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Imaging Single Membrane Fusion Events Mediated by SNARE Proteins

Fix, Marina ; Melia, Thomas J. ; Jaiswal, Jyoti K. ; Rappoport, Joshua Z. ; You, Daoqi ; Söllner, Thomas H. ; Rothman, James E. ; Simon, Sanford M.

Proceedings of the National Academy of Sciences - PNAS, 2004-05, Vol.101 (19), p.7311-7316 [Periódico revisado por pares]

United States: National Academy of Sciences

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  • Título:
    Imaging Single Membrane Fusion Events Mediated by SNARE Proteins
  • Autor: Fix, Marina ; Melia, Thomas J. ; Jaiswal, Jyoti K. ; Rappoport, Joshua Z. ; You, Daoqi ; Söllner, Thomas H. ; Rothman, James E. ; Simon, Sanford M.
  • Assuntos: Base Sequence ; Biological Sciences ; Cell membranes ; Cellular biology ; Divalent cations ; DNA Primers ; Fluorescence ; Lipids ; Liposomes ; Membrane Fusion - physiology ; Membrane Proteins - chemistry ; Membrane Proteins - metabolism ; Membrane Proteins - physiology ; Membranes ; Microscopy, Fluorescence ; P branes ; Phospholipids ; Pixels ; Proteins ; Proteolipids ; Qa-SNARE Proteins ; SNARE Proteins ; Vesicular Transport Proteins
  • É parte de: Proceedings of the National Academy of Sciences - PNAS, 2004-05, Vol.101 (19), p.7311-7316
  • Notas: ObjectType-Article-2
    SourceType-Scholarly Journals-1
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    To whom correspondence should be addressed. E-mail: simon@rockefeller.edu.
    Contributed by James E. Rothman, March 12, 2004
    Abbreviations: SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; v-SNARE, vesicle SNARE; t-SNARE, target SNARE; v-liposome, v-SNARE proteoliposome; NBD, 7-nitrobenz-2-oxa-1,3-diazole; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine; Rh, lissamine rhodamine B; DPPE, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine; RT, room temperature; SUVs, small unilamellar vesicles; TIR-FM, total internal reflection fluorescence microscopy; SNAP-25, synaptosomal-associated protein of 25 kDa; VAMP, vesicle-associated membrane protein.
  • Descrição: 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.
  • Editor: United States: National Academy of Sciences
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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