Prefusion Myoblasts Research Articles

In differentiating prefusion myoblasts connexin43 gap junction coupling is upregulated before myoblast alignment then reduced in post-mitotic cells

Previously we have shown that during in vivo muscle regeneration differentiating rat primary myoblasts transiently upregulate connexin43 (Cx43) gap junctions and leave cell cycle synchronously. Here, we studied the temporal regulation of Cx expression in relation to functional dye coupling in allogenic primary myoblast cultures using western blotting, immuno-confocal microscopy and dye transfer assays. As in vivo, Cx43 was the only Cx isotype out of Cx26, 32, 37, 40, 43 and 45 found in cultured rat myoblasts by immunostaining. Cultured myoblasts showed similar temporal regulation of Cx43 expression and phenotypic maturation to those regenerating in vivo. Cx43 protein was progressively upregulated in prefusion myoblasts, first by the cytoplasmic assembly in sparse myoblast meshworks and then in cell membrane particles in aligned cells. Dye injection using either Lucifer Yellow alone, Cascade Blue with a non-junction permeant FITC-dextran revealed an extensive gap junction coupling between the sparse interacting myoblasts and a reduced communication between the aligned, but still prefused cells. The aligned myoblasts, uniformly upregulate p21(waf1/cip1) and p27(kip1) cell cycle control proteins. Taken together, in prefusion myoblasts less membrane-bound Cx43 was found to mediate substantially more efficient dye coupling in the growing cell fraction than those in the aligned post-mitotic myoblasts. These and our in vivo results in early muscle differentiation are consistent with the role of Cx43 gap junctions in synchronizing cell cycle control of myoblasts to make them competent for a coordinated syncytial fusion.

Actin filament organization in aligned prefusion myoblasts

The organization of the actin cytoskeleton in prefusion aligning myoblasts is likely to be important for their shape and interaction. We investigated actin filament organization and polarity by transmission electron microscopy (TEM) in these cells. About 84% of the filaments counted were either found in a subplasmalemma sheet up to 0.5 microm thick that was aligned with the long axis of the cell, or in protrusions. The remaining filaments were found in the cytoplasm, where they were randomly orientated and not organized into bundles. The polarity of the subplasmalemma filaments changed progressively from one end of the cell to the other. At the ends of the cells and in protrusions, the majority of filaments were organized such that their barbed ends faced the tip of the protrusion. We did not find any actin filament bundles or stress fibres in these cells. Time-lapse phase microscopy demonstrated that aligned cells were still actively migrating at the time of our TEM observations, and their direction of movement was restricted to the long axis of the cell group. The ability of these cells to locomote actively in the absence of actin filament bundles suggests that in these cells the subplasmalemma actin sheet contributes not only to cell shape but also to cell locomotion.

Cytochemical localization of calcium in prefusion myoblasts from the chick embryo myotome.

Myoblast fusion is a Ca2+-dependent process. The aim of this report was to study the localization of Ca2+ in prefusion myoblasts from the brachial somites of chick embryos (51-108 h of incubation), using the potassium pyroantimonate cytochemical method. When observed under a transmission electron microscope, electron-dense precipitates of Ca2+-antimonate were found in the basement membrane of the myotome, which separates the myotome from the adjacent mesenchyma. Within myoblasts, triads and sarcoplasmic reticulum associated with the first newly formed sarcomeres were observed, but a T-tubule network was not found. Moreover, Ca2+-antimonate precipitates were not observed in structures resembling T-tubules or sarcoplasmic reticulum. The results suggest that sarcomerogenesis and sarcoplasmic reticulum development occur simultaneously and that prefusion myoblasts have neither a T-tubule network nor Ca2+ deposits on sarcoplasmic reticulum. Small Ca2+ pools were found in the myoblast nuclei, cytoplasmic vesicles and mitochondrias. Ca2+-antimonate precipitates periodically distributed at the cell periphery, close to the cell membrane, were observed. These precipitates could represent internal Ca2+ stores located in the peripheral couplings and it is proposed that these pools of Ca2+ could be mobilized before fusion, leading to the increase in free intracellular Ca2+ that precedes myoblast fusion.

Drosophila mef2Expression during Mesoderm Development Is Controlled by a Complex Array ofcis-Acting Regulatory Modules

The function of theDrosophila mef2gene, a member of the MADS box supergene family of transcription factors, is critical for terminal differentiation of the three major muscle cell types, namely somatic, visceral, and cardiac. During embryogenesis,mef2undergoes multiple phases of expression, which are characterized by initial broad mesodermal expression, followed by restricted expression in the dorsal mesoderm, specific expression in muscle progenitors, and sustained expression in the differentiated musculatures. In this study, evidence is presented that temporally and spatially specificmef2expression is controlled by a complex array ofcis-acting regulatory modules that are responsive to different genetic signals. Functional testing of ∼12 kb of 5′ flanking region of themef2gene showed that the initial widespread mesodermal expression is achieved through a 280-bptwist-dependent enhancer. The subsequent dorsal mesoderm-restrictedmef2expression is mediated through a 460-bpdpp-responsive regulatory module, which involves the function of the Smad4 homologMedeaand contains several binding sites for Medea and Mad. The analysis also showed that regulatedmef2expression in the caudal and trunk visceral mesoderm, which give rise to longitudinal and circular gut musculatures, respectively, is under the control of distinct enhancer elements. In addition,mef2expression in the cardioblasts of the heart is dependent upon at least two distinct enhancers, which are active at different periods during embryogenesis. Moreover, multiple regulatory elements are differentially activated for specific expression in presumptive muscle founders, prefusion myoblasts, and differentiated muscle fibers. Taken together, the presented data suggest that specific expression of themef2gene in myogenic lineages in theDrosophilaembryo is the result of multiple genetic inputs that act in an additive manner upon distinct enhancers in the 5′ flanking region.

Recognition-alignment and adhesion in myoblast fusion

Myoblast fusion results from a sequence of different stages, previously demonstrated “in vitro”. After withdrawal from the cell cycle, myoblasts align forming long chains, in a process termed “recognition-alignment”. This stage is extracellular Ca2+ and N-Cadherin dependent. Alignment is followed by adhesion, defined as the stage prior to membrane fusion when aggegates are resistant to dispersal by EDTA. Adhesion is extracellular Ca2+-independent and N-CAM-dependent. Membrane fusion originates multinucleate myotubes.We have studied these stages of myoblast fusion at the brachial myotome of chick embryo from 51 to 105 h. of incubation. Samples were obtained by embryo microdissection and included the neural tube, the notochord and the brachial somites. These samples were embedded in araldite by conventional methods. Some samples were embedded in Unicryl, a recently formulated GMA derived resin.The first myoblasts clusters were observed in the ventral-lateral region of the brachial myotome in 22-24 Hamburger and Hamilton stages embryos. Clusters of pre-fusion myoblasts are usually surrounded by “electrondense blast cells” within a basal lamina in process of formation.

Noncoordinate developmental regulation of N-cadherin, N-CAM, integrin, and fibronectin mRNA levels during myoblast terminal differentiation.

N-cadherin, N-CAM, fibronectin, and beta 1-integrins have been implicated in the control of myoblast fusion to form multinucleate myotubes, a critical step in the terminal differentiation of skeletal muscle. We have analyzed the temporal pattern of expression of mRNA transcripts encoding these adhesion molecules during the terminal differentiation of C2 mouse myoblasts. The accumulation of mRNA transcripts encoding N-cadherin, N-CAM, fibronectin, alpha 5-integrin, and beta 1-integrin subunits was developmentally, but not coordinately, regulated. N-cadherin and integrin subunit expression was maximal in prefusion myoblasts and declined thereafter. In contrast, N-CAM mRNA levels were low in prefusion myoblasts, and increased coincident with the onset of terminal differentiation. Fibronectin mRNA levels were also low in myoblasts, and they did not increase until after cell fusion had occurred. The results indicate that despite their lack of coordinate regulation maximal levels of mRNA transcripts encoding adhesion molecules are present at a stage which corresponds to the peak of the active phase of myoblast fusion.

The relationship of nerve to myoblasts and newly-formed secondary myotubes in the fourth lumbrical muscle of the rat foetus.

The formation of normal numbers of skeletal muscle fibres depends on functional innervation of the muscle before and during the period of secondary myotube formation, but little has been known about the physical relationship between nerve terminals and the myoblasts and secondary myotubes over the critical period. This paper reports the results of a serial-section electron microscopic study of the IVth lumbrical muscle of the rat hindlimb, studied on embryonic day 20 (E20), a time when all secondary myotubes are less than 24 h old, and new ones are rapidly forming. Most myoblasts lying within the endplate region of the muscle received some direct neural contact; in almost all cases, the contact originated from an extension of a differentiated nerve terminal present at the endplate of an adjacent primary myotube. At six of 15 neural contact sites on myoblasts, primitive synaptic specialization was present. The newly-formed secondary myotubes were also directly, although sparsely, innervated in nine of ten instances. One secondary myotube was never seen to be innervated, despite extensive serial tracing. Nerve terminals passing to secondary myotubes were also principally derived from the innervation zone of the earlier-formed primary myotubes. Primary myotubes were profusely innervated by multiple axons. The results suggest that most nerve terminals are initially accommodated on the primary generation of myotubes, but progressively transfer to pre-fusion myoblasts or to secondary myotubes as these appear. In general, very young secondary myotubes are innervated by only a single axon, rather than being polyneuronally innervated. The existence of some secondary myotubes which lack any direct innervation suggests that intimate nerve contact may not be obligatory for formation of new secondary myotubes.

Coexpression of α-sarcomeric actin, α-smooth muscle actin and desmin during myogenesis in rat and mouse embryos I. Skeletal muscle

Abstract Expression of vimentin, desmin, α-sarcomeric and α-smooth muscle actins in embryonic tissues of rat and mice was examined using an immunohistochemical approach. The results showed a similarity in the expression of desmin and α-actin isoforms (α-sr and α-sm) in skeletal muscle cells during murine feto-embryonic development. In the two species, coexpression of α-sr and α-sm actins has been observed in cardiomyoblasts, myotomal myoblasts and myotubes. The intensity of α-sm actin expression decreased during the terminal steps of myogenesis and disappeared completely in mature cardiomyocytes and myofibres. Desmin was expressed in all prefusion myoblasts (type 1 and 2 myoblasts), myotubes, and in myofibres. The appearance of desmin in myoblasts of somites preceded by a few hours the expression of the α-actins (α-sr and α-sm). Our study on vimentin expression, limited to rat embryos, revealed that somite premyoblasts expressed only vimentin, type 1 myoblasts expressed vimentin and desmin, and type 2 myoblasts (rhabdomyoblasts) expressed desmin and α-actins (α-sr and α-sm). Our study demonstrates the resemblance between feto-embryonic myogenesis and myogenic neoplastic differentiation: desmin appears before the α-actins in embryonic myoblasts, and can be considered as a marker of an initial step in myogenic differentiation, α-sm actin, considered as a striated muscle cell feto-embryonic actin, is expressed transiently in skeletal myoblasts and cardiomyoblasts during development and reappears during neoplastic transformation of skeletal muscle.

Antibodies to 100- and 60-kDa surface proteins inhibit substratum attachment and differentiation of rodent skeletal myoblasts

A rabbit polyclonal antiserum was raised against membrane vesicles shed from the surface of fusing L6 rat myoblasts. In immunoblots the antiserum recognized fibronectin, a protein of approximately 100,000 Da (100-kDa), and a protein of approximately 60,000 Da (60 kDa). If added prior to cellular alignment, immunoglobulins from this serum inhibited fusion of both rat (L6) and mouse (C2) myoblasts in a dose-dependent fashion. To determine which component of this serum was responsible for fusion inhibition, antibodies against fibronectin, the 100- and 60-kDa proteins were microaffinity purified and tested, individually, for their effects on myoblast fusion. Antibodies against fibronectin had no effect on fusion. Antibodies against the 100-kDa protein released most cells from the substratum. Antibodies against the 60-kDa protein completely inhibited fusion. Fusion inhibition was accompanied by a corresponding inhibition of expression of two differentiation markers, creatine phosphokinase and the acetylcholine receptor. The 60-kDa protein was found, by immunoblot analysis, in smooth muscle-like cells (BC3H1 cells) and in variant L6 cells that do not differentiate and do not fuse. However, in the differentiation incompetent cells, the 60-kDa antigen appeared to be present in reduced amount. Indirect immunofluorescence of unpermeabilized L6 cells revealed alterations in the distribution of all three antigens during development. Fibronectin first appeared in long fibrillar arrays above the surface of cells that were beginning to align and fuse; fibronectin was not present on myotubes. The 100-kDa protein was seen initially in prominent fibrillar projections at the tips of prefusion myoblasts. During fusion the antigen was observed at sites of cell-cell contact and on extracellular vesicles. The 100-kDa protein appeared to be less abundant on myotubes. The 60-kDa protein first appeared in regions of cell-cell contact on cells that were beginning to align and fuse. As. fusion progressed, the 60-kDa protein was also found in extracellular vesicles. The 60-kDa protein was not observed on myotubes. As a result of this study we have identified two previously undescribed cell surface proteins involved in rodent skeletal myogenesis. The first is an approximately 100-kDa protein involved in early interactions of skeletal myoblasts with their substratum. The second is an approximately 60-kDa protein involved in myoblast differentiation. Both proteins are shed from the myoblast surface during myotube formation.

Generation of a stable, posttranslationally modified microtubule array is an early event in myogenic differentiation.

Microtubules (MTs) have been implicated to function in the change of cell shape and intracellular organization that occurs during myogenesis. However, the mechanism by which MTs are involved in these morphogenetic events is unclear. As a first step in elucidating the role of MTs in myogenesis, we have examined the accumulation and subcellular distribution of posttranslationally modified forms of tubulin in differentiating rat L6 muscle cells, using antibodies specific for tyrosinated (Tyr), detyrosinated (Glu), and acetylated (Ac) tubulin. Both Glu and Ac tubulin are components of stable MTs, whereas Tyr tubulin is the predominant constituent of dynamic MTs. In proliferating L6 myoblasts, as in other types of proliferating cells, the level of Glu tubulin was very low when compared with the level of Tyr tubulin. However, when we shifted proliferating L6 cells to differentiation media, we observed a rapid accumulation of Glu tubulin in cellular MTs. By immunofluorescence, the increase in Glu tubulin was first detected in MTs of prefusion myoblasts and was specifically localized to MTs that were associated with elongating portions of the cell. MTs in the multinucleated myotubes observed at later stages of differentiation maintained the elevated level of Glu tubulin that was observed in the prefusion myoblasts. When cells at early stages of differentiation (less than 1 d after switching the culture medium) were immunostained for Glu tubulin and the muscle-specific marker, muscle myosin, we found that the increase in Glu tubulin preceded the accumulation of muscle myosin. Thus, the elaboration of Glu MTs is one of the very early events in myogenesis. Ac tubulin also increased during L6 myogenesis; however, the increase in acetylation occurred later in myogenesis, after fusion had already occurred. Because detyrosination was temporally correlated with early events of myogenesis, we examined the mechanism responsible for the accumulation of Glu tubulin in the MTs of prefusion myoblasts. We found that an increase in the stability of L6 cell MTs occurred at the onset of differentiation, suggesting that the early increase in detyrosination that we observed is a manifestation of a decrease in MT dynamics in elongating myoblasts. We conclude that the establishment of an oriented array of microtubules heightened in its stability and its level of posttranslationally modified subunits may be involved in the subcellular remodeling that occurs during myogenesis.

Myogenesis in the mouse embryo: differential onset of expression of myogenic proteins and the involvement of titin in myofibril assembly.

Antibodies to muscle-specific proteins were used in immunofluorescence to monitor the development of skeletal muscle during mouse embryogenesis. At gestation day (g.d.) 9 a single layer of vimentin filament containing cells in the myotome domain of cervical somites begins to stain positively for myogenic proteins. The muscle-specific proteins are expressed in a specific order between g.d. 9 and 9.5. Desmin is detected first, then titin, then the muscle specific actin and myosin heavy chains, and finally nebulin. At g.d. 9.5 fibrous desmin structures are already present, while for the other myogenic proteins no structure can be detected. Some prefusion myoblasts display at g.d. 11 and 12 tiny and immature myofibrils. These reveal a periodic pattern of myosin, nebulin, and those titin epitopes known to occur at and close to the Z line. In contrast titin epitopes, which are present in mature myofibrils along the A band and at the A-I junction, are still randomly distributed. We propose, that the Z line connected structures and the A bands (myosin filaments) assemble independently, and that the known interaction of the I-Z-I brushes with the A bands occurs at a later developmental stage. After fusion of myoblasts to myotubes at g.d. 13 and 14 all titin epitopes show the myofibrillar banding pattern. The predominantly longitudinal orientation of desmin filaments seen in myoblasts and in early myotubes is transformed at g.d. 17 and 18 to distinct Z line connected striations. Vimentin, still present together with desmin in the myoblasts, is lost from the myotubes. Our results indicate that the putative elastic titin filaments act as integrators during skeletal muscle development. Some developmental aspects of eye and limb muscles are also described.

A role for acetylcholine receptors in the fusion of chick myoblasts.

The role of acetylcholine receptors in the control of chick myoblast fusion in culture has been explored. Spontaneous fusion of myoblasts was inhibited by the nicotinic acetylcholine receptor antagonists alpha-bungarotoxin, Naja naja toxin and monoclonal antibody mcAb 5.5. The muscarinic antagonists QNB and n-methyl scopolamine were without effect. Atropine had no effect below 1 microM, where it blocks muscarinic receptors; at higher concentrations, when it blocks nicotinic receptors also, atropine inhibited myoblast fusion. The inhibitions imposed by acetylcholine receptor antagonists lasted for approximately 12 h; fusion stimulated by other endogenous substances then took over. The inhibition was limited to myoblast fusion. The increases in cell number, DNA content, the level of creatine phosphokinase activity (both total and muscle-specific isozyme) and the appearance of heavy chain myosin, which accompany muscle differentiation, followed a normal time course. Pre-fusion myoblasts, fusing myoblasts, and young myotubes specifically bound labeled alpha-bungarotoxin, indicating the presence of acetylcholine receptors. The nicotinic acetylcholine receptor agonist, carbachol, induced uptake of [14C]Guanidinium through the acetylcholine receptor. Myoblasts, aligned myoblasts and young myotubes expressed the synthetic enzyme Choline acetyltransferase and stained positively with antibodies against acetylcholine. The appearance of ChAT activity in myogenic cultures was prevented by treatment with BUDR; nonmyogenic cells in the cultures expressed ChAT at a level which was too low to account for the activity in myogenic cultures. We conclude that activation of the nicotinic acetylcholine receptor is part of the mechanism controlling spontaneous myoblast fusion and that myoblasts synthesize an endogenous, fusion-inducing agent that activates the nicotinic ACh receptor.

Sequential expression of chicken actin genes during myogenesis.

Embryonic muscle development permits the study of contractile protein gene regulation during cellular differentiation. To distinguish the appearance of particular actin mRNAs during chicken myogenesis, we have constructed DNA probes from the transcribed 3' noncoding region of the single-copy alpha-skeletal, alpha-cardiac, and beta-cytoplasmic actin genes. Hybridization experiments showed that at day 10 in ovo (stage 36), embryonic hindlimbs contain low levels of actin mRNA, predominantly consisting of the alpha-cardiac and beta-actin isotypes. However, by day 17 in ovo (stage 43), the amount of alpha-skeletal actin mRNA/microgram total RNA increased more than 30-fold and represented approximately 90% of the assayed actin mRNA. Concomitantly, alpha-cardiac and beta-actin mRNAs decreased by 30% and 70%, respectively, from the levels observed at day 10. In primary myoblast cultures, beta-actin mRNA increased sharply during the proliferative phase before fusion and steadily declined thereafter. alpha-Cardiac actin mRNA increased to levels 15-fold greater than alpha-skeletal actin mRNA in prefusion myoblasts (36 h), and remained at elevated levels. In contrast, the alpha-skeletal actin mRNA remained low until fusion had begun (48 h), increased 25-fold over the prefusion level by the completion of fusion, and then decreased at later times in culture. Thus, the sequential accumulation of sarcomeric alpha-actin mRNAs in culture mimics some of the events observed in embryonic limb development. However, maintenance of high levels of alpha-cardiac actin mRNA as well as the transient accumulation of appreciable alpha-skeletal actin mRNA suggests that myoblast cultures lack one or more essential components for phenotypic maturation.

Role of Ca 2+ and Ca 2+-activated protease in myoblast fusion

In this report, we have examined the effects of a calcium chelator, EGTA, and a calcium ionophore, A23187, on fusion of a cloned muscle cell line, L 6. Our results confirm that EGTA essentially blocks all myoblast fusion because the lateral alignment of presumptive myoblasts cannot occur in the absence of extracellular calcium. A23187, however, promotes the precocious fusion of myoblasts, apparently by facilitating Ca 2+ transport into myoblasts. We have also demonstrated that a Ca 2+-activated protease, CAP (mM), appears to relocate in response to the Ca 2+ flux, changing from a random, dispersed distribution in proliferative myoblasts to a predominantly peripheral distribution in prefusion myoblasts. Coincident with the mM CAF relocation is an altered distribution of a surface glycoprotein, fibronectin. Extracellular fibronectin is seen in abundance in proliferating myoblasts, but is essentially absent from the surface of fusing myoblasts. We suggest that mM CAF when activated by Ca 2+ influx may act to promote the release of fibronectin from the myoblast cell surface, thus providing a mechanism by which the membrane of the fusing myoblast may be rearranged to accommodate fusion.

Expression of a developmentally regulated antigen on the surface of skeletal and cardiac muscle cells.

H36 is a species-specific, cell-surface antigen on differentiating newborn rat skeletal myoblasts and myogenic lines. This membrane antigen has been defined by a monoclonal antibody raised by the fusion of SP 2/0-Ag14 myeloma cells with spleen cells from mice immunized with myotubes derived from the myogenic E63 line. H36 antigen, isolated by immunoaffinity chromatography, is comprised of two polypeptides with apparent molecular weights of 98,000 and 117,000. Fluorescence photometry and radioimmunoassays have been used to follow quantitative and topographic changes in the H36 determinant during myogenesis. H36 is present at a basal level on replicating myoblasts; it increases on prefusion myoblasts and persists on myotubes. At or near the time of prefusion, it becomes concentrated between adjacent aligned myoblasts and localized on membrane "blebs". H36 is present on both skeletal and cardiac cells but absent from a variety of cells that include fibroblasts, neuronal cells, and smooth muscle. There are approximately 4 x 10(5) determinants per myoblast, and the Ka of the antibody is 3.8 x 10(8) liters/mol. The distributions of H36 on the top and attached surfaces of myoblasts and myotubes are distinct, which suggests localized specialization of these surfaces. H36 is an integral membrane component and upon cross-linking, it associates with the detergent-insoluble cytoskeletal framework. Inhibition of myogenesis by 5-bromodeoxyuridine or by calcium deprivation prevents the developmentally associated changes in the expression of H36. H36 is also absent or markedly reduced on the fu- and Ama102 developmentally defective mutant myoblast lines. We conclude that H36 is a muscle-specific, developmentally regulated cell-surface antigen that may have a role in myoblast differentiation and that can be used to determine the embryonic lineages of skeletal and cardiac muscle.

Distribution of the cell substratum attachment (CSAT) antigen on myogenic and fibroblastic cells in culture.

Previous studies (Neff et al., 1982, J. Cell. Biol. 95:654-666; Decker et al., 1984. J. Cell. Biol. 99:1388-1404) have described a monoclonal antibody (CSAT Mab) directed against a complex of three integral membrane glycoproteins of 120,000-160,000 mol wt (CSAT antigen [ag]) involved in the cell matrix adhesion of myoblasts and fibroblasts. In localization studies on fibroblasts presented here, CSAT ag has a discrete, well-organized distribution pattern. It co-aligns with portions of stress fibers and is enriched at the periphery of, but not directly beneath vinculin-rich focal contacts. In this last location, it co-distributes with fibronectin, consistent with the suggestion that the CSAT ag participates in the mechanism by which fibroblasts attach to fibronectin. In prefusion myoblasts, which are rapidly detached by CSAT Mab, CSAT ag is distributed diffusely as are vinculin, laminin, and fibronectin. After fusion, myotubes become more difficult to detach with CSAT Mab. The CSAT ag and vinculin are organized in a much more discrete pattern on the myotube surface, becoming enriched at microfilament bundle termini and in lateral lamellae which appear to attach myotubes to the substratum. These results suggest that the organization of CSAT ag-adhesive complexes on the surface of myogenic cells can affect the stability of their adhesive contacts. We conclude from the sum of the studies presented that, in both myogenic and fibroblastic cells, the CSAT ag is localized in sites expected of a surface membrane mediator of cell adhesion to extracelluon of CSAT ag-adhesive complexes on the surface of myogenic cells can affect the stability of their adhesive contacts. We conclude from the sum of the studies presented that, in both myogenic and fibroblastic cells, the CSAT ag is localized in sites expected of a surface membrane mediator of cell adhesion to extracellular matrix. The results from studies that use fibroblasts in particular suggest the involvement of CSAT ag in the adhesion of these cells to fibronectin.

Maintenance of DNA repair capacity in differentiating rat muscle cells in vitro.

Abstract— Unscheduled DNA synthesis has been measured at several times during the differentiation of cultured rat skeletal muscle cells in response to exposures to 254nm UV light. There is no change in the amount of repair DNA synthesis as the cells fuse and differentiate from postmitotic prefusion myoblasts to multinucleated contracting myotubes.

Alterations in lipid turnover in developing muscle

The incorporation and turnover of [ 3H] glycerol into skeletal muscle cell cultures derived from embryonic chickens was studied. Both rates of incorporation and turnover of specific lipids were dependent on culture age and lipid species. The pattern of glycerol incorporation showed that prefusion myoblasts primarily synthesized both phosphatidylcholine and triglycerides whereas postfusion myotubes primarily synthesized phosphatidyl choline. This pattern could be modified in postfusion but not prefusion cells by briefly incubating the cells with unilammelar phosphatidyl choline vesicles. Analysis of major lipid species revealed that muscle triglycerides and phospholipids turned over at a higher rate in prefusion cultures compared to the postfusion state. These findings are discussed in light of the marked shift in lipid metabolism which occurs during myogenesis.

Immunological studies of the embryonic muscle cell surface. Antiserum to the prefusion myoblast.

Xenogeneic antisera raised in rabbits have been used to detect compositional changes at the cell surfaces of differentiating embryonic chick skeletal muscle. In this report, we present the serological characterization of antiserum (Anti-M-24) against muscle tissue and developmental stage-specific cell surface antigens of the prefusion myoblast. Cells from primary cultures of 12-d-old embryonic chick hindlimb muscle were injected into rabbits, and the resulting antisera were selectively absorbed to obtain immunological specificity. Cytotoxicity and immunohistochemical assays were used to test this antiserum. Absorption with embryonic or adult chick heart, brain, retina, liver, erythrocytes, or skeletal muscle fibroblasts failed to remove all reactivity of Anti-M-24 for myogenic cells at all stages of development. After absorption with embryonic myotubes, however, Anti-M-24 no longer reacted with differentiated myofibers, but did react with prefusion myoblasts. The myoblast surface antigens detected with Anti-M-24 are components of the muscle cell membrane: (a) these macromolecules are free to diffuse laterally within the myoblast membrane; (b) Anti-M-24, in the presence of complement, induced lysis of the muscle cell membrane; and (c) intact monolayers of viable myoblasts completely absorbed reactivity of Anti-M-24 for myoblasts. These antigens are not loosely adsorbed culture medium components or an artifact of tissue culture because: (a) absorption of Anti-M-24 with homogenized embryonic muscle removed all antibodies to cultured myoblasts; (b) Anti-M-24 reacted with myoblast surfaces in vivo; and (c) absorption of Anti-M-24 with culture media did not affect the titer of this antiserum for myoblasts. We conclude that myogenic cells at all stages of development possess externally exposed antigens which are undetected on other embryonic and adult chick tissues. In addition, myoblasts exhibit surface antigenic determinants that are either masked, absent, or present in very low concentrations on skeletal muscle fibroblasts, embryonic myotubes, or adult myofibers. These antigens are free to diffuse laterally within the myoblast membrane and may be modulated in response to appropriate environmental cues during myodifferentiation.

DNA-dependent RNA polymerases in skeletal muscle cells differentiating in vitro

The main classes of RNA polymerase in cultured chick embryo skeletal muscle cells were identified and their levels determined during differentiation in vitro. Cell cultures contained a high proportion of muscle cells (>80%) and exhibited a high degree of fusion (>80%). The levels of RNA polymerases I and II increased by 1.8 and 1.5 times, respectively, from prefusion myoblasts (24 hr) to postfusion myotubes (120 hr). RNA polymerase III, a single peak on DEAE-Sephadex chromatography, was less than 7% of the total activity. The results suggest that the rates of synthesis of the main RNA types, which reportedly decline during myogenesis, are not determined by the relative levels of the main RNA polymerases and are more likely a reflection of chromatin template availability.