title: Structure-function analysis of myomaker domains required for myoblast fusion.
abstract: During skeletal muscle development, myoblasts fuse to form multinucleated myofibers. Myomaker [Transmembrane protein 8c (TMEM8c)] is a muscle-specific protein that is essential for myoblast fusion and sufficient to promote fusion of fibroblasts with muscle cells; however, the structure and biochemical properties of this membrane protein have not been explored. Here, we used CRISPR/Cas9 mutagenesis to disrupt myomaker expression in the C2C12 muscle cell line, which resulted in complete blockade to fusion. To define the functional domains of myomaker required to direct fusion, we established a heterologous cell-cell fusion system, in which fibroblasts expressing mutant versions of myomaker were mixed with WT myoblasts. Our data indicate that the majority of myomaker is embedded in the plasma membrane with seven membrane-spanning regions and a required intracellular C-terminal tail. We show that myomaker function is conserved in other mammalian orthologs; however, related family members (TMEM8a and TMEM8b) do not exhibit fusogenic activity. These findings represent an important step toward deciphering the cellular components and mechanisms that control myoblast fusion and muscle formation.
authors: Department of Molecular Biology and the Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390; Department of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229 douglas.millay@cchmc.org Eric.Olson@utsouthwestern.edu.
authors: Douglas P
authors: DP
authors: Millay
authors: Department of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229.
authors: Dilani G
authors: DG
authors: Gamage
authors: Department of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229.
authors: Malgorzata E
authors: ME
authors: Quinn
authors: Department of Molecular Biology and the Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors: Yi-Li
authors: YL
authors: Min
authors: Department of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229.
authors: Yasuyuki
authors: Y
authors: Mitani
authors: Department of Molecular Biology and the Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors: Rhonda
authors: R
authors: Bassel-Duby
authors: Department of Molecular Biology and the Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390; douglas.millay@cchmc.org Eric.Olson@utsouthwestern.edu.
authors: Eric N
authors: EN
authors: Olson
mesh_terms: D000818:Animals; D002459:Cell Fusion; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D024510:Muscle Development / Q000502:physiology*; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D032448:Myoblasts, Skeletal / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
chemical_list: D008565:Membrane Proteins; D009124:Muscle Proteins; C000591742:Mymk protein, mouse
keywords: CRISPR/Cas9; cell fusion; muscle development; myogenesis