Insulin regulates the stability of the vesicle fusion pore. (A) pHluorin’s fluorescence brightens (arrowhead) upon fusion pore formation, and then spreads laterally as the vesicle collapses into the PM (asterisk; see Fig. S1 [C and D] for fusion criteria). (B) Three examples of VAMP2-pHluorin fusion events with different durations of pore opening. (C and D) Vesicle “transition times” (arrowhead to asterisk) were measured in basal (C, n = 163) and insulin-stimulated (D, n = 894) cells, plotted as histograms, and fitted with a dual Gaussian distribution. (E) A representative kiss-and-run fusion event, whereby the signal slowly dimmed but did not spread (see Fig. S1 D). Bars, 1 µm. (F–H) Percentage of kiss-and-run events in basal and insulin-stimulated cells (F, n = 734 from three cells; **, P < 0.01), after addition of 1-butanol (G, n = 759 from three cells) or 2-butanol (n = 733 from three cells; ***, P < 0.001), and in FIPI-treated cells, before and after insulin (H, n = 150 from two cells). Error bars indicate mean ± SEM. (I) Working model of the energy landscape of vesicle fusion. Insulin reduces the barrier to full fusion after formation of the fusion pore.