Vesicular ATP activates FACE in an autocrine fashion upon LB exocytosis. (A) Image sequence illustrating analysis of FACE in a cell loaded with Fura-2 and maintained in a bath containing FM1-43 (top left). Fura-2 ratios (top row) and FM1-43 fluorescence (bottom row) were acquired at 3 Hz. Red circle denotes the region where the increase of FM1-43 fluorescence was measured after fusion of LB with the PM. The area within the two white circles represents the ring-like region of interest surrounding the fused LB, where changes in [Ca²⁺]_c (fura-2 ratio) were analyzed. t, time after start of experiment. Bar, 5 µm. (B) Time course of fura-2 ratio (blue) and FM1-43 fluorescence (red) around and within the area of the LB fusion depicted in A. The cell was stimulated with 300 nM PMA. (C) Same as A, recorded in a cell treated with VNUT shRNA for 3 d. t, time after start of experiment. Bar, 5 µm. (D) Time course of fura-2 ratio (blue) and FM1-43 fluorescence (red) around and within the area of the LB fusion depicted in C. The cell was stimulated with 300 nM PMA. Note the absence of FACE. (E) Quantitative analysis of the amplitude of the FACE signal from the experiments in B and D confirm full ablation of FACE in cells treated with VNUT shRNA. n, number of fusions analyzed for each condition. (F) Diffusion of FM1-43 into LBs after fusing with the PM. Δ FM/s represents the slope of the increase in FM1-43 fluorescence of a fused vesicle in the first 10 s after fusion with the PM. This is a measure for initial diffusion of FM1-43 into a fused LB and correlates well with fusion pore diameter (Haller et al., 2001; Miklavc et al., 2011). FM1-43 diffusion was significantly faster (*, P = 0.01) in control cells compared with diffusion in cells treated with VNUT shRNA. n, number of fusions analyzed for each condition. Mean ± SEM is shown.