Prinred in Sweden Copyright @ 1976 by Academic Press, Inc. All rights of reproducrion in any form reserved
Experimental
COORDINATED AND
Cell Research 97 (1976) 387-393
SYNTHESIS
MYOSIN
IN
AND
A VARIETY
NON-MYOGENIC N. RUBINSTEIN, Department
of Anatomy,
University
DEGRADATION
OF ACTIN
OF MYOGENIC
AND
CELLS
J. CHI and H. HOLTZER
of Pennsylvania
School of Medicine,
Philadelphia,
PA 19174, USA
SUMMARY The turnover of myosin and actin in both muscle and non-muscle cells in culture was investigated. By the double-label criterion, myosin and actin were coordinately synthesized and degraded in replicating, mononucleated fibroblasts, chondrocytes, BUdR-suppressed myogenic cells, and in post-mitotic, multinucleated myotubes. Myosin and actin were among the most stable proteins in each cell type. In single label ‘pulse-chase’ experiments, the half-lives of myosin and actin in all replicating, mononucleated cells were 2.5-3 days; in myotubes, however, they were approx. 6 days. Myosin and actin labelled in replicating presumptive myoblasts and chased until the cells ceased replicating and fused into multinucleated myotubes retained the degradation rate of 3 days; this differed from ,the rate of 6 days shown for myosin and actin newly-synthesized in post-mitotic myotubes. The type of myosin synthesized in the mother presumptive myoblast, then, IS transmitted to the postmitotic daughters. This myosin, however, is more rapidly degraded than the definitive myosin that is synthesized in the myotube.
The presence of actin-like and myosin-like molecules in a variety of non-muscle cells has raised interesting questions about the polymorphism of contractile proteins and the diversity of contractile systems [2,7,8, 10, 14, 211. While it is probable that red, white, cardiac, and smooth muscle myosins from one organism are the products of different structural genes [5, 17, 191, it is not yet clear whether all non-muscle myosins are alike or different. Antibodies against chicken skeletal muscle myosin, for example, do not react with extracts of contractile proteins from many kinds of chicken embryonic and mature non-muscle cells [I 1, 121. They only react with contractile proteins from definitive post-mitotic myoblasts, multinucleated myotubes, and mature muscle fibers. Conversely, antibody
prepared against several non-muscle myosins do not react with rabbit skeletal muscle myosin [20,22,25, 271. While obvious differences among myosins exist, few differences have been demonstrated among actins from the various muscle [9] or non-muscle cells [9]. This data suggests that there are several structural genes for the different myosin heavy and light chains. In contrast, there may be only one structural gene for actin which can be linked to the different myosins, or a system of multiple actin genes each with a very similar sequence [ 10, 11, 131. When multiple proteins such as actin and myosin have an intimate relationship within a cell, at least three layers of control can be envisaged: (1) an activating system Expti
Cell
Res 97 (1976)
388
Rub&stein, Chi and Holtzer
which allows expression of the proteins only in certain cell types; (2) a modulating control which varies the total amount of the proteins according to physiological necessities; and (3) an integrating mechanism which coordinates the total amount of each protein synthesized in relationship to the others. With regard to the integration mechanism, many investigators have concluded that actin and myosin in skeletal muscle are not synthesized and degraded as a unit, but are metabolized independently [15,24]. The finding, however, that myosin-like and actin-like molecules are present in fibroblasts and other mononucleated cells within muscle in vivo and in vitro [23] makes it necessary to re-examine this problem, Accordingly, we have begun an investigation into the mechanisms controlling the synthesis of myosin and actin in nonmuscle and muscle cells by demonstrating the coordination of myosin and actin synthesis and degradation in many cell types. MATERIALS
AND METHODS
For the first 36 h, a standard muscle culture consists largely of replicating presumptive myoblasts. Presumptive myoblasts divide and yield post-mitotic myoblasts which are the only cells capable of fusing into multinucleated mvotubes and synthesizing and assembling actin and myosin into inteidigitating-thick and thin filaments. Auurox. 70-80% of the cells in these early cultures &e presumptive myoblasts; the remainder are cells in different precursor compartments of the myogenic and fibrogenic lineages. These early cultures will be referred to as ‘presumptive myoblast cultures’, although, in fact, they contain a heterogeneous population of cells. Fusion of post-mitotic myoblasts into multinucleated myotubes begins after approx. 48 h in vitro. As the cultures mature, there is an increase in the number of nuclei in myotubes, accompanied by a decrease in the number of presumptive myoblasts. At the same time, the proportion and absolute number of tibroblasts in the culture increase. By day 6, when most of the fusion has occurred, the standard muscle cultures consist of large, branched, striated, contracting, multinucleated myotubes, large numbers of replicating, mononucleated fibroblasts, and a small number of replicating, mononucleated, precursor myogenic cells. The heterogeneity of these older cultures is similar Exprl Cell Res 97 (1976)
to the heterogeneity of an adult muscle. Any analysis of these cultures or whole muscles will yield data that is the average of values in different types of cells. To analyse only the myotube population, i.e. the true muscle cells. cultures of oure mvotubes were oreoared from stdndard muscle culturks by adding ‘cyiosine arabinoside. an inhibitor of DNA svnthesis. on davs 2. 3, and 4. On day 5, cultures we;e fed with normal medium, Since this led to the death of most replicating cells and did not affect the post-mitotic myoblasts and myotubes, the resulting cultures had an extremely high mu!tinuc!eated/mononuc!eated cell ratio [23]. The myotubes in these pure myotube cultures contained cross-striations and contracted spontaneously. It should be stressed that it is impossible to obtain a population of comparative purity in vivo. Fig. I shows photomicrographs of standard muscle and pure myotube cultures. Standard muscle cultures were prepared by the method of Bischoff & Holtzer [6] from 1!-day-old chicken embryonic breasts. Cells were plated at a density of 3x IVilOO mm dish and covered with 8 ml of medium 8 : I : I. Fibroblasts, chondrocytes, and BUdRsuppressed myogenic cells were prepared as described by Rubinstein et a!. [23] and Abbott & Holtzer [I]. Twice precipitated actomyosin, prepared by the method of Adelstein et a!. r31, was electrophoresed on 8% SDS-polyacrylamide &is according io Weber & Osborn [26]. Radioactivity in actomyosin was determined by slicing each gel into I mm pieces and placing each slice in a toluene-PPO-POPOP-NCS scintillant. This cocktail was counted in a Beckman Liquid Scintillation Counter. For some experiments, it was important to determine if any cells had been lost from the culture dish during the course of the experiments. Cultures were labelled with [3H]thymidine during the first 24 h in vitro, then washed with balanced salt solution (BSS) and fed normally thereafter. 13HJThymidine content in these culture dishes at any time was an estimate of the percentage of original DNA left on the dish, regardless of replication. Any loss of label from the dish would be-a measuremdnt of cell death. For example, if 75 % of the [3H]thymidine label were left on the dish, then 25% of the originally labelled cells and/ or their descendants would have left the culture dish. This prelabelling technique can also be used to determine the percentage of original protein left on the dish, exclusive of intracellular degradation. In the previous example, if protein had been labelled with [‘Ylleucine at the same time that the DNA had been labelled with [3H]thymidine, 25 % of the labelled protein would also have disappeared because of cell loss. Any additional loss of labelled protein would be a function not of cell death, but of degradation of protein within the cells remaining on thedish. There was very little cell loss in cultures of fibroblasts, chondrocites, and BUdR-suppressed myogenic cells. Only during the preparation of pure myotubes with cytosine arabinoside was there any significant cell death. Fig. 2 shows the loss of prelabelled DNA in chondrocyte cultures and during the preparation of pure myotubes. [3H]Leucine (30-50 Cilmmole), [Ylleucine (280 mCi/mmole), and [3H]thymidine (40-60 Cilmmole)
Synthesis and degradation
Fig. I. Light micrographs of standard muscle (a) and pure myotube (b) cultures, x252. In the standard muscle culture, note that the myotubes are obscured by
of actin and myosin
389
overgrowing mononucleated cell S. In the myotube culture, no mononucleated cells alre evident.
Erptl Cell Res 97 (1976)
390 100,
Rubinstein, Chi and Holtzer 6-a
0
0 0
75.
so. \,
15L-1
23454789
time in culture (days); ordinate: % original DNA. Loss of prelabelled DNA from chondrocyte cultures (O-O) and cytosine arabinoside-treated muscle cultures (0-O). The large loss of DNA in the latter cultures between days 2 and 5 is due to the cytosine arabinoside’s killing of replicating, mononucleated cells. The small loss thereafter is due to the myotubes’ contracting and pulling loose from the surface of the dish.
Fig. 2. Abscissa:
were obtained from New England Nuclear. Cytosine arabinosine and BUdR were purchased from Sigma. All tissue culture materials were from GIBCo.
RESULTS To examine whether actin and myosin in the various cell types were metabolized as a unit or independently, we used the double label technique of Arias et al. [4] as the criterion of coordinated synthesis and degradation. Myotube cultures were labelled for 24 h with 10 $X/plate [3H]leucine on day 5 in vitro, washed with BSS, and grown for four more days in the presence of excess unlabelled leucine. On day 9, the cells were pulsed for 2 h with 3 &i/plate [14C]leucine. The plates were immediately washed -again in BSS, the cells collected, and the actomyosin extracted and electrophoresed. Each gel was sliced and the radioactivity in each slice determined. In this type of experiment, the first label, in this case [3H]leucine, is a measure of degradation. The second label, [ 14C]leucine, is a measure of synthesis, since it is present for only a small fraction of the expected Exptl Cd Res 97 (1976)
half-life of the proteins. [14C]Leucine-labelled proteins, then, have not had a chance to be degraded, while [3H]leucine labelled proteins have had four days for degradation. The [‘“C]/[“H] ratios of a series of proteins are a comparison of their relative rates of synthesis and degradation. For example, if protein A and protein B had the same ratio, they would appear to be synthesized and degraded coordinately. On the other hand, if protein A’s ratio were higher than protein B’s, protein A is less stable. This occurs because more of the [3H]label in protein A was degraded during the 4-day chase period. This type of experiment, however, is only applicable to proteins whose synthesis relative to each other does not change during the time period involved. The ratio of myosin synthesis to actin synthesis was shown to be constant during the course of these experiments in all cell types. For example, in one experiment with pure myotubes, the ratios of myosin synthesis/actin synthesis on days 5, 7, and 9, were 2.7, 2.9, and 2.7, respectively. When myotubes were treated in this manner, the [14C]/[3H] ratio for actin and myosin heavy chain were identical (table 1). The same result was obtained with actin and myosin from chondrocytes, fibroblasts, and Table 1. Relative rates of myosin and actin turnover Myosin
Actin
Cell type
([“C]/[W
(Wl/13Hl)
Myotubes BUdR-suooressed Fibrob& Chondrocytes
1.14+0.10 1.03fO.19 0.43 kO.03 1.29kO.02
1.16+0.03 0.99f0.24 0.42&O. 15 1.3250.12
Cells were labelled with 10 &i/plate [3Hpeucine for 24 h, washed, and chased with excess unlabelled leutine. Four days later, cells were pulsed for 2 h with 3 &i/plate [“Cleucine and the actomyosin was immediately extracted and electrophoresed. The gels were stained and sliced, and the radioactivity in each slice was determined.
Synthesis and degradation
of actin and myosin
391
performed this type of experiment with day 7 myotube cultures by pulsing with [3H]leucine for 2 h before extraction of actomyosin. The actomyosin was electrophoresed and a specific activity (cpmloptical density unit) was calculated by scanning and counting the gel. The specific activity of myosin heavy chain (in arbitrary units) was 1.48kO.28 and that of actin was 1.42& 0.20 (4 experiments). The double label experiments, while indicating a physiological coupling between actin and myosin, do not give an absolute rate of .legradation for these proteins. To determine the half-lives of these molecules, Fig. 3. Abscissa: Slice no; ordinate: (lefr) cpmx lO-3, cultures were labelled for 24 h with 10 &i/ [3H]leucine (O---O); (righf) cpmx IO-*, [‘“C]leucine plate [3H]leucine. Mononucleated cells (he). ’ -Do&e label turnover experiment with BUdR-supwere labelled between days 1 and 2, myopressed myogenic cells. Cells were pulsed with [3H]leucine for 24 h, washed, and chased with excess un- tubes between days 5 and 6 in vitro. The labelled leucine. Four days later, the cells were pulsed plates were washed in BSS and grown in an for 2 h with [‘*C]leucine. The cells were collected; and the actomyosin was isolated and electrophoresed on excess of unlabelled leucine for various an 8% SDS-polyacrylamide gel. periods of time. Samples were taken each day and the actomyosin extracted and electrophoresed. The percentage of labelled BUdR-suppressed myogenic cells. Fig. 3 myosin heavy chain or actin remaining was shows the radioactivity of the 8% gel from calculated. Table 2 gives the results for the a similar experiment with BUdR-suppressed various cell types. While myotube actin and myogenic cells. By this double label cri- myosin had half-lives of approx. 6 days, terion, then, the myosin and actin are coordinately synthesized and degraded in all Table 2. Half-lives of myosin and actin cell types. Myosin Actin If total cell protein from one of these Cell type (days) (days) double label experiments is analyzed on an 6.08fl.30 5.93f0.68 SDS gel, the ratios of non-actomyosin pro- Myotubes 2.93kO.97 Presumptive myoblasts 3.00+0.94 teins vary considerably. In both myotubes BUdR-suppressed 2.50k0.36 2.9OkO.37 2.69tO. I6 2.75*0. IO and non-muscle cells, however, myosin and Fibroblasts 3.07 2.95 actin were among the most stable proteins Chondrocytes Cells were pulsed for 24 h with 10 &/plate r3H]leu(i.e. had the lowest [‘4C]/rH] ratios). washed, and chased with an excess of unlabelled Koizumi [15] has claimed that myosin tine, leucine. Myotubes were labelled between days 5 and 6, and actin synthesis and decay in adult presumptive myoblasts between days 0 and 1, and other cells between days 1 and 2. Each day therechicken muscle in vivo are not coordinated after, cultures were sacrificed; actomyosin was exbecause the specific activities of these pro- tracted and electrophoresed. Gels were stained, sliced, counted, and the amount of labelled myosin reteins in muscle labelled for a short time and and maining was calculated. If necessary, a correction for immediately extracted are not the same. We cell loss was also made. Exptl Cell Res 97(1976)
392
Rubinstein, Chi and Holtzer into myotubes confirms our earlier demonstration that presumptive myoblasts do, indeed, synthesize modest amounts of these two proteins [23]. 0
I
2
3
4
Fig. 4. Abscissa: time after removal of labelled medium (days); ordinate: % initial radioactivity. A-A, Myotube myosin; m-W, presumptive myoblast myosin; O-O, actin. Turnover of myosin from myotubes and presumptive myoblasts. Myotubes were pulsed on day 5 and the radioactive myosin measured through day 9. Presumptive myoblasts were pulsed on day 0 and the radioactivity determined through day 5, when only myotubes were present.
DISCUSSION
In both muscle and non-muscle cells, myosin heavy chains and actins are coordinately synthesized and degraded. The ratio of myosin synthesis/actin synthesis, then, must equal both the ratio of myosin degradationlactin degradation and the steady state ratio of myosinlactin in the cell. We have previously shown this ratio to be approx. 2.8 in myotubes and between 0.6 and the half-lives of myosin and actin in fi- 0.9 in non-muscle cells [7, 231. It should be stressed that the myosin in broblasts, chondrocytes, and BUdR-suppressed myogenic cells were only 2-3 days. myotubes is not the same molecule as the The procedure with the presumptive myosin in non-muscle cells. These proteins myoblasts was somewhat different from have different antigenicities [8, 12, 13J and that just described, since standard muscle different myosin light chains [7]. The actin, cultures contain mononucleated fibroblasts at least in presumptive myoblasts and possibly in other non-myogenic cells, may difwhich have a significant content of rapidlydegrading myosin and actin. Presumptive fer from the actin of myotubes in primary myoblasts were labelled for the first 20 h in structure, method of control, or both. Actin vitro and the amount of radioactive myo- (and myosin) labelled in presumptive myosin and actin remaining was measured on blasts and ‘chased’ into myotubes coneach succeeding day. On days 2 through 5, tinued to be degraded at the ‘mononucytosine arabinoside was added to eliminate cleated cell rate’ and not at the ‘myotube fibroblasts. This led to DNA loss for which rate’. In the myotube, the two sets of myowe had to compensate by the method de- sin and actin (the set synthesized in the scribed in Materials and Methods. Even af- presumptive myoblasts and the set synter the cultures contained only multinu- thesized in the myotube) were being treated cleated myotubes, myosin heavy chain and as independent populations (see fig. 3). This actin-originally labelled in mononucleated might mean that some primary structural presumptive myoblasts-still existed. They difference prevented presumptive myowere, however, being degraded at the same blast myosin and actin from being incorrate as in the other mononucleated cells (i.e. porated into sarcomeres and, thus, preTt=3 days) and not at the same rate as the vented these molecules from being denewly-synthesized myosin and actin in the graded as slowly as the myosin and actin myotubes (Tt=6 days, fig. 4). in myofibrils. The reason for the discrepancy between The fact that myosin and actin labelled in presumptive myoblasts could be chased our findings for skeletal muscle and those Exprl Cell Res 97 (1976)
Synthesis and degradation
of actin and myosin
393
of other investigators [15, 241 is unclear. multimeric particles or feedback loops is Our results were obtained with a pure popu- necessary requires further experiments with lation of myotubes, while others worked intact and fractionated cells. only with whole muscle, a combination of This research was supported by NIH grants HL-15835, myotubes and non-muscle connective tis- HDOO030, HDO0189, NSF-GB-38421X, and by the sue cells. The different myosin and actin Muscular Dystrophy Association. half-lives in various types of cells combined with the different myosin/actin ratios may REFERENCES have complicated the data of earlier invesAbbott, J & Holtzer, H, J cell biol28 (1968) 473. Adelstein, R & Conti, M, Cold Spring Harbor tigators . svmn auant bio137 (1973) 599. Since myosin and actin are synthesized Adeistein, R, Conti; M, Johnson, G, Pastan, I & Pollard, T, Proc natl acad sci US 69 (1972) 3693. on separate polysomes, their synthesis Arias, I, Doyle, D & Schimke, R, J biol them 244 must be synchronized to yield a constant (1969) 3303. Arndt, I & Pepe, F, J histochem cytochem 23 myosin/actin ratio in any cell type. Several (1975) 159. mechanisms for this synchronization can 6. Bischoff, R & Holtzer, H , J cell bio136 (1968) 111. be proposed. A feedback system or a spe- 7. CM., J,Rubinstein, N& Holtzer, H, Proc natl acad sci US 72 (1975). cial interaction between nascent myosin 8. Diensttpan, S & Holtzer, H, Cell cycle and cell differentiation (ed J Reinert & H Holtzer) p. 1. and actin molecules may be possibilities. Springer-Verlag (1975). In addition, preliminary evidence from our 9. Elzinga, M 8c Collins, J, Cold Spring Harbor symp auant biol37 (1973) 1. laboratory shows that it is difficult to unH, Sanger, J, Ishikawa, H & Strahs, K, couple myosin and actin synthesis. This 10. Holtzer, Cold Spring Harbor symp quant bio137 (1973) 549. suggests the possibility that actin and myo- 11. Holtzer, H, Rubinstein, N, Dienstman, S, Chi, J, J & Somlyo, A, Biochimie 56 (1974) 1575. sin polysomes are part of a large, multi- 12. Biehl, Holtzer, H, Strahs, K, Biehl, J, Somlyo, A & Ishikawa. H. Science 188119751.943. merit particle whose composition alone 13. Holtzer, H, Rubinstein, N,.Chi, J, Dienstman, S & regulates the relative rates of synthesis. Biehl, J, Exploratory concepts in muscular dystrophy (ed A Milhorat) p. 3. Excerpta Medica, It is also possible to regulate the relaAmsterdam (1974). tive synthesis of two molecules without 14. Ishikawa, H, Bischoff, R & Holtzer, H, J cell biol 43 (1969) 312. invoking any special mechanisms. Using Koizumi, T, J biochem 76 (1974) 431. reticulocyte lysates, Lodish [16] and 15. 16. Lodish, H, Nature 251 (1974) 385. McKeehan [ 181have demonstrated that the 17. Lowev, S & Risbv, D, Nature 234 (1971) 81. 18. McKeehan, W, J biol them 249 (1974) 6517. relative synthesis of a/P globin can be 19. Nakamura. A. Sreter. F & Gereelv. J. J cell bio149 (1971) 883. - .’ changed by varying the concentrations of R, Pastan, I & Adelstein, R, J biol them ribosomal subunits and initiation factors. 20. Ostlund, 249 (1974) 3903. These alterations occur because of the dif- 21. Pollard, T & Weihing, R, CRC critical rev in bio2 (1974) I. ferent affinities of (Y-and @globin mRNAs 22. them Pollard, T, Thomas, S & Niederman, R, Analyt biochem 60 (1974) 258. for the various initiation factors and riboN, Chi, J & Holtzer, H, Biochem biosomal subunits. In the red blood cell, then, 23. Rubinstein, phys res commun 57 (1974) 438. the 1 : 1 ratio of (Y: p globin synthesis might 24. Velick, S, Biochim biophys acta 20 (1956) 228. K & Groschel-Stewart, U, Proc nati acad be maintained merely by establishing cer- 25. Weber, sci US 71 (1974) 4561. 26. Weber, K & Osborn, M, J biol them 244 (1969) tain concentrations of ribosomes, initiation 4406. factors, and mRNAs. 27. Willingham. M, Ostlund, R & Pastan, I, Proc natl acad sci US 71 (1974) 4144. To discover whether myosin and actin could be coordinated in this manner or Received June 24, 1975 whether a more complex system involving 26-751818
Erptl Cell Res 97 (I 976)
Experimental
COORDINATED AND
Cell Research 97 (1976) 387-393
SYNTHESIS
MYOSIN
IN
AND
A VARIETY
NON-MYOGENIC N. RUBINSTEIN, Department
of Anatomy,
University
DEGRADATION
OF ACTIN
OF MYOGENIC
AND
CELLS
J. CHI and H. HOLTZER
of Pennsylvania
School of Medicine,
Philadelphia,
PA 19174, USA
SUMMARY The turnover of myosin and actin in both muscle and non-muscle cells in culture was investigated. By the double-label criterion, myosin and actin were coordinately synthesized and degraded in replicating, mononucleated fibroblasts, chondrocytes, BUdR-suppressed myogenic cells, and in post-mitotic, multinucleated myotubes. Myosin and actin were among the most stable proteins in each cell type. In single label ‘pulse-chase’ experiments, the half-lives of myosin and actin in all replicating, mononucleated cells were 2.5-3 days; in myotubes, however, they were approx. 6 days. Myosin and actin labelled in replicating presumptive myoblasts and chased until the cells ceased replicating and fused into multinucleated myotubes retained the degradation rate of 3 days; this differed from ,the rate of 6 days shown for myosin and actin newly-synthesized in post-mitotic myotubes. The type of myosin synthesized in the mother presumptive myoblast, then, IS transmitted to the postmitotic daughters. This myosin, however, is more rapidly degraded than the definitive myosin that is synthesized in the myotube.
The presence of actin-like and myosin-like molecules in a variety of non-muscle cells has raised interesting questions about the polymorphism of contractile proteins and the diversity of contractile systems [2,7,8, 10, 14, 211. While it is probable that red, white, cardiac, and smooth muscle myosins from one organism are the products of different structural genes [5, 17, 191, it is not yet clear whether all non-muscle myosins are alike or different. Antibodies against chicken skeletal muscle myosin, for example, do not react with extracts of contractile proteins from many kinds of chicken embryonic and mature non-muscle cells [I 1, 121. They only react with contractile proteins from definitive post-mitotic myoblasts, multinucleated myotubes, and mature muscle fibers. Conversely, antibody
prepared against several non-muscle myosins do not react with rabbit skeletal muscle myosin [20,22,25, 271. While obvious differences among myosins exist, few differences have been demonstrated among actins from the various muscle [9] or non-muscle cells [9]. This data suggests that there are several structural genes for the different myosin heavy and light chains. In contrast, there may be only one structural gene for actin which can be linked to the different myosins, or a system of multiple actin genes each with a very similar sequence [ 10, 11, 131. When multiple proteins such as actin and myosin have an intimate relationship within a cell, at least three layers of control can be envisaged: (1) an activating system Expti
Cell
Res 97 (1976)
388
Rub&stein, Chi and Holtzer
which allows expression of the proteins only in certain cell types; (2) a modulating control which varies the total amount of the proteins according to physiological necessities; and (3) an integrating mechanism which coordinates the total amount of each protein synthesized in relationship to the others. With regard to the integration mechanism, many investigators have concluded that actin and myosin in skeletal muscle are not synthesized and degraded as a unit, but are metabolized independently [15,24]. The finding, however, that myosin-like and actin-like molecules are present in fibroblasts and other mononucleated cells within muscle in vivo and in vitro [23] makes it necessary to re-examine this problem, Accordingly, we have begun an investigation into the mechanisms controlling the synthesis of myosin and actin in nonmuscle and muscle cells by demonstrating the coordination of myosin and actin synthesis and degradation in many cell types. MATERIALS
AND METHODS
For the first 36 h, a standard muscle culture consists largely of replicating presumptive myoblasts. Presumptive myoblasts divide and yield post-mitotic myoblasts which are the only cells capable of fusing into multinucleated mvotubes and synthesizing and assembling actin and myosin into inteidigitating-thick and thin filaments. Auurox. 70-80% of the cells in these early cultures &e presumptive myoblasts; the remainder are cells in different precursor compartments of the myogenic and fibrogenic lineages. These early cultures will be referred to as ‘presumptive myoblast cultures’, although, in fact, they contain a heterogeneous population of cells. Fusion of post-mitotic myoblasts into multinucleated myotubes begins after approx. 48 h in vitro. As the cultures mature, there is an increase in the number of nuclei in myotubes, accompanied by a decrease in the number of presumptive myoblasts. At the same time, the proportion and absolute number of tibroblasts in the culture increase. By day 6, when most of the fusion has occurred, the standard muscle cultures consist of large, branched, striated, contracting, multinucleated myotubes, large numbers of replicating, mononucleated fibroblasts, and a small number of replicating, mononucleated, precursor myogenic cells. The heterogeneity of these older cultures is similar Exprl Cell Res 97 (1976)
to the heterogeneity of an adult muscle. Any analysis of these cultures or whole muscles will yield data that is the average of values in different types of cells. To analyse only the myotube population, i.e. the true muscle cells. cultures of oure mvotubes were oreoared from stdndard muscle culturks by adding ‘cyiosine arabinoside. an inhibitor of DNA svnthesis. on davs 2. 3, and 4. On day 5, cultures we;e fed with normal medium, Since this led to the death of most replicating cells and did not affect the post-mitotic myoblasts and myotubes, the resulting cultures had an extremely high mu!tinuc!eated/mononuc!eated cell ratio [23]. The myotubes in these pure myotube cultures contained cross-striations and contracted spontaneously. It should be stressed that it is impossible to obtain a population of comparative purity in vivo. Fig. I shows photomicrographs of standard muscle and pure myotube cultures. Standard muscle cultures were prepared by the method of Bischoff & Holtzer [6] from 1!-day-old chicken embryonic breasts. Cells were plated at a density of 3x IVilOO mm dish and covered with 8 ml of medium 8 : I : I. Fibroblasts, chondrocytes, and BUdRsuppressed myogenic cells were prepared as described by Rubinstein et a!. [23] and Abbott & Holtzer [I]. Twice precipitated actomyosin, prepared by the method of Adelstein et a!. r31, was electrophoresed on 8% SDS-polyacrylamide &is according io Weber & Osborn [26]. Radioactivity in actomyosin was determined by slicing each gel into I mm pieces and placing each slice in a toluene-PPO-POPOP-NCS scintillant. This cocktail was counted in a Beckman Liquid Scintillation Counter. For some experiments, it was important to determine if any cells had been lost from the culture dish during the course of the experiments. Cultures were labelled with [3H]thymidine during the first 24 h in vitro, then washed with balanced salt solution (BSS) and fed normally thereafter. 13HJThymidine content in these culture dishes at any time was an estimate of the percentage of original DNA left on the dish, regardless of replication. Any loss of label from the dish would be-a measuremdnt of cell death. For example, if 75 % of the [3H]thymidine label were left on the dish, then 25% of the originally labelled cells and/ or their descendants would have left the culture dish. This prelabelling technique can also be used to determine the percentage of original protein left on the dish, exclusive of intracellular degradation. In the previous example, if protein had been labelled with [‘Ylleucine at the same time that the DNA had been labelled with [3H]thymidine, 25 % of the labelled protein would also have disappeared because of cell loss. Any additional loss of labelled protein would be a function not of cell death, but of degradation of protein within the cells remaining on thedish. There was very little cell loss in cultures of fibroblasts, chondrocites, and BUdR-suppressed myogenic cells. Only during the preparation of pure myotubes with cytosine arabinoside was there any significant cell death. Fig. 2 shows the loss of prelabelled DNA in chondrocyte cultures and during the preparation of pure myotubes. [3H]Leucine (30-50 Cilmmole), [Ylleucine (280 mCi/mmole), and [3H]thymidine (40-60 Cilmmole)
Synthesis and degradation
Fig. I. Light micrographs of standard muscle (a) and pure myotube (b) cultures, x252. In the standard muscle culture, note that the myotubes are obscured by
of actin and myosin
389
overgrowing mononucleated cell S. In the myotube culture, no mononucleated cells alre evident.
Erptl Cell Res 97 (1976)
390 100,
Rubinstein, Chi and Holtzer 6-a
0
0 0
75.
so. \,
15L-1
23454789
time in culture (days); ordinate: % original DNA. Loss of prelabelled DNA from chondrocyte cultures (O-O) and cytosine arabinoside-treated muscle cultures (0-O). The large loss of DNA in the latter cultures between days 2 and 5 is due to the cytosine arabinoside’s killing of replicating, mononucleated cells. The small loss thereafter is due to the myotubes’ contracting and pulling loose from the surface of the dish.
Fig. 2. Abscissa:
were obtained from New England Nuclear. Cytosine arabinosine and BUdR were purchased from Sigma. All tissue culture materials were from GIBCo.
RESULTS To examine whether actin and myosin in the various cell types were metabolized as a unit or independently, we used the double label technique of Arias et al. [4] as the criterion of coordinated synthesis and degradation. Myotube cultures were labelled for 24 h with 10 $X/plate [3H]leucine on day 5 in vitro, washed with BSS, and grown for four more days in the presence of excess unlabelled leucine. On day 9, the cells were pulsed for 2 h with 3 &i/plate [14C]leucine. The plates were immediately washed -again in BSS, the cells collected, and the actomyosin extracted and electrophoresed. Each gel was sliced and the radioactivity in each slice determined. In this type of experiment, the first label, in this case [3H]leucine, is a measure of degradation. The second label, [ 14C]leucine, is a measure of synthesis, since it is present for only a small fraction of the expected Exptl Cd Res 97 (1976)
half-life of the proteins. [14C]Leucine-labelled proteins, then, have not had a chance to be degraded, while [3H]leucine labelled proteins have had four days for degradation. The [‘“C]/[“H] ratios of a series of proteins are a comparison of their relative rates of synthesis and degradation. For example, if protein A and protein B had the same ratio, they would appear to be synthesized and degraded coordinately. On the other hand, if protein A’s ratio were higher than protein B’s, protein A is less stable. This occurs because more of the [3H]label in protein A was degraded during the 4-day chase period. This type of experiment, however, is only applicable to proteins whose synthesis relative to each other does not change during the time period involved. The ratio of myosin synthesis to actin synthesis was shown to be constant during the course of these experiments in all cell types. For example, in one experiment with pure myotubes, the ratios of myosin synthesis/actin synthesis on days 5, 7, and 9, were 2.7, 2.9, and 2.7, respectively. When myotubes were treated in this manner, the [14C]/[3H] ratio for actin and myosin heavy chain were identical (table 1). The same result was obtained with actin and myosin from chondrocytes, fibroblasts, and Table 1. Relative rates of myosin and actin turnover Myosin
Actin
Cell type
([“C]/[W
(Wl/13Hl)
Myotubes BUdR-suooressed Fibrob& Chondrocytes
1.14+0.10 1.03fO.19 0.43 kO.03 1.29kO.02
1.16+0.03 0.99f0.24 0.42&O. 15 1.3250.12
Cells were labelled with 10 &i/plate [3Hpeucine for 24 h, washed, and chased with excess unlabelled leutine. Four days later, cells were pulsed for 2 h with 3 &i/plate [“Cleucine and the actomyosin was immediately extracted and electrophoresed. The gels were stained and sliced, and the radioactivity in each slice was determined.
Synthesis and degradation
of actin and myosin
391
performed this type of experiment with day 7 myotube cultures by pulsing with [3H]leucine for 2 h before extraction of actomyosin. The actomyosin was electrophoresed and a specific activity (cpmloptical density unit) was calculated by scanning and counting the gel. The specific activity of myosin heavy chain (in arbitrary units) was 1.48kO.28 and that of actin was 1.42& 0.20 (4 experiments). The double label experiments, while indicating a physiological coupling between actin and myosin, do not give an absolute rate of .legradation for these proteins. To determine the half-lives of these molecules, Fig. 3. Abscissa: Slice no; ordinate: (lefr) cpmx lO-3, cultures were labelled for 24 h with 10 &i/ [3H]leucine (O---O); (righf) cpmx IO-*, [‘“C]leucine plate [3H]leucine. Mononucleated cells (he). ’ -Do&e label turnover experiment with BUdR-supwere labelled between days 1 and 2, myopressed myogenic cells. Cells were pulsed with [3H]leucine for 24 h, washed, and chased with excess un- tubes between days 5 and 6 in vitro. The labelled leucine. Four days later, the cells were pulsed plates were washed in BSS and grown in an for 2 h with [‘*C]leucine. The cells were collected; and the actomyosin was isolated and electrophoresed on excess of unlabelled leucine for various an 8% SDS-polyacrylamide gel. periods of time. Samples were taken each day and the actomyosin extracted and electrophoresed. The percentage of labelled BUdR-suppressed myogenic cells. Fig. 3 myosin heavy chain or actin remaining was shows the radioactivity of the 8% gel from calculated. Table 2 gives the results for the a similar experiment with BUdR-suppressed various cell types. While myotube actin and myogenic cells. By this double label cri- myosin had half-lives of approx. 6 days, terion, then, the myosin and actin are coordinately synthesized and degraded in all Table 2. Half-lives of myosin and actin cell types. Myosin Actin If total cell protein from one of these Cell type (days) (days) double label experiments is analyzed on an 6.08fl.30 5.93f0.68 SDS gel, the ratios of non-actomyosin pro- Myotubes 2.93kO.97 Presumptive myoblasts 3.00+0.94 teins vary considerably. In both myotubes BUdR-suppressed 2.50k0.36 2.9OkO.37 2.69tO. I6 2.75*0. IO and non-muscle cells, however, myosin and Fibroblasts 3.07 2.95 actin were among the most stable proteins Chondrocytes Cells were pulsed for 24 h with 10 &/plate r3H]leu(i.e. had the lowest [‘4C]/rH] ratios). washed, and chased with an excess of unlabelled Koizumi [15] has claimed that myosin tine, leucine. Myotubes were labelled between days 5 and 6, and actin synthesis and decay in adult presumptive myoblasts between days 0 and 1, and other cells between days 1 and 2. Each day therechicken muscle in vivo are not coordinated after, cultures were sacrificed; actomyosin was exbecause the specific activities of these pro- tracted and electrophoresed. Gels were stained, sliced, counted, and the amount of labelled myosin reteins in muscle labelled for a short time and and maining was calculated. If necessary, a correction for immediately extracted are not the same. We cell loss was also made. Exptl Cell Res 97(1976)
392
Rubinstein, Chi and Holtzer into myotubes confirms our earlier demonstration that presumptive myoblasts do, indeed, synthesize modest amounts of these two proteins [23]. 0
I
2
3
4
Fig. 4. Abscissa: time after removal of labelled medium (days); ordinate: % initial radioactivity. A-A, Myotube myosin; m-W, presumptive myoblast myosin; O-O, actin. Turnover of myosin from myotubes and presumptive myoblasts. Myotubes were pulsed on day 5 and the radioactive myosin measured through day 9. Presumptive myoblasts were pulsed on day 0 and the radioactivity determined through day 5, when only myotubes were present.
DISCUSSION
In both muscle and non-muscle cells, myosin heavy chains and actins are coordinately synthesized and degraded. The ratio of myosin synthesis/actin synthesis, then, must equal both the ratio of myosin degradationlactin degradation and the steady state ratio of myosinlactin in the cell. We have previously shown this ratio to be approx. 2.8 in myotubes and between 0.6 and the half-lives of myosin and actin in fi- 0.9 in non-muscle cells [7, 231. It should be stressed that the myosin in broblasts, chondrocytes, and BUdR-suppressed myogenic cells were only 2-3 days. myotubes is not the same molecule as the The procedure with the presumptive myosin in non-muscle cells. These proteins myoblasts was somewhat different from have different antigenicities [8, 12, 13J and that just described, since standard muscle different myosin light chains [7]. The actin, cultures contain mononucleated fibroblasts at least in presumptive myoblasts and possibly in other non-myogenic cells, may difwhich have a significant content of rapidlydegrading myosin and actin. Presumptive fer from the actin of myotubes in primary myoblasts were labelled for the first 20 h in structure, method of control, or both. Actin vitro and the amount of radioactive myo- (and myosin) labelled in presumptive myosin and actin remaining was measured on blasts and ‘chased’ into myotubes coneach succeeding day. On days 2 through 5, tinued to be degraded at the ‘mononucytosine arabinoside was added to eliminate cleated cell rate’ and not at the ‘myotube fibroblasts. This led to DNA loss for which rate’. In the myotube, the two sets of myowe had to compensate by the method de- sin and actin (the set synthesized in the scribed in Materials and Methods. Even af- presumptive myoblasts and the set synter the cultures contained only multinu- thesized in the myotube) were being treated cleated myotubes, myosin heavy chain and as independent populations (see fig. 3). This actin-originally labelled in mononucleated might mean that some primary structural presumptive myoblasts-still existed. They difference prevented presumptive myowere, however, being degraded at the same blast myosin and actin from being incorrate as in the other mononucleated cells (i.e. porated into sarcomeres and, thus, preTt=3 days) and not at the same rate as the vented these molecules from being denewly-synthesized myosin and actin in the graded as slowly as the myosin and actin myotubes (Tt=6 days, fig. 4). in myofibrils. The reason for the discrepancy between The fact that myosin and actin labelled in presumptive myoblasts could be chased our findings for skeletal muscle and those Exprl Cell Res 97 (1976)
Synthesis and degradation
of actin and myosin
393
of other investigators [15, 241 is unclear. multimeric particles or feedback loops is Our results were obtained with a pure popu- necessary requires further experiments with lation of myotubes, while others worked intact and fractionated cells. only with whole muscle, a combination of This research was supported by NIH grants HL-15835, myotubes and non-muscle connective tis- HDOO030, HDO0189, NSF-GB-38421X, and by the sue cells. The different myosin and actin Muscular Dystrophy Association. half-lives in various types of cells combined with the different myosin/actin ratios may REFERENCES have complicated the data of earlier invesAbbott, J & Holtzer, H, J cell biol28 (1968) 473. Adelstein, R & Conti, M, Cold Spring Harbor tigators . svmn auant bio137 (1973) 599. Since myosin and actin are synthesized Adeistein, R, Conti; M, Johnson, G, Pastan, I & Pollard, T, Proc natl acad sci US 69 (1972) 3693. on separate polysomes, their synthesis Arias, I, Doyle, D & Schimke, R, J biol them 244 must be synchronized to yield a constant (1969) 3303. Arndt, I & Pepe, F, J histochem cytochem 23 myosin/actin ratio in any cell type. Several (1975) 159. mechanisms for this synchronization can 6. Bischoff, R & Holtzer, H , J cell bio136 (1968) 111. be proposed. A feedback system or a spe- 7. CM., J,Rubinstein, N& Holtzer, H, Proc natl acad sci US 72 (1975). cial interaction between nascent myosin 8. Diensttpan, S & Holtzer, H, Cell cycle and cell differentiation (ed J Reinert & H Holtzer) p. 1. and actin molecules may be possibilities. Springer-Verlag (1975). In addition, preliminary evidence from our 9. Elzinga, M 8c Collins, J, Cold Spring Harbor symp auant biol37 (1973) 1. laboratory shows that it is difficult to unH, Sanger, J, Ishikawa, H & Strahs, K, couple myosin and actin synthesis. This 10. Holtzer, Cold Spring Harbor symp quant bio137 (1973) 549. suggests the possibility that actin and myo- 11. Holtzer, H, Rubinstein, N, Dienstman, S, Chi, J, J & Somlyo, A, Biochimie 56 (1974) 1575. sin polysomes are part of a large, multi- 12. Biehl, Holtzer, H, Strahs, K, Biehl, J, Somlyo, A & Ishikawa. H. Science 188119751.943. merit particle whose composition alone 13. Holtzer, H, Rubinstein, N,.Chi, J, Dienstman, S & regulates the relative rates of synthesis. Biehl, J, Exploratory concepts in muscular dystrophy (ed A Milhorat) p. 3. Excerpta Medica, It is also possible to regulate the relaAmsterdam (1974). tive synthesis of two molecules without 14. Ishikawa, H, Bischoff, R & Holtzer, H, J cell biol 43 (1969) 312. invoking any special mechanisms. Using Koizumi, T, J biochem 76 (1974) 431. reticulocyte lysates, Lodish [16] and 15. 16. Lodish, H, Nature 251 (1974) 385. McKeehan [ 181have demonstrated that the 17. Lowev, S & Risbv, D, Nature 234 (1971) 81. 18. McKeehan, W, J biol them 249 (1974) 6517. relative synthesis of a/P globin can be 19. Nakamura. A. Sreter. F & Gereelv. J. J cell bio149 (1971) 883. - .’ changed by varying the concentrations of R, Pastan, I & Adelstein, R, J biol them ribosomal subunits and initiation factors. 20. Ostlund, 249 (1974) 3903. These alterations occur because of the dif- 21. Pollard, T & Weihing, R, CRC critical rev in bio2 (1974) I. ferent affinities of (Y-and @globin mRNAs 22. them Pollard, T, Thomas, S & Niederman, R, Analyt biochem 60 (1974) 258. for the various initiation factors and riboN, Chi, J & Holtzer, H, Biochem biosomal subunits. In the red blood cell, then, 23. Rubinstein, phys res commun 57 (1974) 438. the 1 : 1 ratio of (Y: p globin synthesis might 24. Velick, S, Biochim biophys acta 20 (1956) 228. K & Groschel-Stewart, U, Proc nati acad be maintained merely by establishing cer- 25. Weber, sci US 71 (1974) 4561. 26. Weber, K & Osborn, M, J biol them 244 (1969) tain concentrations of ribosomes, initiation 4406. factors, and mRNAs. 27. Willingham. M, Ostlund, R & Pastan, I, Proc natl acad sci US 71 (1974) 4144. To discover whether myosin and actin could be coordinated in this manner or Received June 24, 1975 whether a more complex system involving 26-751818
Erptl Cell Res 97 (I 976)
