Imaging single membrane fusion events mediated by SNARE proteins
- 3 May 2004
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 101 (19), 7311-7316
- https://doi.org/10.1073/pnas.0401779101
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 30-fold compared to the full-length protein, but even in the absence of the Habc 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.Keywords
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