IMM constrictions do not require Oma1 activity but do require the ETC. (A) Western blot of Opa1 isoform pattern in U2OS cells during 4 µM ionomycin stimulation during the period of optimum calcium-induced constrictions. Mass is in kilodaltons. L1, L2, and S3–5 refer to Opa1 isoforms are as defined previously (Anand et al., 2014; Otera et al., 2016). (B) Western blot of Opa1 after live-cell cross-linking with BMH to reveal Opa1 oligomerization changes during 4 µM ionomycin stimulation. Oligomers are labeled with arrowheads and monomers with an arrow. Mass is in kilodaltons. (C) Constrictions in U2OS cells that had been transfected with Oma1 siRNA for 72 h, followed by transfection of mito-R-GECO and mitoBFP for 24 h and stimulation by 4 µM ionomycin for 60 s. Bars: (main) 5 µm; (insets) 2 µm. (D) Effect of 5 µM antimycin A or 2.5 µM rotenone on (left to right) mitochondrial constrictions, actin burst, and mitochondrial calcium spike induced by 4 µM ionomycin. Antimycin A or rotenone was added simultaneously to ionomycin. For mitochondrial constriction assessment: n = 10 ROIs, 200 µm² mitochondrial area (Iono), 11 ROIs, 136 µm² (Iono + rotenone); and 16 ROIs, 300 µm² (Iono + Antimycin A). For actin filament assessment, 10 cells were used for each condition. For mitochondrial calcium assessment, 15–20 cells were used for each condition. P-values are from an unpaired Student’s t test. ***, P < 0.0011. Error bars represent SD (constriction quantification) or SEM (actin filament and mitochondrial calcium assessment).