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Copyright © 2006 by The National Academy of Sciences of the USA Biophysics From the Cover Multiple intermediates in SNARE-induced membrane fusion *Howard Hughes Medical Institute, †Center for Biophysics and Computational Biology, and ¶Department of Physics, University of Illinois at Urbana–Champaign, Urbana, IL 61801; and ‡Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011 §To whom correspondence may be addressed. E-mail: colishin/at/iastate.edu or Email: tjha/at/uiuc.edu Edited by Thomas C. Südhof, University of Texas Southwestern Medical Center, Dallas, TX, and approved October 31, 2006 Author contributions: T.-Y.Y., B.O., and F.Z. contributed equally to this work; Y.-K.S. and T.H. designed research; T.-Y.Y., B.O., and F.Z. performed research; Y.-K.S. and T.H. contributed new reagents/analytic tools; T.-Y.Y. analyzed data; and T.-Y.Y., Y.-K.S., and T.H. wrote the paper. Received July 18, 2006. Freely available online through the PNAS open access option. See commentary "Illuminating membrane fusion" on page 19611. This article has been cited by other articles in PMC.Abstract Membrane fusion in eukaryotic cells is thought to be mediated by a highly conserved family of proteins called SNAREs (soluble N-ethyl maleimide sensitive-factor attachment protein receptors). The vesicle-associated v-SNARE engages with its partner t-SNAREs on the target membrane to form a coiled coil that bridges two membranes and facilitates fusion. As demonstrated by recent findings on the hemifusion state, identifying intermediates of membrane fusion can help unveil the underlying fusion mechanism. Observation of SNARE-driven fusion at the single-liposome level has the potential to dissect and characterize fusion intermediates most directly. Here, we report on the real-time observation of lipid-mixing dynamics in a single fusion event between a pair of SNARE-reconstituted liposomes. The assay reveals multiple intermediate states characterized by discrete values of FRET between membrane-bound fluorophores. Hemifusion, flickering of fusion pores, and kinetic transitions between intermediates, which would be very difficult to detect in ensemble assays, are now identified. The ability to monitor the time course of fusion events between two proteoliposomes should be useful for addressing many important issues in SNARE-mediated membrane fusion. Keywords: FRET, single-molecule spectroscopy, lipid mixing |
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