Comparing the origins and fates of vesicles in the presence and absence of dynasore. (A and B) Vesicle behavior in vehicle control (0.1% DMSO) or dynasore (80 µM) during 6-s trials. Vesicles were loaded with either 3-kD Alexa Fluor 488 (A) or 10-kD Alexa Fluor 488 (B). Synaptic release was stimulated by depolarizing rod terminals with a 2-s puff of 50 mM KCl. With both dyes, significantly more vesicles advanced toward the membrane in vehicle control solutions than in rods treated with dynasore (3 kD, P < 0.0001; 10 kD, P < 0.0001; unpaired t tests). The number of vesicles that began each trial stably associated with the membrane did not differ significantly between dynasore and control cells. The number of vesicles that remained at the membrane surface at the end of each trial was not altered significantly by dynasore treatment with 3 kD–loaded vesicles but was increased slightly with 10-kD vesicles (P = 0.043). Dynasore treatment significantly reduced the frequency of vesicles that fused during depolarizing stimulation with a puff of 50 mM KCl (3 kD, P < 0.0001; 10 kD, P < 0.0001). The number of vesicles that departed the membrane without fusion was also significantly reduced (3 kD, P < 0.0001; 10 kD, P < 0.0001). (C and D) In the subset of vesicles loaded with 3-kD Alexa Fluor 488 (C) or 10-kD Alexa Fluor 488 (D) that began each trial as membrane-associated vesicles, we found no significant difference in the number of nonrelease retreat events (3 kD, P = 0.12; 10 kD, P = 0.25), but dynasore significantly decreased fusion (3 kD, P = 0.014; 10 kD, P = 0.012). The number of vesicles loaded with 10-kD Alexa Fluor 488 that remained at the membrane throughout the entire trial was also increased significantly by dynasore (P < 0.0001). Error bars represent ±SEM. *, P < 0.05; **, P < 0.001; ***, P < 0.0001.