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Changes in protein and glycoprotein biosynthesis during differentiation of satellite cells in vitro
4. DasGupta, C B & Summers; W C, Proc natl acad sci US 75 (1978) 2378. 5. Lytle; C D, Nat1 cancer inst monograph 50 (1978) 145. 6. Lytle. C D & Goddard, J D, Photochem photobiol 29 (1979) 959. 7. D’Ambrosio. S M & Setlow. R B. Proc natl acad sci US 73 (1976) 2396. 8. - Cancer res 38 (1978) 1147. 9. Painter, R B, Biochim biophys acta 609 (1980) 257. 10. - Nature 275 (1978) 243. 11. Nishiyama, Y & Rapp, F. J virol 38 (1981) 164. 12. Yoshida. S, Yamada, M, Masaki. S & Saneyoshi, M, Cancer res 39 (1979) 3955. 13. Yoshida, S & Masaki. S, Biochim biophys acta 561 (1979:)396. 14. Sirover, M A, Cancer res 39 (1979) 2090. 15, Nishiyama, Y & Rapp, FI Virology 100(1980) 189. 16. Painter. R B & Cleaver. J E. Radiation res 37 (1969) 451. 17 Lytle, C D, Int j radiat biol 19 (1971) 329. 18. Lehman. A R. Life sci 15 (1974) 2005. 19. Cleaver, J E & Thomas, GE. Biochem biophys res commun 36 (1969) 203. 20. Lytle, C D. Day III, R S, Hellman, K B & Bockstahler, L E, Mutation res 36 (1976) 257.
muscle tibroblasts; and (4) at least two distinct gtucosamine-containing acidic glycoproteins of aboui 70000 D and pI 5 were synthestzed by myotubes, but not by replicating satellite cells. These data demonstrate that the biosynthetic programs for proteins and glycoproteins of cultured replicating satelhte cells can be distinguished from those of multinucleated myotubes and from those of muscle fibroblasts. These data are interpreted to indicate that during muscle histogenesis in vivo, satellite cells become arrested prior to the expression of terminally differentiated traits.
Skeletal muscles in adult animals are known to have the capacity to regenerate after trauma or during degenerating myopathies [l]. However, both the nature and the source of the cells that form the regenerated adult muscles are still a matter of debate. The prevailing theory is that satellite cells [2] represent such a source [3, 61. This population of mitotically quiescent, mononucleated cells is located along the skeletal Received August 17: 1981 Revised version received December 28, 1981 muscle fiber between the sarcolemma and Accepted December 29, 1981 the basal lamina. The mechanism for the generation of satellite cells is still unclear, as is the stage of myogenesis at which these Copyright % 1982 by Academic Press. inc. cells become arrested. All rights of reproduction in any form reserved 0014.48??:83;010489-06$@~.~,@ In recent years, several laboratories have developed methods for the isolation and Changes in protein and glycoprotein culturing of satellite cells from adult skelebiosynthesis during differentiation of tal muscles; upon proper experimental cunsatellite cells in vitro ditions these cells resume mitotic activity C. BOITANI, G. COSSU.’ IM. BOUCHE. M. and terminally differentiate in vitro [4, 7-MOLINARO and M. PACIFICI,’ Isrir:!to di Istologia ed Embt=io!ogia Generale, Universirti di Roma. 1I]. We have substantially altered the methItaly od for isolating satellite cells to the end cf Srtnrmar~. Satellite cells were isolated from leg skeleobtaining quasi homogeneous populations tal muscles of adult mice and grown in culture. Durof cells [12]. This new method has allowed ing the first few days in culture, satellite cells actively proliferated and starting on day 4 began to fuse into us to analyse the biosynthetic program for multinucleated myotubes. At various time points durproteins and glycoproteins of replicating ing the culture period, the biosynthesis of total cellular proteins and glycoproteins was analysed by puIse- mononucleated satellite cells and to comlabelling with radioactive leucine or sugars followed by electrophoretic analysis on two-dimensional gels. Our pare it with those of multinucleared myofindings are: (1) Replicating mononucleated satellite tubes and muscle frbroblasts. cells on day 1 of culture did not synthesize detectable amounts of a! and /3 tropomyosins, cu-actin, and myosin light chains 1 and 2; (2) the synthesis of these polypeptides was readily detectable in multinucleated myotubes that formed by day 5-6 of culture; (3) other qualitative and quantitative differences in as yet unidentified proteins were observed in replicating cells as compared with multinucleated myotubes as well as to
r To whom offprint requests should be addressed: The Wistar Institute, 36th Street at Spruce, Philadeiphia, PA 19104, USA. e Present address: Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
490
Preliminary notes pCi/ml of L-[“Hlfucose (sp. act. 20 Cilmmole). During the labelling period. incorporation of the isotope into TCA-insoluble material was linear with time up to 24 h. Duplicate aliquots of the cell homogenate h:ere precipitated with 10% cold TCA. The precipitate was collected on Millipore filters and counted for radioactivity in a toluene-based scintillation fluid. Duplicate aliquots of the homogenate were used for protein determination by the method of Lowry et al. [13]. Electroohoretic mocedwes. At the end of the labelIing peridd: cells were washed three times with PBS. harvested in 10 mM Tris-HCI (pH 7.5), 0.15 %Nonidet P40 (NP40): 2 mM phenylmethylsulfonylfluoride IPMSF), and immediatelv boiled for 2 min. The homogenate ‘&as then incubaied for 5 min at 37°C in the presence of DNAse I (1 mglml) and boiled pancreatic RNAse -4 (1 mglml). The homogenate was then lyophilized and dissolved in 9.5 M urea. 2% ampholine (1.6% pH 5-7, and 0.4% pH 8-10) and 5% 2-mercaptoethanol; the concentration of NP40 was adjusted to 2%. Total proteins were analysed by twodimensional gel electrophoresis [14]. For second dimension analysis, samples were run on S-15% slab gels. Approx. 50 pg and 30-50~10~ cpm for [3H]glucosamine-labelled glycoproteins and 100-200~ lo3 cpm (for [3H]leucine-labelled proteins) were loaded onto the isoelectric focusing eel. Slab gels were fixed andprocessed for fluorography rl5l and exposed to X-ray film (Kodak X-Omat) at -70°C for periods ranging from 15 days to 4 months. Puri’cation of contractile proteins. The procedure followed for the puritication of actomyosin from mouse skeletal muscle was identical with that reported previously [ 163.To extract actomyosin from cultures, cells were homogenized in 0.1 M potassium phosphate buffer (pH 7.5), 1 M KCl, 5 mM MgC1, and 1 mM 2-mercaptoethanol, 100 pg of carrier actomyosin was added and the cell homogenate was dialysed against 50 mM phosphate buffer (pH 7). The precipitate was dissolved in 9 M urea, 2% ampholines, 2% NP40 and subjected to two-dimensional electrophoresis.
Fig. I. Phase contrast photographs of (A) l-day-old duplicating satellite cells, and (B) of j-day-old multinucleated myotubes.
Results released from adult The cell population mouse muscle by the sequential enzymatic treatments previously described was comMaterials and Me hods posed mainly of satellite cells with a few Cell cultures. Satellite cells were cultured as pre- muscle fibroblasts. These two cell types viously described [ll]. Muscle fibroblasts were obtained bv subculturing the cells that adhered to the could be readily distinguished during the plate duhng the pre-plating step. initial days of culture [12]. The satellite Cell labellina. Cultures were oulse-labelled with [3H]thymidine on day 1 of secondary cultures for 24 h cells were very small, round or spindleand analysed autoradiographically. Incorporation of shaped, with very little cytoplasm (fig. 1A). the label by 95% of total nuclei indicated that the majority of satellite cells were engaged in DNA syn- The fibroblasts were flat and wide, with thesis on days l-2 of culture (data not shown). At large nuclei and an overall irregular shape. the indicated periods of culture, cells were incubated for 2 or 6 h in the oresence of 50 uCi/ml of r3H1- With time in culture, satellite cells progresleucine (sp. act. 37 dilmmole) or it&bated for 54 h sively enlarged in size and most became in the presence of either 50 &i/ml of D-[3H]g1ucosamine hydrochloride (sp. act. 10 Ci/mmole) or 50 spindle-shaped. Cell fusion began routinely Exp Cell Res 138 (1982)
Preliminury notes
2. Fluorograms of two-dimensional gel electrophoresis of total proteins synthesized by (A) l-day-old satellite cells; (B) Sday-old myotubes; and (C) by muscle fibroblasts. Examples of polppeptides which show major variations among different cell types are indicated by numbers. Fluorograms of purified contractile proteins from (A, imef) l-day-old satellite cells; (I?. inset) S-day-old myotubes.
Fig.
491
between days 3 and 4, and by day 5 approx. 80% of all nuclei were found within multinucleated cells (fig. 1B). Cultures were pulse-labelled on days 1 and 5 of secondary cultures with [3H]leutine. Total newly-synthesized proteins were analysed by two-dimensional gel electrophoresis. Fig. 2A shows the pattern relative to day 1 replicating cells, and fig. 2B, the pattern relative to day 5 multinucleated myotube cultures. Several quantitative and qualitative differences were observed, some of which are indicated by numbered arrows. In particular, the muscle-specific sarcomeric proteins, including myosin light chains 1 and 2, (Yand p tropomyosins and a actin were clearly detectable on fluorographies of radiolabelled proteins synthesized by day 5 myotubes; arrow 3 indicates a protein tentatively identified as the muscle-specific intermediate filament subunit or desmin [17] according to its molecular weight and its isoelectric point. Conversely, day I replicating cells did not synthesize any of the sarcomeric proteins, nor did they synthesize the protein indicated by arrow 3. Myosin heavy chains were not resolved by this electrophoretic system, since they migrated at the basic end of the gel (left). Also myosin light chain 3 was not detected in fluorographies of .labelled myotube samples. The same results are obtained when only the newly synthesized contractile proteins are extracted from day 1 and day 5 cultures (insets of fig. 2A. B), Fig. 2C shows the pattern of proteins synthesized by muscle tibroblasts, which were obtained after the pre-plating step and were grown for 5 days in vitro. ,4 comparison of fig, 2L” with fig. 2A and B revealed a basic similarity in the patterns of proteins synthesized by fibroblasts, satellite cells and myotubes, respectively. However, it was possible to identify some protein species present in
492
Preliminary notes The patterns have some basic similarities to those reported for total cellular glycoproteins synthesized by other types of cells [18] in that they consist of glycoprotein species resolved as discrete spots, as well as some that appear as a series of spots with the same molecular weight but of different charge. Charge heterogeneity in our samples was particularly evident in the acidic, high molecular weight region of the gel (130-80 kD and p1 6-5), which comprises about half of the radioactivity on the gel. When comparing fig. 3A with 3B it is seen that two acidic glycoproteins (nos. 1 and 2) were synthesized by multinucleated myotubes but not by’ replicating satellite cells. These two glycoproteins were not detectable when cells were labelled with fucose. A considerable amount of a high molecular weight glycoprotein (no. 3) was detected in day 1 satellite cells, but was found only in very small amounts in day 5 myotubes.
Discussion The significance of the biochemical data reported here lies primarily in the fact that our cultures are essentially homogeneous. The presence of a conspicuous population of muscle tibroblasts, a common contamifibroblasts and in satellite cells but absent nant in primary muscle cultures, would in myotubes (i.e., nos. 1, 4 and 5); con- have hampered the analysis of the biosynversely, other proteins present in satellite thetic options of replicating mononucleated cells and in myotubes were not detected in satellite cells. Interestingly, we observed tibroblasts (i.e., no. 6). Thus, it is clear that that some of the polypeptides present in each of these three cell types examined is replicating satellite cells but not in myoable to synthesize a specific pattern of pro- tubes were present in cultures of muscle fibroblasts. Conversely, other polypeptides teins. In fig. 3A, B the two-dimensional sepa- common to both replicating satellite cells and myotubes were not found in tibroblasts. ration is seen of total [“H]glucosaminelabelled glycoproteins synthesized by day 1 These data indicate that replicating satellite replicating satellite cells and by day 5 multi- cells are distinguishable phenotypically nucleated myotubes in secondary cultures. from muscle fibroblasts. Although satellite Erp Cell Res 138 (1982)
Preliminary mies cells do not synthesize skeletal musclespecific proteins, they are probably committed to terminal muscle differentiation. Our findings correlate well with reports of low levels of total creatine kinase activity in replicating satellite cells in culture [12, 191and with almost undetectable levels of acetylcholine receptor and acetylcholinesterase (unpublished observations). Conversely, multinucleated myotubes derived from fusion of mononucleated satellite cells readily synthesize the sarcomeric proteins mentioned above and, as reported elsewhere [12], also synthesize high levels of creatine kinase. These myotubes, however, do not synthesize myosin light chain 3, which is normally present in adult fast skeletal muscles [20]. The first conclusion that can be drawn from our experimental observations and from the data reported by others [S, 191 is that the biosynthetic program of replicating satellite cells in vitro has striking similarities to that of cultured embryonic replicating myoblasts of avian origin [21, 221. Similarly, the biosynthetic program of multinucleated myotubes formed by fusion of satellite cells resemble that of embryonic multinucleated myotubes. It is clear, therefore, that during embryogenesis satellite cells become quiescent before the expression of the terminally differentiated program and share at least in part some properties of embryonic presumptive replicating myroblasts. These conclusions are further strengthened by the data obtained from the analysis of total cellular glycoproteins. At least two acidic, glucosamine-containing glycoproteins are synthesized by multinucleated myrotubes: but not by mononucleated satellite cells, whereas other glycoproteins appear to be more prominent in mononucleated cells. The resolution of glycopro-
493
teins by two-dimensional gel electrophoresis is not precise, particularly in the highmolecular weight region of the gel; thus, our data suggest a more complex situation. We present here the first description of qualitative changes in glycoproteins during myogenic cell differentiation in culture. These changes were not detected in earlier studies which used mono-dimensional gel electrophoresis [23-251. Since the nature of these myotube glycoproteins is not known at present, we cannot correlate our data with the changes in glycopeptides reported to occur during the differentiation of satellite cells in culture [26], as well as that of chick embryo skeletal myogenic cells 124. Taken together, the data reported here confirm and extend the morphological observations of Bischoff [4, 281 and Konigsberg [6, 111.These authors isolated and cultivated individual muscle fibers and demonstrated that mononucleated cells, derived from these fibers, did not contain striated myofibrils in their cytoplasm [26]. After myotube formation, the characteristic myofibrillar cross-striation was readily visible. The results of the present study can now be used as background for a detailed biochemical as well as morphological comparison berween mouse embryonic presumptive replicating myoblasts and adult mouse satellite cells. This may lead to a better understanding of the ontogenesis of satellite cells, namely whether these celts represent a distinct lineage of myogenic cells or simply a fraction of a homogeneous population of embryonic myogenic cells.
We wish to thank Drs H. Holtzer and N. Rubinstein for helpful suggestions and critical reading of the manuscript. We also wish to thank Mr M. Coletta for excellent technical assistance and Mrs H. SchGrrand M. Hoffman for editing the manuscript. This work was supported by grants CNR No. 76.01l& and frern Y\1.4TONo. 1620to M. ?*1.
494
Preliminary notes
References 1. Mauro, A, J biophys biochem 9 (1961) 493. 2. Mauro, A, Shaquif, S A & Milhorat, A T, Regeneration of striated muscles and myogenesis. Excerpta Medica, Amsterdam (1970). 3. Carlson, B M, Amj anat 137 (1973) 119. 4. Bischoff, R, Anat ret 183 (1975) 215. 5. Lipton, B H & Schultz, E, Science 205 (1979) 1292. 6. Konigsberg, I R, Muscle regeneration (ed A Mauro) p. 41. Raven Press? New York (1979). 7 Mawatari, S, Miranda, A & Rowland, L P, Neurology 26 (1976) 1021. 8. Yasin. R, Van Beers, G; Bulien, D & Thompson. E J, Exp cell res 102 (1976) 405. 9. Askanas, V & Engel, W K, Neurology 25 (1975) 58.
10. Witkoski, J A, Durbridge; M & Dubowitz, V, In vitro 12 (1976) 98: 11. Konigsberg, U, Lipton. B H & Konigsberg, I R1 Dev biol45 (1975) 260. 12. Cossu, G; Zani, B, Coletta, M1 Bouche, M, Pacifici, M & Molinaro, M, Cell differ 9 (1980) 357. 13. Lowry, 0 H, Rosebrough, NJ, Farr. A L & Randall, R J, J biol them 193 (1951) 265. 14. O’Farrell, P H, J biol them 250 (1975) 4007. 15. Bonner. w M .& Laskey, R A, ‘Eur ; biochem 46 (1974) 83. 16. ?ani,‘B, Cossu, G, Adamo, S & Molinaro, M, Differentiation 10 (1978) 95. 17. Lazarides, E, Nature 283 (1980) 249. 18. Baumann, H & Doyle, D. J biol them 254 (1979)
Copyright Q 19k?-by Academic Press. Inc. All rights of reproduction in any form reserved 0014.4827:8?;040494-05SO1.00;0
Enhancement in ‘apparent’ membrane microviscosity during differentiation of embryonal carcinoma cells induced by retinoids ANTON M. JETTEN? LUIGI M. DE LUCA and ROBERT G. MEEKS.’ Laboraiog of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bldg 37, Rm 2B26, Bethesda, MD 20205, USA Summa~. Upon differentiation of embryonal carcinoma cells induced by retinoids (lo-; M) the ‘apparent’ membrane microviscosity increases dramatically. Only biologically active retinoids induce differentiation and cause an enhancement in microviscosity. Several embryonal carcinoma cell lines have a relatively lower ‘apparent’ microviscosity than their differentiated derivatives, suggesting that this may be a general property of these cells. At higher concentrations retinoids cause a reduction in ‘apparent’ membrane microviscosity of various cells. This change occurs whether the analogue is biologically active or not, indicating the non-specific nature of this action.
Retinoids, analogs of vitamin A, elicit a large number of biological and biochemical 2542. changes in a variety of cell types [l: 21. 19. Yaffe, D & Saxel, 0, Nature 270 (1977) 725. 20. Rubinstein. N A & Holtzer. H. Nature 280 (1979) ~ , In many instances retinoids alter the adhe323. siveness of cells to the substratum. Further21. Chi, J C, Fellini, S A & Holtzer, HI Proc natl acad sci US 72 (1975) 4999. more, this group of compounds inhibit the 22. Devlin, R B & Emerson. C P J, Cell 13 (1978) 599. growth of a large number of transformed 23. Hynes; R 0, Martin, G S, Schear, M, Ciitchiey, D R & Eostein. C J. Dev bio148 (1976) 35. and non-transformed cell lines. Retinoids ’ 24. Chen, ‘LB, Cell lb (1977) 393. ~ 25. Gates, G A & Holland, J J, Biochem j 174 (1978) can also induce differentiation of a variety 873. of embryonal carcinoma cell lines [3, 41. 26. Cossu, G, Pacifici, M, Marino, M, Zani, B, Coletta. M & Molinaro, M. Exp cell res 132 (1981) During this differentiation process a large 349. number of changes in cell surface properties 27. Marino, M, Cossu, G, Neri, G & Molinaro. M, occur such as alterations in glycolipids, fiDev biol78 (1980) 258. 28. Bischoff, R, Muscle regeneration (ed A Mauro) p. bronectin and collagen [4-71, and binding 13. Raven Press, New York (1979). of epidermal growth factor (EGF) [2, 8-j. Received October 5, 1981 Chytil & Ong [9] have proposed that the Revised version received December 15, 1981 specific retinoic acid and retinol-binding Accepted December 28, 1981 proteins present in the cytosol are involved in the mediation of the action of retinoids. It has been shown recently [lo, 111 that these specific binding proteins are likely to ’ Address: Southern Research Institute, Birmingham, 4L 35255. USA. Esp Cell Res I38 (1982)
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muscle tibroblasts; and (4) at least two distinct gtucosamine-containing acidic glycoproteins of aboui 70000 D and pI 5 were synthestzed by myotubes, but not by replicating satellite cells. These data demonstrate that the biosynthetic programs for proteins and glycoproteins of cultured replicating satelhte cells can be distinguished from those of multinucleated myotubes and from those of muscle fibroblasts. These data are interpreted to indicate that during muscle histogenesis in vivo, satellite cells become arrested prior to the expression of terminally differentiated traits.
Skeletal muscles in adult animals are known to have the capacity to regenerate after trauma or during degenerating myopathies [l]. However, both the nature and the source of the cells that form the regenerated adult muscles are still a matter of debate. The prevailing theory is that satellite cells [2] represent such a source [3, 61. This population of mitotically quiescent, mononucleated cells is located along the skeletal Received August 17: 1981 Revised version received December 28, 1981 muscle fiber between the sarcolemma and Accepted December 29, 1981 the basal lamina. The mechanism for the generation of satellite cells is still unclear, as is the stage of myogenesis at which these Copyright % 1982 by Academic Press. inc. cells become arrested. All rights of reproduction in any form reserved 0014.48??:83;010489-06$@~.~,@ In recent years, several laboratories have developed methods for the isolation and Changes in protein and glycoprotein culturing of satellite cells from adult skelebiosynthesis during differentiation of tal muscles; upon proper experimental cunsatellite cells in vitro ditions these cells resume mitotic activity C. BOITANI, G. COSSU.’ IM. BOUCHE. M. and terminally differentiate in vitro [4, 7-MOLINARO and M. PACIFICI,’ Isrir:!to di Istologia ed Embt=io!ogia Generale, Universirti di Roma. 1I]. We have substantially altered the methItaly od for isolating satellite cells to the end cf Srtnrmar~. Satellite cells were isolated from leg skeleobtaining quasi homogeneous populations tal muscles of adult mice and grown in culture. Durof cells [12]. This new method has allowed ing the first few days in culture, satellite cells actively proliferated and starting on day 4 began to fuse into us to analyse the biosynthetic program for multinucleated myotubes. At various time points durproteins and glycoproteins of replicating ing the culture period, the biosynthesis of total cellular proteins and glycoproteins was analysed by puIse- mononucleated satellite cells and to comlabelling with radioactive leucine or sugars followed by electrophoretic analysis on two-dimensional gels. Our pare it with those of multinucleared myofindings are: (1) Replicating mononucleated satellite tubes and muscle frbroblasts. cells on day 1 of culture did not synthesize detectable amounts of a! and /3 tropomyosins, cu-actin, and myosin light chains 1 and 2; (2) the synthesis of these polypeptides was readily detectable in multinucleated myotubes that formed by day 5-6 of culture; (3) other qualitative and quantitative differences in as yet unidentified proteins were observed in replicating cells as compared with multinucleated myotubes as well as to
r To whom offprint requests should be addressed: The Wistar Institute, 36th Street at Spruce, Philadeiphia, PA 19104, USA. e Present address: Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
490
Preliminary notes pCi/ml of L-[“Hlfucose (sp. act. 20 Cilmmole). During the labelling period. incorporation of the isotope into TCA-insoluble material was linear with time up to 24 h. Duplicate aliquots of the cell homogenate h:ere precipitated with 10% cold TCA. The precipitate was collected on Millipore filters and counted for radioactivity in a toluene-based scintillation fluid. Duplicate aliquots of the homogenate were used for protein determination by the method of Lowry et al. [13]. Electroohoretic mocedwes. At the end of the labelIing peridd: cells were washed three times with PBS. harvested in 10 mM Tris-HCI (pH 7.5), 0.15 %Nonidet P40 (NP40): 2 mM phenylmethylsulfonylfluoride IPMSF), and immediatelv boiled for 2 min. The homogenate ‘&as then incubaied for 5 min at 37°C in the presence of DNAse I (1 mglml) and boiled pancreatic RNAse -4 (1 mglml). The homogenate was then lyophilized and dissolved in 9.5 M urea. 2% ampholine (1.6% pH 5-7, and 0.4% pH 8-10) and 5% 2-mercaptoethanol; the concentration of NP40 was adjusted to 2%. Total proteins were analysed by twodimensional gel electrophoresis [14]. For second dimension analysis, samples were run on S-15% slab gels. Approx. 50 pg and 30-50~10~ cpm for [3H]glucosamine-labelled glycoproteins and 100-200~ lo3 cpm (for [3H]leucine-labelled proteins) were loaded onto the isoelectric focusing eel. Slab gels were fixed andprocessed for fluorography rl5l and exposed to X-ray film (Kodak X-Omat) at -70°C for periods ranging from 15 days to 4 months. Puri’cation of contractile proteins. The procedure followed for the puritication of actomyosin from mouse skeletal muscle was identical with that reported previously [ 163.To extract actomyosin from cultures, cells were homogenized in 0.1 M potassium phosphate buffer (pH 7.5), 1 M KCl, 5 mM MgC1, and 1 mM 2-mercaptoethanol, 100 pg of carrier actomyosin was added and the cell homogenate was dialysed against 50 mM phosphate buffer (pH 7). The precipitate was dissolved in 9 M urea, 2% ampholines, 2% NP40 and subjected to two-dimensional electrophoresis.
Fig. I. Phase contrast photographs of (A) l-day-old duplicating satellite cells, and (B) of j-day-old multinucleated myotubes.
Results released from adult The cell population mouse muscle by the sequential enzymatic treatments previously described was comMaterials and Me hods posed mainly of satellite cells with a few Cell cultures. Satellite cells were cultured as pre- muscle fibroblasts. These two cell types viously described [ll]. Muscle fibroblasts were obtained bv subculturing the cells that adhered to the could be readily distinguished during the plate duhng the pre-plating step. initial days of culture [12]. The satellite Cell labellina. Cultures were oulse-labelled with [3H]thymidine on day 1 of secondary cultures for 24 h cells were very small, round or spindleand analysed autoradiographically. Incorporation of shaped, with very little cytoplasm (fig. 1A). the label by 95% of total nuclei indicated that the majority of satellite cells were engaged in DNA syn- The fibroblasts were flat and wide, with thesis on days l-2 of culture (data not shown). At large nuclei and an overall irregular shape. the indicated periods of culture, cells were incubated for 2 or 6 h in the oresence of 50 uCi/ml of r3H1- With time in culture, satellite cells progresleucine (sp. act. 37 dilmmole) or it&bated for 54 h sively enlarged in size and most became in the presence of either 50 &i/ml of D-[3H]g1ucosamine hydrochloride (sp. act. 10 Ci/mmole) or 50 spindle-shaped. Cell fusion began routinely Exp Cell Res 138 (1982)
Preliminury notes
2. Fluorograms of two-dimensional gel electrophoresis of total proteins synthesized by (A) l-day-old satellite cells; (B) Sday-old myotubes; and (C) by muscle fibroblasts. Examples of polppeptides which show major variations among different cell types are indicated by numbers. Fluorograms of purified contractile proteins from (A, imef) l-day-old satellite cells; (I?. inset) S-day-old myotubes.
Fig.
491
between days 3 and 4, and by day 5 approx. 80% of all nuclei were found within multinucleated cells (fig. 1B). Cultures were pulse-labelled on days 1 and 5 of secondary cultures with [3H]leutine. Total newly-synthesized proteins were analysed by two-dimensional gel electrophoresis. Fig. 2A shows the pattern relative to day 1 replicating cells, and fig. 2B, the pattern relative to day 5 multinucleated myotube cultures. Several quantitative and qualitative differences were observed, some of which are indicated by numbered arrows. In particular, the muscle-specific sarcomeric proteins, including myosin light chains 1 and 2, (Yand p tropomyosins and a actin were clearly detectable on fluorographies of radiolabelled proteins synthesized by day 5 myotubes; arrow 3 indicates a protein tentatively identified as the muscle-specific intermediate filament subunit or desmin [17] according to its molecular weight and its isoelectric point. Conversely, day I replicating cells did not synthesize any of the sarcomeric proteins, nor did they synthesize the protein indicated by arrow 3. Myosin heavy chains were not resolved by this electrophoretic system, since they migrated at the basic end of the gel (left). Also myosin light chain 3 was not detected in fluorographies of .labelled myotube samples. The same results are obtained when only the newly synthesized contractile proteins are extracted from day 1 and day 5 cultures (insets of fig. 2A. B), Fig. 2C shows the pattern of proteins synthesized by muscle tibroblasts, which were obtained after the pre-plating step and were grown for 5 days in vitro. ,4 comparison of fig, 2L” with fig. 2A and B revealed a basic similarity in the patterns of proteins synthesized by fibroblasts, satellite cells and myotubes, respectively. However, it was possible to identify some protein species present in
492
Preliminary notes The patterns have some basic similarities to those reported for total cellular glycoproteins synthesized by other types of cells [18] in that they consist of glycoprotein species resolved as discrete spots, as well as some that appear as a series of spots with the same molecular weight but of different charge. Charge heterogeneity in our samples was particularly evident in the acidic, high molecular weight region of the gel (130-80 kD and p1 6-5), which comprises about half of the radioactivity on the gel. When comparing fig. 3A with 3B it is seen that two acidic glycoproteins (nos. 1 and 2) were synthesized by multinucleated myotubes but not by’ replicating satellite cells. These two glycoproteins were not detectable when cells were labelled with fucose. A considerable amount of a high molecular weight glycoprotein (no. 3) was detected in day 1 satellite cells, but was found only in very small amounts in day 5 myotubes.
Discussion The significance of the biochemical data reported here lies primarily in the fact that our cultures are essentially homogeneous. The presence of a conspicuous population of muscle tibroblasts, a common contamifibroblasts and in satellite cells but absent nant in primary muscle cultures, would in myotubes (i.e., nos. 1, 4 and 5); con- have hampered the analysis of the biosynversely, other proteins present in satellite thetic options of replicating mononucleated cells and in myotubes were not detected in satellite cells. Interestingly, we observed tibroblasts (i.e., no. 6). Thus, it is clear that that some of the polypeptides present in each of these three cell types examined is replicating satellite cells but not in myoable to synthesize a specific pattern of pro- tubes were present in cultures of muscle fibroblasts. Conversely, other polypeptides teins. In fig. 3A, B the two-dimensional sepa- common to both replicating satellite cells and myotubes were not found in tibroblasts. ration is seen of total [“H]glucosaminelabelled glycoproteins synthesized by day 1 These data indicate that replicating satellite replicating satellite cells and by day 5 multi- cells are distinguishable phenotypically nucleated myotubes in secondary cultures. from muscle fibroblasts. Although satellite Erp Cell Res 138 (1982)
Preliminary mies cells do not synthesize skeletal musclespecific proteins, they are probably committed to terminal muscle differentiation. Our findings correlate well with reports of low levels of total creatine kinase activity in replicating satellite cells in culture [12, 191and with almost undetectable levels of acetylcholine receptor and acetylcholinesterase (unpublished observations). Conversely, multinucleated myotubes derived from fusion of mononucleated satellite cells readily synthesize the sarcomeric proteins mentioned above and, as reported elsewhere [12], also synthesize high levels of creatine kinase. These myotubes, however, do not synthesize myosin light chain 3, which is normally present in adult fast skeletal muscles [20]. The first conclusion that can be drawn from our experimental observations and from the data reported by others [S, 191 is that the biosynthetic program of replicating satellite cells in vitro has striking similarities to that of cultured embryonic replicating myoblasts of avian origin [21, 221. Similarly, the biosynthetic program of multinucleated myotubes formed by fusion of satellite cells resemble that of embryonic multinucleated myotubes. It is clear, therefore, that during embryogenesis satellite cells become quiescent before the expression of the terminally differentiated program and share at least in part some properties of embryonic presumptive replicating myroblasts. These conclusions are further strengthened by the data obtained from the analysis of total cellular glycoproteins. At least two acidic, glucosamine-containing glycoproteins are synthesized by multinucleated myrotubes: but not by mononucleated satellite cells, whereas other glycoproteins appear to be more prominent in mononucleated cells. The resolution of glycopro-
493
teins by two-dimensional gel electrophoresis is not precise, particularly in the highmolecular weight region of the gel; thus, our data suggest a more complex situation. We present here the first description of qualitative changes in glycoproteins during myogenic cell differentiation in culture. These changes were not detected in earlier studies which used mono-dimensional gel electrophoresis [23-251. Since the nature of these myotube glycoproteins is not known at present, we cannot correlate our data with the changes in glycopeptides reported to occur during the differentiation of satellite cells in culture [26], as well as that of chick embryo skeletal myogenic cells 124. Taken together, the data reported here confirm and extend the morphological observations of Bischoff [4, 281 and Konigsberg [6, 111.These authors isolated and cultivated individual muscle fibers and demonstrated that mononucleated cells, derived from these fibers, did not contain striated myofibrils in their cytoplasm [26]. After myotube formation, the characteristic myofibrillar cross-striation was readily visible. The results of the present study can now be used as background for a detailed biochemical as well as morphological comparison berween mouse embryonic presumptive replicating myoblasts and adult mouse satellite cells. This may lead to a better understanding of the ontogenesis of satellite cells, namely whether these celts represent a distinct lineage of myogenic cells or simply a fraction of a homogeneous population of embryonic myogenic cells.
We wish to thank Drs H. Holtzer and N. Rubinstein for helpful suggestions and critical reading of the manuscript. We also wish to thank Mr M. Coletta for excellent technical assistance and Mrs H. SchGrrand M. Hoffman for editing the manuscript. This work was supported by grants CNR No. 76.01l& and frern Y\1.4TONo. 1620to M. ?*1.
494
Preliminary notes
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10. Witkoski, J A, Durbridge; M & Dubowitz, V, In vitro 12 (1976) 98: 11. Konigsberg, U, Lipton. B H & Konigsberg, I R1 Dev biol45 (1975) 260. 12. Cossu, G; Zani, B, Coletta, M1 Bouche, M, Pacifici, M & Molinaro, M, Cell differ 9 (1980) 357. 13. Lowry, 0 H, Rosebrough, NJ, Farr. A L & Randall, R J, J biol them 193 (1951) 265. 14. O’Farrell, P H, J biol them 250 (1975) 4007. 15. Bonner. w M .& Laskey, R A, ‘Eur ; biochem 46 (1974) 83. 16. ?ani,‘B, Cossu, G, Adamo, S & Molinaro, M, Differentiation 10 (1978) 95. 17. Lazarides, E, Nature 283 (1980) 249. 18. Baumann, H & Doyle, D. J biol them 254 (1979)
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Enhancement in ‘apparent’ membrane microviscosity during differentiation of embryonal carcinoma cells induced by retinoids ANTON M. JETTEN? LUIGI M. DE LUCA and ROBERT G. MEEKS.’ Laboraiog of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institutes of Health, Bldg 37, Rm 2B26, Bethesda, MD 20205, USA Summa~. Upon differentiation of embryonal carcinoma cells induced by retinoids (lo-; M) the ‘apparent’ membrane microviscosity increases dramatically. Only biologically active retinoids induce differentiation and cause an enhancement in microviscosity. Several embryonal carcinoma cell lines have a relatively lower ‘apparent’ microviscosity than their differentiated derivatives, suggesting that this may be a general property of these cells. At higher concentrations retinoids cause a reduction in ‘apparent’ membrane microviscosity of various cells. This change occurs whether the analogue is biologically active or not, indicating the non-specific nature of this action.
Retinoids, analogs of vitamin A, elicit a large number of biological and biochemical 2542. changes in a variety of cell types [l: 21. 19. Yaffe, D & Saxel, 0, Nature 270 (1977) 725. 20. Rubinstein. N A & Holtzer. H. Nature 280 (1979) ~ , In many instances retinoids alter the adhe323. siveness of cells to the substratum. Further21. Chi, J C, Fellini, S A & Holtzer, HI Proc natl acad sci US 72 (1975) 4999. more, this group of compounds inhibit the 22. Devlin, R B & Emerson. C P J, Cell 13 (1978) 599. growth of a large number of transformed 23. Hynes; R 0, Martin, G S, Schear, M, Ciitchiey, D R & Eostein. C J. Dev bio148 (1976) 35. and non-transformed cell lines. Retinoids ’ 24. Chen, ‘LB, Cell lb (1977) 393. ~ 25. Gates, G A & Holland, J J, Biochem j 174 (1978) can also induce differentiation of a variety 873. of embryonal carcinoma cell lines [3, 41. 26. Cossu, G, Pacifici, M, Marino, M, Zani, B, Coletta. M & Molinaro, M. Exp cell res 132 (1981) During this differentiation process a large 349. number of changes in cell surface properties 27. Marino, M, Cossu, G, Neri, G & Molinaro. M, occur such as alterations in glycolipids, fiDev biol78 (1980) 258. 28. Bischoff, R, Muscle regeneration (ed A Mauro) p. bronectin and collagen [4-71, and binding 13. Raven Press, New York (1979). of epidermal growth factor (EGF) [2, 8-j. Received October 5, 1981 Chytil & Ong [9] have proposed that the Revised version received December 15, 1981 specific retinoic acid and retinol-binding Accepted December 28, 1981 proteins present in the cytosol are involved in the mediation of the action of retinoids. It has been shown recently [lo, 111 that these specific binding proteins are likely to ’ Address: Southern Research Institute, Birmingham, 4L 35255. USA. Esp Cell Res I38 (1982)
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