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PubMed2001

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.

Amino Acid Sequence↗
PubMed1992

Differentiation of quail myoblasts transformed with a temperature sensitive mutant of Rous sarcoma virus. II. Relationship of myoblast fusion with calcium and temperature.

The effects of calcium and temperature on fusion of quail embryonic myoblasts were examined using cells transformed with a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV). The transformed quail myoblasts (QM-RSV) fused to form myotubes at 41 degrees C, the non-permissive temperature, but not at 35.5 degrees C, the permissive temperature. On incubation at 41 degrees C, a period of more than 10 hr was needed for the myoblasts to become fusion-competent, but calcium was not needed for development of fusion-competence. Once the cells had become competent, fusion proceeded even at 35.5 degrees C. These results suggest that the src gene product expressed at 35.5 degrees C may control the fusion of cells in the competent stage by inactivating a component(s) that is associated with fusion-competence. However, fusion of even myoblasts in the competent stage was blocked in calcium-deficient medium, suggesting that calcium is essential for the fusion, probably at a step immediately before membrane union. Unlike fusion, other biochemical processes of differentiation proceeded even in calcium-deficient medium, indicating a distinction of fusion from these other processes during myoblast differentiation.

Animals↗
PubMed1994

Preparation and characterization of a monoclonal antibody that inhibits myoblast fusion of avian skeletal myoblasts.

To investigate the mechanism of myoblast fusion using quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (QM-RSV cells), we prepared monoclonal antibodies against a cell surface antigen involved in myogenic differentiation. For this, a Balb/c mouse was immunized with the membrane fraction of QM-RSV cells, and hybridomas producing monoclonal antibodies were raised by fusion of spleen cells from the immunized mouse with myeloma cells. By analysis of the hybridoma supernatants, we obtained a monoclonal antibody, termed H-145, that strongly inhibited myoblast fusion. H-145 inhibited myoblast fusion dose-dependently, and its effect was readily reversed by its removal. H-145 promoted biochemical differentiation of the cells until 48 h. It did not affect a fusion-commitment step to differentiation, but inhibited a later step. Indirect immunofluorescence and immunoblot analyses showed that the antigen reacting with H-145 was a glycoprotein with a molecular weight of approximately 116 kDa. This antigen is present throughout differentiation, but as differentiation progresses, its expression increases and its distribution on the cell surface changes. The antigen purified by H-145 affinity chromatography failed to react with beta 1-integrin, alpha 5-integrin, NCAM, or N-cadherin on immunoblotting. Thus, H-145 antigen differs from these components that are known to be associated with myogenic differentiation. Consequently, the results suggest that H-145 antigen may be a new cell surface antigen associated with cell differentiation.

Animals↗
PubMed1990

Affinity labeling of myoblast surface proteins with 5'-P-fluorosulfonylbenzoyl adenosine: concomitant inhibition of ectoprotein kinase activity and myoblast fusion.

During in vitro myogenesis, mononucleated myoblasts fuse among themselves to form multinucleated myotubes. We have recently reported for the first time in the literature that a Ca2(+)-dependent ectoprotein kinase is responsible for this process, but we had no direct evidence for the role of extracellular ATP. To investigate whether the cells can fuse or not in the absence of this nucleotide, we used a nucleotide affinity label, fluorosulfonylbenzoyl adenosine (FSBA). We report here its use in detecting the nucleotide-binding sites at the cell surface of intact myoblasts in culture. We demonstrate that FSBA blocks fusion by inhibiting the ectoprotein kinase activity of the cells at sublethal concentrations. Radioactive [14C]SBA is incorporated into seven cell surface proteins and into the 48-kDa protein, among others. This species is specific for fusion-competent myoblasts and is implicated in this process. This is the first time that nucleotide-binding molecular species have been identified at the surface of myoblasts.

Adenosine↗
PubMed1997

Drosophila myoblast city encodes a conserved protein that is essential for myoblast fusion, dorsal closure, and cytoskeletal organization.

The Drosophila myoblast city (mbc) locus was previously identified on the basis of a defect in myoblast fusion (Rushton et al., 1995. Development [Camb.]. 121:1979-1988). We describe herein the isolation and characterization of the mbc gene. The mbc transcript and its encoded protein are expressed in a broad range of tissues, including somatic myoblasts, cardial cells, and visceral mesoderm. It is also expressed in the pole cells and in ectodermally derived tissues, including the epidermis. Consistent with this latter expression, mbc mutant embryos exhibit defects in dorsal closure and cytoskeletal organization in the migrating epidermis. Both the mesodermal and ectodermal defects are reminiscent of those induced by altered forms of Drac1 and suggest that mbc may function in the same pathway. MBC bears striking homology to human DOCK180, which interacts with the SH2-SH3 adapter protein Crk and may play a role in signal transduction from focal adhesions. Taken together, these results suggest the possibility that MBC is an intermediate in a signal transduction pathway from the rho/rac family of GTPases to events in the cytoskeleton and that this pathway may be used during myoblast fusion and dorsal closure.

Amino Acid Sequence↗
PubMed1983

Analysis of myogenesis by somatic cell hybridization: III. Myogenic competence of hybrids derived from rat L6 myoblasts and mouse primary fibroblasts and myoblasts.

Hybrid cells derived from rat L6 myoblasts and mouse primary fibroblasts (M x F hybrids), as well as those derived from rat L6 myoblasts and mouse primary myoblasts (M x M hybrids), were examined for their ability to engage in myogenesis as judged by muscle fiber formation plus the expression of skeletal muscle myosin and creatine kinase (CK). Of 172 primary hybrid colonies scored, 59% were myogenic in the M x F fusion and 97% exhibited muscle fiber formation in the M x M fusion. Individual hybrid clones from each cross were isolated, expanded and analyzed for myogenic capabilities as well. All three M x M and all ten M x F isolated clones exhibited preferential elimination of mouse chromosomes. Nonetheless, all were capable of fusing spontaneously and of elaborating skeletal muscle myosin and CK. The three M x M hybrids expressed only MM-CK whereas nine out of ten M x F hybrids produced all three CK isoenzymes (MM, MB, BB). These results suggest that M X M hybrids express CK patterns reminiscent of the rat L6 parental cells while M X F hybrids apparently mimic mouse muscle fiber CK patterns. Various models are discussed which address these phenomena.

Animals↗
PubMed1978

A comparative study of the myosin light chain kinases from myoblast and muscle sources. Studies on the kinases from proliferative rat myoblasts in culture, rat thigh muscle, and rabbit skeletal muscle.

Myosin light chain kinases have been isolated from rat thigh and rabbit skeletal muscle and cultured rat myoblasts. From these preparations, two types of kinases can be distinguished: calcium-dependent and calcium-independent. Both types of kinases can phosphorylate isolated P-light chains of myosin from several sources (skeletal muscle, cardiac muscle, and platelet). Data are shown which support the phosphorylation of the same site on the non-muscle P-light chains by both types of kinases. The rates of these reactins are, however, different for the two types of kinases. Kinetic analysis of the myoblast kinase shows differing affinities for various P-light chains (non-muscle greater than cardiac greater than skeletal). In the proliferative rat myoblast, phosphorylation of myosin is a prerequisite for actin activation of the myosin ATPase activity.

Adenosine Triphosphatases↗
PubMed2005

Hypomorphic Smn knockdown C2C12 myoblasts reveal intrinsic defects in myoblast fusion and myotube morphology.

Dosage of the survival motor neuron (SMN) protein has been directly correlated with the severity of disease in patients diagnosed with spinal muscular atrophy (SMA). It is also clear that SMA is a neurodegenerative disorder characterized by the degeneration of the alpha-motor neurons in the anterior horn of the spinal cord and atrophy of the associated skeletal muscle. What is more controversial is whether it is neuronal and/or muscle-cell-autonomous defects that are responsible for the disease per se. Although motor neuron degeneration is generally accepted as the primary event in SMA, intrinsic muscle defects in this disease have not been ruled out. To gain a better understanding of the influence of SMN protein dosage in muscle, we have generated a hypomorphic series of myoblast (C2C12) stable cell lines with variable Smn knockdown. We show that depletion of Smn in these cells resulted in a decrease in the number of nuclear 'gems' (gemini of coiled bodies), reduced proliferation with no increase in cell death, defects in myoblast fusion, and malformed myotubes. Importantly, the severity of these abnormalities is directly correlated with the decrease in Smn dosage. Taken together, our work supports the view that there is an intrinsic defect in skeletal muscle cells of SMA patients and that this defect contributes to the overall pathogenesis in this devastating disease.

Animals↗
PubMed2004

Nucleofection of muscle-derived stem cells and myoblasts with phiC31 integrase: stable expression of a full-length-dystrophin fusion gene by human myoblasts.

Ex vivo gene therapy offers a potential treatment for Duchenne muscular dystrophy by transfection of the dystrophin gene into the patient's own myogenic precursor cells, followed by transplantation. We used nucleofection to introduce DNA plasmids coding for enhanced green fluorescent protein (eGFP) or eGFP-dystrophin fusion protein and the phage phiC31 integrase into myogenic cells and to integrate these genes into a limited number of sites in the genome. Using a plasmid expressing eGFP, we transfected 50% of a mouse muscle-derived stem cell line and 60% of normal human myoblasts. Co-nucleofection of a plasmid expressing the phiC31 integrase and an eGFP expression plasmid containing an attB sequence produced 15 times more frequent stable expression, because of site-specific integration of the transgene. Co-nucleofection of the phiC31 integrase plasmid and a large plasmid containing the attB sequence and the gene for an eGFP-full-length dystrophin fusion protein produced fluorescent human myoblasts that were able to form more intensely fluorescent myotubes after 1 month of culture. A nonviral approach combining nucleofection and the phiC31 integrase may eventually permit safe autotransplantation of genetically modified cells to patients.

Animals↗
PubMed1999

Effects of IGF-I, IGF-II, bFGF and PDGF on the initiation of mRNA translation in C2C12 myoblasts and differentiating myoblasts.

In order to study the mechanisms by which growth factors stimulate protein synthesis, C2C12 myogenic cells were treated with a variety of growth factors and the recruitment of free ribosomes to polysomes was quantified. All experiments were conducted on C2C12 myoblasts (24 h prior to induction of fusion) and differentiating myoblasts (24 h after induction of fusion). After the 2 h incubation, cells were rinsed with phosphate buffered saline and quickly frozen at -80 degrees C. Cell lysates were fractionated on 15-60% sucrose gradients by centrifugation at 200,000 x g for 1 h. Absorbance at 254 nm was recorded continuously across the gradient. The response to each of the four growth factors, IGF-I and-II, basic fibroblast growth factor (FGF), and platelet-derived growth factor was a decrease (P < 0.05) in monosome peak height and a increase (P < 0.05) in polysome percentage (P < 0.05). All responses were linear, except IGF-I, and the monosome peak height response to FGF which were quadratic (P < 0.05). None of the growth factors had a significant effect (P > 0.05) on RNA concentrations over the 2-h incubation. Protein content did not vary due to growth factor or level of treatment. This corroborates the hypothesis that the acute increase of protein synthesis exhibited by growth factor treated cells is due to an increase in the activity of existing ribosomes rather than an increase in ribosome synthesis. These results suggest that we can study the mechanisms regulating protein synthesis in muscle cells effectively by studying shifts in ribosomal activity. This method gave more consistent results than the H3-tyrosine incorporation and has the added benefit of not requiring the use of radioactivity. The strong correlation between monosome peak heights and percentage polysomes will allow researchers to measure total protein synthetic activity in a culture from the free or cytoplasmic fraction and to reserve the polysomes for other uses. The similarity of response among the various growth factors may indicate a common mechanism for increasing the initiation of protein synthesis.

Animals↗
PubMed1993

Induction of myoblast proliferation in L6 myoblast cultures by fetal serum of double-muscled and normal cattle.

Double-muscled (DM) cattle possess more muscle fibers than do normal-muscled (NM) beef or dairy cattle. Serum-borne growth factors have been shown to modulate myogenesis. Media containing serum from 12 DM and 60 NM fetuses grouped by crown-rump lengths (CRL) of < or = 25, 26 to 50, 51 to 75, or > 75 cm were used to test the effect of fetal serum on L6 myoblast proliferation. Because DM and NM fetuses were similar in CRL at ages corresponding to 20- to 60-cm CRL, CRL was used to determine fetal age. Normal-muscled fetal serum-induced thymidine incorporation in L6 myoblasts was greater (P < .05) at CRL > 50- than at < or = 50-cm CRL. Mean incorporation tended to increase with CRL. Thymidine incorporation was 56, 41, and 41% greater (P < .05) with serum from DM fetuses than with that from NM fetuses at CRL of < or = 25, 26 to 50, and 51 to 75 cm, respectively. Morphological examination of cross-sections of the semitendinosus muscle showed that apparent muscle fiber number was greater (P < .05) for DM fetuses than for NM fetuses. These results confirm greater apparent muscle fiber number in DM cattle and show the existence of greater growth factor activity in serum of DM fetuses during early fetal development. This greater growth factor activity may play a role in bovine muscle fiber hyperplasia.

Animals↗
PubMed1996

Adenylyl cyclase activity in clonally derived human myoblast cultures: evidence for myoblast heterogeneity.

In vitro myogenesis recapitulates the programme of myogenesis in vivo. During the process of muscle differentiation, cAMP plays an important role in the control of gene expression and in the integration of metabolic functions. cAMP generation may be affected by drugs or hormones that interact with the membrane-bound enzyme adenylyl cyclase, including adrenergic agents and glucocorticoids. In this study, adenylyl cyclase activity was evaluated in membranes prepared from human clonally derived muscle cultures. In control cultures, there was considerable inter-clonal variation in basal, sodium-fluoride and forskolin-stimulated adenylyl cyclase activity. Cultures differed in their response to steroids: adenylyl cyclase activity was markedly enhanced in some clones, and was significantly inhibited in other clones. Pre-treatment of cultures with pertussis toxin indicated that the effects of steroids are mediated in part by modulation of G-protein activity. These findings indicate a substantial heterogeneity among myoblast clones with respect to the modulating effect of steroids on adenylyl cyclase activity. This observation may account for the conflicting reports of steroid effects on muscle in vitro, and may be of relevance to the understanding of possible transmembrane signalling alterations in muscle disease.

Adenylate Cyclase Toxin↗
PubMed1999

Effects of serum deprivation, insulin and dexamethasone on polysome percentages in C2C12 myoblasts and differentiating myoblasts.

An increase in the rate of protein synthesis in living cells can be achieved by regulating the quantity of mRNA, ribosomes, and enzymes available for translation or by regulating the efficiency at which existing components are used. Efficiency can be measured by comparing the number of ribosomes actively engaged in the synthesis of protein (polysomes) to the pool of free ribosomes. The objective of this study was to determine the percentage of ribosomes found as polysomes in C2C12 cells deprived of serum or exposed to insulin or dexamethasone 24 h before and after being stimulated to differentiate. Individual 60 mm culture dishes were exposed to serum-free control medium, medium containing serum, insulin, or dexamethasone for a period of 1 h or 2 h and then quickly frozen. The ribosomes and polysomes from these cells were separated by ultracentrifugation on 15 to 60% sucrose gradients and the absorbance across the gradient at 254 nm was recorded. Polysome percentages were determined as the area under the polysome peak divided by the total area under the curve. Serum deprivation caused a 12% decline in the percentage of ribosomes found as polysomes (P < 0.01). Dexamethasone caused a quadratic decline (P < 0.05) in polysome percentage, while insulin yielded a quadratic increase (P < 0.05). Protein synthesis assays measuring 3H-tyrosine uptake showed similar responses. These changes occurred in the absence of any differences in total RNA concentration. It was concluded that differentiation and the absence of serum in the media reduced the rate of recruitment of ribosomes for protein synthesis. Insulin increased ribosome recruitment which was also observed by a similar increase in incorporation of radio-labeled tyrosine.

Animals↗