SEGREGATED ASSEMBLY OF MUSCLE MYOSIN EXPRESSED IN NONMUSCLE CELLS

Skeletal muscle myosin cDNAs were expressed in a simian kidney cell li ne (COS) and a mouse myogenic cell line to investigate the mechanisms controlling early stages of myosin filament assembly. An embryonic chi cken muscle myosin heavy chain (MHC) cDNA was linked to constitutive p romoters from adenovirus or SV40 and transiently expressed in COS cell s. These cells accumulate hybrid myosin molecules composed of muscle M HCs and endogenous, nonmuscle, myosin light chains. The muscle myosin is found associated with a Triton insoluble fraction from extracts of the COS cells by immunoprecipitation and is detected in 2.4 +/- 0.8-mu m-long filamentous structures distributed throughout the cytoplasm by immunofluorescence microscopy. These structures are shown by immunoel ectron microscopy to correspond to loosely organized bundles of 12-16- nm-diameter myosin filaments. The muscle and nonmuscle MHCs are segreg ated in the transfected cells; the endogenous nonmuscle myosin display s a normal distribution pattern along stress fibers and does not coloc alize with the muscle myosin filament bundles. A similar assembly patt ern and distribution are observed for expression of the muscle MHC in a myogenic cell line. The myosin assembles into filament bundles, 1.5 +/- 0.6 mu m in length, that are distributed throughout the cytoplasm of the undifferentiated myoblasts and segregated from the endogenous n onmuscle myosin. In both cell lines, formation of the myosin filament bundles is dependent on the accumulation of the protein. In contrast t o these results, the expression of a truncated MHC that lacks much of the rod domain produces an assembly deficient molecule. The truncated MHC is diffusely distributed throughout the cytoplasm and not associat ed with cellular stress fibers. These results establish that the infor mation necessary for the segregation of myosin isotypes into distinct cellular structures is contained within the primary structure of the M HC and that other factors are not required to establish this distribut ion.