Vesicle fusion generates pLAT synaptic clusters at interacting distances from pSLP76 clusters: signaling nanoterritories. (A–F) Jurkat cells were transfected with siCtr (A), siSyt7 (B), or siMAL (C and E) in the absence (A–D), or presence (E and F) of thapsigargin, and allowed to spread for 3 min on an αCD3- (A–C and E) or αCD45-coated (D and F) coverslips. Cells were stained for pLAT and pSLP76 and analyzed by dSTORM-TIRF imaging. Right panels show a magnified image of a region of interest (frame). pLAT, green; pSLP76, red; pLAT and pSLP76 detections that are distant to each other by less than 20 nm (signaling nanoterritories), white. (G–O) Population analysis of pLAT and pSLP76 signaling nanoterritories in Jurkat cells processed as in A–F. (G–H) Mean percentage per cell of the area occupied by signaling nanoterritories with respect to the total pLAT (G) and pSLP76 (H). (G) Number of clusters per square micrometer is as follows: siCtr, n = 24; siSyt7, n = 21; siMAL, n = 14; siMAL TPS, n = 11; αCD45, n = 20; and αCD45 TPS, n = 22. (H) Number of clusters per square micrometer is as follows: siCtr, n = 18; siSyt7, n = 21; siMAL, n = 14; siMAL TPS, n = 11; αCD45, n = 19; and αCD45 TPS, n = 22. (I) Mean percentage per cell of the area occupied by signaling nanoterritories within each individual pLAT cluster n ≥ 11 cells. (J–O) Percentage of the area occupied by signaling nanoterritories with respect to the total pLAT area (as in G) in function of cluster circularity value bracket for n ≥ 11 cells. Images representative of three experiments. ***, P ≤ 0.0001; **, P ≤ 0.01; *, P ≤ 0.05; Mann-Whitney test. Each circle represents a cell. Bar, 10 µm.