Actin requirement for stimulus-induced mitochondrial calcium spike. (A and B) Effect of 2 μM LatA treatment on ionomycin-induced (A) or histamine-induced (B) actin polymerization burst and mitochondrial calcium spike in U2OS cells; n = 10–15 cells. Error bars represent SEM. (C and D) Mitochondrial calcium spike after ionomycin (C) or histamine (D) stimulus in U2OS-WT, U2OS-INF2-KO, or INF2-KO cells reexpressing GFP-INF2-CAAX or GFP-INF2-nonCAAX. Control U2OS-INF2-KO cells are expressing GFP-Sec61β as an ER marker instead of INF2. Error bars represent SEM. (E) Actin polymerization burst after ionomycin stimulation in U2OS-WT and U2OS-INF2-KO cells. Rescue by transfection of GFP-INF2-CAAX or GFP-INF2-nonCAAX is also shown; n = 15–18 cells. Error bars represent SEM. (F) Ionomycin-induced actin morphology in INF2-KO cells reexpressing GFP-INF2-CAAX or GFP-INF2-nonCAAX. Whole-cell view shown prestimulation. Insets shown prestimulation (0”, left) and after 30 s stimulation (30”, right). Taken in a single confocal plane in an apical region of the cell body, which reduces actin background from stress fibers but causes the ER to appear fragmented. Arrows show examples of actin filament accumulation. Bars: (main) 5 µm; (inset) 2 µm. Corresponds to Video 6.