Chaperone-mediated folding and assembly of myosin in striated muscle

Abstract

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.

Dual function of the UNC-45b Chaperone with myosin and GATA4 in cardiac development

“…The participation of UNC-45b in intermolecular complexes with either cardiac myosin or GATA4 was shown by pull-down experiments with cell lysates. The direct binding between UNC-45b and GATA4 was further confirmed by binding experiments with purified components as has been shown previously for myosin Srikakulam and Winkelmann, 2004;Srikakulam et al, 2008). The luciferase reporter assay indicated that GATA4-mediated transcriptional activation was enhanced by UNC-45b.…”

“…This gene is also expressed in skeletal muscle and is closely related to orthologs in vertebrate organisms from zebrafish to humans. Studies with C. elegans, cultured myocytes and purified muscle proteins have shown the significance of UNC-45b functioning in the myofibrillogenesis and maturation of full contractile functions in multiple striated muscles (Barral et al, 1998;Barral et al, 2002;Hutagalung et al, 2002;Price et al, 2002;Srikakulam and Winkelmann, 2004;Srikakulam et al, 2008).…”

Krp1 (Sarcosin) promotes lateral fusion of myofibril assembly intermediates in cultured mouse cardiomyocytes

“…It has long been known that muscle myosin filaments assemble separately from nascent Z-bodies and actin filaments [35]. Recent studies have shown that nascent myosin filaments are associated with the chaperone proteins Hsc70 and Hsp90 before incorporation into sarcomeres [36] ( figure 10, step 2). The accumulation of abnormally thin striated myofibrils after Krp1 knockdown shows that Krp1 functions downstream of N-RAP, Hsc70 and Hsp90 in the assembly pathway to promote lateral fusion of thin myofibrils ( figure 10, step 4).…”

Functional analysis of homeodomain-containing transcription factor Lbx1 in satellite cells of mouse skeletal muscle

“…Areas rich in folding intermediates were selected and marked on the dishes. Samples were dehydrated through a serial dilution of graded alcohol, embedded in Epon and sectioned with a diamond knife (Srikakulam and Winkelmann, 2004). Thin sections were stained in uranyl acetate and lead citrate, and examined by a JEOL JEM1010 electron microscope operated at 80 kV.…”