ReportMuscle Development

Control of muscle formation by the fusogenic micropeptide myomixer

See allHide authors and affiliations

Science  21 Apr 2017:
Vol. 356, Issue 6335, pp. 323-327
DOI: 10.1126/science.aam9361
  • Fig. 1 Myomixer is a small-membrane protein that is essential for myoblast fusion.

    (A) Western blot showing Myomixer, Myosin, and Gapdh expression in WT or Myomixer KO primary myoblast cultures after 4 days in DM. (B) WT and Myomixer KO primary myoblasts differentiated for 1 week and stained with MY32 antibody (myosin) and Hoechst show the requirement of Myomixer for fusion. Scale bar, 50 μm. (C) Quantification of fusion in WT and Myomixer KO primary myoblast cultures (n = 3 pairs). (D) Amino acid sequence of Myomixer and cross-species homology. Basic residues are blue, acidic residues are red, cysteines are green, and leucines are yellow. H, helix; C, coil. (E) Live-cell staining of Myomixer-transfected C2C12 myoblasts showing cell-surface localization of Myomixer with a C-terminal FLAG tag. Laminin was stained after FLAG staining and permeabilization. (F) Western blot analysis of cytosolic (C) and membrane (M) fractions of retrovirus-Myomixer–infected 293 cells and WT C2C12 cells (day 4 after differentiation). Gapdh blot was used as a positive control of cytosolic proteins. N-Cadherin and epidermal growth factor (EGF) receptor blots were used as positive controls of membrane proteins. *P < 0.05, ***P < 0.001, Student’s t test. Data are mean ± SEM.

  • Fig. 2 Myomixer expression during muscle development and regeneration in mice.

    (A) Western blot showing Myomixer, Myosin, and Gapdh expression during C2C12 differentiation. (B) Western blot showing Myomixer and Gapdh expression in the indicated tissues. SKM, skeletal muscle. (C) In situ hybridization showing Myomixer transcript expression in transverse sections of mouse embryos at E12.5 and E15.5. Scale bar, 250 μm (a), 500 μm (b), and 200 μm (c). Image c is a magnification of the boxed area shown in image b. H, heart; R, radius. (D) Myomixer mRNA expression in CTX-injured skeletal muscle from 1-month-old WT mice, as determined with quantitative PCR. (n = 3 mice/time point). (E) Up-regulation of Myomixer mRNA expression in mdx compared with WT muscle, as detected with quantitative PCR (n = 4 pairs). Data are mean ± SEM.

  • Fig. 3 Myomixer is essential for muscle development in mice.

    (A) WT and Myomixer KO embryos at E17.5 dissected and skinned to reveal the lack of muscle in Myomixer KO limbs. (B) Hematoxylin and eosin (H&E) staining of E17.5 limb muscles shows lack of muscle fibers in Myomixer KO embryos. Scale bar, 50 μm. (C) Immunohistochemistry of E17.5 limb muscles using MY32 (myosin) antibody. WT myofibers show multinucleation, which is absent in Myomixer KO sections. Scale bar, 50 μm. (D) Western blot analysis of Myomixer and Gapdh in forelimb tissues of E17.5 WT and Myomixer KO embryos.

  • Fig. 4 Myomixer binds Myomaker and synergizes to induce cell fusion.

    (A) MY32 (myosin) immunostaining of C2C12 cells infected with retroviruses expressing Myomaker, Myomixer, or both and differentiated for 4 days. Nuclei were counterstained with Hoechst and pseudocolored green. Scale bar, 50 μm. (B and C) Fluorescence images of GFP, mCherry, and Hoechst to counterstain nucleus. Scale bars, 100 μm (B), 50 μm (C). Arrows point to multinucleated GFP+/mCherry+ cells. (D and E) Coimmunoprecipitation assays were performed by using 10T1/2 cells or C2C12 myoblasts infected with retroviruses expressing Myomixer and/or FLAG-Myomaker. IP, immunoprecipitation; IB, immunoblot. (F) MY32 (myosin) immunostaining of C2C12 cells infected with retroviruses expressing Myomaker, together with WT or mutated versions of Myomixer, differentiated for 1 week. Nuclei were counterstained with Hoechst and pseudocolored green. Scale bar, 50 μm. R, arginine; E, glutamic acid; C, cysteine; L, leucine; A, alanine; EEEAA, R34E-R38E-R46E-C52A-L54A.

Supplementary Materials

  • Control of muscle formation by the fusogenic micropeptide myomixer

    Pengpeng Bi, Andres Ramirez-Martinez, Hui Li, Jessica Cannavino, John R. McAnally, John M. Shelton, Efrain Sánchez-Ortiz, Rhonda Bassel-Duby, Eric N. Olson

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Figs. S1 to S7
    • References