Distinct spatiotemporal organization of fusion of different vesicles pools in melanoma cells. (A) Box and whisker plots of frequency of VAMP7-pHluorin– and VAMP3-pHluorin–mediated exocytosis in VMM39 melanoma cells (n = 9 cells/condition; box shows median ± IQR, whiskers reach minimum and maximum values within 1.5 times the IQR). (B) Heat maps of density of VAMP7- and VAMP3-mediated exocytosis in representative VMM39 melanoma cells. Solid white line demarcates longest axis of the cell. Dashed perpendicular lines indicate minor axes 10 µm from the tips of the longest axis. The area encompassed from the edge of the cell to the dotted lines were measured to define a larger and smaller end of the cell. (C) Box and whiskers plots of number of exocytic events normalized to the larger end, middle, and smaller end of the cell. VAMP7-mediated exocytosis was polarized to the larger end of the cell (n = 9 cells). (D) 2D Ripley’s L(r) function applied to the spatial occurrence of VAMP7- and VAMP3-mediated exocytic events (n = 9). VAMP7-mediated events exhibited nonrandom clustering at all distances measured, whereas VAMP3-mediated events were dispersed at larger distances in regularly spaced clusters 1.5–2.5 µm in size. (E) 1D Ripley’s L(r) function applied to the temporal occurrence of VAMP7- and VAMP3-mediated exocytic events. Both VAMP7 and VAMP3-mediated events exhibited random temporal occurrence. See Video 5.