Mitochondrial constrictions are MCU dependent and Drp1 independent. (A) Quantification of mitochondrial constriction frequency in the unstimulated (blue) or ionomycin-stimulated (red) conditions, represented as constrictions per mm mitochondrial length in U2OS-control, Drp1-KD, and MCU-KD cells. P-values were obtained from an unpaired Student’s t test. Error bars represent SEM. Control:, 16 ROI, 2,508 µm mitochondrial length; Drp1 KD, 10 ROI, 1,000 µm; MCU KD: 20 ROI, 2,600 µm. (B) Representative confocal montages of control, Drp1-KD, and MCU-KD U2OS cells transfected with mito-R-GECO (mitochondrial calcium, red) and mitoBFP (mitochondrial matrix, blue) and then treated with 4 µM ionomycin at time 0. Time in seconds. White arrows point to constrictions and yellow arrows to fission events. Bar, 5 µm. Corresponds to Video 8. (C) U2OS cell transfected with GFP-Sec61β (green, ER marker) and mito-R-GECO (red, mitochondrial calcium) and imaged live before ionomycin stimulation (top) and at 35 s after 4 µM ionomycin addition (bottom). Image on the right is an expanded view of the (+) ionomycin condition. Bars: (regular panels) 2 µm; (expanded panel) 1 µm. Arrows denote ER presence at mitochondrial constrictions. Corresponds to Video 9. (D) Quantification of percent mitochondrial constrictions corresponding to ER contact from live-cell confocal microscopy of ionomycin-stimulated cells such as in C and Video 9. 204 constrictions from 26 ROIs from 13 cells assessed, totaling a 461-µm2 mitochondrial area.
