Segregated assembly of muscle myosin expressed in nonmuscle cells.

Abstract

Skeletal muscle myosin cDNAs were expressed in a simian kidney cell line (COS) and a mouse myogenic cell line to investigate the mechanisms controlling early stages of myosin filament assembly. An embryonic chicken muscle myosin heavy chain (MHC) cDNA was linked to constitutive promoters from adenovirus or SV40 and transiently expressed in COS cells. These cells accumulate hybrid myosin molecules composed of muscle MHCs 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-micron-long filamentous structures distributed throughout the cytoplasm by immunofluorescence microscopy. These structures are shown by immunoelectron microscopy to correspond to loosely organized bundles of 12-16-nm-diameter myosin filaments. The muscle and nonmuscle MHCs are segregated in the transfected cells; the endogenous nonmuscle myosin displays a normal distribution pattern along stress fibers and does not colocalize with the muscle myosin filament bundles. A similar assembly pattern 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 micron in length, that are distributed throughout the cytoplasm of the undifferentiated myoblasts and segregated from the endogenous nonmuscle myosin. In both cell lines, formation of the myosin filament bundles is dependent on the accumulation of the protein. In contrast to 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 associated with cellular stress fibers. These results establish that the information necessary for the segregation of myosin isotypes into distinct cellular structures is contained within the primary structure of the MHC and that other factors are not required to establish this distribution.