A single-vesicle content mixing assay for SNARE-mediated membrane fusion.
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.
Department of Physics and Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Jiajie
J
Diao
Zengliu
Z
Su
Yuji
Y
Ishitsuka
Bin
B
Lu
Kyung Suk
KS
Lee
Ying
Y
Lai
Yeon-Kyun
YK
Shin
Taekjip
T
Ha
D001681:Biological Assay / Q000379:methods*; D008561:Membrane Fusion / Q000502:physiology*; D008856:Microscopy, Fluorescence; D050600:SNARE Proteins / Q000378:metabolism*
D050600:SNARE Proteins