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SNARE-Driven, 25-Millisecond Vesicle Fusion In Vitro

Tingting Liu ^*, Ward C. Tucker , Akhil Bhalla , Edwin R. Chapman  and James C. Weisshaar 

Departments of ^* Chemistry and Physiology, University of Wisconsin-Madison, Madison, Wisconsin 53706

Correspondence: Address reprint requests to James C. Weisshaar, E-mail: weisshaar{at}chem.wisc.edu.

Docking and fusion of single proteoliposomes reconstituted with full-length v-SNAREs (synaptobrevin) into planar lipid bilayers containing binary t-SNAREs (anchored syntaxin associated with SNAP25) was observed in real time by wide-field fluorescence microscopy. This enabled separate measurement of the docking rate k_dock and the unimolecular fusion rate k_fus. On low t-SNARE-density bilayers at 37°C, docking is efficient: k_dock = 2.2 x 10⁷ M^–1 s^–1, 40% of the estimated diffusion limited rate. Full vesicle fusion is observed as a prompt increase in fluorescence intensity from labeled lipids, immediately followed by outward radial diffusion (D_lipid = 0.6 µm² s^–1); 80% of the docked vesicles fuse promptly as a homogeneous subpopulation with k_fus = 40 ± 15 s^–1 (_fus = 25 ms). This is 10³–10⁴ times faster than previous in vitro fusion assays. Complete lipid mixing occurs in <15 ms. Both the v-SNARE and the t-SNARE are necessary for efficient docking and fast fusion, but Ca²⁺ is not. Docking and fusion were quantitatively similar on syntaxin-only bilayers lacking SNAP25. At present, in vitro fusion driven by SNARE complexes alone remains 40 times slower than the fastest, submillisecond presynaptic vesicle population response.

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