Chaperone-mediated folding and assembly of myosin in striated muscle.
De novo folding and assembly of striated muscle myosin was analyzed by expressing a GFP-tagged embryonic myosin heavy chain (GFP-myosin) in post-mitotic C2C12 myocytes using replication defective adenoviruses. In the early stages of muscle differentiation, the GFP-myosin accumulates in bright globular foci and short filamentous structures that are later replaced by brightly fluorescent myofibrils. Time-lapse microscopy shows that the intermediates are dynamic and are present in elongating and fusing myocytes and in multinucleated myotubes. Immunostaining reveals the co-localization of the molecular chaperones Hsc70 and Hsp90 with the GFP-myosin in the intermediates, but not in the mature myofibrils. Uninfected cells have similar intermediates suggesting a common pathway for myosin maturation. Two conformation-sensitive antibodies that bind the unfolded motor domain and the coiled-coil conformation of the rod demonstrate that in the intermediates, the myosin rod is folded but the motor domain is not folded. Electron microscopy reveals that the intermediates contain loose filament bundles surrounded by a protein rich matrix. Geldanamycin, a specific inhibitor of Hsp90, reversibly blocks myofibril assembly and triggers accumulation of myosin folding intermediates. We conclude that multimeric complexes of nascent myosin filaments associated with Hsc70 and Hsp90 are intermediates in the folding and assembly pathway of muscle myosin.
Srikakulam
Rajani
R
Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA.
Winkelmann
Donald A
DA
D000257:Adenoviridae Infections / Q000821:virology; D000818:Animals; D002460:Cell Line; D002645:Chickens; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018832:Molecular Chaperones / Q000502:physiology*; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism; D009218:Myosins / Q000235:genetics* / Q000378:metabolism*; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D008164:Luminescent Proteins; D018832:Molecular Chaperones; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009218:Myosins