Fig. 7
From: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation
RhoA/ROCK-mediated actomyosin formation via the PS flippase/PIEZO1 pathway required for myotube morphology. a Schematic diagram of the PS flippase/PIEZO1 axis (black) and activators for the RhoA/ROCK/actomyosin pathway (red). b, c Suppressed cortical accumulation of phosphorylated MLC2 (P-MLC2) in PS flippase-deficient and PIEZO1-deficient myotubes. b Localization of P-MLC2 (anti-P-MLC2 antibody, green), F-actin (phalloidin, red), and nuclei (DAPI, cyan) in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia. Intense co-localization of P-MLC2 and F-actin was observed on the cell periphery of WT myotubes. c Cortex vs. cytoplasm ratio of P-MLC2 signals in b. d, e Rescue of myotube formation by stable expression of a phospho-mimetic form of MLC2 in PIEZO1-deficient C2C12 syncytia. d Syncytia formed by WT and PIEZO1-deficient C2C12 myoblasts stably expressing WT MLC2 (MLC2-WT-GFP) or phospho-mimetic MLC2 (MLC2-DD-GFP) were visualized by immunofluorescent staining with anti-MyHC antibody (differentiated cells, red) and DAPI (nuclei, cyan). e Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei in d. Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3 in d. f Rescue of myotube formation by activation of the RhoA/ROCK/actomyosin pathway in PS flippase- or PIEZO1-deficient C2C12 syncytia. Syncytia formed by WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia after treatment with calyculin A (a myosin II activator) or CN03 (a selective RhoA activator) were visualized by immunofluorescent staining with anti-MyHC antibody and DAPI. Morphologies of the syncytia are shown in Supplementary Figure 6c. Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei. Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3. ****P < 0.0001 (Student’s t-test). NS not significant, n sample number. Bar graphs represent mean ± S.E.M. Box and whiskers graph-line: median, box: upper and lower quartiles, whiskers: maxima and minima. Scale bars: 20 μm (b), 100 μm (d)
