Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion

J Biol Chem. 2009 Nov 13;284(46):32158-66. doi: 10.1074/jbc.M109.047381. Epub 2009 Sep 15.


SNAREs mediate membrane fusion in intracellular vesicle traffic and neuronal exocytosis. Reconstitution of membrane fusion in vitro proved that SNAREs constitute the minimal fusion machinery. However, the slow fusion rates observed in these systems are incompatible with those required in neurotransmission. Here we present a single vesicle fusion assay that records individual SNARE-mediated fusion events with millisecond time resolution. Docking and fusion of reconstituted synaptobrevin vesicles to target SNARE complex-containing planar membranes are distinguished by total internal reflection fluorescence microscopy as separate events. Docking and fusion are SNAP-25-dependent, require no Ca(2+), and are efficient at room temperature. Analysis of the stochastic data with sequential and parallel multi-particle activation models reveals six to nine fast-activating steps. Of all the tested models, the kinetic model consisting of eight parallel reaction rates statistically fits the data best. This might be interpreted by fusion sites consisting of eight SNARE complexes that each activate in a single rate-limiting step in 8 ms.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Exocytosis
  • Kinetics
  • Lipid Bilayers / metabolism*
  • Membrane Fluidity
  • Membrane Fusion*
  • Microscopy, Fluorescence
  • Microscopy, Interference
  • Models, Molecular
  • Protein Binding
  • Proteolipids / chemistry
  • Proteolipids / metabolism
  • R-SNARE Proteins / metabolism*
  • Rats
  • SNARE Proteins / metabolism*
  • Synaptic Vesicles / metabolism*
  • Synaptosomal-Associated Protein 25 / metabolism*


  • Lipid Bilayers
  • Proteolipids
  • R-SNARE Proteins
  • SNARE Proteins
  • Synaptosomal-Associated Protein 25
  • proteoliposomes