Accumulation Of Myosin Research Articles

N-cadherin Promotes the Commitment and Differentiation of Skeletal Muscle Precursor Cells

Cells with the potential to form skeletal muscle are present in the chick embryo prior to gastrulation. Muscle differentiation begins after gastrulation within the somites. The role of cadherin-mediated adhesion in the commitment and differentiation of skeletal muscle precursor cells was examined by analyzing the expression of cell–cell adhesion molecules in cultures of epiblast, segmental plate, and somite cells and by determining the effects of adhesion-perturbing antibodies on the accumulation of MyoD and sarcomeric myosin. Cultured primitive streak stage epiblast cells downregulate E-cadherin and upregulate N-cadherin. This switch in cadherin expression also occursin vivoas epiblast cells enter the primitive streak. Although MyoD protein is present in cells with N- or E-cadherin, only cells with N-cadherin differentiate into skeletal muscle. In contrast to the primitive streak stage epiblast cells, prestreak epiblast cells maintain the expression of E-cadherinin vitro.While the majority of prestreak cells contain MyoD, only a few synthesize myosin. Treatment of primitive streak stage epiblast cells with function-perturbing antibodies to N-cadherin resulted in an inhibition of myosin accumulation and a decrease in the percentage of cells with MyoD. Segmental plate and somite cells are similar to primitive streak stage epiblast cells in that most differentiated into skeletal muscle when cultured in serum-free medium. While function-perturbing antibodies to N-cadherin inhibited the accumulation of myosin in these mesoderm cells, the number of MyoD positive cells was unaffected in somite cultures and only partially reduced in segmental plate cultures. These results suggest that N-cadherin-mediated cell–cell adhesion is involved in both the commitment of muscle precursors and their terminal differentiation.

Signal transduction and motility of Dictyostelium.

This review is concerned with the roles of cyclic GMP and Ca(2+) ions in signal transduction for chemotaxis of Dictyostelium. These molecules are involved in signalling between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Evidence is presented for uptake and/or efflux of Ca(2+) being regulated by cyclic GMP. The link between Ca(2+), cyclic GMP and chemotactic cell movement has been explored using "streamer F" mutants whose primary defect is in the structural gene for the cyclic GMP-specific phosphodiesterase. This mutation causes the mutants to produce an abnormally prolonged peak of cyclic GMP accumulation in response to stimulation with the chemoattractant cyclic AMP. The production and relay of cyclic AMP signals is normal in these mutants, but certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca(2+) uptake, myosin II association with the cytoskeleton and regulation of both myosin heavy and light chain phosphorylation. These changes can be correlated with changes in the shape of the amoebae after chemotactic stimulation. Other mutants in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses. A model is described in which cyclic GMP (directly or indirectly via Ca(2+) regulates accumulation of myosin II on the cytoskeleton by regulating phosphorylation of the myosin heavy and light chain kinases.

Calcium, cyclic GMP and the control of myosin II during chemotactic signal transduction ofDictyostelium

Evidence is presented for Ca2+ and cyclic GMP being involved in signal transduction between the cell surface cyclic AMP receptors and cytoskeletal myosin II involved in chemotactic cell movement. Ca2+ is shown to be required for chemotactic aggregation of amoebae. The evidence for uptake and/or eflux of this ion being regulated by the nucleotide cyclic GMP is discussed. The connection between Ca2+, cyclic GMP and chemotactic cell movement has been explored using “streamer F” mutants. The primary defect in these mutants is in the structural gene for the cyclic GMP-specific phosphodiesterase which results in the mutants producing an abnormally prolonged peak of accumulation of cyclic GMP in response to stimulation with the chernoattractant cyclic AMP. While events associated with production and relay of cyclic AMP signals are normal, certain events associated with movement are (like the cyclic GMP response) abnormally prolonged in the mutants. These events include Ca2+ uptake, myosin II association with the cytoskeleton and inhibition of myosin heavy and light chain phosphorylation. These changes can be correlated with the amoebae becoming elongated and transiently decreasing their locomotive speed after chemotactic stimulation. Other mutants studied in which the accumulation of cyclic GMP in response to cyclic AMP stimulation was absent produced no myosin II responses. Models are described in which cyclic GMP (directly or indirectly via Ca2+) regulates accumulation of myosin II on the cytoskeleton by inhibiting phosphorylation of the myosin heavy and light chain kinases.

A dominant inhibitory version of the small GTP-binding protein Rac disrupts cytoskeletal structures and inhibits developmental cell shape changes in Drosophila

The Rho subfamily of Ras-related small GTP-binding proteins is involved in regulation of the cytoskeleton. The cytoskeletal changes induced by two members of this subfamily, Rho and Rac, in response to growth factor stimulation, have dramatic effects on cell morphology. We are interested in using Drosophila as a system for studying how such effects participate in development. We have identified two Drosophila genes, DRacA and DRacB, encoding proteins with homology to mammalian Rac1 and Rac2. We have made transgenic flies bearing dominant inhibitory (N17DRacA), and wild-type versions of the DRacA cDNA under control of an Hsp70 promoter. Expression of the N17DRacA transgene during embryonic development causes a high frequency of defects in dorsal closure which are due to disruption of cell shape changes in the lateral epidermis. Embryonic expression of N17DRacA also affects germband retraction and head involution. The epidermal cell shape defects caused by expression of N17DRacA are accompanied by disruption of a localized accumulation of actin and myosin thought to be driving epidermal cell shape change. Thus the Rho subfamily may be generating localized changes in the cytoskeleton during Drosophila development in a similar fashion to that seen in mammalian and yeast cells. The Rho subfamily is likely to be participating in a wide range of developmental processes in Drosophila through its regulation of the cytoskeleton.

Myosin heavy chain synthesis is independently regulated in hypertrophy and atrophy of isolated adult cardiac myocytes.

Hypertrophy of isolated adult feline cardiac muscle cells may be induced in culture by either alpha- or beta-adrenergic agonists. However, it has been shown previously that each of these agonists activate different subsets of immediate-early response genes and have different effects on expression of "fetal" protein isoforms and stimulation of protein synthesis. Moreover, in adult feline heart cells, beta-adrenergic agonists, such as isoproterenol, activate sustained synchronous beating and sarcomeric reorganization while alpha-adrenergic agonists, such as phenylephrine, do not. The objective of the present study was to determine whether these differences in proximal signaling events converged in a common signal pathway during activation of contractile protein synthesis. By direct comparisons of actin and myosin heavy chain (HC) synthesis and accumulation following isoproterenol and phenylephrine, it was determined that both agonists stimulate a coordinated accumulation of these proteins during cardiomyocyte growth. However, each agonist stimulated a very different program of contractile protein synthesis. During phenylephrine-induced hypertrophy, actin and myosin HC syntheses were rapidly and coordinately activated and continuously maintained at rates 10-25% greater than untreated cultures. The pattern of myosin HC synthesis following isoproterenol was very much more complex with periods during which it was as much as 40% greater or 25% less than in control cultures. Furthermore, there was no correlation between rates of actin and myosin HC synthesis following isoproterenol. It was concluded that actin and myosin HC syntheses and accumulation were regulated independently and in a very different manner following isoproterenol or phenylephrine. Since protein accumulation was not correlated with synthesis rates during development of hypertrophy, it was also concluded that post-translational mechanisms played a significant role in the maintenance of contractile protein stoichiometry during beta-adrenergic/beating-induced hypertrophy. Myosin HC synthesis also appeared to be independently regulated during cardiomyocyte atrophy induced by the calcium channel blocker nifedipine. Unlike the case in hypertrophy, however, protein balance was not maintained in nifedipine, and the depression of myosin HC synthesis and loss of myosin HC content were much greater than in the case of other contractile proteins.

Thyrotropin Inhibits the Intrinsic Locomotility of Thyroid Cells Organized as Follicles in Primary Culture

The regulation of cell locomotion is a fundamental determinant of tissue architecture. Even in solid tissues of adult organisms cells often retain an intrinsic locomotor capacity which is activated during wound healing or tumor metastasis. In this study we have examined the role of cell locomotion in an in vitro model of thyroid epithelial pattern generation. Primary cultures of adult porcine thyroid cells reorganize to form follicles within three-dimensional cell aggregates when stimulated by thyrotropin (thyroid-stimulating hormone, TSH). Removal of TSH from the culture medium caused established follicles to reorganize into a confluent, two-dimensional epithelioid monolayer. The earliest observed change was the appearance of spreading cells at the peripheries of aggregates. These cells displayed broad lamellipodia whose formation was associated with the redistribution of microfilaments and microtubules and the accumulation of myosin. Spreading cells could migrate into, and fill, artificial wounds several millimeters wide without evidence of cell proliferation, indicating that cells became locomotile as they spread from follicles to form monolayer. Both spreading and migration were inhibited by cytochalasin B. In contrast, cells spread in the presence of colchine, but failed to migrate subsequently. Thyroid cell locomotility from follicles was inhibited by TSH, a cAMP analog, and a cell-free membrane fraction. However, migration from established monolayer cultures was not affected by these regulatory agents. This indicated that cell spreading was an important regulatory locus in thyroid cell patterning. We conclude that the tonic inhibition of thyroid cell locomotility contributes to the maintenance of follicular architecture in vitro. TSH and cell-cell contact may inhibit locomotion by preventing follicular cells from spreading, the earliest step in the morphogenetic conversion of follicles to monolayer.

Over-expression of CUG- or AUG-initiated Forms of Basic Fibroblast Growth Factor in Cardiac Myocytes Results in Similar Effects on Mitosis and Protein Synthesis but Distinct Nuclear Morphologies

Initiation of translation from alternate codons in the same mRNA results in multiple forms of basic fibroblast growth factor (bFGF). High molecular weight species of bFGF make use of leucine translation initiation sites located upstream of the methionine residue used to produce the 18 kiloDalton (kDa) form. Although the addition of exogenous 18 kDa bFGF is known to stimulate DNA synthesis and proliferation of several cell types including embryonic chicken cardiac myocytes, little is known about the role of high molecular weight forms of bFGF. We modified the rat bFGF cDNA to yield high (22/21.5 kDa) or low (18 kDa) molecular weight species of bFGF. Expression of 22/21.5 kDa or 18 kDa bFGF in transfected embryonic chicken ventricular myocyte culture was confirmed by protein blotting. Expression of both high and low molecular weight species of bFGF was associated with (i) a three-fold increase in overall thymidine incorporation as well as cardiomyocyte labelling index (fraction of cardiomyocyte nuclei incorporating tritiated thymidine); (ii) a two- to three-fold increase in cell number; (iii) an eight-fold increase in protein synthesis; and (iv) a three-fold decrease in myosin accumulation. Subcellular localization of bFGF in the transfected myocytes cultures was also assessed by immunofluorescence microscopy. Over-expression of cDNAs yielding high molecular weight bFGF resulted in predominantly nuclear bFGF staining. By contrast, both cytoplasmic and nuclear staining were observed following over-expression of 18 kDa bFGF. Over-expression of 22/21.5 kDa bFGF was associated with the formation of multiple DNA-containing "clumps" resembling condensed chromatin in cardiac myocyte nuclei. These DNA "clumps" were not observed in cardiac myocyte culture over-expressing 18 kDa bFGF. These data indicate that over-expression of high as well as low molecular weight forms of bFGF can stimulate cardiac myocyte proliferative potential and decrease myosin accumulation. However, these forms possess distinct subcellular localizations and can have different biological functions in the nucleus.

Expression and localization of villin, fimbrin, and myosin I in differentiating mouse F9 teratocarcinoma cells

F9 embryonic carcinoma cells are a multipotent cell line which can be induced to differentiate into cells resembling the visceral endoderm, an extraembryonic absorptive epithelium characterized by apical microvilli. We have examined the role of villin, fimbrin, and myosin I, the major actin-binding proteins in the intestinal and visceral yolk sac microvilli, in the development of epithelial polarity and the assembly of the microvillus cytoskeleton in differentiating F9 cells. By immunoblot analysis villin was first detected at 4 days of differentiation. Confocal microscopy localized villin at Day 4 to the apical surface and by Day 6 to the basolateral surfaces as well. In comparison, fimbrin and myosin I were both present in undifferentiated F9 cells and became associated with the apical surface after villin during differentiation to visceral endoderm. The accumulation of villin, fimbrin, and myosin I at the apical surface in differentiating F9 cells correlated with the appearance of microvilli containing organized actin filament bundles. Two mouse villin cDNAs were isolated and characterized to examine villin expression during F9 differentiation. Mouse villin was encoded by two transcripts (3.8 and 3.4 kb) which differ in their 3′-noncoding region. Both villin mRNAs were first detected by Day 4 of differentiation and their appearance coincided with expression of the visceral endoderm marker α-fetoprotein. The pattern of expression and order of accumulation of villin, fimbrin, and myosin I in differentiating F9 cells are common to developing gut and yolk sac epithelium. This suggests that microvillus assembly is directed by a sequence of temporally and spatially regulated localizations of these actin-binding proteins.

A large accumulation of non-muscle myosin occurs at first entry into M phase in rat vascular smooth-muscle cells

Vascular smooth-muscle cells (VSMCs) from rat aortae contained very little non-muscle myosin heavy chain (MHC) immediately after dispersal, and the protein did not accumulate if the cells were held in G0/G1 phase by withholding serum or were held in first S phase by the addition of bromodeoxyuridine (BrdU). However, non-muscle MHC accumulated by greater than 20-fold per cell during first M phase, when over 80% of the cells divided between 48 h and 72 h after addition of serum. Delaying the addition of serum caused a delay in the accumulation of the non-muscle MHC until the cells subsequently entered M phase. If the cells were held in M phase at the metaphase/anaphase boundary by nocadazole, the accumulation of non-muscle myosin still occurred, although division was blocked. When the cells were pulse-labelled with [35S]methionine, it was found that non-muscle MHC was one of the major proteins being made and that its synthesis occurred at similar rates throughout the cell cycle. This implied that the rate of degradation of the protein before first M phase was much faster than in M phase, when the protein accumulated rapidly. This was confirmed by direct measurements of the rate at which [35S]methionine-labelled non-muscle MHC disappeared from the cells, which gave a half-life for the protein of about 8 h before M phase but about 5 days in post-mitotic cells, i.e. an increase of approx. 15-fold. These data are consistent with the hypothesis that there is a mechanism in VSMCs which shortens the half-life of the protein before first M phase and that the accumulation of non-muscle MHC which results from the increase in half-life at first M phase may be necessary for division of these cells.

Expression of slow and fast myosin heavy chains in overloaded muscles of the developing rat

The present study examines the developmental accumulation of slow myosin heavy chain in the extensor digitorum longus, soleus and plantaris muscles of rats after early post-natal imposition of mechanical overload by removal of synergistic muscles. The proportions of slow and fast myosin heavy chain were measured in each muscle by ELISA. Fibres expressing slow myosin were examined immunocytochemically using a monoclonal antibody specific for slow MHC. Between 30 and 60 days of age, MHC increases by 15% (p less than 0.001) in the soleus and by 27% (p less than 0.001) in the plantaris of normally developing, unoperated animals. The effect of overload on the soleus and plantaris is to accelerate the rate of increase in slow MHC accumulation so that levels are respectively 16 and 39% higher than controls by 30 days of age (p less than 0.001). By 60 days, the control soleus and plantaris attain levels of slow MHC roughly equivalent to their overloaded counterparts. In overloaded plantaris the increase in levels of slow myosin does not occur at the expense of fast myosin expression. In the EDL there is a normal developmentally regulated decrease in slow MHC accumulation, reflected by a 40% decrease in levels of slow MHC (p less than 0.0001) and a 50% decrease in the number of slow fibres (p less than 0.001), between 30 days and 20 weeks of age. This elimination of slow myosin accumulation in the EDL is unimpeded by chronic overload. Thus, muscles react to mechanical overload in a tissue specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)

First Entry into M Phase Causes a Large Accumulation of Non-Muscle Myosin in Rat Vascular Smooth Muscle Cells

First Entry into M Phase Causes a Large Accumulation of Non-Muscle Myosin in Rat Vascular Smooth Muscle Cells

Phalloidin‐induced accumulation of myosin in rat hepatocytes is caused by suppression of autolysosome formation

Administration of phalloidin in vivo to rats causes marked changes in the distribution of actin and myosin in hepatocytes, which accompanies reduced bile flow. We have found that in hepatocytes treated with phalloidin for 3 and 7 days, cellular myosin content increased about 1.5-fold and 4.7-fold, respectively. In addition, total cell protein content and several marker enzyme activities were also elevated by 30-120% depending on the duration of phalloidin treatment. These observations allow us to speculate that phalloidin somehow elicits inhibition of cellular protein degradation, which results in the increase of these protein levels. To examine this possibility further, we analyzed leupeptin-induced density shift of phagolysosomes. In normal liver, the injection of leupeptin/E64c caused an increase in the density of both heterolysosomes and autolysosomes, due to retarded digestion of sequestered proteins as a result of the inhibition of lysosomal cathepsins. Accumulation, in these denser autolysosomes, of lactic dehydrogenase, pyruvate kinase, aldolase, and myosin was demonstrated by enzyme assays and immunoblot analysis. In the phalloidin-treated liver, the increase in the density of autolysosomes and the accumulation of above cytoplasmic enzymes were markedly inhibited. However, phalloidin did not affect the shift in the density of heterolysosomes. From these data, we concluded that autolysosome formation was specifically hindered in phalloidin-treated rat hepatocytes, which results in the reduction of autophagic protein degradation and eventual increase in intracellular protein levels.

Generation of a stable, posttranslationally modified microtubule array is an early event in myogenic differentiation.

Microtubules (MTs) have been implicated to function in the change of cell shape and intracellular organization that occurs during myogenesis. However, the mechanism by which MTs are involved in these morphogenetic events is unclear. As a first step in elucidating the role of MTs in myogenesis, we have examined the accumulation and subcellular distribution of posttranslationally modified forms of tubulin in differentiating rat L6 muscle cells, using antibodies specific for tyrosinated (Tyr), detyrosinated (Glu), and acetylated (Ac) tubulin. Both Glu and Ac tubulin are components of stable MTs, whereas Tyr tubulin is the predominant constituent of dynamic MTs. In proliferating L6 myoblasts, as in other types of proliferating cells, the level of Glu tubulin was very low when compared with the level of Tyr tubulin. However, when we shifted proliferating L6 cells to differentiation media, we observed a rapid accumulation of Glu tubulin in cellular MTs. By immunofluorescence, the increase in Glu tubulin was first detected in MTs of prefusion myoblasts and was specifically localized to MTs that were associated with elongating portions of the cell. MTs in the multinucleated myotubes observed at later stages of differentiation maintained the elevated level of Glu tubulin that was observed in the prefusion myoblasts. When cells at early stages of differentiation (less than 1 d after switching the culture medium) were immunostained for Glu tubulin and the muscle-specific marker, muscle myosin, we found that the increase in Glu tubulin preceded the accumulation of muscle myosin. Thus, the elaboration of Glu MTs is one of the very early events in myogenesis. Ac tubulin also increased during L6 myogenesis; however, the increase in acetylation occurred later in myogenesis, after fusion had already occurred. Because detyrosination was temporally correlated with early events of myogenesis, we examined the mechanism responsible for the accumulation of Glu tubulin in the MTs of prefusion myoblasts. We found that an increase in the stability of L6 cell MTs occurred at the onset of differentiation, suggesting that the early increase in detyrosination that we observed is a manifestation of a decrease in MT dynamics in elongating myoblasts. We conclude that the establishment of an oriented array of microtubules heightened in its stability and its level of posttranslationally modified subunits may be involved in the subcellular remodeling that occurs during myogenesis.

Fibroblast growth factor is stored in fiber extracellular matrix and plays a role in regulating muscle hypertrophy

A polyclonal antibody specific to bovine basic fibroblast growth factor (bFGF) localizes bFGF in the extracellular matrix of rat skeletal muscle fibers, where it is bound by the heparin-rich basal lamina. FGF accumulation appears to be augmented in hypertrophied compared to control fibers. In addition, FGF-like growth activity can be isolated from hypertrophied muscle. This growth activity binds to heparin and can be purified by heparin-affinity chromatography. When assayed in a chick myogenic cell culture system, the purified rat growth activity stimulates myobenic cell replication and stimulates myosin accumulation and synthesis. The function, in vivo, of basal lamina-localized FGF in rat muscle is not known, but it is most probably related to increased satellite cell replication seen during normal fiber maturation, during work-induced hypertrophy, and during regeneration. During hypertrophy, we observe the appearance of new fibers in addition to growth of preexisting fibers. It is hypothesized that muscle hypertrophy involves activation of satellite cells which may then fuse with existing fibers and, in addition, also form new fibers.

Adrenocorticotropin is a specific mitogen for mammalian myogenic cells

Peptides derived from proopiomelanocortin (POMC) have been found to stimulate the proliferation of murine myogenic cells. Among these peptides, adrenocorticotropin (ACTH) and alpha-, beta-, and gamma-melanocyte-stimulating hormones (MSH) were found to be active, whereas the opioid peptides were not. At clonal density, both ACTH and MSH caused a three- to fourfold increase in the average number of cells per clone in myogenic but not in fibroblast colonies. At high cell density, ACTH and MSH caused a three- to fourfold increase in proliferation of myogenic cells, reflected by an increased accumulation of skeletal myosin. On the other hand mouse embryo skin or muscle fibroblasts or vertebral chondroblasts did not increase proliferation in response to POMC-derived peptides. The half-maximal dose at which ACTH stimulated myoblast proliferation was around 5 nM, and the mitogenic effect was doubled by suboptimal doses of fibroblast growth factor. The possible physiological significance of the mitogenic effect of ACTH on myogenic cells is discussed.

Heparin inhibits skeletal muscle growth in vitro

Heparin or heparan sulfate proteoglycan (HeSPG), but not chondroitin sulfate or hyaluronic acid, exerts a pronounced inhibitory effect on muscle growth in vitro, as determined by total protein, myosin accumulation or synthesis, and [ 3H]thymidine incorporation studies. Primary muscle fibroblast culture growth is also inhibited by heparin but to a substantially lesser degree compared to muscle (30% and over 90% inhibition of growth, respectively). Heparin-induced inhibition of skeletal muscle growth is a consequence of its interaction with a growth factor(s) present in the media used to support myogenesis; heparin-Sepharose column absorbed horse serum can support muscle growth only in the presence of added heparin-binding growth factors like fibroblast growth factor (FGF) or chicken muscle growth factor (CMGF). Furthermore, heparin prevents the binding of iodinated FGF to the myoblast surface. We also show that the extent of muscle growth is a function of the relative amounts of heparin and FGF in culture. Finally, we provide evidence indicating that FGF can combine with endogenously occurring heparin-like components: immobilized FGF binds sodium-[ 35S]sulfate labeled components secreted in muscle culture conditioned medium, an interaction inhibited by anti-HeSPG antibodies or heparin, but not by other sulfated glycosaminoglycans. Since heparin binding growth factors not only stimulate myoblast proliferation but also actively inhibit the onset of muscle differentiation (G. Spitzz, D. Roman, and A. Strauss 1986. J. Biol. Chem. 261, 9483–9488), their interaction with naturally occurring heparin-like components may be an important physiological mechanism for modulating muscle growth and differentiation in development and regeneration.

Entry of Shigella flexneri into HeLa cells: evidence for directed phagocytosis involving actin polymerization and myosin accumulation.

The enteroinvasive bacterium Shigella flexneri expresses a plasmid-mediated capacity to penetrate into nonphagocytic cells. By using 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin), a fluorescent dye which specifically stains microfilaments, we observed condensations of filamentous actin underneath the plasma membrane of HeLa cells which interacted with the invasive isolate M90T. As demonstrated by indirect immunofluorescence with the antimyosin monoclonal antibody CC-212, myosin accumulated at the same sites. The entry process could be synchronized by using strain SC301, a pIL22 transformant of M90T. pIL22, a recombinant plasmid encoding the Escherichia coli afimbrial adhesin AFA I, rendered shigellae highly adherent to HeLa cells. Using such a system, we demonstrated that the occurrence of bacterial penetration and the appearance of structures brightly stained by NBD-phallacidin were simultaneous events. Such microfilamentous structures resulted from de novo polymerization of the monomeric actin pool in a DNase I inhibition assay, as shown by measurement of the monomeric versus total actin content of infected HeLa cells. These data provide direct evidence that the penetration of S. flexneri into HeLa cells occurs through a mechanism similar to phagocytosis by professional phagocytes.

Mutations of the Drosophila myosin heavy-chain gene: effects on transcription, myosin accumulation, and muscle function.

Mutations of the myosin heavy-chain (MHC) gene of Drosophila melanogaster were identified among a group of dominant flightless and recessive lethal mutants (map position 2-52, 36A8-B1,2). One mutation is a 0.1-kilobase deletion in the 5' region of the MHC gene and reduces MHC protein in the leg and thoracic muscles of heterozygotes to levels found in 36AC haploids. Three mutations are insertions of 8-to 10-kilobase DNA elements within the MHC gene and produce truncated MHC transcripts. Heterozygotes of these insertional mutations possess levels of MHC intermediate between those of haploids and diploids. An additional mutation has no gross alteration of the MHC gene or its RNA transcripts. Although leg and larval muscles function normally in each mutant heterozygote, indirect flight muscles are defective and possess disorganized myofibrils. Homozygous mutants die during embryonic or larval development and display abnormal muscle function prior to death. These findings provide direct genetic evidence that the MHC gene at 36B (2L) is essential for both larval and adult muscle development and function. The results are consistent with the previous molecular evidence that Drosophila, unlike other organisms, has only a single muscle MHC gene per haploid genome. Quantitative expression of both copies of the MHC gene is required for function of indirect flight muscle, whereas expression of a single MHC gene is sufficient for function of larval muscles and adult tubular muscles.

Myogenic growth factor present in skeletal muscle is purified by heparin-affinity chromatography.

A myogenic growth factor has been purified from a skeletal muscle, the anterior latissimus dorsi, of adult chickens. In the range of 1-10 ng, this factor stimulates DNA synthesis as well as protein and muscle-specific myosin accumulation in myogenic cell cultures. Purification is achieved through binding of the factor to heparin. The factor is distinct from transferrin and works synergistically with transferrin in stimulating myogenesis in vitro.

Insulin receptor capping and its correlation with calmodulin-dependent myosin light chain kinase.

Both fluorescence microscopy and fluorometric analysis techniques have been used to characterize insulin receptor capping in IM-9 human lymphoblastoid cells. Morphologically, insulin caps appear similar to lectin or antiimmunoglobulin-induced caps displaying a preferential accumulation of actin, myosin, and actin-binding protein directly underneath the cap structure. Using the fluorescent calcium indicator quin2 we have detected no change in the calcium activity following insulin stimulation. However, in the presence of a number of calmodulin inhibitors, such as W-5, W-7, W-12, and trifluoperazine (TFP), insulin capping is significantly inhibited, which implies that a calmodulin-regulated process is involved. Using double immunofluorescence microscopy, we have found that the calmodulin-dependent myosin light chain kinase (MLCK) is concentrated directly beneath insulin caps. Upon treatment with trifluoperazine (TFP), the redistribution of both MLCK and insulin receptors are inhibited concomitantly. Our data indicate that the calmodulin-dependent myosin light chain kinase may be directly responsible for the activation of actomyosin-mediated contractility during insulin receptor capping.