Fig. 8
Elevated expression of MIEFs reverses hFis1-induced mitochondrial fragmentation through preventing hFis1 binding to pro-fusion proteins. a Elevated levels of MIEF1 or MIEF2 reverse hFis1-induced mitochondrial fragmentation in 293T cells lacking Drp1. Confocal images of mitochondrial morphologies in Drp1−/− 293T cells co-transfected with Myc-hFis1 and either empty vector, MIEF1-V5, MIEF2-V5, MIEF2Δ1-49, or MIEF2Δ50-104 as indicated. The cells were stained with MitoTracker (red) followed by immunostaining with anti-Myc (green) and anti-V5 (blue) antibodies. b Percentages (mean ± SEM) of cells with indicated mitochondrial phenotypes in Drp1−/− 293T cells co-transfected with indicated plasmids in a, in three independent experiments for each condition (n represents the total number of cells analyzed for each condition). ****P < 0.0001; ***P < 0.0005; ns, no significant. c Exogenous expression of MIEFs drastically decreased the interaction between hFis1 and pro-fusion proteins Mfn1 and Mfn2. 293T cells were transfected with empty vector, MIEF1-V5, or MIEF2-V5. Cell lysates were subjected to co-IP with protein G beads pre-bound to rabbit normal IgG (negative control) or anti-hFis1 antibody, followed by immunoblotting with indicated antibodies. d The ratios (mean ± SEM) between Mfn1 or Mfn2 and hFis1 in co-immunoprecipitates in c were analyzed by densitometry. e Elevated expression of hFis1 also impaired the interaction between pro-fusion proteins Mfn1/2 and MIEF1/2. 293T cells were co-transfected with empty vector (as negative control) and MIEF1-V5 or MIEF2-V5, as well as co-transfected with Myc-hFis1 and either MIEF1-V5 or MIEF2-V5 as indicated. Cell lysates were used for co-IP with anti-V5 beads, followed by Western blotting with indicated antibodies. f The ratios (mean ± SEM) between Mfn1 or Mfn2 and MIEF1-V5 or MIEF2-V5 in co-immunoprecipitates in e were analyzed by densitometry