Mouse Myogenic Cell Line Research Articles

PTEN-AKT pathway attenuates apoptosis and adverse remodeling in ponatinib-induced skeletal muscle toxicity following BMP-7 treatment.

Tyrosine kinase inhibitors (TKIs) including ponatinib are commonly used to treat cancer patients. Unfortunately, TKIs induce cardiac as well as skeletal muscle dysfunction as a side effect. Therefore, detailed mechanistic studies are required to understand its pathogenesis and to develop a therapeutic treatment. The current study was undertaken to examine whether ponatinib induces apoptosis and apoptotic mechanisms both in vitro and in vivo models and furthermore to test the potential of bone morphogenetic protein 7 (BMP-7) as a possible treatment option for its attenuation. Sol8 cells, a mouse myogenic cell line was exposed to ponatinib to generate an apoptotic cell culture model and were subsequently treated with BMP-7 to understand its protective effects. For the in vivo model, C57BL/6J mice were administered with ponatinib to understand apoptosis, cell signaling apoptotic mechanisms, and adverse muscle remodeling and its attenuation with BMP-7. TUNEL staining, immunohistochemistry (IHC), and real-time polymerase chain reaction (RT-PCR) methods were used. Our data show significantly (p < 0.05) increased TUNEL staining, caspase-3, BAX/Bcl2 ratio in the in vitro model. Furthermore, our in vivo muscle data show ponatinib-induced muscle myopathy, and loss in muscle function. The observed muscle myopathy was associated with increased apoptosis, caspase-3 staining, and BAX/Bcl-2 ratio as confirmed with IHC and RT-PCR. Furthermore, our data show a significant (p < 0.05) increase in the involvement of cell signaling apoptotic regulator protein PTEN and a decrease in cell survival protein AKT. These results suggest that increased apoptosis following ponatinib treatment showed an increase in skeletal muscle remodeling, sarcopenia, and fibrosis. Furthermore, BMP-7 treatment significantly (p < 0.05) attenuated ponatinib-induced apoptosis, BAX/Bcl2 ratio, decreased PTEN, and increased cell survival protein AKT, decreased adverse muscle remodeling, and improved muscle function. Overall, we provide evidence that ponatinib-induces apoptosis leading to sarcopenia and muscle myopathy with decreased function which was attenuated by BMP-7.

Vitamin D Inhibits Myogenic Cell Fusion and Expression of Fusogenic Genes.

Vitamin D, a fat-soluble vitamin, is an important nutrient for tissue homeostasis and is recently gaining attention for its role in sarcopenia. Although several studies have focused on the role of vitamin D in muscle homeostasis, the molecular mechanism underlying its action on skeletal muscle remains unclear. This study investigated the role of vitamin D in myogenesis and muscle fiber maintenance in an immortalized mouse myogenic cell line. A high concentration of active vitamin D, 1α,25(OH)2D3, decreased the expression of myogenic regulatory factors (MRFs), myf5 and myogenin in proliferating myoblasts. In addition, high concentration of vitamin D reduced myoblast-to-myoblast and myoblast-to-myotube fusion through the inhibition of Tmem8c (myomaker) and Gm7325 (myomerger), which encode muscle-specific fusion-related micropeptides. A similar inhibitory effect of vitamin D was also observed in immortalized human myogenic cells. A high concentration of vitamin D also induced hypertrophy of multinucleated myotubes by stimulating protein anabolism. The results from this study indicated that vitamin D had both positive and negative effects on muscle homeostasis, such as in muscle regeneration and myofiber maintenance. Elderly individuals face a higher risk of falling and suffering fractures; hence, administration of vitamin D for treating fractures in the elderly could actually promote fusion impairment and, consequently, severe defects in muscle regeneration. Therefore, our results suggest that vitamin D replacement therapy should be used for prevention of age-related muscle loss, rather than for treatment of sarcopenia.

The Ror1 receptor tyrosine kinase plays a critical role in regulating satellite cell proliferation during regeneration of injured muscle

The Ror family receptor tyrosine kinases, Ror1 and Ror2, play important roles in regulating developmental morphogenesis and tissue- and organogenesis, but their roles in tissue regeneration in adult animals remain largely unknown. In this study, we examined the expression and function of Ror1 and Ror2 during skeletal muscle regeneration. Using an in vivo skeletal muscle injury model, we show that expression of Ror1 and Ror2 in skeletal muscles is induced transiently by the inflammatory cytokines, TNF-α and IL-1β, after injury and that inhibition of TNF-α and IL-1β by neutralizing antibodies suppresses expression of Ror1 and Ror2 in injured muscles. Importantly, expression of Ror1, but not Ror2, was induced primarily in Pax7-positive satellite cells (SCs) after muscle injury, and administration of neutralizing antibodies decreased the proportion of Pax7-positive proliferative SCs after muscle injury. We also found that stimulation of a mouse myogenic cell line, C2C12 cells, with TNF-α or IL-1β induced expression of Ror1 via NF-κB activation and that suppressed expression of Ror1 inhibited their proliferative responses in SCs. Intriguingly, SC-specific depletion of Ror1 decreased the number of Pax7-positive SCs after muscle injury. Collectively, these findings indicate for the first time that Ror1 has a critical role in regulating SC proliferation during skeletal muscle regeneration. We conclude that Ror1 might be a suitable target in the development of diagnostic and therapeutic approaches to manage muscular disorders.

1H34 微細メッシュ構造を足場とした配向性筋管細胞組織の作製および細胞応答の観察

Tissue engineered cell sheets composed of oriented muscle fibers differentiated from myoblasts can be regarded as more suitable for the regeneration of skeletal muscles. A novel method for fabricating monolayer cell sheets by culturing cells on a suspended mesh patterned structure consisting of narrow mesh lines (< 5 μm) and large apertures (> 100 μm) was developed in our laboratory. In this study, we investigated the influence of mesh shape on the orientation of myoblast and myotubes derived from them. To do this, we cultured C2C12 (mouse myogenic cell line) on fibronectin-coated mesh patterned structure and differentiated them to myotubes after confluency was reached. Using a diamond shaped mesh with a 100 nm long minor axis and a minor angle of 70°, we obtained myoblast cell sheets with cells oriented in the longest axis, in accordance with our previous studies. Importantly, myotubes derived from the oriented myoblasts were also similarly oriented, suggesting that lengthwise fusion as opposed to sidewise fusion occurs predominantly in orientated myoblasts. Using Fluo-4 indicator, we monitored calcium dynamics in the oriented myotubes and found that some myotubes displayed a unique calcium wave that travelled from end-to-end at a speed of 10-30 μm/s. This calcium dynamics was clearly distinct from the typical calcium spike displayed by a majority of myotubes. In our future studies, we will investigate the relationship between this calcium dynamics and the beating motion also observed in some oriented myoblast.

METTL21C Is a Potential Pleiotropic Gene for Osteoporosis and Sarcopenia Acting Through the Modulation of the NF-κB Signaling Pathway

Sarcopenia and osteoporosis are important public health problems that occur concurrently. A bivariate genome-wide association study (GWAS) identified METTL21c as a suggestive pleiotropic gene for both bone and muscle. The METTL21 family of proteins methylates chaperones involved in the etiology of both myopathy and inclusion body myositis with Paget's disease. To validate these GWAS results, Mettl21c mRNA expression was reduced with siRNA in a mouse myogenic C2C12 cell line and the mouse osteocyte-like cell line MLO-Y4. At day 3, as C2C12 myoblasts start to differentiate into myotubes, a significant reduction in the number of myocytes aligning/organizing for fusion was observed in the siRNA-treated cells. At day 5, both fewer and smaller myotubes were observed in the siRNA-treated cells as confirmed by histomorphometric analyses and immunostaining with myosin heavy chain (MHC) antibody, which only stains myocytes/myotubes but not myoblasts. Intracellular calcium (Ca(2+)) measurements of the siRNA-treated myotubes showed a decrease in maximal amplitude peak response to caffeine, suggesting that less Ca(2+) is available for release due to the partial silencing of Mettl21c, correlating with impaired myogenesis. In siRNA-treated MLO-Y4 cells, 48 hours after treatment with dexamethasone there was a significant increase in cell death, suggesting a role of Mettl21c in osteocyte survival. To investigate the molecular signaling machinery induced by the partial silencing of Mettl21c, we used a real-time PCR gene array to monitor the activity of 10 signaling pathways. We discovered that Mettl21c knockdown modulated only the NF-κB signaling pathway (ie, Birc3, Ccl5, and Tnf). These results suggest that Mettl21c might exert its bone-muscle pleiotropic function via the regulation of the NF-κB signaling pathway, which is critical for bone and muscle homeostasis. These studies also provide rationale for cellular and molecular validation of GWAS, and warrant additional in vitro and in vivo studies to advance our understanding of role of METTL21C in musculoskeletal biology.

An efficient method for stable transfection of mouse myogenic C2C12 cell line using a nonviral transfection approach

To describe in detail the use of the T-REx^{TM} System (a tetracycline-regulated mammalian expression system; Invitrogen) for rapid, easy, and effective generation of stable C2C12 cell clones. Tetracycline-inducible stable cell lines are useful tools to study the function of various genes in different types of mammalian cells. However, the generation of stable cell lines is a time-consuming, technically difficult, and expensive process. Materials and methods: Generation of a stable C2C12 cell line was performed by stable transfection of the cells with the pcDNA6/TR plasmid vector, which contains the tetracycline repressor and the blasticidin drug resistance genes. The establishment of the stable cell line and the efficiency of the tetracycline-inducible gene expression system were shown with the transient transfection of reporter plasmid pTO-EGFP and observation of the GFP expression by fluorescence microscopy. Results: We established a stable T-REx^{TM} C2C12 cell line that can be used for muscle research studies by transfecting the gene of interest with a second plasmid. Conclusion: The T-REx^{TM} system is useful in the development of stable cell lines for protein production or regulation of gene expression.

Overexpression of amyloid beta precursor protein enhances expression and secretion of ST6Gal1 in C2C12 myogenic cell line

The Abeta (amyloid-beta) peptide is derived from the sequential cleavage of AbetaPP (amyloid-beta precursor protein) by two enzymes, the β- and γ-secretases. The major β-secretase, identified as the novel transmembrane aspartic protease BACE1 (beta site APP-cleaving enzyme 1), mediates the primary amyloidogenic cleavage of AbetaPP and initiates the production of Abeta. It has been implicated in the proteolytic processing of another substrate, namely ST6Gal1 (β galactoside α2,6-sialyltransferase 1), which is the major α2,6-sialyltransferase responsible for the broad synthesis of glycoproteins and glycolipids. The present study investigated the effect of overexpression of AbetaPP on expression and secretion of ST6Gal1 in skeletal muscle cells by inducing overexpression of wild-type full-length 751-AbetaPP in the mouse myogenic cell line C2C12. Expression and secretion of the ST6Gal1 enzyme were analysed by Western blot and/or immunofluorescence staining. The results of our study demonstrated that AbetaPP overexpression in C2C12 cells increased the expression and the secretion of ST6Gal1 enzyme in vitro.

Oligomers of Mutant Glial Fibrillary Acidic Protein (GFAP) Inhibit the Proteasome System in Alexander Disease Astrocytes, and the Small Heat Shock Protein αB-Crystallin Reverses the Inhibition

The accumulation of the intermediate filament protein, glial fibrillary acidic protein (GFAP), in astrocytes of Alexander disease (AxD) impairs proteasome function in astrocytes. We have explored the molecular mechanism that underlies the proteasome inhibition. We find that both assembled and unassembled wild type (wt) and R239C mutant GFAP protein interacts with the 20 S proteasome complex and that the R239C AxD mutation does not interfere with this interaction. However, the R239C GFAP accumulates to higher levels and forms more protein aggregates than wt protein. These aggregates bind components of the ubiquitin-proteasome system and, thus, may deplete the cytosolic stores of these proteins. We also find that the R239C GFAP has a greater inhibitory effect on proteasome system than wt GFAP. Using a ubiquitin-independent degradation assay in vitro, we observed that the proteasome cannot efficiently degrade unassembled R239C GFAP, and the interaction of R239C GFAP with proteasomes actually inhibits proteasomal protease activity. The small heat shock protein, alphaB-crystallin, which accumulates massively in AxD astrocytes, reverses the inhibitory effects of R239C GFAP on proteasome activity and promotes degradation of the mutant GFAP, apparently by shifting the size of the mutant protein from larger oligomers to smaller oligomers and monomers. These observations suggest that oligomeric forms of GFAP are particularly effective at inhibiting proteasome activity.

Azumolene Inhibits a Component of Store-operated Calcium Entry Coupled to the Skeletal Muscle Ryanodine Receptor

Dantrolene reduces the elevated myoplasmic Ca(2+) generated during malignant hyperthermia, a pharmacogenetic crisis triggered by volatile anesthetics. Although specific binding of dantrolene to the type 1 ryanodine receptor (RyR1), the Ca(2+) release channel of skeletal muscle sarcoplasmic reticulum, has been demonstrated, there is little evidence for direct dantrolene inhibition of RyR1 channel function. Recent studies suggest store-operated Ca(2+) entry (SOCE) contributes to skeletal muscle function, but the effect of dantrolene on this pathway has not been examined. Here we show that azumolene, an equipotent dantrolene analog, inhibits a component of SOCE coupled to activation of RyR1 by caffeine and ryanodine, whereas the SOCE component induced by thapsigargin is not affected. Our data suggest that azumolene distinguishes between two mechanisms of cellular signaling to SOCE in skeletal muscle, one that is coupled to and one independent from RyR1.

The effect of galectin-1 on the differentiation of fibroblasts and myoblasts in vitro.

Normal murine dermal fibroblasts implanted into the muscles of the mdx mouse, a model for Duchenne muscular dystrophy, not only participate in new myofibre formation but also direct the expression of the protein dystrophin which is deficient in these mice. We have reported that the lectin galectin-1 is implicated in the conversion of dermal fibroblasts to muscle. In the current work we confirm the presence of galectin-1 in the medium used for conversion. Furthermore we report that exposure of clones of dermal fibroblasts to this lectin results in 100% conversion of the cells. Conversion was assessed by the expression within the cells of the muscle-specific cytoskeletal protein desmin. We also investigate the effects of galectin-1 on cells of the C2C12 mouse myogenic cell line and on primary mouse myoblasts. Exposing both transformed and primary myoblasts to the lectin resulted in an increase in fusion of cells to the terminally differentiated state in both types of cultures. Galectin-1 does not cause the myogenic conversion of murine muscle-derived fibroblasts.

Myosin II Folding Is Mediated by a Molecular Chaperonin

The folding pathway of the heavy meromyosin subfragment (HMM) of a skeletal muscle myosin has been investigated by in vitro synthesis of the myosin heavy and light chains in a coupled transcription and translation assay. Analysis of the nascent translation products for folding intermediates has identified a major intermediate that contains all three myosin subunits in a complex with the eukaryotic cytosolic chaperonin. Partially folded HMM is released from this complex in an ATP-dependent manner. However, biochemical and functional assays reveal incomplete folding of the myosin motor domain. Dimerization of myosin heavy chains and association of heavy and light chains are accomplished early in the folding pathway. To test for other factors necessary for the complete folding of myosin, a cytoplasmic extract was prepared from myotubes produced by a mouse myogenic cell line. This extract dramatically enhanced the folding of HMM, suggesting a role for muscle-specific factors in the folding pathway. We conclude that the molecular assembly of myosin is mediated by the eukaryotic cytosolic chaperonin with folding of the motor domain as the slow step in the pathway.

The proto-oncogene Bc16 inhibits apoptotic cell death in differentiation-induced mouse myogenic cells.

The Bc16 gene is located at chromosomal band 3q27, a breakpoint for translocation that frequently occurs in B cell lymphomas. Bc16 has been found to be preferentially expressed in germinal center B cells, and expression of this gene has been shown to be essential for germinal center formation in vivo. The physiological function of Bc16 and its role in lymphomagenesis, however, are not yet known. Since significant expression of Bc16 has been demonstrated in skeletal muscle, we have utilized a differentiation-inducible mouse myogenic cell line, C2C12, to elucidate the function of the Bc16 gene product. Expression of Bc16 mRNA was very low in growing myocytes, but was increased in differentiating myocytes cultured in serum-starved medium. Incubation of these cells with cytokines or chemicals that are known to block differentiation suppressed this increased Bc16 message abundance, indicating that Bc16 induction is related to the process of terminal differentiation in muscle cells. While a fraction of myocytes is known to undergo apoptosis after serum-starvation to induce differentiation, adenovirus-mediated overexpression of Bc16 enhanced the viability of the differentiating cells by preventing the apoptosis. High levels of Bc16 antisense mRNA expression induced substantial apoptosis during the differentiation of C2C12 cells, but this was effectively prevented by infection with adenovirus that expressed Bc16 sense mRNA. These results indicate that Bc16 acts to prevent apoptotic cell death in differentiating myocytes. The deregulation of expression of this antiapoptotic gene may also contribute to the development of B cell lymphomas.

Over Expression of the Murine Myotonic Dystrophy Protein Kinase in the Mouse Myogenic C2C12 Cell Line Leads to Inhibition of Terminal Differentiation

Myotonic dystrophy (DM) is an autosomal dominant human disorder, caused by the abnormal expansion of a CTG trinucleotide repeat in the 3′ untranslated region of a protein kinase gene (DMPK). Muscle symptoms are a common feature of the disorder and in the adult onset cases there are increased patterns of muscle fibre degeneration and regeneration. In the congenitally affected infants there is a failure of muscle maturation, with the histological presence of numerous immature fibres. However, the pathological mechanism in both forms of the disease is unclear. We report that over-expression of the murinedmpkgene, in a murine myogenic cell line, leads to markedly reduced levels of fusion to the terminally differentiated state. These findings complement recently published data using a heterologous expression/cell system and may have implications for the understanding of the disease process in this disorder.

The glucocorticoid receptor and AP-1 are involved in a positive regulation of the muscle regulatory gene myf5 in cultured myoblasts.

The muscle regulatory factor, myf5, is involved in the establishment of skeletal muscle precursor cells. Little is known, however, about the control of the expression of the gene encoding this basic helix-loop-helix (bHLH) factor. We have addressed this question in the mouse myogenic cell line, C2, and in a derivative of this cell line where the myf5 gene is the only muscle-specific bHLH factor to be expressed at the myoblast stage. We present evidence that the synthetic glucocorticoid dexamethasone, and the pharmacological agent anisomycin, act synergistically to rapidly up-regulate the levels of myf5 transcript and protein. The glucocorticoid antagonist RU 486 abolishes this synergy, demonstrating the involvement of the glucocorticoid receptor. The expression of a dominant negative mutant of c-jun which interferes with the transactivating properties of all AP-1 family members also blocks the induction of myf5 by anisomycin and dexamethasone. An activator of protein kinase C (PKCs), 12-O-tetradecanoyl phorbol 13-acetate (TPA), abolishes the up-regulation of myf5 gene expression by dexamethasone and anisomycin, and its effect is counteracted by an inhibitor of PKCs, GF 109203X. These results point to the possible involvement of PKCs in the negative control of myf5. Evidence that both positive and negative regulation of myf5 transcripts, described here, does not require the fresh synthesis of transcription factors suggests that myf5 may behave like an immediate early gene.

Gene transfer and expression of human alpha-galactosidase from mouse muscle in vitro and in vivo.

Lysosomal storage disorders are amenable to treatment by enzyme replacement. Genetic modification of muscle via direct injection of expression vectors might represent an alternative method of providing the defective enzymes, if adequate and long-lasting expression levels can be achieved in muscle. We have used the C2C12 mouse myogenic cell line to study the effect of combination of muscle-specific regulatory elements on the expression of the human lysosomal enzyme alpha-galactosidase (alpha-gal). In differentiated myotubes, a construct containing the myosin light chain 1/3 enhancer in combination with the human cytomegalovirus promoter resulted in higher expression than constructs combining the same enhancer with the rabbit beta-myosin heavy chain promoter, or containing the CMV promoter only. Increased enzymatic activity was detectable both in cell extracts and in supernatants. Furthermore, human fibroblasts deficient in alpha-gal were able to take up the enzyme from medium conditioned by transfected myoblasts. This did not occur in the presence of mannose-6-phosphate which indicates that the uptake was via mannose-6-phosphate receptors. To our knowledge, this is the first report in which a correctly processed form of human alpha-gal was expressed and secreted from differentiated muscle cells. Direct injection of a plasmid expression vector into mouse tibialis anterior muscle showed significantly increased levels of alpha-gal 7 days after injection.

Autonomous differentiation in the mouse myogenic cell line, C2, involves a mutual positive control between insulin-like growth factor II and MyoD, operating as early as at the myoblast stage.

We have studied the contribution of the endogenous production of insulin-like growth factor II (IGFII) and of the muscle regulatory factor, MyoD, to the autonomy of differentiation in isolated skeletal myoblasts. Inhibition of MyoD and IGFII gene expression in myoblasts of the mouse myogenic cell line, C2, was achieved by transfection and selection of stably transfected cells (anti-MyoD and anti-IGFII cells) with vectors producing MyoD or IGFII antisense RNA. We observed that inhibiting either MyoD or IGFII has multiple and similar consequences. In addition to the inhibition of the target gene, expression of MyoD transcripts in anti-IGFII myoblasts and expression of IGFII in anti-MyoD myoblasts were also abolished, whereas accumulation of transcripts for the muscle regulatory factor, Myf5, was markedly increased in both cell types. However, despite this Myf5 up-regulation, both anti-IGFII and anti-MyoD myoblasts lost the ability to undergo autonomous differentiation (differentiation in the absence of added IGF), further indicating that Myf5 and MyoD are not strictly interchangeable. Additional evidence of a link between MyoD and IGFII was obtained: (1) forced expression of the MyoD cDNA stimulated IGFII gene expression, and (2) treatment of C2 myoblasts with fibroblast growth factor, not only diminished MyoD expression and compromised differentiation as previously shown by others, but also abolished IGFII expression. These experiments showing loss or gain of function argue in favor of a mutual positive control between IGFII and MyoD operating as early as the myoblast stage.

Cloning and sequencing of mouse skeletal muscle alpha-dystroglycan.

alpha-Dystroglycan has attracted much interest due to its important function in linking laminin in the extracellular matrix and dystrophin in the muscle. The complete sequence of mouse skeletal muscle alpha-dystroglycan was produced and cloned by applying the reverse transcriptase-polymerase chain reaction approach to the total RNA extracted from the mouse myogenic cell line C2C12. The results demonstrate the usefulness of this approach and the high degree of conservation of alpha-dystroglycan between different species, and they provide the basis for additional work with the murine species.

A Retroviral Vector Containing a Muscle-Specific Enhancer Drives Gene Expression Only in Differentiated Muscle Fibers

Genetically modified myogenic cells have a number of potentially relevant applications for gene therapy of genetic defects. Retroviral vectors proved to be a safe and efficient tool to transfer and express genes into satellite cells and their differentiated progeny, although muscle-specific regulation of the transferred gene is very difficult to achieve in a conventional vector framework. We modified a Moloney murine leukemia virus (MoMLV)-derived retroviral vector containing a bacterial beta-galactosidase (beta-Gal) reporter gene by inserting a muscle creatinine kinase (MCK) enhancer element into the U3 region of the viral long terminal repeat (LTR). The resulting vector (mLBSN) was transferred into cells of different histological origin, including undifferentiated murine and human myogenic cells, which were unable to express the transgene at detectable levels. Instead, gene expression from the modified LTR was obtained in a mouse myogenic cell line and in human primary satellite cells upon induction of differentiation into myotubes in culture, and correlated with the activation of the muscle differentiation program. beta-Gal-negative, mLBSN-transduced human satellite cells were also transplanted into the quadricep muscle of immunodeficient mice, where activation of the transgene expression was observed in vivo after differentiation and fusion into muscle fibers. These results show that retroviral vectors carrying LTRs modified in the enhancer sequences may be used to target tissue- and differentiation-specific gene expression into the muscle. For practical purposes, satellite cells engineered by muscle-specific retroviral vectors might represent an effective tool to deliver expression of a given gene product specifically into the muscle tissue, avoiding undesired protein accumulation in mononucleated cells. More generally, this type of vector might be useful whenever regulated expression of a transferred gene is necessary in a target cell or tissue.

Cytolocation of prosome antigens on intermediate filament subnetworks of cytokeratin, vimentin and desmin type

ABSTRACT Analysis by double-label indirect immunofluorescence of PtK1 and HeLa cells had previously demonstrated that prosome* antigens form networks that superimpose on those of the intermediate filaments of the cytokeratin type. We show here that in PtK1 cells various prosomal antigens also reside to a variable extent on intermediate filaments subnetworks of the vimentin type. In proliferating human fibroblasts the prosome and vimentin networks were found to coincide, while in proliferating myoblasts of the C2.7 mouse myogenic cell line the prosomal antigens seem to superimpose on the intermediate filaments of the desmin type. Thus, the prosomes, which are RNP particles of variable composition and subcomplexes of untranslated mRNP, and carry a multicatalytic proteinase activity, seem to co-localize with the specific kind of cytoplasmic intermediate filament in relation to the cell type. These results, which generalize the previous data, are discussed in view of possible role(s) for prosomes in mRNA metabolism and/or intermediate filaments remodelling.

Segregated assembly of muscle myosin expressed in nonmuscle cells.

Skeletal muscle myosin cDNAs were expressed in a simian kidney cell line (COS) and a mouse myogenic cell line to investigate the mechanisms controlling early stages of myosin filament assembly. An embryonic chicken muscle myosin heavy chain (MHC) cDNA was linked to constitutive promoters from adenovirus or SV40 and transiently expressed in COS cells. These cells accumulate hybrid myosin molecules composed of muscle MHCs and endogenous, nonmuscle, myosin light chains. The muscle myosin is found associated with a Triton insoluble fraction from extracts of the COS cells by immunoprecipitation and is detected in 2.4 +/- 0.8-micron-long filamentous structures distributed throughout the cytoplasm by immunofluorescence microscopy. These structures are shown by immunoelectron microscopy to correspond to loosely organized bundles of 12-16-nm-diameter myosin filaments. The muscle and nonmuscle MHCs are segregated in the transfected cells; the endogenous nonmuscle myosin displays a normal distribution pattern along stress fibers and does not colocalize with the muscle myosin filament bundles. A similar assembly pattern and distribution are observed for expression of the muscle MHC in a myogenic cell line. The myosin assembles into filament bundles, 1.5 +/- 0.6 micron in length, that are distributed throughout the cytoplasm of the undifferentiated myoblasts and segregated from the endogenous nonmuscle myosin. In both cell lines, formation of the myosin filament bundles is dependent on the accumulation of the protein. In contrast to these results, the expression of a truncated MHC that lacks much of the rod domain produces an assembly deficient molecule. The truncated MHC is diffusely distributed throughout the cytoplasm and not associated with cellular stress fibers. These results establish that the information necessary for the segregation of myosin isotypes into distinct cellular structures is contained within the primary structure of the MHC and that other factors are not required to establish this distribution.