Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 101, Issue 19, Pages 7311-7316Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0401779101
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Funding
- NIDDK NIH HHS [R01 DK027044, R01 DK 27044] Funding Source: Medline
- NIGMS NIH HHS [F32 GM069200, 1F32 GM 069200] Funding Source: Medline
- NINDS NIH HHS [R01 NS043391, R91 NS 043391] Funding Source: Medline
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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.
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