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

Jiajie Diao, Zengliu Su, Yuji Ishitsuka, Bin Lu, Kyung Suk Lee, Ying Lai, Yeon-Kyun Shin () and Taekjip Ha ()
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Jiajie Diao: University of Illinois at Urbana-Champaign
Zengliu Su: Biophysics, and Molecular Biology, Iowa State University
Yuji Ishitsuka: University of Illinois at Urbana-Champaign
Bin Lu: Biophysics, and Molecular Biology, Iowa State University
Kyung Suk Lee: University of Illinois at Urbana-Champaign
Ying Lai: Biophysics, and Molecular Biology, Iowa State University
Yeon-Kyun Shin: Biophysics, and Molecular Biology, Iowa State University
Taekjip Ha: University of Illinois at Urbana-Champaign

Nature Communications, 2010, vol. 1, issue 1, 1-6

Abstract: Abstract 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.

Date: 2010
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DOI: 10.1038/ncomms1054

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