(A) Stills from DCV fusion assay. Electrical stimulation starts at second 10. Yellow arrowheads indicate stationary vesicles that fuse, red arrowhead indicates a stationary vesicle that does not fuse and white arrow shows a moving DCV that fuses. Kymograph shows the trajectories of the stationary and moving vesicle over time. (B) Fusing DCVs classified as stationary or moving showing that CAPS deletion strongly affects fusion from stationary vesicles (CAPS-2KO stationary prior to fusion: 12.2±2.8, moving: 4±1.0, n=10, *p<0.05; CAPS DKO stationary: 2.3±1.3, moving: 4.0±1.7, n=3, ns, N=3). (C) Non-fusing DCVs classified as stationary or moving showing that CAPS deletion does not affect general trafficking behavior of non-fusing vesicles (CAPS-2KO stationary: 33.8±1.2, moving: 17.6±0.7, n=5, *p<0.05; CAPS DKO stationary: 33.7±3.0, moving: 19.3±2.4, n=3, *p<0.05). (D) Average velocity of DCVs classified as moving prior to fusion in B, before stimulation (PreStim = 30 s; CAPS-2KO: 193.6±9.9 nm/s, n=169 vesicles, CAPS DKO: 180.4±9.2 nm/s, n=18) and during electrical stimulation (Stim: from second 30 to the onset of fusion; CAPS-2KO = 180.9±6.6 nm/s, n=169; CAPS DKO: 169.2±7.5, n=18). (E) Average velocity of non-fusing DCVs in C, before stimulation (PreStim = 30 s; CAPS-2KO: 190.8±13.7 nm/s, n=168, CAPS DKO 186.8±13.5 nm/s, n=91), during stimulation (Stim = 24 s; CAPS-2KO: 175.3±6.7 nm/s, n=168; CAPS DKO 201.8±11.8 nm/s, n=91) and after stimulation (PostStim = 36 s CAPS-2KO: 182.2±6.2 nm/s, n=168, CAPS DKO 196.2±8.5 nm/s, n=91). (F) Typical examples of electron micrographs of neuronal DCVs in synapses. Scale bar 50 nm. (G) Number of synapses containing one or more DCVs (CAPS-2KO: 53.9±12.1%, n=198 synapses; CAPS DKO 47.6±5.4%, n=152 synapses, N=4). (H) Percentage of docked DCVs per synapse (CAPS-2KO: 10.3±1.7%, DCVs = 285, CAPS DKO: 11.1±3.7%, DCVs = 201). (I) Average distance of DCVs to the closest plasma membrane (CAPS-2KO: 81.2±4.8; CAPS DKO: 84.9±10.4). (J) Average number of DCVs per synapse (CAPS-2KO: 1.6±0.2; CAPS DKO: 1.5±0.2). (K) Frequency distribution of number of DCVs per synapse (% synapses, normalized to the total number of DCVs per group).