TY - JOUR
T1 - Structure-function analysis of myomaker domains required for myoblast fusion
AU - Millay, Douglas P.
AU - Gamage, Dilani G.
AU - Quinn, Malgorzata E.
AU - Min, Yi Li
AU - Mitani, Yasuyuki
AU - Bassel-Duby, Rhonda
AU - Olson, Eric N.
N1 - Funding Information:
We thank J. Cabrera for graphical assistance, A. Fernandez-Perez for technical assistance, and D. Rosenbaum for helpful suggestions. D.P.M. was funded by a Development Grant from the Muscular Dystrophy Association. This work was supported by grants from the Cincinnati Children''s Trustee Program (to D.P.M.); NIH Grants HL-077439, HL-111665, HL- 093039, DK-099653, and U01-HL-100401; and Robert A. Welch Foundation Grant 1-0025 (to E.N.O.).
PY - 2016/2/23
Y1 - 2016/2/23
N2 - 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.
AB - 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.
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U2 - 10.1073/pnas.1600101113
DO - 10.1073/pnas.1600101113
M3 - Article
C2 - 26858401
AN - SCOPUS:84959440135
SN - 0027-8424
VL - 113
SP - 2116
EP - 2121
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 8
ER -