Fig. 1.

Munc13 and DHG promote the formation of stably, docked “immobile” vesicles. (A) To reconstitute the Munc13/Munc18-dependent vesicle priming and fusion process under cell-free conditions, we included a 1:1 Syntaxin/Munc18 complex and palmitoylated SNAP25 in the suspended lipid membrane, as well as VAMP2/Synaptotagmin in SUVs, with CPX and Munc13 in solution. We monitored the fate of a large number of individual vesicles (~200 per condition) using fluorescent labels in the SUVs. (B) The number of vesicles attached or “docked” to the suspended bilayer did not appreciably change without or with Munc13 (gray and blue bar, respectively), but DHG activation (pink bar) of Munc13 significantly increased the number of docked vesicles. (C) The fate of the docked vesicles strongly depended on the availability of Munc13 and its activation by DHG. In the absence of Munc13 or DHG, most vesicles undock (brown bar), with only a small proportion converting into the immobile docked stage (green bar). The inclusion of Munc13 increased the number of immobile vesicles, which was further enriched by the addition of DHG. In all cases, we observed a small percentage of spontaneous fusion events (yellow bar). (D) DHG and Munc13 also accelerated the formation of the immobile docked stage. Without Munc13 or DHG, the docked vesicles reached the immobile state over 1 to 2 s. The addition of Munc13 lowered this transition time to ~0.5 s, which was further reduced by DHG to ~0.25 s. The average values and SDs from three to four independent experiments (with ~200 vesicles per condition) are shown. **P < 0.05; ***P < 0.005 using the Student’s t test.