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

Author

Listed:
  • 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)

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.

Suggested Citation

  • Debasis Das & Huan Bao & Kevin C. Courtney & Lanxi Wu & Edwin R. Chapman, 2020. "Resolving kinetic intermediates during the regulated assembly and disassembly of fusion pores," Nature Communications, Nature, vol. 11(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:11:y:2020:i:1:d:10.1038_s41467-019-14072-7
    DOI: 10.1038/s41467-019-14072-7
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    Cited by:

    1. Kevin C. Courtney & Taraknath Mandal & Nikunj Mehta & Lanxi Wu & Yueqi Li & Debasis Das & Qiang Cui & Edwin R. Chapman, 2023. "Synaptotagmin-7 outperforms synaptotagmin-1 to promote the formation of large, stable fusion pores via robust membrane penetration," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
    2. Bhavya R. Bhaskar & Laxmi Yadav & Malavika Sriram & Kinjal Sanghrajka & Mayank Gupta & Boby K. V & Rohith K. Nellikka & Debasis Das, 2024. "Differential SNARE chaperoning by Munc13-1 and Munc18-1 dictates fusion pore fate at the release site," Nature Communications, Nature, vol. 15(1), pages 1-18, December.

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