S A Fellini

The distribution of intermediate filament (IF) subunits during maturation of skeletal myotubes in vitro was examined by immunofluorescence, using antibodies against two different types of chick IF subunits: (a) 58-kdalton subunits of fibroblasts (anti-58K), and (b) 55-kdalton subunits of smooth muscle (anti-55K). Anti-58K bound to a filament network in replicating presumptive myoblasts and fibroblasts, as well as in immature myotubes. The distribution in immature myotubes was in longitudinal filaments throughout the cytoplasm. With maturation, staining of myotubes by anti-58K diminished and eventually disappeared. Anti-55K selectively stained myotubes, and the fluorescence localization underwent a drastic change in distribution with maturation--from dense, longitudinal filaments in immature myotubes to a cross-striated distribution in mature myotubes that was associated with the I--Z region of myofibrils. However, the emergence of a cross-striated anti-55K pattern did not coincide temperally with the emergence of striated myofibrils, but occurred over a period of days thereafter.

The protein subunit of 100-A filaments constitutes approximately 50% of the cytoskeleton protein of chick fibroblasts. In addition to the 43,000-dalton protein (constitutive actin) common to all cell types, fibroblast cytoskeletons contain a 58,000-dalton protein likely to be the 100-A filament subunit, whereas smooth muscle contains, instead, a 55,000-dalton protein. Additional differences among 100-A filaments are shown by immunofluorescence using antibodies angainst chick fibroblast 58,000-dalton component (anti-F58K) and against chick brain 100-A filament subunits (anti-BF). Anti-F58K binds to 100-A filaments in chick fibroblasts, presumptive myoblasts, chondroblasts, pigment cells, and neurons, but not to 100-A filaments in mouse or human fibroblasts. This antibody stains cables of 100-A filaments induced by sequentially treating cells with cytochalasin B and Colcemid. Anti-BF binds only to neurofilaments and not to 100-A filaments of other cell types studied. Absorption or antibodies with purified subunits from gizzard 100-A filaments eliminates binding of anti-F58K to the filaments of all cell types but does not diminish binding of anti-BF to neurofilaments. Various IgGs also bind nonspecifically to induced cables of 100-A filaments. The problem of nonspecific binding of labeled antibodies, as well as the problem of cell and species specificity of the 100-A filaments, is discussed.

Most theories of determination or differentiation assume that embryonic cells differ from mature cells. Embryonic cells are thought to have metastable control mechanisms. These labile controls are believed to become progressively more stabilized as the cells differentiate. Zygote, blastula, neural plate, limb bud, somite, or ‘stem’ cells are conceived of as undifferentiated, totipotent, or multipotential cells. As such, these cells supposedly have available for activation a larger repertoire of phenotypic programmes than their progeny. A necessary corollary to this view is that the activation of one particular phenotypic programme out of the many available is a function of instructive exogenous inducing molecules.

Myosins synthesized in non-myogenic cells and replicating presumptive myoblasts differ from those synthesized in postmitotic mononucleated myoblasts and myotubes. Myoblasts and myotubes synthesize the definitive light chains, MLC1 and MLC2. These light chains display different molecular weights in sodium dodecyl sulfate-polyacrylamide gels from the fibroblast light chains FLC1 and FLC2 synthesized in non-myogenic cells and presumptive myoblasts. There are immunological differences between the myosin heavy chains synthesized in myoblasts and myotubes and those synthesized in non-myogenic cells and presumptive myoblasts. Fluorescein-labeled antibodies against skeletal light meromyosin are bound only along the lateral edges of emerging and definitive A-bands. This antibody to light meromyosin is not bound to the outside of, or the microfilaments subtending, the plasma membrane in non-myogenic cells or in myoblasts or in myotubes. These findings suggest that: (1) non-myogenic cells and replicating presumptive myoblasts synthesize similar myosin heavy and light chains; (2) replicating presumptive myoblasts synthesize a different set of myosins from those synthesized by their postmitotic daughters, the myoblasts; (3) the myosins associated with the plasma membranes of non-myogenic and myogenic cells are products of structural genes distinct from those coding for the myosins for skeletal myofibrils.