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A single-vesicle content mixing assay for SNARE-mediated membrane fusion

Shin, Yeon-Kyun ; Ha, Taekjip ; Diao, Jiajie ; Su, Zengliu ; Ishitsuka, Yuji ; Lu, Bin ; Lee, Kyung Suk ; Lai, Ying

Nature communications, 2010-08, Vol.1 (5), p.1-6, Article 54 [Periódico revisado por pares]

England: Nature Publishing Group

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A single-vesicle content mixing assay for SNARE-mediated membrane fusion
Autor: Shin, Yeon-Kyun ; Ha, Taekjip ; Diao, Jiajie ; Su, Zengliu ; Ishitsuka, Yuji ; Lu, Bin ; Lee, Kyung Suk ; Lai, Ying
Assuntos: Assays ; Biological Assay - methods ; Complementary DNA ; DNA ; Energy transfer ; Expansion ; Fluorescence ; Fluorescence resonance energy transfer ; Fusion protein ; Lipids ; Membrane fusion ; Membrane Fusion - physiology ; Membranes ; Microscopy, Fluorescence ; N-Ethylmaleimide-sensitive protein ; Pore formation ; Proteins ; Recycling ; SNAP receptors ; SNARE Proteins - metabolism ; Vesicle fusion ; Waste management ; Yeast ; Yeasts
É parte de: Nature communications, 2010-08, Vol.1 (5), p.1-6, Article 54
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These authors contributed equally to this work.
Descrição: The in vitro studies of membrane fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have primarily been conducted by following the mixing of lipids. However, the formation of a fusion pore and its expansion has been difficult to detect directly because of the leakiness of proteoliposomes, vesicle aggregation and rupture that often complicate the interpretation of ensemble fusion experiments. Fusion pore expansion is an essential step for full-collapse fusion and for recycling of fusion mechanisms. Here, we demonstrate a method to detect the inter-vesicular mixing of large cargoes at the single-molecule and -vesicle level. The change in fluorescence resonance energy transfer signal when a DNA hairpin encapsulated in a surface-tethered vesicle encounters a complementary DNA strand from another vesicle indicates content mixing. We found that the yeast SNARE complex alone without any accessory proteins can expand the fusion pore large enough to transmit ~11 kDa cargoes.
Editor: England: Nature Publishing Group
Idioma: Inglês

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A single-vesicle content mixing assay for SNARE-mediated membrane fusion

Shin, Yeon-Kyun ; Ha, Taekjip ; Diao, Jiajie ; Su, Zengliu ; Ishitsuka, Yuji ; Lu, Bin ; Lee, Kyung Suk ; Lai, Ying

England: Nature Publishing Group

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