title: Dynamics of myosin replacement in skeletal muscle cells.
abstract: Highly organized thick filaments in skeletal muscle cells are formed from ~300 myosin molecules. Each thick-filament-associated myosin molecule is thought to be constantly exchanged. However, the mechanism of myosin replacement remains unclear, as does the source of myosin for substitution. Here, we investigated the dynamics of myosin exchange in the myofibrils of cultured myotubes by fluorescent recovery after photobleaching and found that myofibrillar myosin is actively replaced with an exchange half-life of ~3 h. Myosin replacement was not disrupted by the absence of the microtubule system or by actomyosin interactions, suggesting that known cytoskeletal systems are dispensable for myosin substitution. Intriguingly, myosin replacement was independent of myosin binding protein C, which links myosin molecules together to form thick filaments. This implies that an individual myosin molecule rather than a thick filament functions as an exchange unit. Furthermore, the myosin substitution rate was decreased by the inhibition of protein synthesis, suggesting that newly synthesized myosin, as well as preexisting cytosolic myosin, contributes to myosin replacement in myofibrils. Notably, incorporation and release of myosin occurred simultaneously in myofibrils, but rapid myosin release from myofibrils was observed without protein synthesis. Collectively, our results indicate that myosin shuttles between myofibrils and the nonmyofibrillar cytosol to maintain a dynamic equilibrium in skeletal muscle cells.
authors.affiliation: Animal Products Research Division, NARO Institute of Livestock and Grassland Science, Ibaraki, Japan; and.
authors.forename: Koichi
authors.initials: K
authors.lastname: Ojima
authors.affiliation: Research Faculty of Agriculture, Graduate School of Agriculture, Hokkaido University, Hokkaido, Japan.
authors.forename: Emi
authors.initials: E
authors.lastname: Ichimura
authors.affiliation: Research Faculty of Agriculture, Graduate School of Agriculture, Hokkaido University, Hokkaido, Japan.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Yasukawa
authors.affiliation: Research Faculty of Agriculture, Graduate School of Agriculture, Hokkaido University, Hokkaido, Japan.
authors.forename: Jun-Ichi
authors.initials: J
authors.lastname: Wakamatsu
authors.affiliation: Research Faculty of Agriculture, Graduate School of Agriculture, Hokkaido University, Hokkaido, Japan nishi@anim.agr.hokudai.ac.jp.
authors.forename: Takanori
authors.initials: T
authors.lastname: Nishimura
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D049452:Green Fluorescent Proteins; D018485:Muscle Fibers, Skeletal / Q000378:metabolism* / Q000502:physiology; D009218:Myosins / Q000235:genetics / Q000378:metabolism*
chemical_list: D049452:Green Fluorescent Proteins; D009218:Myosins
keywords: fluorescence recovery after photobleaching; myofibril; myosin; skeletal muscle; thick filament