Fig. 3.

Munc13 and DHG promote and accelerates SNARE complex formation under docked vesicles. The formation of SNAREpins under the docked vesicles (labeled with ATTO465) was visualized by the binding of Alexa Fluor 647-labeled Complexin (CPX). (A) Colocalization analysis revealed that in the absence of Munc13 and DHG (gray bar), only about 25% of vesicles contained CPX signals. The proportion of vesicles colocalized with CPX improved to ~55% when Munc13 was included (blue bar) and to ~90% with DHG activation. This indicated that Munc13 directly promotes the SNARE complex formation, and its chaperone function is further stimulated by DHG binding. (B) Munc13 and DHG also significantly reduced the delay between vesicle docking and CPX binding, with CPX arrival within ~0.3 s with DHG activation, compared to a 2 to 3 s delay with Munc13 alone and an 8 to 10-s delay without Munc13 or DHG. (C) Representative fluorescence traces of vesicle (black) docking and CPX (red) arrival in the presence of Munc13 without or with DHG are shown. (A and B) The average values and SDs from three independent experiments (with ~100 vesicles per condition) are shown. **P < 0.05, ***P < 0.005 using the Student’s t test.