GPCR activation triggers MVB–PM fusion in single cells in a calcium-independent manner. (a) Schematic model of imaging setup. (b) Fusion activity of HeLa cells stimulated with KCl (70 mM), caffeine (20 mM), or histamine (100 µM). n ≥ 8 cells per condition. (c) Total projection of fusion events over a 60-s time course onto cells before (top) and after (bottom) stimulation with histamine (100 µM). Pseudocolored as in Fig. 1 j. (d) Measurement of individual HeLa cells (n = 14) before and during stimulation with histamine (100 µM). (e) Mean fusion kinetics of CD63-pHluorin HeLa cells (n = 6) showing the distribution of fusion events over time (dark blue line; SD is in light blue) and the calcium levels (red) during histamine stimulation (gray-shaded block). (f) Heat maps revealing calcium responses (measured by Fluo-4) upon histamine stimulation obtained by subtracting the fluorescent intensity values before stimulation from those after 8-s stimulation. Cells were nontreated or incubated with a buffer with fast (BAP​TA) or slow (EGTA) calcium-binding kinetics. Bars, 10 µm. (g) Quantification of fusion activity of histamine-stimulated HeLa cells in the presence of EGTA (top) or BAP​TA (bottom) buffers. n ≥ 10 cells per condition. (h) Measurement of individual HUV​EC cells (n = 30) before and after stimulation with histamine (100 µM). *, P < 0.05; **, P < 0.01 using Student’s two-tailed two-sample t test. All t tests were unpaired except for d and h. Whiskers in the box plots (b and g) represent 1.5 times the interquartile distance or the highest or lowest point, whichever is shorter.