title: Imaging single membrane fusion events mediated by SNARE proteins.
abstract: Using total internal reflection fluorescence microscopy, we have developed an assay to monitor individual fusion events between proteoliposomes containing vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and a supported planar bilayer containing cognate target SNAREs. Approach, docking, and fusion of individual vesicles to the target membrane were quantified by delivery and subsequent lateral spread of fluorescent phospholipids from the vesicle membrane into the target bilayer. Fusion probability was increased by raising divalent cations (Ca2+ and Mg2+). Fusion of individual vesicles initiated in <100 ms after the rise of Ca2+ and membrane mixing was complete in 300 ms. Removal of the N-terminal H(abc) domain of syntaxin 1A increased fusion probability >30-fold compared to the full-length protein, but even in the absence of the H(abc) domain, vesicle fusion was still enhanced in response to Ca2+ increase. Our observations establish that the SNARE core complex is sufficient to fuse two opposing membrane bilayers at a speed commensurate with most membrane fusion processes in cells. This real-time analysis of single vesicle fusion opens the door to mechanistic studies of how SNARE and accessory proteins regulate fusion processes in vivo.
authors.affiliation: Laboratory of Cellular Biophysics, The Rockefeller University, 1230 York Avenue, Box 304, New York, NY 10021, USA.
authors.forename: Marina
authors.initials: M
authors.lastname: Fix
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Melia
authors.forename: Jyoti K
authors.initials: JK
authors.lastname: Jaiswal
authors.forename: Joshua Z
authors.initials: JZ
authors.lastname: Rappoport
authors.forename: Daoqi
authors.initials: D
authors.lastname: You
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Söllner
authors.forename: James E
authors.initials: JE
authors.lastname: Rothman
authors.forename: Sanford M
authors.initials: SM
authors.lastname: Simon
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008856:Microscopy, Fluorescence; D011510:Proteolipids; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins
chemical_list: D017931:DNA Primers; D008565:Membrane Proteins; D011510:Proteolipids; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; C020485:proteoliposomes