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-,um-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 ,um 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.

Chaperone-mediated folding and assembly of myosin in striated muscle

“…Areas rich in folding intermediates were selected and marked on the dishes. The samples were dehydrated through a graded alcohol series and embedded in epon and sectioned (Birk et al, 1988;Moncman et al, 1993). Gold sections were cut with a diamond knife, picked up on formvar-coated grids and stained with 2% uranyl acetate followed by 1% phosphotungstic acid, pH 3.2.…”

Myosin II Folding Is Mediated by a Molecular Chaperonin

“…We analyzed the folding of S2 region using anti-S2 mAb 10F12.3, a conformation-sensitive antibody that preferentially binds S2 in its coiled-coil conformation (39,40). In Western blot analysis, this antibody reacts strongly with native HMM resolved on native gels, but shows very weak recognition of the same fragment blotted from denaturing gels (data not shown).…”

Modulation of myosin filament organization by C-protein family members.

“…Sarcomeric MyHC assembles into arrays of thick filaments when expressed in nonmuscle cells (Moncman et al, 1993;Vikstrom et al, 1993). Because MyBPs have been postulated to play a role in the organization of thick filaments, we investigated the interactions of these two sarcomeric components through transfection of nonmuscle cells.…”

“…The same distribution pattern is seen when rat a-or ,3-cardiac MyHC is expressed in COS cells (Vikstrom et al, 1993), and occurs even when protein expression levels vary over 100-fold (Seiler and Leinwand, unpublished observations). These structures fail to form when a construct lacking the myosin rod is expressed, indicating that the structures represent assemblages of myosin, and not aggregates of protein (Vikstrom et al, 1993 It had previously been shown by electron microscopy that sarcomeric MyHC expressed in COS cells assembles into arrays of thick filaments (Moncman et al, 1993;Vikstrom et al, 1993). To examine the organization of the cable structures, immunogold electron microscopy was carried out.…”

“…The absence of endogenous muscle proteins in COS cells allows the cellular distribution of sarcomeric proteins, alone or in pairwise combinations, to be assessed. When sarcomeric myosin is expressed in COS cells, it assembles into bundles of thick filaments (Moncman et al, 1993;Vikstrom et al, 1993). The experiments described below have allowed us to address the role of MyBPs in modulating thick filament formation and organization.…”