Chick Embryonic Muscle Research Articles

Partial purification of a ribonucleic acid cAMP-independent protein kinase from embryonic chicken muscle.

A cAMP-indepedent protein kinase (P38 kinase) from embryonic chicken muscle with ability to phosphorylate a 38,000 molecular weight polypeptide and to bind to RNAs has been further characterized. An approximately 2000-fold purification of this enzyme was achieved by a combination of affinity and ion-exchange chromatography. Our studies indicate that this protein kinase can not phosphorylate the small subunit of rabbit reticulocyte initiation factor eIF-2 in the presence of its normal endogenous substrate, nor is it activated over a wide range of concentrations of double-stranded RNA. This P38 kinase is, therefore, distinct from the hemin-regulated translational inhibitor of protein synthesis in rabbit reticulocytes and from the interferon-induced protein kinase identified In several systems.

Similarity of Acetylcholine Receptors of Denervated, Innervated and Embryonic Chicken Muscles

The native (9-S) form of the acetylcholine receptor, purified from chick embryonic or adult innervated or denervated chicken muscles, was shown in each case to contain three subunits of Mr about 40000 (predominant), 50000 and 54000. The 40000-Mr subunit (termed alpha) appears to exist in two forms; the minor variant form of it, with Mr about 41000, may have a different carbohydrate content. Bromo[3H]acetylcholine, an affinity alkylating reagent for this receptor, labels the 40000-Mr subunit and the accompanying 41000-Mr variant. The apparent ratio of the subunits varies with the several methods of detection used (protein staining, radio-iodination or tritiation) but the alpha subunit always predominates. The alpha subunit was separated in urea gel isoelectric focusing; its isoelectric point was identical there for the three receptors from innervated, denervated and embryonic muscles. Their respective peptide maps for alpha subunit were identical, and likewise for the 54000-Mr subunit. All of the evidence obtained shows that the subunit structures of the junctional, extra-junctional and embryonic chick muscle receptors are identical. Although the alpha subunit in the receptor from Torpedo electric organ, cat muscle and chicken muscles is distinguishable in all three cases in dodecylsulphate gel electrophoresis, it is affinity-labelled in each case. The peptide map of this subunit after limited proteolysis is also very similar in all three cases. It is concluded that the alpha subunits from these three diverse sources are closely related structurally.

Cloning, partial sequencing, and expression of glyceraldehyde-3-phosphate dehydrogenase gene in chick embryonic heart muscle cells.

Two recombinant plasmids containing structural gene sequences of chick embryonic heart glyceraldehyde-3-phosphate dehydrogenase (GAP dehydrogenase) were constructed and characterized. The plasmids pGAP 30 and pGAP 36 have inserts of 1200 and 950 base pairs, respectively. The identity of the clones was established by hybrid-arrested and hybrid-selection translation assays, and by immunoprecipitation of hybrid-selected translation product with GaP dehydrogenase antiserum. Hybridization of labeled pGAP 30 DNA to size-fractionated chick heart poly(A) RNA occurred at the region on the gel corresponding to the mobility of GAP dehydrogenase mRNA. Base sequence analysis of plasmid pGAP 30 and the comparison of the amino acid sequence derived from it with that of pig muscle GAP dehydrogenase revealed that the amino acid sequence of GAP dehydrogenase is strictly conserved between the chick and pig muscle tissues. Expression of GAP dehydrogenase mRNA in developing chick heart cells in cultures was monitored by in situ hybridization. The GAP dehydrogenase mRNA was present in 5-h-old dividing myoblasts, in contrast to mRNAs specific for contractile proteins, which appear late in myoblast development paralleling morphogenetic differentiation of myoblasts into myocytes (Jakowlew, S. B., Khandekar, P., Datta, K., Narula, S. K., Arnold, H. H., and Siddiqui, M. A. Q. (1982) J. Mol. Biol. 156, 673-682).

Quantitation of muscle-specific mRNAs by using cDNA probes during chicken embryonic muscle development in ovo.

The emergence of abundant-class mRNAs specific for contractile muscle proteins and their distribution between polysomal and free mRNP fractions were studied in skeletal muscle excised from chicken embryos during the transition from myoblasts (day 9) to myotubes (day 18). Muscle-specific cDNA was selectively prepared by hybridizing cDNA to template RNA (polysomal poly(A)+ mRNA) from day-14 embryos followed by isolation of the abundant class, which represents approximately 20% of total mRNA. The specificity of the cDNA probe for this class was confirmed by the differential degree of hybridization to cytoplasmic RNA from cultured myotube and myoblast cells and by its inability to hybridize with mRNA from nonmuscle cells such as liver. Except for muscle from day-9 embryos, the concentrations of the abundant-class muscle-specific mRNAs were higher in polysomes than in free mRNP fractions. Furthermore, the levels of these mRNAs in polysomes increased 12-fold from day 9 (myoblast) to day 14 (intermediate) with a further 3.6-fold increase from day 14 to day 18 (myotube). In contrast to this 45-fold net increase in the polysomal level of these mRNAs from day 9 to day 18, the levels in the free mRNP fraction showed only a 3-fold decrease during this period. Because the amount of mRNA lost from the mRNP fraction is much less than the net increase in the polysome fraction, mRNP does not serve as a reservoir of untranslated muscle-specific mRNA for transfer to polysomes. Consequently, the emergence of muscle-specific polysomal mRNA for contractile proteins during myogenesis in ovo appears to be regulated primarily by transcriptional control.

Isolation of a new high molecular weight protein associated with desmin and vimentin filaments from avian embryonic skeletal muscle

Filaments with a diameter of 80-120 A have been prepared from 14-d-old chick embryonic skeletal muscle, using a physiological salt solution and gel filtration chromatography. The filaments obtained are composed of the two known muscle intermediate-filament proteins, vimentin and desmin, as well as the vimentin- and desmin-associated high molecular weight protein, synemin (230,000 mol. wt). In addition, they contain a previously unidentified high molecular weight protein (280,000 mol wt) which differs from synemin by isoelectric point, molecular weight, and immunological reactivity. Immunofluorescence on cultured myogenic cells,using antisera to the 280,000-dalton polypeptide, has revealed that this protein has the same spatial distribution as desmin, vimentin, and synemin in both early myotubes, where it associates with cytoplasmic filaments, and late in myotubes, where it is associated with myofibril Z lines. Examination by immunofluorescence of frozen sections of developing embryonic skeletal muscle reveals a gradual diminution in the presence of the 280,000-dalton protein. The 280,000-dalton protein is undetectable in adult skeletal and smooth muscle, as shown by immunofluorescence and immunoautoradiography. In chick embryonic fibroblasts grown in tissue culture, only a subpopulation of the cells is reactive with antibodies to the 280,000-dalton protein even though all these cells contain vimentin. In the reactive cells, vimentin and the 280,000-dalton polypeptide exhibit an indistinguishable cytoplasmic filamentous network, which aggregates into filamentious bundles when the cells are exposed to colcemid. These results suggest that this newly identified high molecular weight protein is closely associated with intermediate filaments containing either vimentin alone or vimentin, desmin and synemin. The expression of this protein appears to be developmentally regulated and does not appear to parallel the expression of any of the other three intermediate-filament proteins. The absence of the 280,000-dalton polypeptide in adult muscle cells and its gradual reduction during development implies that is probably not required for the maintenance of Z-disk structure after the assembly of the sarcomere.

Changes in tropomyosin during development of chick embryonic skeletal muscles in vivo and during differentiation of chick muscle cells in vitro.

We have studied the changes in tropomyosin subunits and their pattern of phosphorylation during embryonic development and also during in vitro differentiation of cultured muscle cells. We have found that in 10-day-old embryos tropomyosin consists of 75% beta subunit and 25% alpha subunit. However, only 60% of the beta subunit is phosphorylated while the alpha subunit is almost exclusively present in its phosphorylated form. During subsequent development, the amount of alpha-tropomyosin increases so that by hatching it represents 50% of the total subunits. During the same time, the amount of phosphorylated variants for both the alpha and the beta subunits decreases so that after hatching they represent less than 20% of each subunit. In cultures of differentiating myoblasts, the alpha subunit is synthesized before the beta subunit, and within each subunit, the unphosphorylated variants are the first to be synthesized. The reason for this discrepancy is not known at the present time.

The translational inhibitor 10 S cytoplasmic ribonucleoprotein of chick embryonic muscle. Dissociation and reassociation.

Cytoplasmic 10S ribonucleoprotein (iRNP) is a potent inhibitor of mRNA translation in vitro and contains a 4 S translation inhibitory RNA species (iRNA) (Sarkar, S., Mukherjee, A. K., and Guha, C. (1981) J. Biol. Chem. 256, 5077-5086). This ribonucleoprotein has now been resolved into protein and RNA components by DEAE-cellulose chromatography in the absence of both K+ and Mg2+ ions. These cations are required for maintaining the nucleoprotein structure of iRNP. Incubation of the dissociated protein and RNA components in the presence of K+ and Mg2+ at 35 degrees C reconstitutes a 10 S particle which is indistinguishable from native iRNP with respect to the elution profile by gel filtration, UV spectra, buoyant density, resistance to pancreatic RNase, and ability to inhibit exogenous mRNA translation in vitro. Chick muscle tRNA and globin mRNA could not form an RNP complex with the protein moieties of iRNP. The separated proteins, unlike iRNA and iRNP, do not inhibit mRNA translation. Their function may be to protect iRNA from ribonuclease digestion, since iRNP is ribonuclease-resistant. The ability to dissociate the iRNP particle and to specifically reconstitute it from the separated components indicates that it is a unique cellular entity which is distinct from other ribonucleoproteins.

Paraoxon increases the rate of synthesis of acetylcholinesterase in cultured muscle

Acetylcholinesterase (AChE) activity of cultured chick embryonic pectoral muscle was significantly increased above control activity during recovery from brief treatments with paraoxon ( O, O-diethyl- p-nitrophenyl phosphate), the anticholinesterase metabolite of parathion. Paraoxon also increased the rate of degradation of the enzyme, suggesting the higher AChE activity of paraoxon-treated cells was due to a stimulation of enzyme synthesis.

Unmasking of cryptic binding sites for α-bungarotoxin in membrane fractions from chick retina

Summary Phospholipase C treatment, low pH or detergent extraction double the number of high affinity binding sites for [ 125 I] α-bungarotoxin in membrane preparations from retina of newly hatched chickens. The cryptic toxin binding sites unmasked by these treatments exhibit the same affinity for the iodinated toxin and for differen cholinergic ligands as those present in untreated membranes. [ 125 I]α-bungarctoxin binding to the cryptic sites is inhibited by 1 mM Co 2+ . Membrane preparations from chick embryonic muscle show only a moderate increase in the number of toxin binding sites upon phospholipase C treatment. The existence of cholinergic ligand sites in neuronal membranes which normally are inaccessible to α-bungarotoxin may explain the resistance of neuronal nicotinic-cholinergic transmission to α-toxins.

The translational inhibitory 10 S cytoplasmic ribonucleoprotein of chicken embryonic muscle is distinct from messenger ribonucleoproteins

The translational inhibitory 10 S cytoplasmic ribonucleoprotein of chicken embryonic muscle is distinct from messenger ribonucleoproteins

A ribonuclease-resistant cytoplasmic 10 S ribonucleoprotein of chick embryonic muscle. A potent inhibitor of cell-free protein synthesis.

A ribonucleoprotein (RNP) particle sedimenting at 10 S in sucrose gradients had been isolated from the post-polysomal fraction of homogenates of 14-day-old chick embryonic leg and breast muscle by sucrose gradient fractionation and gel filtration. The 10 S RNP contains a 4 S RNA species (base composition: AMP, .3%; GMP, 22.2%; CMP, 24.2%; and UMP, 23.2%), and shows three major bands in the 70-90-nucleotide size range by polyacrylamide gel electrophoresis in 99% formamide. The 4 S RNA does not contain oligo(U)- and oligo(A)-rich tracts. The RNP has a characteristic buoyant density of 1.410 g/ml, which corresponds to an RNA/protein ratio of about 1:4. The UV absorption spectra of the RNP is very distinct from that of its RNA component. Both 4 S RNA and the 10 S RNP are potent inhibitors of translation of a variety of mRNAs such as chick muscle poly(A)+ mRNA, rabbit globin mRNA, EMC virus RNA, and poly(A)- and mRNA of rat liver in micrococcal nuclease-treated rabbit reticulocyte lysate. The inhibitory action of the RNA and the RNP on mRNA translation appears to involve the initiation process. The RNA and RNP do not have a nuclease activity associated with them. The hyperchromicity profile of the inhibitory RNA with increasing temperature indicates that it does not contain a significant amount of double-stranded structure. This is also supported by the complete loss of biological activity of the RNA by treatment with pancreatic RNase. In contrast, the inhibitory activity of the RNP was resistant to RNase. Electrophoresis of the protein moieties of the inhibitory RNP using both one- and two-dimensional gel techniques in the presence of sodium dodecyl sulfate shows a complex pattern of polypeptides of Mr = 12,000-150,000. The protein pattern of the 10 S particle is quite different from those of free and polysomal mRNP and poly(A)-protein complexes of chick embryonic muscles, indicating that most, if not all of the mRNA-associated proteins, are absent in the 19 S RNP. The properties of the inhibitory RNA indicate that it is different from the various low molecular weight RNA species which are involved in the modulation of protein synthesis in cell-free systems. It is concluded that the 10 S particle represents a novel class of RNP, which may be involved in posttranscriptional regulation of protein synthesis in embryonic muscles.

Cytoplasmic low molecular weight ribonucleic acid species of chick embryonic muscles, a potent inhibitor of messenger ribonucleic acid translation in vitro.

Cytoplasmic low molecular weight ribonucleic acid species of chick embryonic muscles, a potent inhibitor of messenger ribonucleic acid translation in vitro.

Lysosomes and proteolytic enzyme activities in cultured striated muscle cells.

Primary cell cultures prepared from chick embryonic skeletal muscle and the rat myogenic line L6 were examined morphologically and biochemically during several stages of development. The L6 cells were cultured to provide three morphologically distinct populations: prefusion, postfusion, and a subclone of cells that did not fuse even at high density. Ultrastructural studies revealed the characteristic morphology of healthy myoblasts. Acridine orange staining and cytochemical localization of acid phosphatase suggest the presence of presumptive lysosomal material. Enzymatic studies of lysosomal cathepsins B, D, H, and L revealed unusually high enzyme specific activities in these homogeneous myoblast populations. No activity was detected for the two nonlysosomal enzymes Ca2+-proteinase and serine proteinase. It is suggested that the lysosomal apparatus and its complement of enzymes play a significant role in the differentiation of muscle myotubes.

Heparin-inhibitable lectin. Purification from chicken liver and embryonic chicken muscle.

The heparin-inhibitable lectin activity from adult chicken liver and embryonic chick pectoral muscle has been purified by gel filtration on Sepharose CL-2B followed by affinity chromatography on heparin-Sepharose. Specific activity of this abundant material was increased about 70-fold with about 28% recovery of activity. The purified lectin, which behaved as a large aggregate, could be dissociated into two proteins with apparent molecular weights of 13,000 and 16,000 upon electrophoresis in sodium dodecyl sulfate. 125I-tryptic peptide maps of these proteins showed that they were distinct from each other and also from lectins of similar molecular weight previously purified from chicken. Agglutination activity of the lectin, tested with specially prepared rabbit erythrocytes, was very sensitive to heparin which inhibited 50% at concentrations of approximately 10(-5) mM. Dermatan sulfate and heparan sulfate were also potent inhibitors, whereas other glycosaminoglycans were not. The most potent monosaccharide hapten inhibitor was N-acetyl-D-galactosamine. Because of its properties, we operationally refer to this material as chicken heparin-lectin, recognizing that this does not necessarily indicate that it functionally interacts with heparin in vivo.

Quantitation of changes in cell surface determinants during skeletal muscle cell differentiation using monospecific antibody

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate antigenic changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS-1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion, myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty-one clones were obtained which produced antibodies specific for myogenic cells. Thirty-five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitatively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.

Nonselective inhibition of messenger RNA translation by highly purified low molecular weight RNA species from ribosomal salt wash of chick embryonic muscle.

A low molecular weight RNA species, in the 70-90 nucleotide size range (iRNA), has been purified from the ribosomal salt wash of chick embryonic muscle by a combination of DEAE-cellulose and hydroxyapatite chromatography. This method yields iRNA free from contaminating tRNA and gives better and more reproducible yields than those obtained with our previous method involving lengthy dialysis of the salt wash. The iRNA at the concentration of 20-80 ng range strongly inhibits the translation of homologous and heterologous mRNAs i.e. chick muscle poly(A)+mRNA and rabbit globin mRNA; uncapped mRNA; and poly(A)-mRNA in micrococcal nuclease-treated reticulocyte lysate indicating that inhibition by iRNA is nonselective in nature. The translation of endogenous globin mRNA and polysomes in the lysate is strikingly less sensitive to iRNA suggesting that the initiation step is primarily affected by iRNA. The iRNA does not appear to be double-stranded RNA. It is concluded that iRNA is distinct from other low molecular weight RNA species described in the literature which modulate protein synthesis in cell-free systems.

The effects of Con A on cell surface shedding in cell cultures.

A cell surface immobilizing concentration of Con A inhibits the shedding of [3H]fucose-containing glycoproteins from the surface of chick embryonic leg and breast muscle cell cultures and cultures of the rat skeletal muscle cell line L6. The Con A-induced inhibition of shedding is much less in the mouse fibroblast cell line 3T3. In all 4 cell types the lectin inhibits the shedding of some fucosyl-glycoproteins more than others, especially those of a lipid-containing fraction which is excluded in Biogel A-5m chromatography. This differential nature of the Con A effect is not changed by the cytoskeletal disruptors cytochalasin B or colchicine. Con A causes an increase in the amount of trypsin-sensitive surface fucosylglycoprotein in the cell surface and appears to decrease the overall amount of cell surface degradation suggesting the inhibition of shedding caused by Con A is not due to an increase in internalization and degradation. The data suggest that some shedding may occur at specific cell surface sites to which surface materials must laterally migrate.

Regulation of phosphatidylcholine biosynthesis in cultured chick embryonic muscle treated with phospholipase C.

Cultures of embryonic chick muscle cells grown in medium containing phospholipase C from Clostridium perfringens incorporated [3H]choline into lipid at a rate 3- to 5-fold higher than control cultures. To determine the mechanism by which stimulation of phosphatidylcholine synthesis occurred in phospholipase C-treated cells, activities of enzymes and levels of intermediates in the biosynthetic pathway for phosphatidylcholine were examined. Activities of choline kinase, choline phosphotransferase, glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate acyltransferase, acylglycerol-3-phosphate acyltransferase, and phosphatidic acid phosphatase in phospholipase C-treated cells were the same or only slightly higher than in control cells. CTP:phosphocholine cytidylyltransferase, on the other hand, was 3 times as active in homogenates from phospholipase C-treated cells. Levels of phosphocholine decreased and levels of CDP-choline increased in phospholipase C-treated cells, and a calculation of the disequilibrium ratio indicated that the cytidylyltransferase reaction was not at equilibrium. The cytidylyltransferase was, thus, identified as the regulatory enzyme for choline flux in these cells. The cytidylyltransferase was located in both the cytosolic and particulate fractions from cultured muscle cells and a much larger portion of enzyme activity was associated with the particulate fraction in cells treated with phospholipase C. Sonicated preparations of total chick lipids, phosphatidylethanolamine, and phosphatidylserine greatly stimulated the cytosolic cytidylyltransferase activity but had no effect on the particulate enzyme. Neither stimulation of incorporation of [3H]choline into lipid nor activation of the cytidylyltransferase was dependent on protein synthesis. A model for the mechanism of regulation of phosphatidylcholine synthesis in embryonic chick muscle is presented.

Myogenesis of normal and dystrophic chick embryonic skeletal muscle cells in vitro: biosynthesis of plasma membrane proteins.

Plasma membranes were prepared from primary cell cultures of normal and genetically dystrophic chick embryonic pectoral muscle. These membranes were analyzed both by one-dimensional sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis and by two-dimensional electrophoresis using isoelectric focusing in the first dimension. No marked and reproducible abnormalities could be detected in the synthesis, or accumulation, of plasma membrane proteins of dystrophic muscle cells maintained in culture for periods of up to 6 days. Analysis of the relative rates of protein turnover, analysis of fucose incorporation into plasma membrane proteins, and comparison of iodinated cell surface proteins also failed to reveal distinct abnormalities in plasma membranes derived from cultured dystrophic muscle cells. Although the results obtained do not rule out an early defect in plasma membrane protein biosynthesis during the development of dystrophic skeletal muscle in vivo, they do demonstrate that the synthesis and assembly of at least the major plasma membrane proteins occur normally during the initial phases of terminal differentiation of isolated dystrophic skeletal muscle cells in tissue culture.

Anchorage-independent muscle cell differentiation

Cells from embryonic chicken muscle were cultivated in serum-free medium. After two days, the suspended cells (almost all of which were nondividing myoblasts) were subcultured in serum-containing medium, either in gelatincoated tissue culture dishes (to promote reattachment) or in bacteriological dishes (to prevent reattachment). The extent of fusion was high in both suspended and reattached cultures. Newly synthesized proteins from day-5 cultures were resolved by two-dimensional electrophoresis and detected by autoradiography. Not only were the same protein species synthesized, but also the relative intensities of the spots corresponding to known muscle-specific proteins as well as the patterns of the many unidentified spots were similar. Synthesis of creatine kinase subunits B and M at different times was determined. In both suspended and reattached cells there was, as expected for differentiating myogenic cells, a marked increase by day 5 in the ratio of M to B subunits synthesized. Immunofluorescent staining with antibodies against an M-line protein with M(r) 165,000 revealed myofibrils partially wound about the nuclei of suspended cells; these became strung out in the axis of the cell as reattached cells elongated. The synthesis of muscle proteins, their assembly into myofilaments, and formation of well-organized myofibrils is evidently not anchorage dependent. However, proper alignment of parallel arrays of myofibrils in register does appear to require cell attachment to substrate.