Rescue of the Li+-induced delay of embryonic myogenesis in vitro by added inositol

Signaling between embryonic myoblasts to coordinate gene expression is part of normal skeletal muscle development in the embryo. An unanswered question is the nature of the second messengers carrying the information to the nucleus. We have investigated the cell membrane events associated with the binding of prostaglandin to a transient receptor on the embryonic chick myoblast membrane in vitro. The membrane events include a transient change in membrane order seen by electron paramagnetic resonance (EPR), a change in cell-cell adhesion, a rapid decrease in membrane permeability and fusion of the membrane bilayers. The addition of 20 mM Li+, an inhibitor of inositol phosphate phosphatase, perturbed the transient change in membrane order and delayed the change in cell-cell adhesion and conductivity for 2-6 h. Other alkali metal ions had no such effects. The addition of inositol to the culture medium in the continued presence of Li+ restored the normal timing of the two latter events. We interpret this as evidence for an inositol phosphate second messenger system which might connect the activation of the prostaglandin receptor with the change in cell-cell adhesion, the changes in membrane conductivity and perhaps bilayer fusion. We suggest that Li+, by blocking the regeneration of polyphosphatidylinositol from inositol phosphate, reduced the efficiency of the second messenger system such that further differentiation of the myoblast membrane was delayed. The exogenous inositol provided an alternative source and membrane differentiation was unaffected.     

Prostaglandin binding does not require direct cell-cell contact during chick myogenesis in vitro

Myogenic differentiation in vitro involves at least three events at the cell surface: binding of prostaglandin to the cells, contact-mediated cell-cell recognition, and fusion of the myoblast membranes into myotubes. While the earlier events are thought to be necessary for subsequent fusion, the sequence of events has not been determined. A major impediment to determining the initial event has been the lack of synchrony of cell differentiation in vitro. To overcome this, we cultured chick embryo myoblasts as a suspension of single cells in gyratory rotation in medium without added Ca2+. Under these conditions, myoblasts exhibited characteristic prostaglandin binding at 34 h. Within 30 min, the cells began to aggregate. Because this occurred without change of medium or conditions of rotation, we termed the process autoaggregation. Within 8-10 h. cells within these autoaggregates began to fuse into syncytia. These results suggest that an early cell surface event in embryonic myogenesis is the characteristic binding of prostaglandin to the myoblasts. The results demonstrate that this binding precedes any direct cell-cell contact and suggest that it causes the subsequent change in myoblast cell-cell adhesion.     

Changes in myoblast membrane order during differentiation as measured by EPR

The events which make possible the characteristic fusion of the cell membranes of embryonic myoblasts are known to involve modification of the cell membrane (Hausman, R.E., Dobi, E.T., Woodford, E.J., Petrides, S., Ernst, M. and Nichols E.B. (1986) Dev. Biol. 113, 40-48). Myoblasts from chick embryos were allowed to differentiate in gyrotory aggregate culture and the order of their membranes was measured by EPR. Two spin-labels which insert at different depths into the lipid bilayer were used. Measurement with the 5-nitroxystearate label showed an increase in myoblast membrane order (2T' parallel) from 0-15 h of culture and again from 26-38 h of culture. Measurement with the 12-nitroxystearate label showed the 0-15 h increase in order but the second increase was greatly reduced and shifted in time. While the specific sources of these changes in membrane order cannot yet be identified, the changes observed correlated well with known events of myogenic differentiation in vitro. The initial increase in membrane order occurred while the myoblasts were recovering from the effects of trypsin dissociation and undergoing gyrotory aggregation. The second increase in membrane order occurred during the known period of prostaglandin receptor activity and increased cell-cell adhesion.     

Morphological changes and spatial regulation of diacylglycerol kinase-zeta, syntrophins, and Rac1 during myoblast fusion

The fusion of mononuclear myoblasts into multinucleated myofibers is essential for the formation and growth of skeletal muscle. Myoblast fusion follows a well-defined sequence of cellular events, from initial recognition and adhesion, to alignment, and finally plasma membrane fusion. These processes depend upon coordinated remodeling of the actin cytoskeleton. Our recent studies suggest diacylglycerol kinase-zeta (DGK-zeta), an enzyme that metabolizes diacylglycerol to yield phosphatidic acid, plays an important role in actin reorganization. Here, we investigated whether DGK-zeta has a role in the fusion of cultured C2C12 myoblasts. We show that DGK-zeta and syntrophins, scaffold proteins of the dystrophin glycoprotein complex that bind directly to DGK-zeta, are spatially regulated during fusion. Both proteins accumulated with the GTPase Rac1 at sites where fine filopodia mediate the initial contact between myoblasts. In addition, DGK-zeta codistributed with the Ca(2+)-dependent cell adhesion molecule N-cadherin at nascent, but not previously established cell contacts. We provide evidence that C2 cells are pulled together at cell-cell junctions by N-cadherin-containing filopodia reminiscent of epithelial adhesion zippers, which guide the advance of lamellipodia from apposing cells. At later times, vesicles with properties of macropinosomes formed close to cell-cell junctions. Reconstruction of confocal optical sections showed these form dome-like protrusions from the dorsal surface of contacting cells. Collectively, these results suggest DGK-zeta and syntrophins play a role at multiple stages of the fusion process. Moreover, our findings provide a potential link between changes in the lipid content of the membrane bilayer and reorganization of the actin cytoskeleton during myoblast fusion.     

Requirement for G protein activity at a specific time during embryonic chick myogenesis

Signaling between embryonic myoblasts involves prostaglandin metabolism, the activation of a membrane receptor and changes in polyphosphatidyl inositol metabolism. Many of these membrane-localized events occur between 33 to 35 h of differentiation, concomitant with a dramatic change in membrane organization, in myoblast aggregates in culture. Since many receptors affect inositol phosphate metabolism by activating a GTP-binding protein (G protein), we asked if there was evidence for such a protein in myogenic signaling. We show that during the period of differentiation in culture when prostaglandin is needed to bind to a transient receptor, a pertussis toxin-sensitive but cholera toxin-insensitive G protein must act. If this activation is blocked, the characteristic change in myoblast cell adhesion and subsequent membrane fusion do not occur. We suggest that a G protein couples the activated prostaglandin receptor and the change in polyphosphatidyl inositol metabolism and that this membrane transduction step is necessary for subsequent membrane differentiation events during myogenesis.     

A role for the Ca2(+)-dependent adhesion molecule, N-cadherin, in myoblast interaction during myogenesis

The formation of multinucleate skeletal muscle cells (myotubes) is a Ca2(+)-dependent process involving the interaction and fusion of mononucleate muscle cells (myoblasts). Specific cell-cell adhesion precedes lipid bilayer union during myoblast fusion and has been shown to involve both Ca2(+)-independent (CI)2 and Ca2(+)-dependent (CD) mechanisms. In this paper we present evidence that CD myoblast adhesion involves a molecule similar or identical to two known CD adhesion glycoproteins, N-cadherin and A-CAM. These molecules were previously identified by other laboratories in brain and cardiac muscle, respectively, and are postulated to be the same molecule. Antibodies to N-cadherin and A-CAM immunoblotted a similar band with a molecular weight of approximately 125,000 in extracts of brain, heart, and pectoral muscle isolated from chick embryos and in extracts of muscle cells grown in vitro at Ca2+ concentrations that either promoted or inhibited myotube formation. In assays designed to measure the interaction of fusion-competent myoblasts in suspension, both polyclonal and monoclonal anti-N-cadherin antibodies inhibited CD myoblast aggregation, suggesting that N-cadherin mediates the CD aspect of myoblast adhesion. Anti-N-cadherin also had a partial inhibitory effect on myotube formation likely due to the effect on myoblast-myoblast adhesion. The results indicate that N-cadherin/A-CAM plays a role in myoblast recognition and adhesion during skeletal myogenesis.     

Involvement of cell surface phosphatidylinositol-anchored glycoproteins in cell-cell adhesion of chick embryo myoblasts

During myogenesis myoblasts fuse to form multinucleate cells that express muscle-specific proteins. A specific cell-cell adhesion process precedes lipid bilayer union during myoblast fusion (Knudsen, K. A., and A. F. Horwitz. 1977. Dev. Biol. 58:328-338) and is mediated by cell surface glycoproteins (Knudsen, K. A., 1985. J. Cell Biol. 101:891- 897). In this paper we show that myoblast adhesion and myotube formation are inhibited by treating fusion-competent myoblasts with phosphatidylinositol-specific phospholipase C (PI-PLC). The effect of PI-PLC on myoblast adhesion is dose dependent and inhibited by D-myo- inositol 1-monophosphate and the effect on myotube formation is reversible, suggesting a specific, nontoxic effect on myogenesis by the enzyme. A soluble form of adhesion-related glycoproteins is released from fusion-competent myoblasts by treatment with PI-PLC as evidenced by (a) the ability of phospholipase C (PLC)-released material to block the adhesion-perturbing activity of a polyclonal antiserum to intact myoblasts; and (b) the ability of PLC-released glycoprotein to stimulate adhesion-perturbing antisera when injected into mice. PI-PLC treatment of fusion-competent myoblasts releases an isoform of N-CAM into the supernate, suggesting that N-CAM may participate in mediating myoblast interaction during myogenesis.     

Dependence of myoblast fusion on a cortical actin wall and nonmuscle myosin IIA

Cell-cell fusion is a fundamental cellular process that is essential for development as well as fertilization. Myoblast fusion to form multinucleated skeletal muscle myotubes is a well studied, yet incompletely understood example of cell-cell fusion that is essential for formation of contractile skeletal muscle tissue. Studies in this report identify several novel cytoskeletal events essential to an early phase of myoblast fusion among cultured murine myoblasts. During myoblast pairing and alignment, cortical actin filaments organize into a dense actin wall structure that parallels and extends the length of the plasma membrane of the bipolar, aligned cells. As fusion progresses, gaps appear within the actin wall at sites of vesicle accumulation, the vesicles pair across the aligned myoblasts, cell-cell contacts and fusion pores form. Inhibition of nonmuscle myosin IIA (NM-MHC-IIA) motor activity prevents formation of this cortical actin wall, as well as the appearance of vesicles at a membrane proximal location, and myoblast fusion. These results suggest that early formation of a subplasmalemmal actin wall during myoblast alignment is a critical event for myoblast fusion that supports bipolar membrane alignment and temporally regulates trafficking of vesicles to the nascent fusion sites during skeletal muscle myoblast differentiation.     

Colocalization of N-CAM and N-cadherin in avian skeletal myoblasts

The cell-cell adhesion molecules, N-CAM and N-cadherin, have been shown previously to mediate myoblast interaction during cell fusion accompanying skeletal myogenesis. To study the localization of both molecules in fusion-competent myoblasts, we used antigen-specific primary antibodies and a double-labeling preembedding immuno-electron microscopy technique. Ultrastructural observations and quantitative analysis of the results reveal that N-CAM and N-cadherin frequently colocalize in clusters on the myoblast plasma membrane. The data provide morphological evidence that the two adhesion glycoproteins cooperate in mediating myoblast interaction during myoblast fusion.     

Electron spin resonance detection of free radicals in the mercaptan-activation and UV-inactivation of neocarzinostatin

Differentiation of chick embryo myoblasts $\text{in}\text{,}\text{vitro}$ requires both cell-cell recognition and cell-cell fusion. Prostaglandin E₁ is known to play a role in controlling fusion, and a specific receptor has been postulated. We demonstrate two peaks of specific binding activity for prostaglandin E₁ during myoblast differentiation $\text{in}\text{,}\text{vitro}$: one at 36 hours and one at 44 hours of culture. The prostaglandin binding activity of both peaks is sensitive to the inhibitors of prostaglandin synthesis, indomethacin and aspirin, and to the antibiotic tunicamycin. The 36 hour peak of binding activity occurs at the same time as the process of cell-cell recognition (24–36 hours) and recognition and prostaglandin binding exhibit similar sensitivity to indomethacin, aspirin and tunicamycin.     

The Arf-GEF Schizo/Loner regulates N-cadherin to induce fusion competence of Drosophila myoblasts

Myoblast fusion is a key process in multinucleated muscle formation. Prior to fusion, myoblasts recognize and adhere to each other with the aid of cell-adhesion proteins integrated into the membrane. Their intracellular domains participate in signal transduction by binding to cytoplasmic proteins. Here we identified the calcium-dependent cell-adhesion protein N-cadherin as the binding partner of the guanine-nucleotide exchange factor Schizo/Loner in Drosophila melanogaster. N-cadherin was expressed in founder cells and fusion-competent myoblasts of Drosophila during the first fusion phase. Our genetic analyses demonstrated that the myoblast fusion defect of schizo/loner mutants is rescued in part by the loss-of-function mutation of N-cadherin, which suggests that Schizo/Loner is a negative regulator of N-cadherin. Based on our findings, we propose a model where N-cadherin must be removed from the myoblast membrane to induce a protein-free zone at the cell-cell contact point to permit fusion.     

M-cadherin and beta-catenin participate in differentiation of rat satellite cells

Cadherins belong to a large family of membrane glycoprotein adhesion receptors that mediate homophilic, calcium-dependent cell adhesion. During myogenesis, cadherins are involved in initial cell-to-cell recognition; and it has also been suggested that they play a role in the initiation of myoblast fusion into multinuclear myotubes. One of the members of the cadherin family, M-cadherin, has been detected during embryogenesis in myogenic cells of somitic origin and in adult muscles. We investigated the distribution and function of M-cadherin and beta-catenin during differentiation of myoblasts in primary cultures of rat satellite cells. We found that M-cadherin was accumulated at the areas of contact between fusing myoblasts and that it colocalized with beta-catenin. Moreover, beta-catenin colocalized with actin in pre-fusing myoblasts. We show that myoblast differentiation is accompanied by an increase in the amounts of M-cadherin and beta-catenin both at the mRNA and the protein level. Flow cytometry analysis showed that M-cadherin expression was highest in fusing myoblasts. In addition, an antibody specific for the extracellular domain of M-cadherin inhibited the fusion of cultured myoblasts. These data suggest that regulation of the M-cadherin level plays an important role in the differentiation of satellite cells and in myoblast fusion in primary cultures.     

Essential genes for myoblast fusion in Drosophila embryogenesis.

In Drosophila, as in vertebrates, each muscle is a syncytium and arises from mesodermal cells by successive fusion. This requires cell-cell recognition, alignment, formation of prefusion complexes, followed by electron-dense plaques and membrane breakdown. Because muscle development in Drosophila is rapid and well-documented, it has been possible to identify several genes essential for fusion. Molecular analysis of two of these genes revealed the importance of cytoplasmic components. One of these, Myoblast city, is expressed in several tissues and is homologous to the mammalian protein DOCK180. Myoblast city is presumably involved in cell recognition and cell adhesion. Blown fuse, the second cytoplasmic component, is selectively expressed in the mesoderm and essential in order to proceed from the prefusion complex to electron-dense plaques at opposed membranes between adjacent myoblasts. The rolling stone gene is transiently expressed during myoblast fusion. The Rost protein is located in the membrane and thus might be a key component for cell recognition. The molecular characterization of further genes relevant for fusion such as singles bar and sticks and stones will help to elucidate the mechanism of myoblast fusion in Drosophila.     

Developmental regulation of a cadherin during the differentiation of skeletal myoblasts

Cadherins are a family of integral membrane glycoproteins which mediate calcium-dependent intercellular adhesion in vertebrate species. Here we present evidence that fusion-competent rat L6 myoblasts express a cadherin (Mr 127 kDa). The levels of this cadherin were found to be developmentally regulated. Maximal levels were expressed prior to fusion. The increase in cadherin levels observed during differentiation was prevented by the differentiation inhibitor, 5-bromo-2'-deoxyuridine. L6 myoblasts grown in the presence of anti-cadherin antibodies exhibited an altered morphology in comparison to control cultures, coupled with decreased myoblast fusion. These data indicate that the developmental regulation of cadherin is part of the program of terminal differentiation of skeletal myoblasts, and that cadherins are involved in the process of myoblast fusion.     

Integrin alpha3 subunit participates in myoblast adhesion and fusion in vitro

Satellite cells are myogenic precursor cells, participating in growth, and regeneration of skeletal muscles. The proteins that play a role in myogenesis are integrins. In this report, we show that the integrin alpha3 subunit is expressed in quiescent satellite cells and activated myoblasts. We also find that in myoblasts the integrin alpha3 subunit is localized at cell-cell and cell-extracellular matrix contacts. We notice that increase in protein and mRNA encoding the integrin alpha3 subunit accompanies myoblast differentiation. Using double immunofluorescence and immunoprecipitation experiments, we demonstrate that the integrin alpha3 subunit co-localizes with actin, and binds the integrin beta1 subunit and ADAM12, suggesting that the complex alpha3beta1/ADAM12 is probably involved in myoblast fusion. Importantly, overexpression of the full-length integrin alpha3 subunit increases myoblast fusion whereas an antibody against its extracellular domain inhibits fusion. These data demonstrate that the integrin alpha3 subunit may contribute to satellite cell activation and then myoblast adhesion and fusion.     

Is Intercellular Communication Via Gap Junctions Required for Myoblast Fusion?

Fusion of myoblasts to form syncitial muscle cells results from a complex series of sequential events including cell alignment, cell adhesion and cell communication. The aim of the present investigation was to assess whether intercellular communication through gap junctions would be required for subsequent membrane fusion. The presence of the gap junction protein connexin 43 at areas of contact between prefusing rat L6 myoblasts was established by immunofluorescent staining. These myoblasts were dye-coupled, as demonstrated by the use of the scrape-loading/dye transfer technique. L6 myoblast dye coupling was reversibly blocked by heptanol in short term experiments as well as after chronic treatment. After a single addition of 3.5 mM heptanol, gap junctions remained blocked for up to 8 hours, then this inhibitory effect decreased gradually, likely because the alcohol was evaporated. Changing heptanol solutions every 8 hours during the time course of L6 differentiation resulted in a lasting drastic inhibition of myoblast fusion. We further investigated the effect of heptanol and of other uncoupling agents on the differentiation of primary cultures of embryonic chicken myoblasts. These cells are transiently coupled by gap junctions before myoblast fusion and prolonged application of heptanol, octanol and 18-β-glycyrrhetinic acid also inhibited their fusion. The effect of heptanol and octanol was neither due to a cytotoxic effect nor to a modification of cell proliferation. Moreover, heptanol treatment did not alter myoblast alignment and adhesion. Taken together these observations suggest that intercellular communication might be a necessary step for myoblast fusion.     

Is Intercellular Communication Via Gap Junctions Required for Myoblast Fusion?

Fusion of myoblasts to form syncitial muscle cells results from a complex series of sequential events including cell alignment, cell adhesion and cell communication. The aim of the present investigation was to assess whether intercellular communication through gap junctions would be required for subsequent membrane fusion. The presence of the gap junction protein connexin 43 at areas of contact between prefusing rat L6 myoblasts was established by immunofluorescent staining. These myoblasts were dye-coupled, as demonstrated by the use of the scrape-loading/dye transfer technique. L6 myoblast dye coupling was reversibly blocked by heptanol in short term experiments as well as after chronic treatment. After a single addition of 3.5 mM heptanol, gap junctions remained blocked for up to 8 hours, then this inhibitory effect decreased gradually, likely because the alcohol was evaporated. Changing heptanol solutions every 8 hours during the time course of L6 differentiation resulted in a lasting drastic inhibition of myoblast fusion. We further investigated the effect of heptanol and of other uncoupling agents on the differentiation of primary cultures of embryonic chicken myoblasts. These cells are transiently coupled by gap junctions before myoblast fusion and prolonged application of heptanol, octanol and 18-β-glycyrrhetinic acid also inhibited their fusion. The effect of heptanol and octanol was neither due to a cytotoxic effect nor to a modification of cell proliferation. Moreover, heptanol treatment did not alter myoblast alignment and adhesion. Taken together these observations suggest that intercellular communication might be a necessary step for myoblast fusion.     

The calcium-dependent myoblast adhesion that precedes cell fusion is mediated by glycoproteins

Presumptive myoblasts from explants of chick embryo pectoral muscle proliferate, differentiate, and fuse to form multinucleate myotubes. One event critical to multinucleate cell formation is the specific adhesion of myoblasts before union of their membranes. In the studies reported here five known inhibitors of myotube formation--trifluoperazine, sodium butyrate, chloroquine, 1,10 phenanthroline, and tunicamycin--were tested for their effect on the Ca++-dependent myoblast adhesion step. The first four inhibitors of myotube formation do not perturb myoblast adhesion but rather block fusion of aggregated cells, which suggests that these agents perturb molecular events required for the union of the lipid bilayers. By contrast, tunicamycin exerts its effect by inhibiting the myoblast adhesion step, thereby blocking myotube formation. The effect of tunicamycin can be blocked by a protease inhibitor, however, which implies that the carbohydrate residues protect the glycoproteins from proteolytic degradation rather than participate directly in cell-cell adhesion. Whereas trypsin treatment of myoblasts in the absence of Ca++ destroys the cells' ability to exhibit Ca++-dependent adhesion, the presence of Ca++ during trypsin treatment inhibits the enzyme's effect, which suggests that myoblast adhesion is mediated by a glycoprotein(s) that has a conformation affected by Ca++. Finally, myoblast adhesion is inhibited by an antiserum raised against fusion-competent myoblasts. The effect of the antiserum is blocked by a fraction from the detergent extract of pectoral muscle that binds to immobilized wheat germ agglutinin, which again suggests that glycoproteins mediate Ca++-dependent myoblast adhesion.     

Prostaglandin binding activity and myoblast fusion in aggregates of avian myoblasts

Myoblast aggregates provide a system for studying cell interactions which have several advantages over standard, stationary cultures. In gyrotory rotation, aggregate size can be controlled and is independent of cell migration. In muscle aggregates, fibroblasts are excluded, yet myoblast differentiation and fusion occur in a highly synchronous fashion. Specific PG binding occurs in chick or quail myoblast aggregates: in chick the peak of binding is at 35-36 hr. Aggregation is complete 16 hr before PG binding activity appears. This suggests either that gyrotory aggregation is not identical to myoblast recognition, or that PG binding activity occurs subsequent to myoblast recognition. Myoblast aggregates begin to release PG before 18 hr. The amount detected remains constant until binding begins at 34 hr when PG binding to the aggregates begins. Thus, both the release of PG and PG receptor activity are characteristics of the myoblasts and release of prostaglandin precedes appearance of the binding activity. As a first step in identifying the PG receptor and determining its appearance on the myoblast cell surface, we have prepared antisera against myoblast surfaces which blocks receptor-ligand interaction and have absorbed it against both peripheral and intrinsic membrane fractions. The results indicate that the PG receptor is a myoblast peripheral membrane macromolecule.