Single Vesicle Fusion Assay in Model Membranes to Study SNARE-medicated Membrane Fusion

Domanska, Marta Katarzyna, Department of Molecular Physiology and Biological Physics, University of Virginia
Tamm, Lukas, Department of Molecular Physiology and Biological Physics, University of Virginia
Nakamoto, Robert, Department of Molecular Phys and Biological Physics, University of Virginia
Cafiso, David, Department of Chemistry, University of Virginia
Castle, David, Department of Cell Biology, University of Virginia

The fusion of synaptic vesicles at the active zones with the neuronal plasma membrane (PM) is the best described example of exocytosis. Neurosecretion is tightly regulated and the fastest membrane fusion event in mammalian cells. It involves a cascade of protein - protein interactions between soluble and membrane proteins that include soluble N - ethylmaleimide - sensitive factor attachment proteins receptors (SNAREs). It is widely accepted that zippering interactions between the PM acceptor SNARE proteins syntaxin 1A and SNAP25, and vesicular SNARE synaptobrevin 2 (Syb2), lead to vesicle docking and fusion. We established a - single vesicle planar supported membrane fusion assay. The individual SNARE - mediated docking and fusion events were observed with millisecond time resolution by TIRF microscopy. We optimized the SNARE protein concentration to observe robust docking and fusion events in our assay. We showed that Syb2 vesicle docking was dependent on SNAP25 and on the density of the acceptorSNARE complex in the phosphatidylcholine/cholesterol (PC/Chol) membranes. The subsequent vesicle fusion did not require Ca2+ and was efficient at room temperature. To better depict the lipid environment of the PM, phosphatidylserine (PS) and phosphatidylethanolamine (PE) lipids were added to our PC/Chol model membranes and their influence on vesicle docking and fusion was tested. PS and PE lipids were added in different proportions either to the Syb2 vesicles or to both the supported bilayers and Syb2 vesicles. We showed that PS and PE reduce the probability of fusion. However, we ii observed an increase in the docking efficiency when the PE content in both membranes was raised from 0 to 30%. A detailed kinetic analysis of the single fusion events revealed that in PC/Chol bilayers 6 to 9 SNARE complexes are optimal for fast membrane fusion occurring ~18 ms after docking. In PC/PS/PE/Chol membranes the number of SNARE complexes necessary to drive fast membrane fusion decreased approximately to 3 to 5 complexes depending on the PE content. The presented work significantly enhances our understanding of the neuronal SNARE - mediated membrane fusion process. The developed assay is a powerful tool for future work on the role of late regulatory proteins, lipids and hybrid fusion studies with neuronal synaptic vesicles.

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PHD (Doctor of Philosophy)
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