Imaging single membrane fusion events mediated by SNARE proteins Journal Article
| Authors: | Fix, M.; Melia, T. J.; Jaiswal, J. K.; Rappoport, J. Z.; You, D.; Söllner, T. H.; Rothman, J. E.; Simon, S. M. |
| Article 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 Habc domain of syntaxin 1A increased fusion probability > 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: | protein domain; protein function; complex formation; membrane proteins; in vivo study; assay; imaging system; amino terminal sequence; base sequence; microscopy, fluorescence; fluorescence spectroscopy; dna primers; calcium cell level; bilayer membrane; magnesium ion; phospholipid; cell vacuole; vesicular transport proteins; calcium ion; membrane fusion; snare protein; qa-snare proteins; snare proteins; priority journal; article; proteoliposome; total internal reflection fluorescence microscopy; proteolipids |
| Journal Title: | Proceedings of the National Academy of Sciences of the United States of America |
| Volume: | 101 |
| Issue: | 19 |
| ISSN: | 0027-8424 |
| Publisher: | National Academy of Sciences |
| Date Published: | 2004-05-11 |
| Start Page: | 7311 |
| End Page: | 7316 |
| Language: | English |
| DOI: | 10.1073/pnas.0401779101 |
| PROVIDER: | scopus |
| PMCID: | PMC409915 |
| PUBMED: | 15123811 |
| DOI/URL: | |
| Notes: | Proc. Natl. Acad. Sci. U. S. A. -- Cited By (since 1996):90 -- Export Date: 16 June 2014 -- CODEN: PNASA -- Source: Scopus |
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