Resolving kinetic intermediates during the regulated assembly and disassembly of fusion pores

Debasis Das, Huan Bao, Kevin C. Courtney, Lanxi Wu and Edwin R. Chapman ()
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Debasis Das: University of Wisconsin-Madison
Huan Bao: University of Wisconsin-Madison
Kevin C. Courtney: University of Wisconsin-Madison
Lanxi Wu: University of Wisconsin-Madison
Edwin R. Chapman: University of Wisconsin-Madison

Nature Communications, 2020, vol. 11, issue 1, 1-12

Abstract: Abstract The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the formation of these dynamic structures, and their kinetic transitions are tightly regulated by accessory proteins at the synapse. Here, we utilize two single molecule approaches, nanodisc-based planar bilayer electrophysiology and single-molecule FRET, to address the relationship between SNARE complex assembly and rapid (micro-millisecond) fusion pore transitions, and to define the role of accessory proteins. Synaptotagmin (syt) 1, a major Ca2+-sensor for synaptic vesicle exocytosis, drove the formation of an intermediate: committed trans-SNARE complexes that form large, stable pores. Once open, these pores could only be closed by the action of the ATPase, NSF. Time-resolved measurements revealed that NSF-mediated pore closure occurred via a complex ‘stuttering’ mechanism. This simplified system thus reveals the dynamic formation and dissolution of fusion pores.

Date: 2020
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DOI: 10.1038/s41467-019-14072-7

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