MCU suppression inhibits mitochondrial division, but not Drp1 oligomerization. (A) U2OS cells were transfected with scrambled siRNA (control), MCU siRNA (MCU KD), and Drp1 siRNA (Drp1 KD) for 72 h Cells were then fixed and mitochondria stained using anti-Tom20 (red) and DAPI (blue). ROIs of fixed dimension were analyzed for mitochondrial length and number as described in Materials and methods. Images of control (left), Drp1 KD (middle), or MCU KD cells (right) are shown. Bars: (main) 5 µm; (insets) 2 µm. (B) Mean mitochondrial length quantification represented as area (square micrometers) per mitochondrion (left) and mean mitochondrial number quantification (right) for 80, 53, and 50 cells for control, MCU KD, and Drp1 KD cells, respectively. Errors bars represent SEM. P-values were obtained from an unpaired Student’s t test. (C) Quantification of mitochondrial division rate in control and MCU KD U2OS cells by measuring the number of division events in peripheral ROIs from live-cell videos of mitochondrial matrix marker (mito-BFP), either in unstimulated cells or cells in the first 10 min of 4 µM ionomycin stimulation; n = 19 cells [control (−) ionomycin], 21 cells [control (+) ionomycin], 20 cells [MCU KD (−) ionomycin], or 22 cells [MCU KD (+) ionomycin]. Each point represents one ROI per cell. P-values were obtained from an unpaired Student’s t test. *, P < 0.015; ***, P < 0.0001. Error bars represent SD. (D) Quantification of mitochondrially associated Drp1 oligomers and mitochondrial calcium in response to 4 µM ionomycin. GFP-Drp1 knock-in cells were treated with scrambled siRNA or MCU siRNA for 48 h and then transfected with mito-R-GECO and mito-BFP. Cells were imaged at 72 h after siRNA treatment; n = 20 cells in each case. Ionomycin addition at 0 s. Error bars represent SEM. (E) Micrographs from GFP-Drp1 knock-in U2OS cells (control and MCU siRNA treated) transfected with mito-BFP (red) and treated with 4 µM ionomycin as in B. Prestimulation (0”) and 300-s stimulations are shown. Bar, 10 µm. (F) Drp1 oligomerization kinetics in MCU KD cells, as measured in the whole cell. GFP-Drp1 knock-in U2OS cells were treated with control siRNA or MCU siRNA for 72 h and then stimulated with 4 µM ionomycin while acquiring GFP images. The amount of oligomerized Drp1 was assessed as GFP signal above the cytoplasmic background signal, as described in Materials and methods; n = 20 cells in each case. Error bars represent SEM. (G) Quantification of mitochondrial calcium increase in control or Drp1 KD U2OS cells upon 4 µM ionomycin stimulation; n = 14 cells in each case. Error bars represent SEM.