Fig. 7: Actomyosin tension inhibits fission by promoting MIEF1 phosphorylation.
a, Volcano plot showing differentially phosphorylated peptides in MCF10A-RAS cells treated with ROCK–MLCK inhibitors (YM) for 2 h (n = 5 samples per condition in a single experiment). b, Heat maps showing the levels of phosphorylated S55/59 MIEF1 in fibroblasts cultured on hydrogels of differential stiffness77 (n = 3 per condition; P < 0.0001), of induced pluripotent stem cell (iPS)-derived cardiomyocytes (CMs) bearing the hyperactive MHY7 R403Q substitution (n = 6 per condition; P = 0.019) and of myectomy samples from patients with hypertrophic cardiomyopathy (HCM)78 (n = 5 per condition; P = 0.0015). c, Immunoblotting for HA-MIEF1 isoforms (WT, S55/59A (SA) and S55/59E (SE)) expressed in MCF10A-RAS cells. Where indicated, cells were treated with ROCK–MLCK inhibitors (YM). Asterisks denote electrophoretic shifts. GFP was co-transfected. Consistent results were obtained in n = 4 experiments. d, Immunoblotting with two anti-phospho-MIEF1 antisera on HA-MIEF1 variants immunopurified from MCF10A-RAS cell extracts. Where indicated, cells were treated with ROCK–MLCK inhibitors (YM). Consistent results were obtained in n = 3 experiments. unt, untransfected. e, Immunoblotting with two anti-phospho-MIEF1 antisera on endogenous MIEF1 after immunoprecipitation from MCF10A-RAS cell extracts. Consistent results were obtained in n = 3 experiments. f, Representative immunofluorescence staining for HA-MIEF1 variants in MCF10A-RAS cells treated with ROCK–MLCK inhibitors (YM). Scale bar, 2 μm. Consistent results were obtained in n = 3 experiments. g, Quantification of MIEF1 puncta in MCF10A-RAS cells as in f (n = 36 (MIEF1 WT DMSO), 36 (MIEF1 WT YM) and 43 cells (MIEF1 SA) from two experiments). h, Quantification of endogenous DRP1 puncta in MCF10A-RAS cells as in f (n = 30 (MIEF1 WT DMSO), 27 (MIEF1 WT YM) and 31 cells (MIEF1 SA) from two experiments). i, Mitochondrial length analysis in MCF10A-RAS cells depleted of MIEF1/2 (siMIEF1/2a) and transfected to reconstitute different levels of the WT, phosphorylation-mimicking (SE) and phosphorylation-null (SA) MIEF1 variants. Where indicated, cells were treated with ROCK–MLCK inhibitors (YM) (n = 39 (siCO DMSO), 41 (siCO YM), 39 (siMIEF1/2a DMSO), 40 (siMIEF1/2a YM), 43 (siMIEF1/2a MIEF1 WT low level YM), 46 (siMIEF1/2a MIEF1 WT medium level YM), 41 (siMIEF1/2a MIEF1 WT high level YM), 37 (siMIEF1/2a MIEF1 SA low level YM), 43 (siMIEF1/2a MIEF1 SA medium level YM), 38 (siMIEF1/2a MIEF1 SA high level YM), 21 (siMIEF1/2a MIEF1 SE low level YM), 23 (siMIEF1/2a MIEF1 SE medium level YM) and 25 cells (siMIEF1/2a MIEF1 SE high level YM) from three experiments). Scale bars, 10 μm. j, Mitochondrial length analysis in MCF10A-RAS cells with overexpression of WT and SA MIEF1 variants on stiff glass (n = 43 (untransfected), 34 (WT low level), 36 (WT high level), 35 (SA low level) and 42 cells (SA high level) from three experiments). Scale bars, 5 μm. The data are presented as means and single points (g and h) or means ± s.d (i and j). Statistical significance was determined by two-tailed Student’s t-test. In i and j, this was calculated on punctate mitochondria. Source numerical data and unprocessed blots are available (see source data and Supplementary Table 1). IP, immunoprecipitate.
