Highly homologous filamin polypeptides have different distributions in avian slow and fast muscle fibers

The high molecular weight actin-binding protein filamin is located at the periphery of the Z disk in the fast adult chicken pectoral muscle (Gomer, R. H., and E. Lazarides, 1981, Cell, 23: 524-532). In contrast, we have found that in the slow anterior latissimus dorsi (ALD) muscle, filamin was additionally located throughout the l band as judged by immunofluorescence with affinity-purified antibodies on myofibrils and cryosections. The Z line proteins desmin and alpha-actinin, however, had the same distribution in ALD as they do in pectoral muscle. Quantitation of filamin and actin from the two muscle types showed that there was approximately 10 times as much filamin per actin in ALD myofibrils as in pectoral myofibrils. Filamin immunoprecipitated from ALD had an electrophoretic mobility in SDS polyacrylamide gels identical to that of pectoral myofibril filamin and slightly greater than that of chicken gizzard filamin. Two-dimensional peptide maps of filamin immunoprecipitated and labeled with 125I showed that ALD myofibril filamin was virtually identical to pectoral myofibril filamin and was distinct from chicken gizzard filamin.     

Identification of a high molecular weight actin-binding protein in skeletal muscle.

A large polypeptide having a molecular weight of 240,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate has been identified in whole cell homogenates from chick skeletal muscle myoblasts and the rat myoblast L6 cell line. A similar polypeptide was identified in both thigh and breast chicken skeletal muscle, but the latter contained less of this protein per g of tissue. Antibodies made to gizzard filamin (an actin-binding protein having a molecular weight of 240,000) cross-reacted with the partially purified Mr = 240,000 protein from chicken skeletal muscle. With use of the indirect immunofluorescence technique, the filamin antibody localized in the Z-line region of chicken skeletal muscle myofibrils. These results indicate that skeletal muscle contains a filamin-like protein that may form an integral part of the myofibril structure.     

The synthesis and deployment of filamin in chicken skeletal muscle

During myogenesis in vitro the actin-binding protein filamin is present in myoblasts and early fused cells and is associated with α-actinin-containing filament bundles, as judged by double immunofluorescence using antibodies specific for these two proteins. Approximately one day after cell fusion, yet before the development of a-actinin-containing Z line striations, filamin disappears from the cells. Later in myogenesis, several days after the appearance of α-actinin-containing Z line striations, filamin reappears and accumulates in the cells. Double immunofluorescence with antibodies to filamin and vimentin (or desmin) reveals that the newly appearing filamin localizes now to the myofibril Z line and is visible there shortly before vimentin or desmin becomes associated with the Z line. Immunofluorescent localization of filamin in isolated chicken skeletal myofibrils and Z disc sheets indicates that filamin has the same distribution as desmin and vimentin; it surrounds each myofibril Z disc and forms honeycomb-like networks within each Z plane of the muscle fiber. Filamin may thus be involved in the transition of desmin and vimentin to the Z disc. Analysis of whole-cell extracts by SDS-polyacrylamide gel electrophoresis and by immunoautoradiography shows that filamin is present in myoblasts and in myotubes early after cell fusion. Concomitant with the absence of filamin fluorescence during the subsequent few days of myogenesis, the quantity of filamin is markedly reduced. During this time, metabolic pulse-labeling with ³⁵S-methionine reveals that the synthetic rate of filamin is also markedly reduced. As filamin fluorescence appears at the Z line, the quantity of filamin and its synthetic rate both increase. The removal of filamin from the cells suggests that filamin either may not be required, or may actually interfere with a necessary process, during the early stages of sarcomere morphogenesis. These results also indicate that the periphery of the Z disc is assembled in at least two distinct steps during myogenesis.     

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.     

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

The tissue specificity of chicken 165,000 M-protein, tentatively names "myomesin", a tightly bound component of the M-line region of adult skeletal and heart myofibrils, was investigated by immunological techniques. Besides skeletal and heart muscle, only thymus (known to contain myogenic cells) was found to contain myomesin. No myomesin could however, be detected in smooth muscle or any other tissue tested. This result was confirmed in vitro on several cultured embryonic cell types. Only skeletal and heart muscle cells, but not smooth muscle or fibroblast cells, showed the presence of myomesin. When the occurrence and the distribution of myomesin during differentiation of breast muscle cells in culture were studied by the indirect immunofluorescence technique, this protein was first detected in postmitotic, nonproliferating myoblasts in a regular pattern of fluorescent cross- striations. In electron micrographs of sections through young myotubes, it could be shown to be present within the forming H-zones of nascent myofibrils. In large myotubes the typical striation pattern in the M- line region of the myofibrils was observed. Synthesis of myomesin measured by incorporation of [35S]methionine into immunoprecipitable protein of differentiating cells increased sharply after approximately 48 h in culture, i.e., at the time when the major myofibrillar proteins are accumulated. No significant amounts of myomesin were, however, found in cells prevented from undergoing normal myogenesis by 5'- bromodeoxyuridine. The results indicate that myomesin (a) is a myofibrillar protein specific for cross-striated muscle, (b) represents a highly specific marker for cross-striated muscle cell differentiation and (c) might play an important role in myofibril assembly and/or maintenance.     

Gelsolin sensitivity of microfilaments as a marker for muscle differentiation

The ability of porcine smooth muscle gelsolin to sever actin filaments was used to study alterations in the organization of F-actin containing structures during skeletal myogenesis. In permeabilized fibroblasts and unfused myoblasts, gelsolin induced complete degradation of the actin cytoskeleton. After fusion of myoblasts to multinucleated myotubes, gelsolin removed a substantial amount of actin, revealing fibers with a sarcomere-like arrangement of gelsolin-insensitive actin. These fibrils were much thinner and had shorter sarcomeres than fully differentiated myofibrils. The proportion of gelsolin-resistant fibrils increased during differentiation, resulting in almost complete inertness of mature myofibrils. Fibrils isolated from adult muscle were also found nearly resistant to gelsolin. Extraction of tropomyosin and myosin in buffer of high ionic strength prior to gelsolin treatment reestablished the susceptibility to the severing protein, both in myotubes and isolated myofibrils. Only small remnants of phalloidin-stainable material were retained. We therefore conclude that during myotube differentiation either an increased interaction of actin with actin-binding proteins (e.g., myosin and tropomyosin), or the assembly of muscle-specific isoforms of these proteins protect the filaments against degradation by actin severing proteins.     

Ultrastructural studies of lizard (Anolis carolinensis) myogenesis in vitro

In vitro differentiation of lizard (Anolis carolinensis) skeletal muscle cells was studied by electron microscopy. Myogenesis was studied under conditions in which large numbers of postmitotic prefusion myoblasts accumulate (Growth Medium) and under conditions which are permissive for myotube formation (Fusion Medium). In Growth Medium, myogenic cells proliferate, then assume a characteristic spherical morphology which permits definitive identification of prefusion myoblasts. During the early stages of culture, these round myoblasts resemble myoblasts described in other systems; ultrastructural similarities and differences are discussed. After longer periods of culture in Growth Medium, a continuum of differentiation from isolated myofilaments to assembled myofibrils was seen in these mononucleated cells. These observations confirm the dissociability of contractile protein assembly and myoblast fusion Cultures maintained in Fusion Medium or transferred from Growth Medium to Fusion Medium form multinucleated myotubes on a predictable time scale. Myogenesis was followed in these cultures with particular reference to the early events in myofilament assembly and myofibril formation.     

Chicken gizzard filamin, retina filamin and cgABP260 are respectively, smooth muscle-, non-muscle- and pan-muscle-type isoforms: distribution and localization in muscles

We determined the full cDNA sequences of chicken gizzard filamin and cgABP260 (chicken gizzard actin-binding protein 260). The primary and secondary structures predicted by these sequences were similar to those of chicken retina filamin and human filamins. Like mammals, chickens have 3 filamin isoforms. Comparison of their amino acid sequences indicated that gizzard filamin, retina filamin, and cgABP260 were the counterparts of human FLNa (filamin a), b, and c, respectively. Antibodies against the actin-binding domain (ABD) of these 3 filamin isoforms were raised in rabbits. Using immunoabsorption and affinity chromatography, we prepared the monospecific antibody against the ABD of each filamin. In immunoblotting, the antibody against the gizzard filamin ABD detected a single band in gizzard, but not in striated muscles or brain. In brain, only the antibody against the retina filamin ABD produced a strong single band. The antibody against the cgABP260 ABD detected a single peptide band in smooth, skeletal, and cardiac muscle. In immunofluorescence microscopy of muscular tissues using these antibodies, the antibody against the gizzard filamin ABD only stained smooth muscle cells, and the antibody against the retina filamin ABD strongly stained endothelial cells of blood vessels and weakly stained cells in connective tissue. The antibody against the cgABP260 ABD stained the Z-lines and myotendinous junctions of breast muscle, the Z-lines and intercalated disks of cardiac muscle, and dense plaques of smooth muscle. These findings indicate that chicken gizzard filamin, retina filamin, and cgABP260 are, respectively, smooth muscle-type, non-muscle-type, and pan-muscle-type filamin isoforms.     

Drosophila rolling pebbles: a multidomain protein required for myoblast fusion that recruits D-Titin in response to the myoblast attractant Dumbfounded.

The fusion of myoblasts leading to the formation of myotubes is an integral part of skeletal myogenesis in many organisms. In Drosophila, specialized founder myoblasts initiate fusion through expression of the receptor-like attractant Dumbfounded (Duf), which brings them into close contact with other myoblasts. Here, we identify Rols7, a gene expressed in founders, as an essential component for fusion during myotube formation. During fusion, Rols7 localizes in a Duf-dependent manner at membrane sites that contact other myoblasts. These sites are also enriched with D-Titin, which functions to maintain myotube structure and morphology. When Rols7 is absent or its localization is perturbed, the enrichment of D-Titin fails to occur. Rols7 integrates the initial event of myoblast attraction with the downstream event of myotube structural organization by linking Duf to D-Titin.     

Dynamic distribution of an antigen involved in the differentiation of avian myoblasts: II. Possible association of beta1 integrin with myofibril organization

Previous studies have shown that a monoclonal antibody, H-145, inhibits myotube formation of quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells) [Hyodo and Kim, 1994: Exp. Cell Res. 212:120-131]. The antigen recognized by H-145 (H-145 antigen), which is a glycoprotein with a molecular mass of about 116 kDa, is related to a step immediately before myoblast fusion. To determine the functional significance of H-145 antigen, we examined its dynamic state during myogenic differentiation of QM-RSV cells. H-145 antigen showed a unique and discrete distribution. In immature myotubes immediately after myoblast fusion, many ring-like structures of H-145 antigen appeared on the ventral surface of the cells, encircling the actin dots detected simultaneously by immunofluorescence and interference reflection microscopy. The core of the ring-like structures was filled with the termini of actin bundles, mainly formed by alpha-actin. Other cytoskeletal-associated proteins, such as vinculin and alpha-actinin, were also associated with these structures. The ring-like structures of H-145 antigen were observed only during a restricted period when myoblasts fused actively, suggesting their relationships to myotube formation and an early stage of myofibril formation. With maturation of the myotubes, most of the H-145 antigen became redistributed in linear arrays on the apical cell surface and was probably associated with the termini of actin bundles to organize myofibrils, suggesting that the antigen was also related to maturation of myotubes. Experiments using monoclonal antibodies against chick beta1 integrin showed that H-145 antigen is beta1 integrin or a very closely related derivation. Thus H-145 antigen (beta1 integrin) is possibly involved in both myoblast fusion and the myofibril organization in myotubes.     

The multiple ADP/ATP translocase genes are differentially expressed during human muscle development.

The expression of the genes encoding the three isoforms of the human ADP/ATP translocase (T1, T2, and T3) has been analyzed at different stages of myogenic differentiation in an in vitro muscle cell system and compared with that in mature muscle. The results indicate that the three stages of muscle differentiation corresponding to myoblast proliferation, myotube formation, and mature muscle fibers are characterized by a different pattern of expression of the ADP/ATP translocase genes. In particular, the two T2-specific mRNAs are present at high, similar levels in myoblasts and myotubes and markedly decrease in amount in mature adult muscle. By contrast, the T3-specific mRNA is present in high amount in growing myoblasts, decreases markedly in myotubes, and is barely detectable in adult muscle. Finally, the T1-specific mRNA is present at a high level in adult muscle and is not detectable in either myoblasts or myotubes. Therefore, T1 gene expression appears to be a marker of a late stage in myogenesis. A parallel investigation of expression of the myosin heavy chain mRNA revealed absence of hybridization with the specific probe in RNA from proliferating myoblasts, a significant hybridization in myotube RNA, and a strong signal in adult muscle RNA.     

Distinct roles for classical nuclear import receptors in the growth of multinucleated muscle cells

Proper muscle function is dependent on spatial and temporal control of gene expression in myofibers. Myofibers are multinucleated cells that are formed, repaired and maintained by the process of myogenesis in which progenitor myoblasts proliferate, differentiate and fuse. Gene expression is dependent upon proteins that require facilitated nuclear import, however little is known about the regulation of nucleocytoplasmic transport during the formation of myofibers. We analyzed the role of karyopherin alpha (KPNA), a key classical nuclear import receptor, during myogenesis. We established that five karyopherin alpha paralogs are expressed by primary mouse myoblasts in vitro and that their steady-state levels increase in multinucleated myotubes, suggesting a global increase in demand for classical nuclear import during myogenesis. We used siRNA-mediated knockdown to identify paralog-specific roles for KPNA1 and KPNA2 during myogenesis. KPNA1 knockdown increased myoblast proliferation, whereas KPNA2 knockdown decreased proliferation. In contrast, no proliferation defect was observed with KPNA4 knockdown. Only knockdown of KPNA2 decreased myotube growth. These results identify distinct pathways involved in myoblast proliferation and myotube growth that rely on specific nuclear import receptors suggesting that regulation of classical nuclear import pathways likely plays a critical role in controlling gene expression in skeletal muscle.     

Stac3 Inhibits Myoblast Differentiation into Myotubes

The functionally undefined Stac3 gene, predicted to encode a SH3 domain- and C1 domain-containing protein, was recently found to be specifically expressed in skeletal muscle and essential to normal skeletal muscle development and contraction. In this study we determined the potential role of Stac3 in myoblast proliferation and differentiation, two important steps of muscle development. Neither siRNA-mediated Stac3 knockdown nor plasmid-mediated Stac3 overexpression affected the proliferation of C2C12 myoblasts. Stac3 knockdown promoted the differentiation of C2C12 myoblasts into myotubes as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA and protein expression of myogenic markers including myogenin and myosin heavy chain. In contrast, Stac3 overexpression inhibited the differentiation of C2C12 myoblasts into myotubes as evidenced by decreased fusion index, decreased number of nuclei per myotube, and decreased mRNA and protein expression of myogenic markers. Compared to wild-type myoblasts, myoblasts from Stac3 knockout mouse embryos showed accelerated differentiation into myotubes in culture as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA expression of myogenic markers. Collectively, these data suggest an inhibitory role of endogenous Stac3 in myoblast differentiation. Myogenesis is a tightly controlled program; myofibers formed from prematurely differentiated myoblasts are dysfunctional. Thus, Stac3 may play a role in preventing precocious myoblast differentiation during skeletal muscle development.     

AN ANALYSIS OF MYOGENESIS BY THE USE OF FLUORESCENT ANTIMYOSIN

Antibodies against myosin of adult chicken skeletal muscle were labelled with fluorescein and used as staining reagents to analyze the development of trunk myoblasts in the chick embryo. Myoblasts from the brachial myotomes were studied in three ways: (a) Specimens were fixed, sectioned, and stained with iron-hematoxylin. (b) Living myoblasts, and myoblasts prepared by glycerol extraction, were teased and examined by phase contrast microscopy. (c) Embryo trunks were treated with fluorescent antimyosin or with a control solution of fluorescent normal globulin, and were examined by fluorescence and phase contrast microscopy. Both glycerol-extracted and fixed materials were used. Cross-striated myofibrils appeared first in stage 16 to 17 embryos in the series studied by antimyosin staining and fluorescence microscopy. Striated myofibrils appeared first in stage 18 to 19 embryos, in the series stained by iron-hematoxylin, and at stage 22 to 23, in the series studied by glycerol extraction and phase contrast microscopy. In each series, myofibrils without apparent cross-striations were detected shortly before cross-striations were observed. Specific staining by antimyosin occurred only in differentiating myoblasts. Within the myoblasts antimyosin staining was confined to the A bands of the slender myofibrils. The following observations suggest that the first delicate striated structure to appear in the early 3 day myoblast was remarkably mature: (1) The sarcomere pattern both in length and in internal detail, was similar to that of adult muscle. (2) The distribution of myosin, as revealed by antimyosin staining, was the same in the embryonic as in the mature myofibril. (3) Glycerol-extracted myoblasts contracted vigorously on exposure to ATP. The changes in sarcomere band pattern were indistinguishable from those occurring during contraction of adult muscle induced by ATP. (4) ATP contraction was blocked by prior antimyosin staining in embryonic myoblasts as in mature muscle. It is suggested that the early myofibril grows laterally as a thin sheet associated with the sarcolemma, and that growth in length occurs in the growth tips of the elongating myoblast.     

Myoblast structure affects subsequent skeletal myotube morphology and sarcomere assembly

Skeletal myogenesis is a precise procedure marked by specific changes in muscle cell morphology and cytoarchitecture. Cessation of proliferation by skeletal muscle precursor cells (myoblasts) coincides with the induction of fusion to form multinucleated myotubes and the initiation of differentiation, the process through which sarcomeres are formed. Concurrently, there is a distinct upregulation in expression of muscle-specific isoforms and an extreme downregulation of non-muscle-specific cytoskeletal isoforms. The sarcomere is the contractile unit of the cell and is comprised of a number of different proteins aggregated and aligned in very ordered arrays along the myotube. It is this rigorously controlled alignment that gives striated muscle its characteristic "striped" appearance. Previous studies, conducted predominantly in cardiac muscle, propose models for the development of the sarcomere that attribute little of the differentiative process to the myoblast morphology and cytoskeletal arrangement. In this study, perturbation of myoblast morphology and cytoskeletal arrangement by transfection with nonmuscle actin genes in the mouse skeletal muscle cell line C2 resulted in myotubes of both varied morphology and sarcomeric structure. The results presented herein not only provide novel insights into the formation of the sarcomere in skeletal muscle, but also suggest a role for myoblast morphology and cytoskeletal structure in the subsequent differentiation of the myotube.     

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.