DEVELOPMENTAL
BIOLOGY
48, 431-437
Myosin Synthesis
(1976)
by Fusion-Arrested Chick Embryo Culture PAUL S. Moss
Department
of Zoology,
AND RICHARD
University Accepted
of California, September
Myoblasts
in Cell
C. STROHMAN Berkeley,
California
94720
19, 1975
The synthesis and accumulation of myosin was studied in subcultures of fusion-blocked, postmitotic embryonic chicken myogenic cells. Electron micrographs and fluorescent microscopy with antimyosin revealed that most, if not all, of these cells contain myosin. It was also found that these cells are capable of accumulating myosin at rates comparable to fused cells. Incipient T-tubule formation was also present in some of the blocked cells. It is concluded that cell fusion is not a prerequisite for myosin synthesis and accumulation or T-tubule formation during myogenesis in vitro.
the synthesis and assembly of myosin filaments (Paterson and Strohman, 1972). In this report, however, we show that if postmitotic fusion-blocked myogenic cells are maintained in subculture, they synthesize and accumulate myosin at rates comparable to normally differentiated myotubes. It is also demonstrated that some of these fusion-arrested cells are capable of assembling an incipient T-tubule system. These results, and those of the accompanying paper by Vertel and Fischman (1975) and the recent report by Emerson and Beckner (1975) demonstrate that myogenic cell fusion is not a prerequisite for the activation of myosin synthesis during skeletal muscle myogenesis.
INTRODUCTION
The timing of myosin synthesis during chicken skeletal muscle myogenesis has been the subject of many reports. Most in vitro studies of muscle differentiation have demonstrated that myogenic cell fusion normally precedes the bulk accumulation of myosin and the appearance of myofilaments (for review, see Fischman, 1972). However, Holtzer et al. (1957) demonstrated the elaboration of myofilaments in mononucleated somitic myoblasts by stage 16 in viuo, and Okazaki and Holtzer (1965, 1966) found that at least some single myogenic cells can synthesize myosin prior to fusion in vitro (see also Coleman and Coleman, 1968; and Dienstman, 1974). In an earlier communication from this laboratory (Paterson and Strohman, 19721, the high rates of myosin synthesis normally associated with fully differentiated myotubes were not observed in culture until after cell fusion, and even then not until after a lag period of from 8-12 hr. In addition, when fusion is blocked by lowering the external calcium concentration, myogenic cells become postmitotic but do not enter into the accelerating rate of myosin synthesis observed for normal fused cultures. These considerations led to the conclusion that cell fusion is a normal prerequisite for further differentiation, including
MATERIALS
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Cells. Primary cultures of 12-day embryonic muscle cells were prepared as previously described (Paterson and Strohman, 1972). Fusion-blocked cells were obtained by feeding the cells at 24 hr with a low calcium medium (Chelex medium) prepared by the method of Paterson and Prives (1973). Subcultures of these fusionarrested cells were prepared by rinsing the monolayers with calcium-free MEM, followed by a 5- to B-min incubation at room temperature in 0.02% trypsin (Difco) diluted in calcium-free MEM. The cell sus431
Copyright All rights
AND
432
DEVELOPMENTAL
BIOLOGY
pension was rinsed twice in fresh Chelex medium, filtered through four layers of Nitex in a double Swinney, and preplated (Fambrough and Rash, 1971) in 5 ml at 2-3 x lo6 cells/ml for 20-30 min on noncollagen coated loo-mm Falcon dishes. The cells were then plated in fresh Chelex medium on collagen-coated Falcon dishes at the concentration indicated in the figure legend. Cultures of chick embryo fibroblasts were prepared from overgrown (6day) myogenic cultures. Methods for cell counts, fusion indices, autoradiography, and electron microscopy were those standard in this laboratory (Paterson and Strohman, 1972). Antiserum. Preparation and testing of antiserum to column purified chicken breast myosin essentially followed the procedures of Lowey and Steiner (1972). Each rabbit was immunized with 4 mg of antigen in complete Freund’s Adjuvant injected subcutaneously in the rear footpads and back of the neck. The animals became positive after 2 weeks and were boosted with 2 mg at 1 month. Thereafter, blood was collected from the marginal ear vein at 2-week intervals and assayed for antimyosin activity by the quantitative precipitin test in high salt buffer (Lowey and Steiner, 1972). The IgG fraction was isolated and purified by precipitation in 40% ammonium sulfate at room temperature and chromatography on Whatman DE-52. Fluorescence microscopy. Cells were examined for myosin content by the indirect fluorescent antibody technique (Weller and Coons, 1954). All procedures were carried out directly on 35mm collagen-coated Falcon culture dishes. After fixation in acetone:ethanol (1:l) for 3 min at o”C, the monolayer-s were rinsed three times in phosphate buffered saline (PBS) and incubated with immune (or preimmune) globulin (1-3 mg/ml) in PBS at 37°C for 20 min. After rinsing extensively in PBS, the cells were treated with fluorescein-conjugated goat anti-rabbit IgG (GAR-FITC; Antibodies, Inc.) diluted 1:7 in PBS for 20 min at
VOLUME
48, 1976
37°C. The cells were then rinsed thoroughly in PBS and mounted in glycerol:water (9:l) prior to examination with an epi-illuminated Leitz Orthopan fluorescent microscope. Myosin synthesis and accumulation. The rates of synthesis and accumulation of myosin heavy chain (MHC) were determined by the method of Paterson and Strohman (1972) with the following modifications: (1) The cells were pulsed in 4 ml of labeling medium consisting of 1 part fresh complete or Chelex medium and 1 part conditioned medium (centrifuged medium from sister cultures of approximately the same age) containing 15 &i/ml of [4,5H311eucine (30-50 Ci/mmole) (Schwartz/ Mann). (2) The SDS-polyacrylamide gels were run without the addition of carrier myosin to the high salt extract. The gels were stained for 3 hr, destained for 20 hr with one change, and the region of the gel containing the band comigrating with a column chromatographed MHC standard was scanned at 550 nm on a Gilford Linear Gel Scanner. Complete and specific precipitation of the 220,000 MW protein from the high salt extract by antimyosin demonstrated that this band is definitely MHC (Moss, unpublished results). The amount of MHC accumulated was determined by comparing the absorbance with that of a series of standard gels loaded with l-10 pg of column-purified myosin. To determine the rate of synthesis of MHC, the gels were frozen, sliced, and counted as previously described (Paterson and Strohman, 1972). RESULTS
Cell counts and autoradiographs of fusion blocked cells subcultured at 68 hr demonstrated that only 5-6% of these cells were labeled when grown continuously in a calcium deficient medium and tritiated thymidine (Fig. 11, confirming the postmitotic nature of these cells (Paterson and Strohman, 1972). After l-2 days in subculture, most of the myogenic cells appeared
Moss
AND
STROHMAN
Myosin Synthesis by Myoblasts
FIG. 1A. Growth and [H”]thymidine labeling of myogenic cultures (2 x lo5 tells/60-mm dish) transferred at 68 hr and maintained in low calcium medium. Growth of: O-O, myoblasts; O-O, fibroblasts. x 750.
by phase microscopy to thicken and become more refractile as compared to their earlier, thinner and more elongated shape. Electron microscopy of eight of these cells revealed that all of them contained thick filaments (Fig. 2). Interestingly, although the filaments appeared to be fairly wellaligned, all but one of the cells lacked obvious Z bands. Two of these cells had also developed what appears to be the incipient T-tubule system similar to that observed in young myotubes by Ezerman and Ishikawa (1967) (Fig. 3). To demonstrate that the eight cells examined with the electron microscope were representative of all of the single cells, the cultures were assayed for myosin by the indirect fluorescent antibody technique. Figure 4 demonstrates the ability of the antiserum to react specifically with the A bands of cross-striated myotubes. When the same experiment was done with subcultures of fusion-blocked cells, more than 90% of the myogenic cells stained with the antimyosin (Fig. 4). Cross striation in these cells, however, was only rarely observed.
FIG. 1B. Autoradiograph of subcultured cells labeled for 5 days (post-subculture) in 2 &i:ml [H:‘]thymidine. 89% of the fibroblasts and Vi of the myoblasts were labeled. Myoblast and fibroblast morphology is easily distinguished in low calcium medium (Paterson and Strohman, 19721 M. myoblast; F, fibroblast.
When 4- to !&day-old cultures of fusion arrested cells (subcultured at 72 hr) were assayed for myosin, the rates of MHC synthesis and accumulation were observed to increase linearly with time (Fig. 5). The most interesting result of this experiment was that releasing the cells from the fusion block by the addition of 2 mM CaCl, to the culture medium did not promote an increase in either the rate of synthesis or accumulation of MHC, although extensive cell fusion occurred. Furthermore, we observed that the rate of accumulation of MHC by subcultured postmitotic single cells is comparable to that of myotubes obtained by either subculturing cells at 24 hr and allowing them to fuse without prior exposure to a low calcium environment, or by blocking fusion after subculturing at 24 hr and then releasing them with CaCl, at 78 hr (Fig. 6). Since these cultures contain fibroblasts (Fig. 1) and it has been shown that fibroblasts synthesize myosin (Adelstein et al.,
434
DEVELOPMENTAL
BIOLOGY
VOLUME
46,
1976
19721, the rates of synthesis and accumulation of MHC by chick embryo breast muscle fibroblasts were determined. Figure 5 shows that these cells do synthesize MHC, but the rates of synthesis and accumulation do not increase significantly on a per cell basis and therefore cannot account for the linearly increasing rates observed in the muscle cultures. DISCUSSION
An earlier report from this laboratory demonstrated that the increase in the rate of myosin synthesis during myogenesis in vitro was coupled to cell fusion; blocking fusion with EGTA (ethyleneglycolbis(aminoethyl etherlN,N-tetraacetic acid) prevented this increase (Paterson and Strohman, 19721. Furthermore, electron micrographs did not reveal thick filaments in mononucleated postmitotic myoblasts. In this communication, we have demonstrated that if the fusion-arrested cells are maintained by subculture, the myoblasts are capable of accumulating myosin heavy chain at a rate comparable to that of fused cells. This linearly increasing rate of accumulation cannot be accounted for by
FIG. 2. Electron micrograph of a &day-old blast (inset) subcultured at 72 hr and maintained low calcium medium. N, nucleus; MF, myosin ments. x 9000.
myoin tila-
FIG. 3. Electron micrograph of a myoblast subcultured at 72 hr and maintained in low calcium for an additional 3 days. The vesicular network adjacent to the nucleus, presumably the developing T-tubule system, is similar to those seen in young myotubes. N, nucleus; T, T-tubules. x 24,000.
Moss
AND
STROHMAN
Myosm
Synthesis
by Myoblasts
435
FIG. 4. Indirect fluorescent staining of myogenic cultures with antimyosin. (A) 4-day control cultures. x 400. (B) Stained A bands of a myotube in a 4-day control culture. x 4000. (Ci 5-day-old fusion arrested cells subcultured at 68 hr in low calcium medium. All of the elongated myogenic cells are positive, x 400. Background fluorescence with preimmune serum and GAR-FITC was weak relative to the specific staining with antimyosin.
FIG. 5. Rates of synthesis and accumulation of MHC in subculture. The cells were transferred at 72 hr at 1 x 106cells/100-mm dish. At 96 hr, half of the cultures were released from the fusion block by the addition of 2 nN CaCl, to the nutrient medium. In this experiment, only myogenic cells were counted in determining the fusion index. (A) Accumulation of MHC by O----O, released cells; O-O, blocked cells; m-m, tibroblasts. Percentage fusion of a---a blocked cells, A-A released cells. (B) Incorporation of [H”lleucine into MHC. Legend as in Fig. 5A. The fibroblast data was adjusted for cell replication during the experiment.
DEVELOPMENTAL BIOLOGY
436
Time
In culture
(hrs!
FIG. 6. Accumulation of MHC by myogenic cells. Cells subcultured at 24 hr at 1 x lo6 cells/lOO-mm dish in complete medium (O-0, control) or low calcium medium (O-O, blocked; O-0, released).
fibroblasts or the small number (8-11%) of fused cells in the culture, and is assumed to be an average rate of all the myosin containing cells detected by the indirect fluorescent antibody assay. Furthermore, releasing the cells from the prolonged fusion block results in rapid cell fusion, but no increase in the rate of MHC synthesis or accumulation. It is clear therefore that in a calcium deficient medium, cell fusion is bypassed without preventing further differentiation. Not only do the single cells synthesize myosin, thick and thin filament alignment is elaborate, and the beginnings of the T-tubule network are seen. Missing or at least retarded are Z bands, and their absence most probably accounts for the lack of cross-striations in most of these cells. For the moment, we must assume that the myogenic cells used here have programs of differentiation that can be activated by events other than cell fusion. We may speculate that the single fusionblocked cells are in the G, or G, stage of the cell cycle and are retarded in moving
VOLUME 46, 1976
from the beginning to the end of this stage. We know, for example, that the cell cycle time of a number of cells is extended by growth in low calcium (Shainberg et al., 1969; Whittfield, 1973). It is possible that myogenic cells in a calcium deficient medium require an extended period of time to move to the critical postmitotic stage during which regulation for myosin synthesis is activated. Furthermore, this time period may be dependent on changes in the nutritional medium. These considerations may account, in part, for varying reports on rates of MHC synthesis or accumulation. For example, a relatively low rate of myosin synthesis by nonfed, fusion-arrested cells was observed by Paterson and Strohman (19721, while Vertel and Fischman (1975) report a significant accumulation of MHC by fusion-arrested, periodically fed cells that were maintained for a similar length of time. Two final points should be mentioned. First, the present study uses Chelex medium to lower the external Ca2+ concentration, while in the earlier studies of Paterson and Strohman (1972), EGTA was added directly to the culture medium. It is unlikely, however, that our results would vary significantly inasmuch as Vertel and Fischman (1975) and Emerson and Beckner (1975) were able to obtain essentially the same findings with EGTA. Second, the use of Chelex medium involves refeeding the cells, while with EGTA, as used by Paterson and Strohman (19721, changing the medium was not part of the experimental protocol. It is possible that feeding and/or subculturing brings about some synchrony in the blocked cells since myosin synthesis is observed to increase rather abruptly (preliminary results in this laboratory suggest that feeding blocked cells after 1 day in culture results in a slightly sharper rise in MHC accumulation). Although it is now clear that the onset of myosin accumulation may proceed without cell fusion, the metabolic events that trigger fusion and/or myosin accumulation
Moss
AND STROHMAN
Myosin
remain unknown. One may speculate that in myogenic cells some specific interaction at the cell surface is operating, which constitutes the stimulus for terminal differentiation. This leads to the prediction that specific factors might be found that would interact at the myogenic cell surface and would there regulate cell fusion and/or other aspects of the metabolism of the myogenic cell. Indeed, experimental evidence that such factors exist has been presented elsewhere (Leung et al., 1973). In any case, we believe the observations reported here and elsewhere (Paterson and Prives, 1973; Turner et al ., 1974; Emerson and Beckner, 1975; Vertel and Fischman, 1975) demonstrate that myogenic cell fusion per se is not an essential regulatory mechanism for the expression of at least some differentiative events that occur during myogenesis. We wish to thank J. M. Eastwood for valuable technical assistance and sions. We also gratefully acknowledge tion of B. Vertel and D. Fischman, discussed our results and exchanged course of this work (see accompanying work was supported by Grant No. the U.S. National Institute of Health Bay Area Heart Research Committee fellowship (P.S.M.1.
and E. Hughes helpful discusthe cooperawith whom we data during the paper). This GM-13882 from (R.C.S.1 and a predoctoral
REFERENCES ADELSTEIN, R. S., CONTI, M. A., JOHNSON, G. S., PASTAN, I., and POLLARD, T. D. (1972). Isolation and characterization of myosin from cloned mouse fibroblasts. Proc. Nat. Acad. 5%. USA 69, 36933697. COLEMAN, J. R., and COLEMAN, A. W. (1968). Muscle differentiation and macromolecular synthesis. J. Cell. Physiol. 72, Supp. 1, 19-34. DIENSTMAN, S. R. (1974). Skeletal myogenesis without fusion in uitro. J. Cell Bid. 63, 83a. EMERSON, C. P., and BECKNER, S. K. (19751. Activation of myosin synthesis in fusing and mononucleated myoblasts. J. Mol. Biol. 93, 431-448. EZERMAN, E. B., and ISHIKAWA, H. (19671. Differentiation of the sarcoplasmic reticulum and T system in developing chick skeletal muscle in vitro.
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by Myoblasts
437
J. Cell Biol. 35, 405-420. FAMBROUGH, D., and RASH, J. E. (19711. Development of acetylcholine sensitivity during myogenesis. Develop. Biol. 26, 55-68. FISCHMAN, D. / 1972). Development of striated muscle. In “The Structure and Function of Muscle” (G. H. Bourne, ed.1, pp. 75-149, Academic Press, New York. HOLTZER, H., MARSHALL, J., and FINK, H. (1957). An analysis of myogenesis by the use of fluorescent antimyosin. J. Biophys. B&hem. Cytol. 3, 705723. HOLTZER, H. (1970). Myogenesis. In “Cell Differentiation” (0. Schjeide and J. de Vellis, eds.), Chap. 17, pp. 476-502. LEUNG, J., MUNAR, E., and NAMEROFF, M. (19731. Release of myoblast recognition factor(s) by phospholipase C. J. Cell Biol. 59, 191a. LOWEY, S., and STEINER, L. A. (1972). An immunological approach to the structure of myosin and the thick filament. J. Mol. Biol. 65, 111-126. OKAZAKI, K., and HOLTZER, H. (1965). An analysisof myogenesis in citro using fluorescein-labeled antimyosin. J. Histochem. Cytochem. 13, 727-739. OKAZAKI, K., and HOLTZER, H. (1966). Myogenesis: fusion, myosin synthesis, and the mitotic cycle. Proc. Nat. Acad. Sci. USA 56, 148441490. PATERSON, B., and STROHMAN, R. C. (1972). Myosin synthesis in cultures of differentiating chicken skeletal muscle. Develop. Biol. 29, 113-138. PATERSON, B., and PRIVES, J. (1973). Appearance of acetylcholine receptor in differentiating cultures of embryonic chick breast muscle. J. WE Biol. 59, 241-245. SHAINBERG, A., YAGIL, G., and YAFFE, D. 11969). Control of myoge.iesis in vitro by Ca’- concentration in nutritional medium. Exptl. Cell Res. 58. 163-167. TURNER, D. C., MAIER, V., and EPPENBERGER, H. (1974). Creatine kinase and aldolase isoenzyme transitions in cultures of chick skeletal muscle cells. Develop. Biol. 37, 63-89. VERTEL, B., and FISCHMAN, D. (1976). Myosin accumulation in mononucleated cells of chick muscle cultures. Develop. Biol. 48, 438-446. WELLER, T. H., and COONS, A. H. (1954). Fluorescent antibody studies with agents of Varicella and Herpes Zoster prepared in vitro. Proc. Sot. Exptl. Biol. Med. 86, 789. WHITFIELD, J. F., RIXON, R. H., MACMANUS, J. P., and BALK, S. D. (1973). Calcium, cyclic adenosine 3’5’-monophosphate and the control ofcell proliferation: a review. In Vitro 8, 257-278.
BIOLOGY
48, 431-437
Myosin Synthesis
(1976)
by Fusion-Arrested Chick Embryo Culture PAUL S. Moss
Department
of Zoology,
AND RICHARD
University Accepted
of California, September
Myoblasts
in Cell
C. STROHMAN Berkeley,
California
94720
19, 1975
The synthesis and accumulation of myosin was studied in subcultures of fusion-blocked, postmitotic embryonic chicken myogenic cells. Electron micrographs and fluorescent microscopy with antimyosin revealed that most, if not all, of these cells contain myosin. It was also found that these cells are capable of accumulating myosin at rates comparable to fused cells. Incipient T-tubule formation was also present in some of the blocked cells. It is concluded that cell fusion is not a prerequisite for myosin synthesis and accumulation or T-tubule formation during myogenesis in vitro.
the synthesis and assembly of myosin filaments (Paterson and Strohman, 1972). In this report, however, we show that if postmitotic fusion-blocked myogenic cells are maintained in subculture, they synthesize and accumulate myosin at rates comparable to normally differentiated myotubes. It is also demonstrated that some of these fusion-arrested cells are capable of assembling an incipient T-tubule system. These results, and those of the accompanying paper by Vertel and Fischman (1975) and the recent report by Emerson and Beckner (1975) demonstrate that myogenic cell fusion is not a prerequisite for the activation of myosin synthesis during skeletal muscle myogenesis.
INTRODUCTION
The timing of myosin synthesis during chicken skeletal muscle myogenesis has been the subject of many reports. Most in vitro studies of muscle differentiation have demonstrated that myogenic cell fusion normally precedes the bulk accumulation of myosin and the appearance of myofilaments (for review, see Fischman, 1972). However, Holtzer et al. (1957) demonstrated the elaboration of myofilaments in mononucleated somitic myoblasts by stage 16 in viuo, and Okazaki and Holtzer (1965, 1966) found that at least some single myogenic cells can synthesize myosin prior to fusion in vitro (see also Coleman and Coleman, 1968; and Dienstman, 1974). In an earlier communication from this laboratory (Paterson and Strohman, 19721, the high rates of myosin synthesis normally associated with fully differentiated myotubes were not observed in culture until after cell fusion, and even then not until after a lag period of from 8-12 hr. In addition, when fusion is blocked by lowering the external calcium concentration, myogenic cells become postmitotic but do not enter into the accelerating rate of myosin synthesis observed for normal fused cultures. These considerations led to the conclusion that cell fusion is a normal prerequisite for further differentiation, including
MATERIALS
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
METHODS
Cells. Primary cultures of 12-day embryonic muscle cells were prepared as previously described (Paterson and Strohman, 1972). Fusion-blocked cells were obtained by feeding the cells at 24 hr with a low calcium medium (Chelex medium) prepared by the method of Paterson and Prives (1973). Subcultures of these fusionarrested cells were prepared by rinsing the monolayers with calcium-free MEM, followed by a 5- to B-min incubation at room temperature in 0.02% trypsin (Difco) diluted in calcium-free MEM. The cell sus431
Copyright All rights
AND
432
DEVELOPMENTAL
BIOLOGY
pension was rinsed twice in fresh Chelex medium, filtered through four layers of Nitex in a double Swinney, and preplated (Fambrough and Rash, 1971) in 5 ml at 2-3 x lo6 cells/ml for 20-30 min on noncollagen coated loo-mm Falcon dishes. The cells were then plated in fresh Chelex medium on collagen-coated Falcon dishes at the concentration indicated in the figure legend. Cultures of chick embryo fibroblasts were prepared from overgrown (6day) myogenic cultures. Methods for cell counts, fusion indices, autoradiography, and electron microscopy were those standard in this laboratory (Paterson and Strohman, 1972). Antiserum. Preparation and testing of antiserum to column purified chicken breast myosin essentially followed the procedures of Lowey and Steiner (1972). Each rabbit was immunized with 4 mg of antigen in complete Freund’s Adjuvant injected subcutaneously in the rear footpads and back of the neck. The animals became positive after 2 weeks and were boosted with 2 mg at 1 month. Thereafter, blood was collected from the marginal ear vein at 2-week intervals and assayed for antimyosin activity by the quantitative precipitin test in high salt buffer (Lowey and Steiner, 1972). The IgG fraction was isolated and purified by precipitation in 40% ammonium sulfate at room temperature and chromatography on Whatman DE-52. Fluorescence microscopy. Cells were examined for myosin content by the indirect fluorescent antibody technique (Weller and Coons, 1954). All procedures were carried out directly on 35mm collagen-coated Falcon culture dishes. After fixation in acetone:ethanol (1:l) for 3 min at o”C, the monolayer-s were rinsed three times in phosphate buffered saline (PBS) and incubated with immune (or preimmune) globulin (1-3 mg/ml) in PBS at 37°C for 20 min. After rinsing extensively in PBS, the cells were treated with fluorescein-conjugated goat anti-rabbit IgG (GAR-FITC; Antibodies, Inc.) diluted 1:7 in PBS for 20 min at
VOLUME
48, 1976
37°C. The cells were then rinsed thoroughly in PBS and mounted in glycerol:water (9:l) prior to examination with an epi-illuminated Leitz Orthopan fluorescent microscope. Myosin synthesis and accumulation. The rates of synthesis and accumulation of myosin heavy chain (MHC) were determined by the method of Paterson and Strohman (1972) with the following modifications: (1) The cells were pulsed in 4 ml of labeling medium consisting of 1 part fresh complete or Chelex medium and 1 part conditioned medium (centrifuged medium from sister cultures of approximately the same age) containing 15 &i/ml of [4,5H311eucine (30-50 Ci/mmole) (Schwartz/ Mann). (2) The SDS-polyacrylamide gels were run without the addition of carrier myosin to the high salt extract. The gels were stained for 3 hr, destained for 20 hr with one change, and the region of the gel containing the band comigrating with a column chromatographed MHC standard was scanned at 550 nm on a Gilford Linear Gel Scanner. Complete and specific precipitation of the 220,000 MW protein from the high salt extract by antimyosin demonstrated that this band is definitely MHC (Moss, unpublished results). The amount of MHC accumulated was determined by comparing the absorbance with that of a series of standard gels loaded with l-10 pg of column-purified myosin. To determine the rate of synthesis of MHC, the gels were frozen, sliced, and counted as previously described (Paterson and Strohman, 1972). RESULTS
Cell counts and autoradiographs of fusion blocked cells subcultured at 68 hr demonstrated that only 5-6% of these cells were labeled when grown continuously in a calcium deficient medium and tritiated thymidine (Fig. 11, confirming the postmitotic nature of these cells (Paterson and Strohman, 1972). After l-2 days in subculture, most of the myogenic cells appeared
Moss
AND
STROHMAN
Myosin Synthesis by Myoblasts
FIG. 1A. Growth and [H”]thymidine labeling of myogenic cultures (2 x lo5 tells/60-mm dish) transferred at 68 hr and maintained in low calcium medium. Growth of: O-O, myoblasts; O-O, fibroblasts. x 750.
by phase microscopy to thicken and become more refractile as compared to their earlier, thinner and more elongated shape. Electron microscopy of eight of these cells revealed that all of them contained thick filaments (Fig. 2). Interestingly, although the filaments appeared to be fairly wellaligned, all but one of the cells lacked obvious Z bands. Two of these cells had also developed what appears to be the incipient T-tubule system similar to that observed in young myotubes by Ezerman and Ishikawa (1967) (Fig. 3). To demonstrate that the eight cells examined with the electron microscope were representative of all of the single cells, the cultures were assayed for myosin by the indirect fluorescent antibody technique. Figure 4 demonstrates the ability of the antiserum to react specifically with the A bands of cross-striated myotubes. When the same experiment was done with subcultures of fusion-blocked cells, more than 90% of the myogenic cells stained with the antimyosin (Fig. 4). Cross striation in these cells, however, was only rarely observed.
FIG. 1B. Autoradiograph of subcultured cells labeled for 5 days (post-subculture) in 2 &i:ml [H:‘]thymidine. 89% of the fibroblasts and Vi of the myoblasts were labeled. Myoblast and fibroblast morphology is easily distinguished in low calcium medium (Paterson and Strohman, 19721 M. myoblast; F, fibroblast.
When 4- to !&day-old cultures of fusion arrested cells (subcultured at 72 hr) were assayed for myosin, the rates of MHC synthesis and accumulation were observed to increase linearly with time (Fig. 5). The most interesting result of this experiment was that releasing the cells from the fusion block by the addition of 2 mM CaCl, to the culture medium did not promote an increase in either the rate of synthesis or accumulation of MHC, although extensive cell fusion occurred. Furthermore, we observed that the rate of accumulation of MHC by subcultured postmitotic single cells is comparable to that of myotubes obtained by either subculturing cells at 24 hr and allowing them to fuse without prior exposure to a low calcium environment, or by blocking fusion after subculturing at 24 hr and then releasing them with CaCl, at 78 hr (Fig. 6). Since these cultures contain fibroblasts (Fig. 1) and it has been shown that fibroblasts synthesize myosin (Adelstein et al.,
434
DEVELOPMENTAL
BIOLOGY
VOLUME
46,
1976
19721, the rates of synthesis and accumulation of MHC by chick embryo breast muscle fibroblasts were determined. Figure 5 shows that these cells do synthesize MHC, but the rates of synthesis and accumulation do not increase significantly on a per cell basis and therefore cannot account for the linearly increasing rates observed in the muscle cultures. DISCUSSION
An earlier report from this laboratory demonstrated that the increase in the rate of myosin synthesis during myogenesis in vitro was coupled to cell fusion; blocking fusion with EGTA (ethyleneglycolbis(aminoethyl etherlN,N-tetraacetic acid) prevented this increase (Paterson and Strohman, 19721. Furthermore, electron micrographs did not reveal thick filaments in mononucleated postmitotic myoblasts. In this communication, we have demonstrated that if the fusion-arrested cells are maintained by subculture, the myoblasts are capable of accumulating myosin heavy chain at a rate comparable to that of fused cells. This linearly increasing rate of accumulation cannot be accounted for by
FIG. 2. Electron micrograph of a &day-old blast (inset) subcultured at 72 hr and maintained low calcium medium. N, nucleus; MF, myosin ments. x 9000.
myoin tila-
FIG. 3. Electron micrograph of a myoblast subcultured at 72 hr and maintained in low calcium for an additional 3 days. The vesicular network adjacent to the nucleus, presumably the developing T-tubule system, is similar to those seen in young myotubes. N, nucleus; T, T-tubules. x 24,000.
Moss
AND
STROHMAN
Myosm
Synthesis
by Myoblasts
435
FIG. 4. Indirect fluorescent staining of myogenic cultures with antimyosin. (A) 4-day control cultures. x 400. (B) Stained A bands of a myotube in a 4-day control culture. x 4000. (Ci 5-day-old fusion arrested cells subcultured at 68 hr in low calcium medium. All of the elongated myogenic cells are positive, x 400. Background fluorescence with preimmune serum and GAR-FITC was weak relative to the specific staining with antimyosin.
FIG. 5. Rates of synthesis and accumulation of MHC in subculture. The cells were transferred at 72 hr at 1 x 106cells/100-mm dish. At 96 hr, half of the cultures were released from the fusion block by the addition of 2 nN CaCl, to the nutrient medium. In this experiment, only myogenic cells were counted in determining the fusion index. (A) Accumulation of MHC by O----O, released cells; O-O, blocked cells; m-m, tibroblasts. Percentage fusion of a---a blocked cells, A-A released cells. (B) Incorporation of [H”lleucine into MHC. Legend as in Fig. 5A. The fibroblast data was adjusted for cell replication during the experiment.
DEVELOPMENTAL BIOLOGY
436
Time
In culture
(hrs!
FIG. 6. Accumulation of MHC by myogenic cells. Cells subcultured at 24 hr at 1 x lo6 cells/lOO-mm dish in complete medium (O-0, control) or low calcium medium (O-O, blocked; O-0, released).
fibroblasts or the small number (8-11%) of fused cells in the culture, and is assumed to be an average rate of all the myosin containing cells detected by the indirect fluorescent antibody assay. Furthermore, releasing the cells from the prolonged fusion block results in rapid cell fusion, but no increase in the rate of MHC synthesis or accumulation. It is clear therefore that in a calcium deficient medium, cell fusion is bypassed without preventing further differentiation. Not only do the single cells synthesize myosin, thick and thin filament alignment is elaborate, and the beginnings of the T-tubule network are seen. Missing or at least retarded are Z bands, and their absence most probably accounts for the lack of cross-striations in most of these cells. For the moment, we must assume that the myogenic cells used here have programs of differentiation that can be activated by events other than cell fusion. We may speculate that the single fusionblocked cells are in the G, or G, stage of the cell cycle and are retarded in moving
VOLUME 46, 1976
from the beginning to the end of this stage. We know, for example, that the cell cycle time of a number of cells is extended by growth in low calcium (Shainberg et al., 1969; Whittfield, 1973). It is possible that myogenic cells in a calcium deficient medium require an extended period of time to move to the critical postmitotic stage during which regulation for myosin synthesis is activated. Furthermore, this time period may be dependent on changes in the nutritional medium. These considerations may account, in part, for varying reports on rates of MHC synthesis or accumulation. For example, a relatively low rate of myosin synthesis by nonfed, fusion-arrested cells was observed by Paterson and Strohman (19721, while Vertel and Fischman (1975) report a significant accumulation of MHC by fusion-arrested, periodically fed cells that were maintained for a similar length of time. Two final points should be mentioned. First, the present study uses Chelex medium to lower the external Ca2+ concentration, while in the earlier studies of Paterson and Strohman (1972), EGTA was added directly to the culture medium. It is unlikely, however, that our results would vary significantly inasmuch as Vertel and Fischman (1975) and Emerson and Beckner (1975) were able to obtain essentially the same findings with EGTA. Second, the use of Chelex medium involves refeeding the cells, while with EGTA, as used by Paterson and Strohman (19721, changing the medium was not part of the experimental protocol. It is possible that feeding and/or subculturing brings about some synchrony in the blocked cells since myosin synthesis is observed to increase rather abruptly (preliminary results in this laboratory suggest that feeding blocked cells after 1 day in culture results in a slightly sharper rise in MHC accumulation). Although it is now clear that the onset of myosin accumulation may proceed without cell fusion, the metabolic events that trigger fusion and/or myosin accumulation
Moss
AND STROHMAN
Myosin
remain unknown. One may speculate that in myogenic cells some specific interaction at the cell surface is operating, which constitutes the stimulus for terminal differentiation. This leads to the prediction that specific factors might be found that would interact at the myogenic cell surface and would there regulate cell fusion and/or other aspects of the metabolism of the myogenic cell. Indeed, experimental evidence that such factors exist has been presented elsewhere (Leung et al., 1973). In any case, we believe the observations reported here and elsewhere (Paterson and Prives, 1973; Turner et al ., 1974; Emerson and Beckner, 1975; Vertel and Fischman, 1975) demonstrate that myogenic cell fusion per se is not an essential regulatory mechanism for the expression of at least some differentiative events that occur during myogenesis. We wish to thank J. M. Eastwood for valuable technical assistance and sions. We also gratefully acknowledge tion of B. Vertel and D. Fischman, discussed our results and exchanged course of this work (see accompanying work was supported by Grant No. the U.S. National Institute of Health Bay Area Heart Research Committee fellowship (P.S.M.1.
and E. Hughes helpful discusthe cooperawith whom we data during the paper). This GM-13882 from (R.C.S.1 and a predoctoral
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