Anchorage-independent muscle cell differentiation

Abstract

Cells from embryonic chicken muscle were cultivated in serum-free medium. After two days, the suspended cells (almost all of which were nondividing myoblasts) were subcultured in serum-containing medium, either in gelatincoated tissue culture dishes (to promote reattachment) or in bacteriological dishes (to prevent reattachment). The extent of fusion was high in both suspended and reattached cultures.Newly synthesized proteins from day-5 cultures were resolved by two-dimensional electrophoresis and detected by autoradiography. Not Most normal cell types exhibit anchorage-dependent proliferation; by contrast, many transformed cell lines proliferate well in suspension, a characteristic that appears to be consistently correlated with tumorigenicity (1, 2). For 3T6 cells, which show strict anchorage dependence of proliferation, forced suspension has been shown to result in a rapid decrease in both mRNA synthesis and mRNA degradation and in a slower decrease in total protein synthesis (3). Some proteins may be synthesized in near-normal amounts in suspension (4) and some mRNA species, and the proteins they code for, are synthesized in greater-than-normal amounts during recovery after reattachment (4). For these cells, then, there is reason to believe that their pattern of gene expression is regulated by cell-substrate interactions.Myogenic precursor cells from avian or mammalian embryos proliferate readily in monolayer culture. With time, they give rise to nondividing, terminally differentiating "myoblasts" that synthesize and accumulate muscle-specific molecules and form syncytial "myotubes." These cells elongate, elaborate the specialized subcellular structures characteristic of functional muscle, and contract (for review, see refs. 5-7).The influence of the culture substrate on muscle differentiation has received much attention (5). In clonal cultures myotube formation is favored by collagen-coated substrates. At high density this requirement is overcome, probably by cellderived collagen (5). Holtzer et al. (7) state that myogenic cells "remain alive when cultured on a surface to which they are unable to stick but will not differentiate unless they become attached." On the other hand, it has been reported that, under conditions that prevent normal cell-substrate interaction, myogenic cells did not divide and migrate, but were able to fuse and to synthesize and assemble myofibrillar proteins (8). Recently, several authors have observed that chicken myogenic cells can fuse in suspension, forming multinucleated "myoballs" (9-12). These myoballs possess acetylcholine receptors, generate action potentials, contract, and, when allowed to attach to a substrate, elongate into myotubes (10,11).In this paper, we reexamine the question of the anchorage dependence of skeletal muscle differentiation. Samples of the same cell suspension are cultivated in the same medium either in suspension or attached to culture dishes and then compared directly with respect to: (i) extent of fusion; (ii) pattern of synthesized prote...

Notch Pathway Activation Contributes to Inhibition of C2C12 Myoblast Differentiation by Ethanol

“…We recognized that quantifying the effects of a small molecule on the size, shape and other properties of cells that stain positively for the myotube marker Troponin T (TnT) and counterstained for the nuclear stain DAPI is labor intensive, “low throughput”, and limited by investigator fatigue [25]. While anchorage-independent myoblast differentiation has been demonstrated [26], we did not favor automated flow assisted analysis of myotube properties because the asymmetric shape of myotubes is dependent on attachment to a tissue culture surface. Here, we demonstrate a method for systematic image recording of each well of a 96-well tissue culture dish coupled with computer-based image analysis capable of measuring the size and nuclear features of more than one thousand myotubes per well.…”

The Mr 165,000 M-protein myomesin: a specific protein of cross-striated muscle cells.

“…Obviously, the antigen is highly concentrated in the M-line region, thus little or no myomesin is available anywhere else within the cell . This is also true for cells differentiating in suspension cultures where M-line-containing myofibrils wound about the nucleus can be detected as long as the cells are not allowed to attach to a substrate (36) . Furthermore, myomesin can be used as a sensitive marker for the localization of muscle cells in somites and in myotomes during early embryonic development in vivo as has been shown on cryostat sections of as early as stage 24 (15) chick embryos (M .…”

“…Becton, Dickinson & Co., Oxnard, Calif.) containing four to six sterilized and gelatinized glass coverslips. Breast muscle cells in suspension were grown according to Puri et al (36) . Incubation conditions were as those already described (47) .…”

“…Accumulation ofa number of muscle-specific isoproteins is thought to begin after withdrawal of myogenic cells from the mitotic cycle and can occur independent of fusion into myotubes (7,9,27,29,33,46,49). Myogenic cells were cultured in suspension and later allowed to attach and spread for 24 h according to Puri et al (36). As can be seen in Fig.…”

Extracellular-matrix synthesis by skeletal muscle in culture. Major secreted collagenous proteins of clonal myoblasts

“…The importance of the extracellular matrix in skeletal-muscle development has been well established (Hauschka & Konigsberg, 1966;Konigsberg, 1970; Hauschka, 1972;Hauschka & White, 1972;De La Haba et al, 1975;Lipton, 1977;Puri et al, 1980;Chiquet et al, 1981;Ehrisman et al, 1981;Popiela et al, 1982;Beach et al, 1983).…”