Spontaneous myoblast fusion is mediated by cell surface Ca-dependent protein kinase(s)

Mononucleated myoblasts divide in vitro until they attain confluency and fuse, forming multinucleated myotubes. Fusion is an extracellular Ca2+-dependent process. We used for our studies an established line of skeletal myoblasts (L6) as well as a non-fusing Myo- alpha-amanitin-resistant mutant of this line (Ama102). Our results show that extracellular calcium at concentrations which elicit myoblast fusion activates the phosphorylation of a protein species of 48 kD, present at the surface of mononucleated myoblasts of the fusing wild type (L6). At fusion, as the cells become independent of the extracellular calcium concentration for their further differentiation, this activation can no longer be observed. In fusion inhibition experiments, where we used lowered calcium levels, the phosphorylation of the 48 kD protein band is clearly decreased. When the myoblasts are fed with standard medium, they fuse rapidly and the phosphorylation of the 48 kD species is markedly increased. The above-described phenomenon takes place at the cell surface and is completed in a short time. The use of Myo- mutant showed that it is developmentally regulated. In view of our results, it is reasonable to postulate that Ca2+-activated phosphorylation of the cell surface could be on the basis of spontaneous myoblast fusion.     

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.     

PKC-induced sensitization of Ca2+-dependent exocytosis is mediated by reducing the Ca2+ cooperativity in pituitary gonadotropes

The highly cooperative nature of Ca2+-dependent exocytosis is very important for the precise regulation of transmitter release. It is not known whether the number of binding sites on the Ca2+ sensor can be modulated or not. We have previously reported that protein kinase C (PKC) activation sensitizes the Ca2+ sensor for exocytosis in pituitary gonadotropes. To further unravel the underlying mechanism of how the Ca2+ sensor is modulated by protein phosphorylation, we have performed kinetic modeling of the exocytotic burst and investigated how the kinetic parameters of Ca2+-triggered fusion are affected by PKC activation. We propose that PKC sensitizes exocytosis by reducing the number of calcium binding sites on the Ca2+ sensor (from three to two) without significantly altering the Ca2+-binding kinetics. The reduction in the number of Ca2+-binding steps lowers the threshold for release and up-regulates release of fusion-competent vesicles distant from Ca2+ channels.     

Antisocial, an intracellular adaptor protein, is required for myoblast fusion in Drosophila.

Somatic muscle formation in Drosophila requires fusion of muscle founder cells with fusion-competent myoblasts. In a genetic screen for genes that control muscle development, we identified antisocial (ants), a gene that encodes an ankyrin repeat-, TPR repeat-, and RING finger-containing protein, required for myoblast fusion. In ants mutant embryos, founder cells and fusion-competent myoblasts are properly specified and patterned, but they are unable to form myotubes. ANTS, which is expressed specifically in founder cells, interacts with the cytoplasmic domain of Dumbfounded, a founder cell transmembrane receptor, and with Myoblast city, a cytoskeletal protein, both of which are also required for myoblast fusion. These findings suggest that ANTS functions as an intracellular adaptor protein that relays signals from Dumbfounded to the cytoskeleton during myoblast fusion.     

Opposing effects of calcium entry and phorbol esters on fusion of chick muscle cells

Studies utilizing cultured muscle cells have shown that myoblast fusion requires extracellular Ca2+ and involves transient coordinated changes in cell membrane topography and cytoskeletal organization. However, neither the mechanisms by which Ca2+ influences these changes nor its cellular sites of action are known. We have investigated the effects of Ca2+ channel modulators and phorbol esters on fusion of embryonic chick myoblasts in culture. Myoblast fusion was inhibited by the Ca2+ channel blockers D600 and nitrendipine and stimulated by the Ca2+ channel activator Bay K 8644. We have obtained evidence that the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits fusion through activation of protein kinase C. Myoblasts prevented from fusing by Ca2+ channel blockers or TPA display a distinctive elongated morphology that is characteristic of cells prevented from fusion by Ca2+ deprivation. The inhibition of fusion by D600 and TPA is significantly diminished in the presence of the Ca2+ ionophore A23187. TPA arrest of myoblast fusion was found to be accompanied by an increase in phosphorylation of the 20-kDa light chain of cytoplasmic myosin in a dose- and time-dependent manner. The effects of TPA on myoblast fusion and phosphorylation of myosin light chain were mimicked by the cell permeant diacylglycerol sn-1,2-dioctanoylglycerol, a potent activator of protein kinase C. The present results suggest that activators of protein kinase C block fusion by interfering with a Ca2+ signal transduction pathway and that this interference may be associated with a protein kinase C catalyzed inhibitory phosphorylation of myosin light chain.     

The immunoglobulin-like protein Hibris functions as a dose-dependent regulator of myoblast fusion and is differentially controlled by Ras and Notch signaling.

Hibris (Hbs) is a transmembrane immunoglobulin-like protein that shows extensive homology to Drosophila Sticks and stones (Sns) and human kidney protein Nephrin. Hbs is expressed in embryonic visceral, somatic and pharyngeal mesoderm among other tissues. In the somatic mesoderm, Hbs is restricted to fusion competent myoblasts and is regulated by Notch and Ras signaling pathways. Embryos that lack or overexpress hbs show a partial block of myoblast fusion, followed by abnormal muscle morphogenesis. Abnormalities in visceral mesoderm are also observed. In vivo mapping of functional domains suggests that the intracellular domain mediates Hbs activity. Hbs and its paralog, Sns, co-localize at the cell membrane of fusion-competent myoblasts. The two proteins act antagonistically: loss of sns dominantly suppresses the hbs myoblast fusion and visceral mesoderm phenotypes, and enhances Hbs overexpression phenotypes. Data from a P-homed enhancer reporter into hbs and co-localization studies with Sns suggest that hbs is not continuously expressed in all fusion-competent myoblasts during the fusion process. We propose that the temporal pattern of hbs expression within fusion-competent myoblasts may reflect previously undescribed functional differences within this myoblast population.     

Comparison of Ca2+ -dependent phosphorylation in viable dispersed brain cells with calmodulin-dependent protein kinase activity in cell-free preparations of rat brain.

Using two depolarizing agents, veratrine and high concentrations of extracellular KCl, we studied depolarization-stimulated phosphorylations in 32P-labelled dispersed brain tissue in order to identify phosphoprotein substrates for Ca2+ - and calmodulin-dependent protein kinase activity at the cellular level, for comparison with findings in cell-free preparations. In intact brain cells, the only prominent depolarization-stimulated phosphorylation was a 77 kDa protein separated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. This phosphorylation was dependent on external Ca2+, since chelation of Ca2+ in media with 6 mM-EGTA or the presence of verapamil (a Ca2+ -channel blocker) in the incubation media inhibited depolarization-stimulated phosphorylation of the 77 kDa protein. Phosphorylation of the 77 kDa protein also appeared to be dependent on calmodulin, because depolarization-stimulated phosphorylation was significantly decreased (P less than 0.05) when 100 microM-trifluoperazine was present in the incubation media. Polymyxin B, an inhibitor of Ca2+- and phospholipid-dependent phosphorylation, and 12-O-tetradecanoylphorbol 13-acetate, the phorbol ester enhancing Ca2+- and phospholipid-dependent phosphorylation, had no effect on the phosphorylation of the 77 kDa protein. The 77 kDa phosphoprotein was identified as a protein previously named synapsin I [Ueda, Maeno & Greengard (1973) J. Biol. Chem 248, 8295-8305] on the basis of similar migration of native and proteolytic fragments of the 77 kDa protein with those of authentic synapsin I on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Whereas several studies with cell-free preparations showed that 57 kDa and 54 kDa endogenous phosphoproteins were the most prominent species phosphorylated in a Ca2+ and calmodulin-dependent manner, these results indicate that synapsin is the most prominent Ca2+-and calmodulin-dependent phosphorylation in intact cells. The phosphorylations of 54 kDa and 57 kDa proteins may not be as important in vivo, but instead occur as a result of the disruption of cellular integrity inherent in preparation of cell-free subfractions of brain tissue.     

Fusion-inhibiting monoclonal antibodies and their relevant antigens in relation to sexual process of Dictyostelium discoideum

Sexual cell fusion occurs between NC4 and HM1, the heterothallic strains in Dictyostelium discoideum. Cells of these strains are fusion incompetent when cultured on agar plates in the light and become fusion competent upon cultivation in a liquid medium in darkness. Two cell-surface components, gp70 and gp138, have been identified and characterized as being relevant to sexual cell fusion. Both are glycoproteins, and the former is detected only in fusion-competent HM1 cells, while the latter is detected both in fusion-competent HM1 and fusion-competent NC4 cells. We therefore suspect gp 70 to be responsible for cell recognition and gp138, for membrane fusion. Therefore, NC4 cells are expected to possess specific surface molecule(s) that can be recognized by HM1 cells. In the present study, we raised monoclonal antibodies (mAbs) against membrane fractions of NC4 cells and selected fusion-inhibiting mAbs to identify novel molecules related to sexual cell fusion in D. discoideum. Out of the five mAbs we obtained three, DE1, GG6, and HH9, were characterized. DE1 recognized antigens that specifically existed in fusion-competent NC4 cells but not in fusion-incompetent NC4 or HM1 cells. GG6 recognized cell-surface proteins with approximate molecular weights of 125 and 32 kDa in both fusion-competent NC4 and fusion-competent HM1 cells. In addition GG6 also recognised other proteins commonly present in fusion-incompetent cells. The 125 kDa protein appeared to be the same as gp138. The epitope recognized by HH9 was sodium dodecyl sulfate (SDS)-sensitive.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Myoblast determination in the somatic and visceral mesoderm depends on Notch signalling as well as on milliways(mili(Alk)) as receptor for Jeb signalling

The visceral muscles of the Drosophila midgut consist of syncytia and arise by fusion of founder and fusion-competent myoblasts, as described for the somatic muscles. A single-step fusion results in the formation of binucleate circular midgut muscles, whereas a multiple-step fusion process produces the longitudinal muscles. A prerequisite for muscle fusion is the establishment of myoblast diversity in the mesoderm prior to the fusion process itself. We provide evidence for a role of Notch signalling during establishment of the different cell types in the visceral mesoderm, demonstrating that the basic mechanism underlying the segregation of somatic muscle founder cells is also conserved during visceral founder cell determination. Searching for genes involved in the determination and differentiation of the different visceral cell types, we identified two independent mutations causing loss of visceral midgut muscles. In both of these mutants visceral muscle founder cells are missing and the visceral mesoderm consists of fusion-competent myoblasts only. Thus, no fusion occurs resulting in a complete disruption of visceral myogenesis. Subsequent characterisation of the mutations revealed that they are novel alleles of jelly belly (jeb) and the Drosophila Alk homologue named milliways (mili(Alk)). We show that the process of founder cell determination in the visceral mesoderm depends on Jeb signalling via the Milliways/Alk receptor. Moreover, we demonstrate that in the somatic mesoderm determination of the opposite cell type, the fusion-competent myoblasts, also depends on Jeb and Alk, revealing different roles for Jeb signalling in specifying myoblast diversity. This novel mechanism uncovers a crosstalk between somatic and visceral mesoderm leading not only to the determination of different cell types but also maintains the separation of mesodermal tissues, the somatic and splanchnic mesoderm.     

Blown fuse regulates stretching and outgrowth but not myoblast fusion of the circular visceral muscles in Drosophila

Circular visceral muscles of Drosophila are binuclear syncytia arising from fusion of two different kinds of myoblasts: a circular visceral founder cell and one visceral fusion-competent myoblast. In contrast to fusion leading to the somatic body-wall musculature, myoblast fusion for the circular visceral muscles does not result in massive syncytia but instead in syncytia interconnected with multiple cytoplasmic bridges, which differentiate into large web-shaped muscles. Here, we show that these syncytial circular visceral muscles build a gut-enclosing network with the interwoven longitudinal visceral muscles. At the ultrastructural level, during circular visceral myoblast fusion and the first step of somatic myoblast fusion prefusion complexes and electron-dense plaques were not detectable which was surprising as these structures are characteristic for the second step of somatic myoblast fusion. Moreover, we demonstrate that Blown fuse (Blow), a cytoplasmic protein essential for the second step of somatic myoblast fusion, plays a different role in circular visceral myogenesis. Blow is known to be essential for progression beyond the prefusion complex in the somatic mesoderm; however, analysis of blow mutants established that it has a restricted role in stretching and outgrowth of the syncytia in the circular visceral muscles. Furthermore, we also found that in the visceral mesoderm, Blow is expressed in both the fusion-competent myoblasts and circular visceral founders, while expression in the somatic mesoderm is initially restricted to fusion-competent myoblasts. We also demonstrate that different enhancer elements in the first intron of blow are responsible for this distinct expression pattern. Thus, we propose a model for Blow in which this protein is involved in at least two clearly differing processes during Drosophila muscle formation, namely somatic myoblast fusion on the one hand and stretching and outgrowth of circular visceral muscles on the other.     

Role of tyrosine phosphorylation of phospholipase C gamma1 in the signaling pathway of HMG-CoA reductase inhibitor-induced cell death of L6 myoblasts

Our previous studies have shown that the HMG-CoA reductase (HCR) inhibitor (HCRI), simvastatin, kills L6 myoblasts by involving Ca2+ mobilization from the Ca2+ pool in the cells but not by influx from extracellular space. More recently, we found that HCRI induced tyrosine phosphorylation of several cellular proteins, followed by apoptotic cell death of L6 myoblasts. The present study was aimed to elucidate the molecular target(s) of these tyrosine phosphorylations induced by HCRI and demonstrated that simvastatin induces tyrosine phosphorylation of phospholipase C (PLC) gamma1. This tyrosine phosphorylation of PLC-gamma1 caused the increment of the intracellular inositol triphosphate (IP3) levels in L6 myoblasts. Pretreatment of the cells with herbimycin A, a specific inhibitor of protein tyrosine kinase, inhibited a simvastatin-induced increase in IP3 level in the cells as well as tyrosine phosphorylation of PLC-gamma1. Interestingly, pretreatment of the cells with U-73122, a specific inhibitor of PLC, prevented simvastatin-induced cell death. Thus, these results strongly suggest that simvastatin-induced tyrosine phosphorylation of PLC-gamma1 plays, at least in part, an important role for the development of simvastatin-induced cell death.     

Role of Ca2+ and Ca2+-activated protease in myoblast fusion

In this report, we have examined the effects of a calcium chelator, EGTA, and a calcium ionophore, A23187, on fusion of a cloned muscle cell line, L6. Our results confirm that EGTA essentially blocks all myoblast fusion because the lateral alignment of presumptive myoblasts cannot occur in the absence of extracellular calcium. A23187, however, promotes the precocious fusion of myoblasts, apparently by facilitating Ca2+ transport into myoblasts. We have also demonstrated that a Ca2+-activated protease, CAF (mM), appears to relocate in response to the Ca2+ flux, changing from a random, dispersed distribution in proliferative myoblasts to a predominantly peripheral distribution in prefusion myoblasts. Coincident with the mM CAF relocation is an altered distribution of a surface glycoprotein, fibronectin. Extracellular fibronectin is seen in abundance in proliferating myoblasts, but is essentially absent from the surface of fusing myoblasts. We suggest that mM CAF when activated by Ca2+ influx may act to promote the release of fibronectin from the myoblast cell surface, thus providing a mechanism by which the membrane of the fusing myoblast may be rearranged to accommodate fusion.     

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.     

Drosophila dumbfounded: a myoblast attractant essential for fusion

Aggregation and fusion of myoblasts to form myotubes is essential for myogenesis in many organisms. In Drosophila the formation of syncytial myotubes is seeded by founder myoblasts. Founders fuse with clusters of fusion-competent myoblasts. Here we identify the gene dumbfounded (duf) and show that it is required for myoblast aggregation and fusion. duf encodes a member of the immunoglobulin superfamily of proteins that is an attractant for fusion-competent myoblasts. It is expressed by founder cells and serves to attract clusters of myoblasts from which myotubes form by fusion.     

Ca2+/calmodulin-dependent phosphorylation of the 100-kDa protein in chick embryonic muscle cells in culture.

The pattern of protein phosphorylation was found to change in differentiating chick embryonic myoblasts in culture. The extent of phosphorylation of 42-, 50-, and 100-kDa proteins increased while that of a 63-kDa protein declined in extracts of myoblasts that had been cultured for increasing periods. Of these, the increase in phosphorylation of the 100-kDa protein occurred most dramatically in extracts of myoblasts in an early stage of differentiation and was specifically inhibited by trifluoperazine (TFP) and other calmodulin (CaM) antagonists including chlorpromazine and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). Treatment of increasing concentrations of TFP to culture medium also decreased the phosphorylation state of the 100-kDa protein and the degree of myoblast fusion in parallel. In addition, levels of both the kinase activity and the 100-kDa protein but not of CaM appeared to rise in the cells cultured for longer periods. These results suggest that (1) a Ca2+/CaM-dependent protein kinase is responsible for phosphorylation of the 100-kDa protein, (2) the TFP-mediated myoblast fusion block may be associated with the inhibitory effect of the drug against the kinase activity, and (3) the increase in phosphorylation state of the 100-kDa protein during myogenic differentiation is due to the rise in levels of the kinase and its substrate.     

A membrane protein with possible relevance to sexual cell fusion in Dictyostelium discoideum

The molecular mechanism of sexual cell fusion in Dictyostelium discoideum was studied using the heterothallic strains HM1 and NC4. Monovalent antibodies (Fab) prepared from rabbit antiserum against a crude membrane preparation of fusion-competent HM1 cells inhibited fusion between HM1 and NC4 cells. Six out of 43 antigenic proteins were found in fusion-competent HM1 cells but not in fusion-incompetent cells. Among them, only one protein with a molecular mass of 70 kDa was able to neutralize the fusion-inhibiting activity of Fab, suggesting its possible participation in sexual cell fusion.