# Regulation of Membrane Fusion in Exocytosis

> **NIH NIH R01** · YALE UNIVERSITY · 2021 · $672,306

## Abstract

Project Summary/Abstract
How can virtually the same SNARE machine operate at dramatically different speeds depending on context,
often far faster than a single SNAREpin? This is one of the central questions driving the field today, and the
problem it embodies stands in boldest relief at the neuronal synapse, so it is here that we focus on the
structures, biophysics, and physiological properties of the key protein machinery. Our overall hypothesis is that
multiple SNAREpins released synchronously, each already close to the point of triggering fusion, co-operate to
achieve fusion dramatically faster than any one alone. During the current period of support we discovered that
the calcium sensor Synaptotagmin (normally anchored in synaptic vesicle) can self-assemble in vitro into Ca2+-
sensitive, ring-like oligomers ~30 nm in diameter and have suggested that such rings forming between the
synaptic vesicle (or insulin secretory vesicle) and the plasma membrane would prevent release until they are
disrupted by Ca2+. Our specific hypothesis is that such ring oligomers of Synaptotagmin (Syt) are a central
organizing principle for exocytosis, enabling the clamping and rapid synchronous release of multiple
SNAREpins. This hypothesis is strongly supported by recent experiments in which a targeted mutation (F349A)
that de-stabilizes Syt1 rings dramatically increases spontaneous and evoked release and in hippocampal
neurons, and dramatically reduces the synchronicity of release with the action potential. We propose to 1)
Test the hypothesis that ring-like oligomers of Synaptotagmins regulate exocytosis; 2) Test the hypothesis that
Syt1 and Syt7 play distinct structural and functional roles in synchronous and asynchronous release from the
same docked vesicles; 3) Elucidate the dynamics and topology of Munc13 and its proposed oligomers and the
posited dual roles as vesicle tether and outer ring chaperone templating SNAREpins; and 4) Obtain by single
particle cryo-EM and cryo EM tomography high resolution structures of functional release sites in vitro and in
situ trapped in defined functional states. Similar machinery mediates neuroendocrine secretory physiology,
including pancreatic insulin secretion, so we expect the answers will be highly relevant to the mission of
NIDDK. Further, there is little doubt in the post-leptin era of the key role of the nervous system in metabolic
balance and diseases.

## Key facts

- **NIH application ID:** 10246265
- **Project number:** 5R01DK027044-45
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** SHYAM S KRISHNAKUMAR
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $672,306
- **Award type:** 5
- **Project period:** 1991-09-30 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10246265

## Citation

> US National Institutes of Health, RePORTER application 10246265, Regulation of Membrane Fusion in Exocytosis (5R01DK027044-45). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10246265. Licensed CC0.

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