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.     

Glycosphingolipids during skeletal muscle cell differentiation: Comparison of normal and fusion-defective myoblasts

The regulation of glycosphingolipid (GSL) synthesis in culture by fusion-competent (E63) myoblasts and fusion-defective (fu-1) cells was examined. Upon reaching confluency E63 cells fused to form multinucleated myotubes and demonstrated many characteristics of developing skeletal muscle including induction of creatine kinase activity and a shift in creatine kinase isozymes to the MM isoform. The fu-1 cells displayed none of these characteristics, despite the fact that both cells were cloned from the same parental myoblast line (rat L8). There was a transient increase in the synthesis of total neutral GSLs by E63 cells at the time of membrane fusion. In contrast, neutral GSL synthesis by fu-1 cells gradually decreased with time in culture. The major GSLs synthesized by both cell types were lactosylceramide and ganghoside GM3, with more complex structures being observed with prolonged time in culture. Several glycosyltransferase activities were assayed at varying times in culture. Generally, the changes in activities fell into three groups. One group was maximally activated at the end of the culture period (GalT-3, GalNAcT-1 and GalT-6). Another group was maximally activated during the time of active membrane fusion (GlcT and SAT-1). A third group was maximally activated at the time of cell contact and the beginning of membrane fusion (GlcNAcT-1 and GalT-2). In terms of the times of maximal activation there were few differences between E63 and fu-1 cells, with one notable exception. The activity of GalT-2 (lactosylceramide synthase) in E63 cells increased dramatically upon contact and the beginning of membrane fusion, whereas there were no changes in GalT-2 activity in fu-1 cells during time in culture. These results support our hypothesis that membrane glycosphingolipids play an important role in the differentiation of skeletal muscle cells.Abbreviations GSL glycosphingolipid - CK creatine kinase - HPTLC high performance thin layer chromatography - PMSF phenylmethylsulfonyl fluoride - CTH ceramide trihexoside (GbOse₃Cer) - GlcCer glycosylceramide - LacC N-acetylglucosamine - NeuNAc N-acetylneuraminic acid (sialic acid)     

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.     

Expression of myoblast and myocyte antigens in relation to differentiation and the cell cycle

Cell cycle parameters and expression of myoblast and myocyte antigens were investigated during exponential growth and during the differentiation phase of rat L8( E63 ) myoblasts by an integrated approach involving microspectrophotometry with DNA fluorochromes, [3H]thymidine autoradiography, and immunofluorescent staining with monoclonal antibodies. In addition to the majority of cells which are recruited into myotubes, two distinct populations of mononucleate cells were resolved in cultures of rat myoblasts undergoing differentiation. These mononucleate cells consist of (1) a population of proliferating cells with a prolonged G1 transit time; (2) a population of non-proliferating cells which remain arrested in G1 for more than 72 h. The latter group was examined with respect to the expression of two marker antigens recognized by two monoclonal antibodies: antibody B58 reacts with a macromolecular component present in undifferentiated myoblasts but not in mature myotubes, and antibody XMlb reacts with a muscle-specific isoform of myosin. All four possible combinations of expression of these antigens by single cells were found: B58 +XM1b -, B58 +XM1b +, B58 - XM1b -, and B58 - XMlb +. The implication of these findings with respect to the transition from the proliferative to the differentiative phase of myogenesis is discussed.     

Possible role of calpain I and calpain II in differentiating muscle

The variable distribution of the 80-kD subunit of two calcium-activated proteases, calpain I and calpain II, has been examined in L8 and L6 myoblasts, and their non-fusing variants, fu-1 and M3A using non-cross-reacting monoclonal antibodies to both subunits. Immunofluorescence results have shown that while the 80-kD subunit of calpain I is localized in the cytoplasm of all the myoblasts, the 80-kD subunit of calpain II appears to be predominantly associated with the plasma membranes of L8 and L6 myoblasts. The distribution of the 80-kD subunit of calpain II in non-fusing myoblasts, fu-1 and M3A, is generally cytoplasmic and diffuse. Immunoblot analysis of membrane and cytosol fractions of all the myoblasts using the monoclonal antibodies described above essentially confirms the immunofluorescence findings. Because calpain II exhibits a peripheral distribution in cells which are fusion-competent, L6 and L8 myoblasts, but not in fu-1 and M3A myoblasts, we suggest that calpain II may play a role in the Ca2+-mediated fusion events of differentiating (prefusion) myoblasts.     

Developmentally regulated cell surface structures on mouse and human embryonal carcinoma cell lines

Three monoclonal antibodies 5.1.H, 8.7.D and 13.7.A raised against semi-purified Tera 1 membrane fractions recognize distinct onco-foetal antigens which are developmentally regulated on cells such as Tera 2 clone 13 and appear to be restricted in their expression to undifferentiated ectoblastic cells and certain organized cystic structures mimicking the foetal intestine. These antigens, absent from normal adult tissues, differ markedly from glycosidic stage-specific antigens such as 75.12 which, while functioning as embryonal carcinoma differentiation markers, are also expressed on certain adult tissues. No evidence for a role of fucosyltransferases in regulating either 75.12 or SSEA-1 antigen expression on embryonal carcinoma cells or for the presence of lectin-like structures recognizing these antigens on such cells was found.     

Quantitation of changes in cell surface determinants during skeletal muscle cell differentiation using monospecific antibody

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate anti-genie changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS-1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty-one clones were obtained which produced antibodies specific for myogenic cells. Thirty-five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.     

Endocytosis of alpha 2-macroglobulin is developmentally regulated during myogenesis

A monoclonal antibody, H143, reacts with an intracellular antigen present and accumulated in E63 rat myoblasts. H143 is directed against a species-specific determinant on purified equine serum alpha 2-macroglobulin. Immunofluorescence analyses of differentiating myoblasts grown in horse serum demonstrate that the capacity to take up alpha 2-macroglobulin is stage-specific: the rapid uptake of alpha 2-macroglobulin characteristic of myoblasts ceases prior to their fusion to form multinucleate fibers (myotubes). Neither rat fibroblasts nor a developmentally defective mutant of E63 exhibit this change in alpha 2-macroglobulin uptake. The temperature and calcium requirements for the uptake of H143 antigen, and its accumulation as effected by lysosomotropic amines, indicate that alpha 2-macroglobulin is taken up by myoblasts via a developmentally regulated endocytic process. Electron microscopy using equine alpha 2-macroglobulin labeled with colloidal gold supports this finding.     

Mechanism of murine leukemia virus-induced fusion in rat myoblasts defective in differentiation.

fu-1 cells, a line of rat myoblasts defective in differentiation, can be fused into multinucleate syncytia by Moloney murine leukemia virus. The effects of treating the virus with specific antibody, UV irradiation, and elevated temperature and the requirements for cellular RNA and protein synthesis have been studied as they relate to this virus-induced fusion. The results indicate that intact, but not necessarily infectious, virions are required to promote fusion of fu-1 cells. Neither actinomycin D nor cycloheximide altered the formation of syncytia; thus, neither viral nor cellular RNA or protein synthesis is required for fusion. fu-1 cells infected with the ts3 temperature-sensitive mutant of Moloney murine leukemia virus accumlate large amounts of budding virus on their cell membrane; however, this membrane-associated virus failed to induce syncytia. Upon release of the virus at the permissive temperature, fusion did occur. We conclude that contact or attachment of the immature virus to the cell membrane is not sufficient to promote murine leukemia virus-induced cell fusion; complete virions are required. From these data, we propose that adsorption and penetration of the virus may induce a change in the cell membrane that subsequently promotes the fusion of susceptible cells.     

The stage-specific expression of TEC-1, -2, -3, and -4 antigens on bovine preimplantation embryos

The preimplantation developmental period is associated with constant changes within the embryo, and some of these changes are apparent on the embryo cell surface. For example, during transition from maternal to embryonic genome control and the compaction and differentiation of embryonic cells, the cell surface undergoes morphologic alterations that reflect changes in gene control. In order to gain insight into the events occurring during embryonic development and cellular differentiation, monoclonal antibodies specific for cell surface antigens (TEC antigens) of embryonic cells have been generated previously and shown to recognise either the carbohydrate moiety of embryoglycan or a developmentally regulated protein epitope. The TEC antigens have been identified on mouse preimplantation embryos, and their expression is specific to particular developmental stages. To determine whether these antigens are conserved in higher mammals, we examined the expression of four TEC antigens (TEC-1 to TEC-4) on in vitro–derived bovine and murine embryos during the preimplantation stage of development. It was found that bovine oocytes and embryos derived from in vitro maturation (IVM) and in vitro fertilisation (IVF) showed stage-specific expression of each of the TEC antigens investigated, with the pattern of expression overlapping but not identical to that seen in the mouse. Immunoprecipitation together with Western blot analysis showed that the TEC monoclonal antibodies recognised a single glycoprotein band with an apparent molecular weight of 70 kDa. Confocal microscopy of immunofluorescence staining of the bovine cells showed this protein to be located on the cell surface. The apparent negative expression of these TEC antigens by immunohistochemistry and immunoprecipitation at particular stages of development appears to be due to the epitopes being inaccessible to the TEC antibodies, since Western blotting revealed the TEC antigens to be present at all stages of development examined. Antibodies identifying stage-specific antigens will provide useful markers to characterise early embryonic cells, monitor normal embryonic development in vitro, and identify cell surface structures having a function in cell-cell interactions during embryogenesis and differentiation. Mol. Reprod. Dev. 49:19–28, 1998. © 1998 Wiley-Liss, Inc.     

A laminin substrate promotes myogenesis in rat skeletal muscle cultures: analysis of replication and development using antidesmin and anti-BrdUrd monoclonal antibodies

Cells from newborn rat hindlimb show greatly enhanced myogenicity when grown on surfaces coated with poly-L-lysine followed by laminin (PLL/Lam) instead of the collagens routinely used. Coating with poly-L-lysine (PLL) alone or with PLL followed by collagen does not enhance myogenicity. Both myogenic and nonmyogenic cells, as distinguished by a monoclonal antibody specific for desmin, attach equally well to collagen- and laminin-coated surfaces, but there is a two- to five-fold increase in the number of myogenic cells on PLL/Lam by 72 hr, followed by increased myotube formation. To determine whether this increase in myogenic cells was a consequence of a selective increase in proliferation on PLL/Lam, incorporation of 5-bromodeoxyuridine into DNA followed by labeling with anti-BrdUrd antibody was used as an index of cell proliferation. The results indicate that desmin is expressed in replicating rat myoblasts, and that replication of myogenic cells is greatly enhanced on laminin compared to collagen. The rate of replication of nonmyogenic cells is the same on both substrates. Addition of 10 micrograms/ml laminin to the medium of cells seeded on PLL or collagen has no effect on myogenicity. We conclude that a laminin substrate enhances skeletal myogenesis in vitro by promoting selectively the replication of myoblasts. Cultures prepared from fetuses at 17 and 19 days gestation also show enhanced myogenicity when grown on PLL/Lam, while those from 15-day fetuses do not. Growth and development of fetal myoblasts on collagen were very poor, whereas myoblasts from the newborn rat do proliferate and differentiate on this substrate. Thus myogenic cells at different stages of fetal and neonatal development may require and respond to different extracellular environments. Myotube formation in the E63 clone of L8 rat myoblasts is inhibited by PLL/Lam.     

The Physical Interaction of Myoblasts with the Microenvironment during Remodeling of the Cytoarchitecture

Integrins, focal adhesions, the cytoskeleton and the extracellular matrix, form a structural continuum between the external and internal environment of the cell and mediate the pathways associated with cellular mechanosensitivity and mechanotransduction. This continuum is important for the onset of muscle tissue generation, as muscle precursor cells (myoblasts) require a mechanical stimulus to initiate myogenesis. The ability to sense a mechanical cue requires an intact cytoskeleton and strong physical contact and adhesion to the microenvironment. Importantly, myoblasts also undergo reorientation, alignment and large scale remodeling of the cytoskeleton when they experience mechanical stretch and compression in muscle tissue. It remains unclear if such dramatic changes in cell architecture also inhibit physical contact and adhesion with the tissue microenvironment that are clearly important to myoblast physiology. In this study, we employed interference reflection microscopy to examine changes in the close physical contact of myoblasts with a substrate during induced remodeling of the cytoarchitecture (de-stabilization of the actin and microtubule cytoskeleton and inhibition of acto-myosin contractility). Our results demonstrate that while each remodeling pathway caused distinct effects on myoblast morphology and sub-cellular structure, we only observed a ∼13% decrease in close physical contact with the substrate, regardless of the pathway inhibited. However, this decrease did not correlate well with changes in cell adhesion strength. On the other hand, there was a close correlation between cell adhesion and β1-integrin expression and the presence of cell-secreted fibronectin, but not with the presence of intact focal adhesions. In this study, we have shown that myoblasts are able to maintain a large degree of physical contact and adhesion to the microenvironment, even during shot periods (<60 min) of large scale remodeling and physiological stress, which is essential to their in-vivo functionality.     

Changes in cell surface antigens during in vitro lizard myogenesis

Indirect immunofluorescence has been used to examine surface antigens of lizard myogenic cells during in vitro differentiation. At least two developmental stage-specific surface alterations have been identified. One of these is a compositional change and involves the appearance of a cell-surface antigen(s) as the cells differentiate. This antigen(s) (Ag1422) is muscle specific and is characteristic of some rounded-up G0 myosin-positive myocytes, all stretched-back, G0 myosin-positive myocytes, and all identifiable myotubes. The antigen is not found on proliferating myoblasts, extended G1 (myosin-negative) cell-cycle-competent myoblasts or newly differentiated rounded-up, G0 myosin-positive myocytes. Pretreatment of cells with neuraminidase, trypsin, or proteinase K indicates the antigen is not present in "masked" form on normally nonreactive cells. Proteinase K is effective in the removal or destruction of the antigen, indicating it is at least partially protein in nature. The antigen is expressed in a similar developmental stage-specific fashion on early-passage myogenic cells taken from both adult lizard tail regenerates and embryonic muscle. The antibodies identifying Ag1422 can be removed by adsorption with homogenates of mature skeletal muscle. Therefore, Ag1422 is not an artifact due to in vitro conditions or the expression of a transformation antigen unique to the continuous culture line. The second alteration is an apparent restriction in the mobility of surface components (antigens and lectin receptors). Upon treatment with multivalent ligands, undifferentiated myosin-negative myoblasts exhibit rapid patching and capping of cell surface components while well-differentiated myocytes and myotubes do not. This mobility restriction is evident after the appearance of Ag1422. Treatment with cytochalasin B (15 micrograms/ml) and/or colchicine (100 microM) does not alter the restricted mobility of surface components seen on differentiated cells. Therefore, neither microfilaments nor microtubules seem to be involved in the mobility restriction. These observations are discussed in relation to current views of myogenesis.     

Inhibition of adhesion of F9 embryonal carcinoma cells to substratum by a novel monoclonal antibody, TEC-05, reactive with a developmentally regulated carbohydrate epitope

Embryonal carcinoma cells carry on their surfaces carbohydrate antigens that are also expressed in early embryonic cells. We report here the expression and properties of a new developmentally regulated carbohydrate epitope, which is defined by a monoclonal antibody TEC-05. This antibody was generated by immunization of a rat with mouse embryonal carcinoma cells P19S1801A1. By immunofluorescence, the TEC-5 epitope was first detected on 8-cell-stage mouse embryos and was present on all subsequent stages of preimplantation development. Absorption analysis revealed that TEC-5 epitope was expressed only on a limited number of adult mouse tissues. In the direct radioantibody binding assay, TEC-05 reacted strongly with OTF9-63 cells and with some of the mouse embryonal carcinoma cell lines tested. Its reaction with differentiated cell lines was weak or undetectable. In the course of differentiation of OTF9-63 cells induced by retinoic acid, the epitope disappeared with the onset of morphological differentiation. The binding of the antibody to OTF9-63 cells was inhibited to 50% by 10-50 microM N-acetyllactosamine and lactose. Immunolabelling of extracts from OTF9-63 cells separated by sodium-dodecyl-sulfate (SDS) polyacrylamide gel electrophoresis revealed that TEC-5 epitope was carried by high-molecular-weight glycoconjugates (molecular weight greater than 100,000). Molecules, isolated from [3H]-fucose-labelled OTF9-63 cells by indirect immunoprecipitation with TEC-05 antibody, were degraded by extensive pronase digestion or mild alkaline treatment to large carbohydrate chains that were excluded from a Sephadex G-50 column. Direct evidence that TEC-05 antibody bound to embryoglycan was obtained using a modified Farr's assay. The antibody was found to inhibit adhesion of F9 and OTF9-63 cells to substratum. The inhibitory effect, which could be abrogated by lactose, seemed to be specific, because another IgM monoclonal antibody which also binds to embryoglycan had no effect. Combined data indicated that TEC-05 antibody recognizes a carbohydrate epitope which is involved in cell-substratum adhesion of F9 cells and which provides a new marker for structure-function studies of stage-specific embryonic antigens.     

Changes in intramembranous particle topography and concanavalin A receptor mobility associated with myoblast differentiation.

These studies have examined the distribution of plasma membrane intramembranous particles (PMP) visualized by freeze fracture and concanavalin A receptors seen by ultrastructural cytochemistry of differentiated and undifferentiated L6 myoblasts. Undifferentiated mononucleated cells have a clustered distribution of PMP on the majority of the fracture faces. Associated with cell differentiation and cell fusion a more uniform distribution of PMP is observed. Changes also occur with myoblast differentiation in the topography and dynamics of receptors bound to concanavalin A. If undifferentiated or differentiated cells are fixed with glutaraldehyde and then reacted with con-A a uniform distribution of con-A is seen on the cell surfaces. In contrast to this if unfixed live cells are reacted at 37 degrees C with con-A a profound redistribution occurs on differentiated cells (greater than 99% showing redistribution) while receptors remain in a uniform array on undifferentiated cells (approximately 95% uniform distribution). In addition to the membrane binding, con-A is observed to bind to an extracellular filamentous matrix seen in high density undifferentiated cultures which then appears to be degraded with differentiation and myoblast fusion. These studies show that a number of membrane changes, both structural and dynamic occur with myoblast differentiation.     

Changes in Intramembranous Particle Topography and Concanavalin A Receptor Mobility Associated with Myoblast Differentiation

These studies have examined the distribution of plasma membrane intramembranous particles (PMP) visualized by freeze fracture and concanavalin A receptors seen by ultrastructural cytochemistry of differentiated and undifferentiated L6 myoblasts. Undifferentiated mononucleated cells have a clustered distribution of PMP on the majority of the fracture faces. Associated with cell differentiation and cell fusion a more uniform distribution of PMP is observed. Changes also occur with myoblast differentiation in the topography and dynamics of receptors bound to concanavalin A. If undifferentiated or differentiated cells are fixed with glutaraldehyde and then reacted with con-A a uniform distribution of con-A is seen on the cell surfaces. In contrast to this if unfixed live cells are reacted at 37° C with con-A a profound redistribution occurs on differentiated cells (greater than 99% showing redistribution) while receptors remain in a uniform array on undifferentiated cells (approximately 95% uniform distribution). In addition to the membrane binding, con-A is observed to bind to an extracellular filamentous matrix seen in high density undifferentiated cultures which then appears to be degraded with differentiation and myoblast fusion. These studies show that a number of membrane changes, both structural and dynamic occur with myoblast differentiation.