Fig. 1.
Granules increase their motions just before exocytosis. (A and B) Tracks of two granules before stimulation (black path) and during stimulation with DMPP (red path) until fusion (marked with X). The large black circles represent a 300-nm granule centered on the last position before fusion. The blue circles in A and B are used in the visit density analysis (see Methods) and have radii of 17 and 47 nm, respectively. (C) (Upper) The motion of granules destined to fuse during stimulation was expressed relative to the average motion before stimulation, ΔR/ΔRprestimulation. Paths of 72 granules immediately before fusion were aligned at the time of fusion (right side) or at the time of stimulation (left side). The average ± SEM of ΔRprestimulation was 25.3 ± 1.1 nm. The time course was subjected to a sliding three-position smoothing algorithm. The last motion before fusion was not smoothed. There was an increase in motion in the last few hundred milliseconds of fusion, with the motion during the last 100 ms of fusion being twice that of the prestimulation motions. (Lower) The same analysis was performed on nonfusing granules. Nonfusing granules were selected from the same cells as those in Upper. Their motions were aligned during stimulation to the average time at which granules were fusing in the same cell. Nonfusing granules did not substantially increase their motion when neighboring granules are fusing. (D) Time courses of increases in ΔR/ΔRprestimulation for 35 granules that fused after 8 s of stimulation (Upper) and for 37 granules that fused during the first 8 s of stimulation (Lower).