The stimulus-induced actin burst precedes the mitochondrial calcium spike. (A and D) Quantification of cytoplasmic calcium (Cyto-R-GECO), cytoplasmic actin (GFP-Ftractin), or mitochondrial calcium (Mito-R-GECO) in U2OS cells after 4 µM ionomycin (A) or 100 µM histamine (D) stimulation. Rapid acquisition mode (4.8 frames/s). Values on the y axis represent the value at time X normalized to the value at time 0 (F/F0); n = 10–16 cells (also given in C). Error bars represent SEM. Corresponds to Videos 1 and 2. (B and E) Zoom of early time points from graphs in A (ionomycin) and D (histamine) showing the distinct kinetics of cytoplasmic calcium, actin, and mitochondrial calcium. Error bars have been removed for clarity. (C and F) Box-and-whiskers plots of stimulation half-times based on the curves in A and D. Number of cells for each reading is 14 (cytoplasmic calcium, ionomycin), 10 (actin, ionomycin), 14 (mitochondrial calcium, ionomycin), 12 (ER calcium, ionomycin), 14 (cytoplasmic calcium, histamine), 11 (actin, histamine), 16 (mitochondrial calcium, histamine), and 10 (ER calcium, histamine). Each point represents one cell. Error bars represent SD (G and H) Time-lapse montages of actin (green, GFP-Ftractin) and mitochondrial calcium (red, mito-R-GECO) changes in the same cell after 4 µM ionomycin (G) or 100 µM histamine (H) stimulation. Mitochondrial calcium alone shown in the top panels and merge in the bottom panels; taken at one frame per 1.03 s (G) and 1.2 s (H). Arrows in the merged panel indicate regions of the cytoplasm displaying the cytoplasmic actin burst. Bars: (G) 5 µm; (H) 10 µm. Corresponds to Videos 3 and 4.