# Development of a high resolution assay to characterize exocytotic vesicle fusion

> **NIH NIH R21** · CORNELL UNIVERSITY · 2020 · $95,524

## Abstract

Transmitter release is mediated by fusion of neurosecretory vesicles with the plasma membrane. While it is
known that Soluble NSF Attachment REceptor (SNARE) proteins form the core complex of the molecular fusion
machine, the precise molecular rearrangements leading to fusion pore formation are still unknown. We will
develop a highly innovative technology that will enable experiments to achieve a precise mechanistic
understanding of structural molecular rearrangements associated with the fusion of neurosecretory vesicles at
the plasma membrane. The approach combines electrochemical detector (ECD) arrays, with reconstituted
supported membranes to study fusion of isolated chromaffin granules simultaneously by amperometry and total
internal reflection fluorescence (TIRF) imaging. We have previously performed combined ECD and TIRF
experiments using intact chromaffin cells and discovered a rapid conformational change in SNAP25 associated
with fusion events. However, these measurements were performed using a FRET construct incorporating
CFP/Venus and the actual nature of the structural change remains unknown. Proceeding to the reconstituted
system will make it possible to incorporate small labels at arbitrary sites in the SNARE proteins or other
accessory proteins, a technology that will make it possible to identify precisely which amino acids in the SNARE
complex and accessory proteins move and change distance at specific times during the fusion process. The
amperometric recordings can be performed with a time resolution of a millisecond or less and by averaging
fluorescence changes from multiple fusion events, the time of such fluorescence changes relative to the fusion
event can be determined with very high precision, not limited by the exposure time used in the fluorescence
image acquisition. This has become possible with the time super-resolution approach named Event Correlation
Microscopy (ECOM), developed in the Lindau Lab. The technology we propose to develop is high risk because
we need to establish a protocol to form the supported bilayers on top of the ECD arrays and explore how a
sufficiently high rate of fusion events at a given ECD array can be achieved to perform the required averaging of
large numbers of fusion events (as we did in the cells). It is known, that when supported bilayers incorporating
SNARE proteins are formed on a quartz or silicon oxide surface, fusion of dense core vesicles does occur. The
project will be performed in collaboration between Dr. Lindau at Cornell who has developed the ECD and ECOM
methods and Dr. Kiessling who has pioneered the study of SNARE protein conformations in the supported bilayer
system. If successful, this technology will enable the experimental identification of the detailed molecular steps
in vesicle fusion and to test the predictions from structural work as well as molecular dynamics simulations.

## Key facts

- **NIH application ID:** 10041876
- **Project number:** 1R21NS118319-01
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** Manfred LINDAU
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $95,524
- **Award type:** 1
- **Project period:** 2020-06-01 → 2021-10-01

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10041876

## Citation

> US National Institutes of Health, RePORTER application 10041876, Development of a high resolution assay to characterize exocytotic vesicle fusion (1R21NS118319-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10041876. Licensed CC0.

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