Fate of microtubule-organizing centers during myogenesis in vitro

Microtubule organization and nucleation were studied during in vitro human myogenesis by immunocytology that used monoclonal and polyclonal antitubulin antibodies and a rabbit nonimmune serum that reacts with human centrosomes. In myoblasts, we observed a classical microtubule network centered on juxtanuclear centrosomes. Myotubes possessed numerous microtubules organized in parallel without any apparent nucleation centers. Centrosomes in these cells were not associated one to each nucleus but were often clustered in the vicinity of nuclei groups. They were significantly smaller than those of the mononucleated cells. The periphery of each nucleus in myotubes was labeled with the serum that labels centrosomes suggesting a profound reorganization of microtubule-nucleating material. Regrowth experiments after Nocodazole treatment established that microtubules were growing from the periphery of the nuclei. The redistribution of nucleating material was shown to take place early after myoblast fusion. Such a phenomenon appears to be specific to myogenic differentiation in that artificially induced polykaryons behaved differently: the centrosomes aggregated to form only one or a few giant nucleating centers and the nuclei did not participate directly in the nucleation of microtubules. The significance of these results is discussed in relation to the possible role of the centrosome in establishing cell polarity.     

Switching of filamin polypeptides during myogenesis in vitro

During chicken skeletal myogenesis in vitro, the actin-binding protein filamin is present at first in association with actin filament bundles both in myoblasts and in myotubes early after fusion. Later in mature myotubes it is found in association with myofibril Z disks. These two associations of filamin are separated by a period of several days, during which the protein is absent from the cytoplasm of differentiating myotubes (Gomer, R., and E. Lazarides, 1981, Cell, 23:524-532). To characterize the two classes of filamin polypeptides we have compared, by two-dimensional peptide mapping, 125I-labeled filamin immunoprecipitated from myoblasts and fibroblasts to filamin immunoprecipitated from mature myotubes and adult skeletal myofibrils. Myoblast filamin is highly homologous to fibroblast and purified chicken gizzard filamins. Mature myotube and adult myofibril filamins are highly homologous but exhibit extensive peptide differences with respect to the other three classes of filamin. Comparison of peptide maps from immunoprecipitated 35S-methionine-labeled filamins also shows that fibroblast and myoblast filamins are highly homologous but show substantial peptide differences with respect to mature myotube filamin. Filamins from both mature myotubes and skeletal myofibrils exhibit a slightly higher electrophoretic mobility than gizzard, fibroblast, and myoblast filamins. Short pulse-labeling studies show that mature myotube filamin is synthesized as a lower molecular weight variant and is not derived from a higher molecular weight precursor. These results suggest that myoblast and mature myotube filamins are distinct gene products and that during skeletal myogenesis in vitro one class of filamin polypeptides is replaced by a new class of filamin polypeptides, and that the latter is maintained into adulthood.     

Ultrastructural differentiation during embryonic Drosophila myogenesis in vitro

Summary Cultures of embryonicDrosophila melanogaster cells were examined by electron microscopy and events in myogenesis were recorded. Thick and thin myofilaments, T-tubules and sarcoplasmic reticulum all appeared at about the same time, 10.5 hr. This was about 5 hr after the final division of myoblasts and about the time that muscle cells were elongating, aligning and fusing. Sarcoplasm typical of insect muscle was detected by 18.5 hr, as were myotendonal and tendocuticular junctions. Two populations of myocytes were detected, the cytoplasm of one more electron-dense than the other. The only previous report of myofibrilogenesis in invertebrate embryos had described novel mechanisms. InDrosophila embryonic material, however, the sequence of myofibrilogenesis resembled that in post-embryonic insect or vertebrate material. Mrs. Pilar Toribio-Fiorio provided excellent technical assistance, and Patricia Minter, the secretarial expertise. This investigation was supported, in part, by NIH Grant NS9330 and the James Douglas Research Fund to Robert L. Seecof and NIH Grant No. 1 RO1 CA17223-01 to Raymond L. Teplitz.     

Activation of the interferon system by short-interfering RNAs

RNA interference (RNAi) is a powerful tool used to manipulate gene expression or determine gene function. One technique of expressing the short double-stranded (ds) RNA intermediates required for interference in mammalian systems is the introduction of short-interfering (si) RNAs. Although RNAi strategies are reliant on a high degree of specificity, little attention has been given to the potential non-specific effects that might be induced. Here, we found that transfection of siRNAs results in interferon (IFN)-mediated activation of the Jak-Stat pathway and global upregulation of IFN-stimulated genes. This effect is mediated by the dsRNA-dependent protein kinase, PKR, which is activated by 21-base-pair (bp) siRNAs and required for upregulation of IFN-beta in response to siRNAs. In addition, we show by using cell lines deficient in specific components mediating IFN action that the RNAi mechanism itself is independent of the interferon system. Thus, siRNAs have broad and complicating effects beyond the selective silencing of target genes when introduced into cells. This is of critical importance, as siRNAs are currently being explored for their potential therapeutic use.     

Change in a nuclear phosphoprotein during in vitro myogenesis

Synthesis and phosphorylation of total myoblast nuclear proteins have been studied during three stages of in vitro myogenesis: myoblast proliferation (22 h), pre-fusion mitotic arrest (44 h), and in myotubes (68 h). Phosphorylation in intact cells with [³²P]orthophosphate followed by SDS slab gel electrophoresis and radioautography of nuclear proteins reveals a striking decline in phosphorylation of a 100 000 D band at 44 h. This phosphoprotein band is not detectable at 68 h. Phosphorylation of isolated nuclei with γ-[³²P]ATP reveals the 100 000 D band as the major phosphoprotein which declines to 40% by 68 h. Assessment of content and synthesis of individual nuclear proteins by Coomassie Blue staining and [-³⁵S]methionine labelling reveals no appreciable changes in co-migrating 100 000 D bands, suggesting that the decline in phosphorylation is caused by either decreased kinase or increased phosphatase activity.     

Changes in the mRNA population of chick myoblasts during myogenesis in vitro

We have analyzed the sequence complexity, frequency distribution and coding capacity of the mRNA populations of primary chick embryo muscle cultures at different stages of myogenesis. Prefusion cultures, fused myofibrillar cultures and cultures blocked for both fusion and myogenesis all contain about 17,000 different mRNA sequences, arranged in three of four abundance classes. The myofibril (96 hr) cultures, however, contain about 2500 sequences in higher concentration and six sequences in exceptionally high concentration, each present in about 15,000 copies per nucleus. These sequences are shown to be 10 times less common in premyogenic (26 hr) cultures and 40 times less common in cultures that have been blocked by BUdR against both fusion and myogenesis. The concentration of these sequences in cultures developing toward myofibril formation correlates well with the capacity of the mRNA to stimulate the cell-free synthesis of muscle-specific proteins. A more direct approach to the identity of the abundant class of myofibril mRNA indicates that it contains the templates for the synthesis of seven polypeptides that are synthesized in particularly large amounts in myogenic cultures, including myosin, actin and tropomyosin. Between 20 and 30% of the abundant mRNA is transcribed from moderately repetitive DNA sequences. The remainder of the abundant, and all of the less-abundant, mRNA is transcribed from single-copy DNA.     

Prostaglandin dependence of membrane order changes during myogenesis in vitro

Myogenic differentiation in vitro involves at least three events at the cell surface: binding of prostaglandin to cells, cell-cell adhesion, and fusion of the myoblast membranes into syncytia. Previous work has suggested that binding of prostaglandin is causal to the change in cell-cell adhesion and that both are accompanied by a characteristic reorganization of the myoblast membrane detected as a transient increase in membrane order by electron paramagnetic resonance. We show here that this membrane order change, which reaches a maximum at 38 h of development in vitro, was the last membrane order change before bilayer fusion which begins several hours later. This membrane order change, which accompanies the change in cell-cell adhesion, was dependent on the availability of prostaglandin. In myoblasts maintained in indomethacin, where further differentiation is known to be blocked at the prostaglandin binding step, the membrane order change did not occur. However, if myoblasts are provided with exogenous prostaglandin, the membrane order change occurred and differentiation proceeded. The results indicate that the basis of the membrane order change was the reorganization of myoblast membranes to allow increased adhesion and prepare the membrane for bilayer fusion. They also demonstrate that, like the increase in myoblast adhesion, the membrane order change was dependent on prostaglandin being available to bind to its receptor.     

A re-evaluation of DNA repair during skeletal myogenesis in vitro

In previous studies on DNA repair during myogenesis, comparisons made of repair in post-replication myoblasts and in myotubes led to the conclusion that the capacity to repair damage in DNA decreased during myoblast differentiation. Using unscheduled DNA synthesis in response to UV-induced damage as an indicator of DNA repair in a myogenic line of rat skeletal muscle, it is demonstrated that nuclei in myotubes possess identical repair capacity as that in proliferating myoblasts. Furthermore, a brief increase in DNA repair capacity was observed to immediately follow the cessation of replicative DNA synthesis. This transient increase in repair capacity is consistent with the data of earlier reports and explains the previous but inappropriate conclusion that repair diminishes during myogenic differentiation. This transient increase in the capacity to repair DNA was not observed in a developmentally defective, non-differentiating line of similar myogenic origin.     

Alternative promoter usage by aldolase A during in vitro myogenesis

Aldolase A in the mouse, as in human and rat, shows tissue-specific variability of message size. In addition, in muscle tissue the mRNA size is also developmentally regulated. In order to determine whether this muscle-specific regulatory mechanism can be reproduced in vitro, we have examined the mRNA species of aldolase A isolated from mouse C2C12 myoblasts and myotubes on Northern blots and by primer extension. We show that aldolase A mRNA increases during in vitro myogenesis; that this induction is accompanied by a change in the message population; and that this change is due to activation of a muscle-specific alternative promoter.     

Developmental regulation of a secreted gelatin-binding protein during myogenesis in vitro

Collagen has a stimulatory effect on the differentiation of skeletal muscle cells in culture. Putative collagen-binding proteins were isolated from detergent-solubilized cultures of the L6 rat muscle cell line and primary clonal cultures of human skeletal muscle satellite cells, using gelatin-Sepharose affinity chromatography. In addition to fibronectin, which has been reported by others to be synthesized by cultured muscle cells, we found that muscle cultures synthesized gelatin-binding proteins of lower apparent molecular weight. Only one of these proteins was secreted into the growth medium and bound to type I collagen. Binding of this protein to gelatin and collagen-Sepharose was resistant to repeated washing with 1 M NaCl and nonionic detergent. The secreted gelatin-binding protein had an apparent molecular weight of 63,000-72,000, depending upon the conditions of electrophoresis. The lack of reactivity of the secreted protein with polyclonal antisera against fibronectin, the lack of effect of protease inhibitors on its appearance in the medium, and the rapid de novo production of the protein during pulse labeling with radioactive methionine indicated that it was not a fibronectin fragment. The rate of synthesis of the secreted gelatin-binding protein increased markedly during the myogenesis of rat and human cultures.     

Glycosphingolipid biosynthesis during myogenesis of rat L6 cells in vitro

Summary Glycosphingolipid biosynthesis was examined using [³H]-galactose as a precursor as rat L6 myoblasts fused to form multinucleated myotubes. Incorporation of label into neutral glycolipids decreased steadily as the population of myotubes increased, so that final biosynthesis was one-half that observed with myoblasts (p < 0.02). Conversely, ganglioside biosynthesis doubled during myoblast confluency (p < 0.02) and then decreased as myotubes formed. Qualitatively, L6 cells synthesized large amounts of ganglioside GM3 during all myogenic phases. The major neutral glycosphingolipid products were lactosylceramide and paragloboside (nLcOse₄Cer). Few changes in TLC autoradiographic patterns were noted during differentiation, with the exception of a slight decrease in ganglioside GM1. The results indicate that the biosynthesis of glycosphingolipids is tightly regulated during myogenesis in vitro and suggest a role for membrane gangliosides in muscle cell differentiation.Abbreviations GM1 II³NeuAc-GgOse₄Cer - GM3 II³NeuAc-GgOse²Cer - MG4 IV³NeuAc-nLcOse₄Cer - MG6 VI³NeuAc V⁴Gal-IV³GlcNAc-nLcOse⁴Cer - TLC Thin-Layer Chromatography - DMEM Dulbecco's Modified Eagles' Medium     

Ontogeny of insulin binding during chick skeletal myogenesis in vitro.

Our studies show that insulin receptors exist on chicken skeletal muscle cells at all developmental stages in culture. ¹²⁵I-labeled insulin binding at physiological concentrations to mature myotubes demonstrated saturability, binding proportional to cell number, reversibility, and specificity by competition with native hormone which reduced specific binding by 40% with 1 ng/ml and was maximal with 10 μg/ml. Further evidence for specificity was shown by no competition of insulin specific binding with insulin A chain, insulin B chain, growth hormone, and thyrotropin. Two binding sites were detected, with affinity constants of 10¹⁰M^?1 and 2 × 10⁹M^?1. The hormone receptor complex showed rapid dissociation (70% in 30 min) after equilibrium binding. During myogenesis, an increase in insulin receptors occurs from 500 per proliferating myoblast to 3000 per cell equivalent in mature (6 day) myotubes. Since these studies demonstrate that insulin receptors are present and other studies have shown that insulin is present during most of chicken embryogenesis, insulin may regulate muscle development in vivo to a greater degree than previously suspected.     

Fibroblasts promote the formation of a continuous basal lamina during myogenesis in vitro

Analyses were made of the requirements for the formation of a continuous basal lamina during myogenesis of quail muscle in vitro. A culture system was developed in which mass cultures of differentiating muscle cells were embedded in a native gel of rat tail collagen. Fibroblastic cells, which were also present in the cultures, migrated into the gel and within a few days surrounded the newly formed myotubes. In this environment, a continuous basal lamina was formed at the surface of the myotubes as demonstrated by immunofluorescent staining with monoclonal antibodies against type IV collagen, laminin, and heparan sulfate, as well as by electron microscopic immunolocalization. To distinguish between the role of the fibroblasts and the collagen gel in promoting basal lamina formation, clones of quail muscle cells lacking fibroblasts were subsequently embedded in a native rat tail collagen gel. Under these conditions, only very limited fluorescent staining for basement membrane components was observed associated with the myotubes. However, the introduction of chick muscle or skin fibroblasts into the clonal cultures just before gel formation resulted in the formation of an extensive basal lamina on the surface of the myotubes. Conditioned medium from fibroblast cultures by itself was not effective in promoting basal lamina formation. These results clearly show that during myogenesis in vitro fibroblasts must be in close proximity to the myotubes for a continuous basal lamina to form. These results probably relate closely to the interactions that must occur during myogenesis in vivo between the muscle cells and the surrounding connective tissue including the developing tendons.     

Location of myostatin expression during bovine myogenesis in vivo and in vitro

Mutations in the myostatin gene lead to double-muscling in cattle indicating that it is a negative regulator of the total number of muscle fibres. Myostatin expression was analysed by RT-PCR in three developing bovine muscles. It decreased during differentiation in Semitendinosus and Biceps femoris, and increased in the late differentiating Masseter during gestation. A combination of in situ hybridisation and immuno-histochemical detection of myosin heavy chains (MHC) allowed us to locate the expression in myofibres containing only developmental MHC at different stages and in fast IIA fibres at the end of gestation. In vitro, myostatin was undetectable during proliferation, peaked at the onset of fusion and decreased during terminal differentiation. It was not detected in myotubes by in situ hybridisation. The inhibition of differentiation by BrdU prevented the decrease in expression. Our results show that the peak in myostatin expression coincides with early differentiation indicating a regulatory role in cattle myogenesis.     

Proteoglycan synthesis by primary chick skeletal muscle during in vitro myogenesis

The proteoglycans synthesized by primary chick skeletal muscle during in vitro myogenesis were compared with those of muscle-specific fibroblasts. Cultures of skeletal muscle cells and muscle fibroblasts were separately labeled using [35S] sulfate as a precursor. The proteoglycans of the cell layer and medium were separately extracted and isolated by ion-exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sepharose CL-2B. Two cell layer-associated proteoglycans synthesized both by skeletal muscle cells and muscle fibroblasts were identified. The first, a high molecular weight proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.07 and contained exclusively chondroitin sulfate chains with an average molecular weight greater than 50,000. The second, a relatively smaller proteoglycan, eluted from Sepharose CL-2B with a Kav of 0.61 and contained primarily heparan sulfate chains with an average molecular weight of 16,000. Two labeled proteoglycans were also found in the medium of both skeletal muscle and muscle fibroblasts. A high molecular weight proteoglycan was found with virtually identical properties to that of the high molecular weight chondroitin sulfate proteoglycan of the cell layer. A second, smaller proteoglycan had a similar monomer size (Kav of 0.63) to the cell layer heparan sulfate proteoglycan, but differed from it in that this molecule contained primarily chondroitin sulfate chains with an average molecular weight of 32,000. Studies on the distribution of these proteoglycans in muscle cells during in vitro myogenesis demonstrated that a parallel increase in the relative amounts of the smaller proteoglycans occurred in both the cell layer and medium compared to the large chondroitin sulfate proteoglycan in each compartment. In contrast, muscle-derived fibroblasts displayed a constant ratio of the small proteoglycans of the cell layer and medium fractions, compared to the larger chondroitin sulfate proteoglycan of the respective fraction as a function of cell density. Our results support the concept that proteoglycan synthesis is under developmental regulation during skeletal myogenesis.     

Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro

The distribution of intermediate filament (IF) subunits during maturation of skeletal myotubes in vitro was examined by immunofluorescence, using antibodies against two different types of chick IF subunits: (a) 58-kdalton subunits of fibroblasts (anti-58K), and (b) 55-kdalton subunits of smooth muscle (anti-55K). Anti-58K bound to a filament network in replicating presumptive myoblasts and fibroblasts, as well as in immature myotubes. The distribution in immature myotubes was in longitudinal filaments throughout the cytoplasm. With maturation, staining of myotubes by anti-58K diminished and eventually disappeared. Anti-55K selectively stained myotubes, and the fluorescence localization underwent a drastic change in distribution with maturation--from dense, longitudinal filaments in immature myotubes to a cross-striated distribution in mature myotubes that was associated with the I--Z region of myofibrils. However, the emergence of a cross-striated anti-55K pattern did not coincide temperally with the emergence of striated myofibrils, but occurred over a period of days thereafter.     

Multiple effects of interferon on myogenesis in chicken myoblast cultures

Effects of chicken interferon on the differentiation of chicken skeletal muscle in vitro were examined. Continuous treatment of chicken myoblast culture with 200 IU/ml of interferon (10 IU/mg protein) resulted in significant inhibition of cell fusion and subsequent myotube formation. However, treatment of myoblast culture with 2 to 200 IU/ml of interferon increased activities of creatine kinase and myokinase in 4- or 6-day cultured muscle cells in a dose-dependent fashion. The effect of interferon on myokinase was less than on creatine kinase. Three-fold increase in creatine kinase activity induced by interferon was not accompanied by the accelerated transition of creatine kinase isozyme from BB- to MM-type. On the other hand, accumulation of acetylcholinesterase in interferon-treated cells at day 6 was suppressed to nearly half the level of control cells. Rates of actin and myosin synthesis in 4-day cultures estimated by pulse-labelling with [35S]methionine were also suppressed to 85% of control cultures. However, a proportion of 35S-labelled actin and myosin in labelled proteins associated with glycerinated cells was not changed by interferon treatment. These results indicate that partially purified interferon has multiple effects on the process of the myogenic differentiation of chicken myoblast in vitro.