Fig. 3

MIEFs promote mitochondrial fusion independent of Drp1. a Confocal images of mitochondrial morphology in wild-type (WT) and Drp1^−/− 293T cells. b, c Confocal images of mitochondrial fusion in WT and Drp1^−/− 293T polykaryons from the PEG-mediated cell fusion assay. Cells stably expressing mitoRFP (red) or mitoGFP (green) were co-cultured and subjected to PEG-mediated cell fusion. Mitochondrial fusion is indicated by co-localization of mitoRFP and mitoGFP (yellow mitochondria) (b). The extent of mitochondrial fusion in individual hybrid cells was analyzed by Pearson’s correlation coefficient (PCC) in three independent experiments for each condition and data are summarized in (c). d, e The mito-PAGFP-based mitochondrial fusion assay in WT and Drp1^−/− 293T cells. Cells co-transfected with mito-PAGFP (0.5 μg) and mito-DsRed (0.2 μg) were photoactivated in the ROI (white circle, 3 μm diameter) in preactivation images of mitochondria (red). After photoactivation, mito-PAGFP fluorescence (green) intensity and mitochondrial marker (mito-DsRed) were collected at indicated time points (d). Mitochondrial fusion was quantified by analyzing changes in fluorescence intensity of photoactivated mito-PAGFP in ROIs at 40 s and 10, 20, and 30 min. The dilution rates (percentage) of the GFP fluorescence intensity at different time points were normalized by the fluorescence intensity at 40 s after photoactivation (e). (n represents the total number of cells analyzed for each condition). n.s., not significant. f, g Confocal images of mitochondrial fusion from the PEG-based fusion assay in WT and Drp1^−/− 293T polykaryons transfected with empty vector, MIEF1-V5, or MIEF2-V5. Cells with stable expression of mitoRFP or mitoGFP were co-cultured and transfected with empty vector (control), MIEF1-V5, or MIEF2-V5 as indicated and then subjected to PEG-mediated cell fusion. Mitochondrial fusion is indicated by co-localization of mitoRFP (red) and mitoGFP (green) (yellow mitochondria) (f). The extent of mitochondrial fusion in individual hybrid cells was analyzed via the Pearson’s correlation coefficient (PCC) in three independent experiments for each condition and data summarized in (g). Data are expressed as means ± SEM, n represents the number of cells analyzed (c, e, and g)