and activates calcium-triggered vesicle fusion in our synthetic system, consistent with
complexin's function as observed in neuronal cultures. However, we found no effect of Munc18 on intrinsic
fusion rates, suggesting that its primary function is to facilitate SNARE complex formation. The specific aims
for the next 5 year period are as follows: (1) Decipher the molecular mechanism of NSF-mediated SNARE
disassembly. We plan to determine structures of NSF and of the complex of NSF, SNAREs and SNAPs upon
hydrolyzing ATP by using single- particle cryo-EM. (2) Study how complexin and synaptotagmin-1 cooperate in
fast synchronous release. Our recent work revealed a conserved, calcium-independent interface between
synaptotagmin-1 and the neuronal SNARE complex. Following up on this result, we plan to investigate the
interplay between synaptotagmin-1, and complexin-1, and the neuronal SNARE complex at the atomic level.
We plan to crystalize this supercomplex, along with functional studies in neuronal cultures in order to test the
new interfaces that we may discover in the crystal structure. (3) Investigate the role of Munc13. We will test the
hypothesis that Munc13 is a facilitator for efficient SNARE complex formation. (4) Establish a hybrid fusion
assay to study the fusion kinetics of purified endogenous synaptic vesicles obtained from mice brains. We will
extend our single vesicle fusion assay to use purified synaptic vesicles in combination with synthetic plasma
membrane mimicking “acceptor” vesicles. We hypothesize that different pools of synaptic vesicles may result
in different fusion kinetics.

## Key facts

- **NIH application ID:** 9853048
- **Project number:** 5R37MH063105-20
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** AXEL T BRUNGER
- **Activity code:** R37 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $536,061
- **Award type:** 5
- **Project period:** 2016-03-08 → 2021-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9853048

## Citation

> US National Institutes of Health, RePORTER application 9853048, Single molecule studies of SNARE-induced vesicle fusion (5R37MH063105-20). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9853048. Licensed CC0.

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