Coordinate regulation of contractile protein synthesis during myoblast differentiation

Cell,

Vol. 13, 599-611,

April

1978,

Copyright

0 1978 by MIT

Coordinate Regulation of Contractile Protein Synthesis during Myoblast Differentiation Robert B. Devlin and Charles P. Emerson, Department of Biology Gilmer Hall University of Virginia Charlottesville, Virginia 22903

Jr.

Summary The synthesis of contractile proteins has been studied during the differentiation of quail skeletal muscle myoblasts in culture. Myoblast differentiation was synchronized by transferring secondary cultures of rapidly dividing myoblasts into medium lacking cell division-promoting factors. Cultures at various stages of differentiation were then pulse-labeled with %-methionine, and cell extracts were resolved by electrophoresis on twodimensional gels. Incorporation into specific proteins was quantitated by autoradiography and fluorography using a scanning densitometer. Contractile proteins synthesized by muscle cultures were identifed by their co-electrophoresis on two-dimensional gels with contracile proteins purified from quail breast muscle. Our results show that the synthesis of myosin heavy chain, two myosin light chains, two subunits of troponin and two subunits of tropomyosin is first detected at the time of myoblast fusion and then rapidly increases at least 500 fold to maximum rates which remain constant in muscle fibers. Both the kinetics of activation and the molar rates of synthesis of these contractile proteins are virtually identical. Muscle-specific actin (LY) synthesis also increases at the time of myoblast fusion, but this actin (a) is synthesized at 3 times the rate of the other contractile proteins. The synthesis of 30 other muscle cell proteins was quantitated, and most of these are shown to follow different patterns of regulation. From these results, we conclude that the contractile proteins are regulated coordinately during myoblast differentiation. Introduction Differentiated cells often synthesize characteristic proteins which are expressed at specific times during their differentiation. Some differentiated cell types synthesize large amounts of only one or a few such proteins, whereas other cell types synthesize a complex array of characteristic proteins. The complexity of the proteins and mRNAs in these latter cell types suggests that there are mechanSims which coordinate gene expression during cellular differentiation, and several of the current models of gene regulation are based on this assumption (Georgiev, 1972; Davidson and Britten, 1973).

Skeletal muscle is an ideal cell type for examining the possibility of coordinate regulation of gene expression during cellular differentiation. Muscle cells synthesize numerous contractile proteins in large quantities, as well as other muscle-specific proteins such as creatine phosphokinase and acetylcholine receptors. The most abundant of the contractile proteins in muscle are actin, myosin, tropomyosin and troponin, which are present in a well defined stoichiometry in the myofibrils of skeletal muscle fibers (Potter, 1974). These proteins alone are composed of at least nine different polypeptide components which are structurally distinct from the “contractile” proteins present in nonmuscle cell types (Fine and Blitz, 1975; Chi, Fellini and Holtzer, 1975; Storti and Rich, 1976). The contractile proteins are therefore logical candidates for a group of cell type-specific proteins whose synthesis might be regulated coordinately during differentiation. Our previous studies have shown that the synthesis of one of the muscle contractile proteins, the heavy chain subunit of myosin, is highly regulated during the differentiation of quail myoblasts in cell culture (Emerson and Beckner, 1975; Emerson, 1977). Specifically, dividing myoblasts do not synthesize myosin heavy chain. Its synthesis is then activated at least 2000 fold when myoblasts fuse, although fusion per se is not required to initiate this activation process. Other studies also have shown that the synthesis of myosin heavy chain increases during myoblast differentiation (Coleman and Coleman, 1966; Paterson and Strohman, 1972; Yaffe and Dym, 1972), and in addition indicate that the synthesis of other contractile proteins may also be regulated (Yablonka and Yaffe, 1976; Whalen, Butler-Browne and Gros, 1976). The extent to which these proteins are regulated coordinately, however, has not been examined. These considerations therefore led us to undertake a quantitative study of synthesis of all the contractile proteins during myoblast differentiation to determine the temporal sequence of their activation and to compare their rates of synthesis. In this study, we have examined the synthesis of contractile proteins and 30 other muscle proteins in cultures of quail myoblasts undergoing highly synchronous fusion to form muscle fibers. Synthesis was measured by pulse-labeling cultures at various stages of differentiation with %-methionine, and then resolving proteins by electrophoresis on two-dimensional gels (O’Farrell, 1975). Incorporation of radioactivity into specific proteins was quantitated by autoradiography and computerized densitometry. Most of the contractile proteins can be readily resolved and identified on twodimensional gels, and this technique has facilitated analysis of the synthesis of the contractile proteins

Cell 600

as a group. In addition, we have been able to examine the precise temporal sequence of synthesis of these proteins during myoblast differentiation by making use of step-down procedures which we have developed for synchronizing myoblast differentiation (Emerson, 1977). These procedures involve simply transferring cultures of dividing myoblasts from growth medium into a medium from which cell division-promoting activities have been removed. Under these conditions, myoblasts undergo synchronous fusion and myosin heavy chain synthesis is rapidly activated. We now show that all the major contractile proteins are synthesized coordinately during myoblast differentiation. Results Characterization of Contractile Proteins by TwoDimensional Gel Electrophoresis Actin, myosin, tropomyosin and troponin were purified from quail breast muscle. Each of these proteins was then characterized by electrophoresis on two-dimensional gels. The gel system which gives optimal resolution of all the subunit components of these contractile proteins involves electrophoresis of proteins first on an isoelectric focusing gel with a pH range of 4-7 and then on an SDS slab gel containing a lo-15% gradient of acrylamide. By staining such gels with Amido black, we were able to identify and mark the positions of the musclespecific actin (a), myosin heavy chain (MHC), three myosin light chains (LC1, LC2, LC3), a minor component of LC2 (L&J, two troponin subunits (TNt, TN,) and two forms of tropomyosin (TM1, TM,). The subunit composition and molecular weights of these contractile proteins are similar to those of other vertebrate muscles of the fast (white) fiber type (Sarkar, Streter and Gergely, 1971). The positions of muscle actin and tropomyosin on twodimensional gels also are similar to those reported using calf or chick muscle cells (Storti and Rich, 1976; Whalen et al., 1976; lzant and Lazarides, 1977). The only major contractile protein which we

Figure

1. Two-Dimensional

Gel Electrophoresis

of %-Labeled

Proteins

were unable to characterize was troponin TN,. Even though this subunit was present on one-dimensional SDS gels, it was not detectable on twodimensional gels. It has been reported that TNi and TN, migrate on urea/acrylamide gels as a complex in the presence of minute amounts of calcium (Head and Perry, 1974). We tested this possibility by electrophoresis of troponin in the presence of EGTA, but TN, was still not evident. TN, probably does not enter the isoelectric focusing gel under our conditions because of its high isoelectric point, and is lost when the isoelectric focusing gel is equilibrated with SDS buffer. Contractile Protein Synthesis by Cultured Muscle Cells Having established that the muscle-specific forms of the contractile proteins of quail breast muscle can be resolved on two-dimensional gels, we used this technique to examine contractile protein synthesis in secondary cultures of embryonic quail breast muscle. For these experiments, we compared proteins synthesized by cultures of dividing myoblasts and by cultures of differentiated muscle fibers. Secondary cultures at these two stages were labeled for 2 hr with ?3-methionine. Cell extracts were then fractionated by electrophoresis on two-dimensional gels in the presence of purified contractile proteins which were used as markers. Gels were stained with Amido black to locate the positions of each of the contractile protein components and then dried for autoradiography. Figures 1 and 2 show autoradiograms of twodimensional gels of the proteins synthesized by dividing myoblasts and by differentiated muscle fibers. Figure 1B shows that differentiated muscle fibers actively incorporate ?3-methionine into proteins which migrate at positions coincident with actin (A), myosin light chains (LC,, LC,, LC,,), the small troponin subunit (TN,) and tropomyosins (TM). Myosin light chain LC3 was the only contractile protein whose synthesis was not detected in

Synthesized

by Cultures

of Dividing

Myoblasts

and Muscle

Fibers

%-methionine (A) immediately Cultures of dividing myoblasts were shifted to l-10 CM medium and then pulsed for 2 hr with 60 &i/ml after the shift and (B) 63 hr later, when cultures consist of muscle fibers. Cell extracts from both stages were mixed with contractile protein markers and fractionated by two-dimensional gel electrophoresis using pH 4-7 isoelectric focusing gels in the first dimension. lo6 cpm were applied to each gel. The gels were stained and dried, and autoradiograms were exposed for 166 hr. The positions of the contractile proteins on the autoradiograms were identified by their co-migration with stained markers of purified contractile proteins and are designated by the following abbreviations: myosin heavy chain (MHC), myosin light chains (LC,, LC,, LC,,), tropomyosin (TM), troponin (TN,, TN,) and actin (A). Other muscle cell proteins are designated by numbers (see text). The photographs shown in this figure were printed at high contrast to visualize accurately the overall distribution of proteins on these autoradiograms. It should be noted, however, that the intensities of the lighter spots are disproportionately enhanced, and that the actin and tropomyosin spots are overdeveloped on these photographs. See Figure 2 for photographs of the actin and tropomyosin regions of autoradiograms, and Figures 3-5 for quantitative measurements of the intensities of proteins on autoradiograms. (A) Autoradiogram of 9 proteins from cultures of dividing myoblasts. Arrows indicate positions of proteins which are not synthesized by myoblasts but are synthesized by muscle fibers. (B) Autoradiogram of 9 proteins from cultures of muscle fibers.

Coordinate 601

Synthesis

of Contractile

Proteins

Cell 602

Figure 2. Actin, Tropomyosin, Troponin and Myosin Heavy Chain Regions of Two-Dimensional Gels of the 35S-Labeled Proteins from Cultures of Dividing Myoblasts and Muscle Fibers Autoradiograms of *?S-labeled proteins from dividing myoblasts and myofibers were prepared as described in Figure 1. except that 500,000 cpm were applied to each gel and autoradiography was for 250 hr. Only those portions of the autoradiograms containing actin, tropomyosin, troponin TNt and myosin heavy chain are shown. (A) The actin region of a gel of proteins synthesized by dividing myoblasts. The arrow indicates the position of the muscle-specific actin (a); p and y indicate the positions of the nonmuscle actins. (6) The actin region of a gel of proteins synthesized by myofibers. (C) The tropomyosin region of a gel of proteins synthesized by dividing myoblasts. The arrows indicate the positions of TM1 and TM*. (D) The tropomyosin region of a gel of proteins synthesized by myofibers. (E) The troponin TN, and MHC regions of a gel of proteins synthesized by dividing myoblasts and resolved in the first dimension on isoelectric focusing gals having a pH range of 3-10

muscle fibers. Figures 2B and 2D show that the muscle fibers synthesize the muscle form of actin (a), which is resolved from two nonmuscle actins (p and r), as well as the two muscle tropomyosins, TM1 and TM2. Radioactivity at the positions of myosin heavy chain (MHC) and the large subunit of troponin (TN3 was also present on the pH 7 edge of isoelectric focusing gels (Figure 1B). Figure 2F shows that MHC and TNt could be more clearly resolved on isoelectric gels having a pH range of 3-10. Under these conditions, many of the proteins at the pH 7 end of the pH 4-7 gel enter the isoelectric focusing gel leaving MHC and TN, isolated at the pH 10 end. (This broader pH range was not used routinely since it does not provide adequate resolution of the different actins and tropomyosins. These results therefore show that the muscle fibers formed in cell culture actively synthesize at least eight of the muscle-specific contractile proteins. In contrast to muscle fibers, dividing myoblasts do not incorporate Y-methionine into any of the muscle contractile proteins, with the possible exception of muscle actin (a). Figure IA, 2C and 2E show that myosin light chains (LC1 and LG), troponins (TNt and TN,) and tropomyosins (TM1 and TM%) are not detectable on autoradiograms of %methionine-labeled myoblast proteins. Radioactivity in the region of MHC is present on autoradiograms of gels with an isoelectric focusing pH range of 4-7 (Figure 1A). Most of this radioactivity, however, is resolved from MHC on gels having a pH range of 3-10 (Figure 2E). Our previous studies have shown that the low level of radioactivity remaining in the MHC region on gels of myoblast proteins does not react with myosin antibodies, and that MHC synthesis can be assayed quantitatively by immunoprecipitation (Emerson, 1977). Consequently in this study, we have used this immunoprecipitation assay to quantitate MHC synthesis during myoblast differentiation (see Figure 3A). Figure 2A shows the presence of a low level of radioactivity in the actin (a) region of an autoradiogram of myoblast proteins. This radioactivity, however, is not localized as a discrete spot, but rather coalesces with the radioactivity of the adjacent nonmuscle actins (p and r), which are intensely labeled. Thus we do not rule out the possibility that this radioactivity within the actin ((Y) position represents spreading from the nonmuscle actin region rather than a low level of muscle actin (01) synthesis. In either case, the results shown in instead of 4-7. The arrows indicate the positions of MHC and TNt. (F) The troponin and MHC regions of a gal of proteins synthesized by myofibers and resolved on pH 3-10 isoelectric focusing gels as in(E).

Coordinate

Synthesis

of Contractile

Proteins

603

IA

1

50

40

40 30 II ‘0 I

50; ‘0

iz

1

2OI ?a b

f z” 0

2 100 IO

y 0 Figure

IO HOURS 3. Kinetics

20

30

AFTER of Synthesis

40 MEDIUM

50

60

70

70

CHANGE

of Contractile

Proteins

during

Myoblast

Differentiation

Cultures of dividing myoblasts in growth medium were shifted to l-10 CM medium and then pulsed for 2 hr with 80 &i/ml %-methionine at six times: at 0 hr, immediately after the shift; at 12 hr, when muscle cell fusion is initiated; at 19 hr; at 27 hr; at 39 hr, when fusion is complete: and at 63 hr, when muscle fibers are cross-striated. Labeled proteins from each time were separated by two-dimensional gel electrophoresis. 500,000 cpm were applied to each gel and the autoradiographs were exposed for 250 hr. Autoradiograms were then scanned with the computerized densitometer, and the optical density of each protein spot was calculated. Each value represents the average from three separate experiments. MHC synthesis was measured by immunoprecipitation of cell extracts with antimyosin antibodies. (A) L&(O); LG( 0); TN,(A); TN,(A); MHC(W). The ordinate for MHC is on the right; the ordinate for the other proteins is on the left. (B) TM,(O); TM,(O); actin (a)(A). The ordinate for actin (a) is on the right; the ordinate for the tropomyosins is on the left.

Figures 1 and 2 clearly demonstrate that at least eight contractile proteins are actively synthesized after myoblasts fuse to form multinucleated fibers. Comparison of the autoradiograms in Figures 1A and 1 B also shows that, except for these contractile proteins, the other proteins synthesized by myoblasts and muscle fibers are remarkably similar-that is, most of the noncontractile proteins detected on autoradiograms are synthesized by both myoblasts and fibers, although, as discussed below, the majority of these proteins undergo quantitative changes in synthesis during differentiation. Kinetics of Activation of Contractile Protein Synthesis Synthesis of the contractile proteins was quantitated in cultures of myoblasts undergoing highly synchronous differentiation. For these experiments, myoblast differentiation was synchronized by transferring dividing myoblasts into a “conditioned” medium (l-10 CM) which does not support cell division, but rather promotes fusion and the rapid activation of MHC synthesis (Emerson, 1977). To examine the kinetics of activation of contractile protein synthesis, cultures of dividing myoblasts

were transferred from growth medium into l-10 CM medium and then pulse-labeled for 2 hr with 35S-methionine at six times during the process of myoblast differentiation: at 0 hr, immediately after this transfer when cultures consist of dividing myoblasts; at 12 hr, when myoblast fusion is initiated; at 19 hr; at 27 hr; and at 39 hr, when fusion is virtually complete and 90-95% of the nuclei in cultures are within multinucleated fibers; and at 64 hr, when muscle fibers appear cross-striated. Cell extracts from cultures labeled at each of these six times were then fractionated by electrophoresis on twodimensional gels. An equal amount of TCA-precipitable radioactivity was applied to each gel to normalize for a 4 fold increase in the overall rate of 35S-methionine incorporation by cultures during the time course of the experiment. This increased incorporation is the result of a final doubling of myoblasts during the 10 hr period immediately following addition of l-10 CM medium (Emerson, 1977) and a subsequent 2 fold increase in the cellular rate of protein synthesis after myoblast fusion (Bowman and Emerson, 1977; unpublished observations). Incorporation of ?S-methionine into MHC was quantitated by immunoprecipitation of cell extracts

Cell 604

with myosin antibodies. We have previously shown that this is a quantitative and sensitive procedure for assaying MHC synthesis (Emerson, 1977). Incorporation into the other contractile proteins was quantitated by two-dimensional gel electrophoresis of cell extracts followed by autoradiography of dried gels. Autoradiograms were scanned at 100 with a computerized densitometer P ’ intervals which quantitated the optical density at the positions of the contractile proteins. The autoradiograms used for these measurements were developed from gels on which all the contractile proteins were resolved optimally, as shown in Figure 2. Autoradiography and densitometry have previously been shown to be a quantitative method for determinging radioactivity in acrylamide gels (Fairbanks, Levinthal and Reeder, 1965), and quantitation of higher energy isotopes such as ?S by autoradiography does not require the use of preflashed X-ray film (Laskey and Mills, 1975). In some experiments, we also quantitated radioactivity on two-dimensional gels by fluorography and densitometry using preflashed X-ray film as described by Laskey and Mills (1975). Fluorography is also a quantitative method for measuring radioactivity in gels, and in our hands, reduced the time required for development of X-ray film by about 6 fold. The spots formed on fluorograms, however, are considerably more diffuse than those on autoradiograms. This loss of resolution between closely resolved proteins such as the a, p and yactins and TM1 and TM2 tropomyosins limits the use of fluorography to analysis of proteins such as myosin light chains, LC1 and LCZ, which are well resolved from other proteins on two-dimensional gels. Quantitation of radioactivity in gels by autoradiography and fluorography was rountinely monitored by excising regions of gels corresponding to the contractile proteins and measuring radioactivity by scintillation counting. These measurements confirmed that there is a quantitative relationship between the optical densities developed on autoradiograms and fluorograms and the levels of radioactivity localized in the gels for these experiments (Fairbanks, et al., 1964; Laskey and Mills, 1975). Figure 3 shows the results of our analysis of the kinetics of activation of contractile protein synthesis during myoblast differentiation. The synthesis of the myosin, troponin and tropomyosin subunits is detected 12 hr after cultures of dividing myoblasts are transferred into l-10 CM medium. At this time, myoblast fusion is first observed. Incorporation into these proteins then rapidly increases and reaches a maximum by 30 hr. Actin (a) synthesis also increases at the time of myoblast fusion, but continues to increase after the synthesis of the other contractile proteins reaches maximum rates

at 30 hr. Quantitation of incorporation by scintillation counting of radioactivity in gels has shown that after 64 hr in l-10 CM, the myosin light chains, troponins and tropomyosins each account for 1.8% of the ?S-methionine incorporation, and MHC and actin (01) for 9% and 13%, respectively. These eight concentractile proteins therefore represent a major fraction of the protein synthetic activity of muscle fibers. The synthesis of the myosin light chains during myoblast differentiation was also quantitated by fluorography of two-dimensional gels. Figure 4 shows that the kinetics of activation of LCI and LC2 synthesis are identical to those obained using autoradiography. Furthermore, myosin light chains, as well as troponin and tropomyosin, were not detected on fluorograms of proteins from cultures of dividing myoblasts, even when these fluorograms were exposed for up to 7 days. Under these exposure conditions, fluorograms were developed to 4 times the overall intensities of the autoradiograms used to calculate the data shown in Figures 3 and 4. (Many of the regions on these fluorograms were overexposed after this length of time, and otpical densities in these regions were beyond the range of linearity.) We have found that ~20 dpm of YS can be quantitated when localized in protein bands (CO.1 cm*) on two-dimensional gels (Laskey

n‘0

6

iT5

z

a4 0

“0 3 \

02 0

I

0

IO HOURS

20

30 AFTER

Figure 4. Fluorographic Analysis sis during Myoblast Differentiation

40 MEDIUM of Myosin

50

60

70

CHANGE LC1 and LC2 Synthe-

Cultures at six times during myoblast differentiation were labeled with 9-methionine, and cell extracts were fractionated on twodimensional gels as described in Figure 3. 500,000 cpm were appled to each gel, and duplicate gels were run for each time point. One set of gels was quantitated by fluorography, and for comparison, the other set was developed by autoradiography. The total development time for autoradiograms was 6 times longer than for the fluorograms to achieve comparable development of X-ray films. The optical densitities of myosin LCI and LC, regions on fluorograms and autoradiograms were determined by scanning densitometry. Fluorography: LG(O), L&(A). Autoradiography: LC1( 0), L&(a).

Coordinate

Synthesis

of Contractile

Proteins

605

and Mills, 1975). This level of radioactivity (20 dpm) would be expected at the positions of the myosin light chains, troponins and tropomyosins on gels of myoblast proteins if myoblasts incorporate YSmethionine into the contractile proteins at l/500 the rate observed after myoblasts fuse and the activation of contractile protein synthesis is complete. Since we were unable to detect radioactivity at these positions, we conclude that incorporation of 35S-methionine into these contractile proteins increases at least 500 fold during myoblast differentiation. This is a minimum estimate of the actual increase. We also found that incorporation into muscle actin (a) increases at least 250 fold above the level detected on gels of myoblast proteins assuming that all the radioactivity within the (a) position is muscle actin. Incorporation into MHC, as assayed by immunoprecipitation, increases at least 1000 fold above the background level of detection in myoblasts. Previously, we quantitated MHC synthesis from measurements of amino acid incorporation and the specific activities of the intracellular amino acid pools, and these studies showed that the cellular rates of MHC synthesis increase at least 2000 fold after myoblast fusion Table

1. Relative

Molar

Rates

of Contractile

Protein

Protein

Molar Rates from Optical Density Measurements

Molar Rates Radioactivity Measurements

G

1 .oo

1 .oo

LC,

1.14

1 .lO

TM1

0.81

1.09

TM,

0.80

1.07

TN,

0.93

TN,

0.84

0.91

2.45

3.75

h

1.05c

Actin MHC

(a)

Synthesis from

a Radioactivity in TN, was not measured because of the high background radioactivity in this region of the gel. b Densitometry was not used to quantitate MHC synthesis because MHC does not migrate as a discrete spot on two-dimensional gels. c The radioactivity present in MHC was determined by antibody precipitation of MHC, as described in Experimental Procedures. Cultures of myofibers after 63 hr in l-10 CM medium were pulsed with 3*S-methionine for 2 hr. The 35S-labeled proteins were resolved by two-dimensional gel electrophoresis, and radioactivity in contractile proteins was determined by computerized densitometry and by scintillation counting as described in Experimental Procedures. The relative molar rates of contractile protein synthesis were determined by correcting the incorporation into each of the contractile proteins for their methionine contents [actin (a) is known to contain 16 methionines; LC,, LC,, TM,, TM, and TN, each contain six methionines; TN, contains nine methionines; and MHC contains 24 methionines] and then normalizing these values to LC,. All values shown are averages from at least two experiments.

(Emerson and Beckner, 1975; Emerson, 1977). The large increase in 35S-methionine incorporation into MHC and the other contractile proteins following myoblast fusion must therefore reflect at least a 500 fold increase in the rates of synthesis of each of these contractile proteins. The kinetics of synthesis of muscle actin (a) and the tropomyosins (Figure 36) differ somewhat from those of the other contractile proteins (Figure 3A). Actin (a) synthesis increases much faster than the other contractile proteins, reaching a rate which is 55% of its final rate shortly after fusion begins. At this time, the myosin light chains are being synthesized at only 10% of their final rate and the tropomyosins at only 35% of their final rate. Furthermore, the synthesis of actin (a) does not level off in muscle fibers as does the synthesis of the other contractile proteins, but rather increases at a reduced rate. It is also apparent from Figure 3B that the synthess of the two tropomyosin subunits does not increase in a precisely coordinated fashion. TM1 is initially synthesized at nearly twice the rate of TM,, but by the time the cells are completely fused, both tropomyosin subunits are made at the same rate. Despite these minor differences in actin and tropomyosin synthesis, however, it is clear that as a group, the contractile proteins are activated simultaneously at the time of myoblast fusion and that their rates of synthesis increase with remarkably similar kinetics. Stoichiometry of Contractile Protein Synthesis The relative rates of synthesis of contractile proteins were determined from measurements of Yjmethionine incorporation into each of the contractile proteins and from their known methionine contents. For these experiments, cultures of differentiated muscle fibers (63 hr in I-10 CM medium) were labeled for 2 hr with 35S-methionine, and the proteins were resolved by electrophoresis on twodimensional gels. 35S incorporation into each of the contractile proteins was determined by densitometry of autoradiograms as well as by scintillation counting of the contractile protein regions of these gels. The results of these measurements, presented in Table 1, show that all the contractile proteins are synthesized at virtually identical molar rates except for actin (a), which is synthesized at approximately 3 times the molar rate of the other contractile proteins. Rates determined by densitometry and scintillation counting are in good agreement, although the scintillation counting method gave somewhat higher values for actin synthesis, possibly reflecting our difficulty in accurately excising the OLregion free from adjacent radioactivity at the

Cell 606

/3 actin position. By either method, however, actin (a) is synthesized at a higher rate than the other contractile proteins. The evidence that all the other contractile proteins are synthesized at identical rates and activated with similar kinetics therefore indicates that these proteins are regulated coordinately during myoblast differentiation. Synthesis of Other Muscle Cell Proteins during Myoblast Differentiation It is possible that the kinetics of synthesis exhibited by the contractile proteins during myoblast differentiation is not confined to this group of proteins alone, but rather is characteristic of the majority of the proteins in differentiating muscle cells. Thirty proteins were therefore selected at random from autoradiograms (see Figure l), and their rates of synthesis during myoblast differentiation were quantitated by densitometry of the same autoradiograms used for analysis of contractile protein synthesis. We grouped these proteins into five general classes based on their kinetics of synthesis during myoblast differentiation. The results are shown in Figure 5. Class I Representatives of this class are shown in Figure 5A. The kinetics of synthesis of these proteins are very similar to those for the contractile proteins. These proteins are not synthesized at detectable rates in dividing myoblasts, and their synthesis is initiated at the time of myoblast fusion. Most of these proteins, however, are not synthesized at a constant maximal rate in muscle fibers, and with one exception (14), YS-methionine is incorporated into these proteins at only about one fifth the rate of the contractile proteins. Class II These proteins, shown in Figure 5B, are synthesized at increasing rates as fusion progresses, but are also synthesized by dividing myoblasts. The relative rate of synthesis of these proteins increases only 4-5 fold as compared to the 500 fold increase in contractile protein synthesis. %-methionine, however, is incorporated into most of these proteins at higher rates than the contractile proteins. These proteins are probably the major structural proteins of muscle cells, since they and the contractile proteins are the only proteins visible on stained two-dimensional gels of muscle fiber cell extracts. Two of these proteins, D1 and Dz, migrate on gels at the same positions as the desmids, which are major components of the 100 A filaments in smooth, cardiac and skeletal muscle (Izant and Lazarides, 1977). Although the synthesis of D1 and Ds is detectable at very low levels in dividing myoblasts, their synthesis increases during myoblast differentiation with kinetics similar to

those of the contractile proteins, suggesting a similar mode of regulation. Class 111 These proteins (Figure 5C) are synthesized at rates which vary less than 2 fold during myoblast differentiation. These proteins are therefore synthesized at rates which are relatively constant compared with the synthesis of contractile proteins and most of the other proteins examined. %S--methionine is incorporated into class III proteins at generally very low rates, and these proteins are not detectable on stained gels, indicating that they are not abundant proteins in muscle cells. Class IV The synthesis of these proteins (Figure 5D) decreases during myoblast differentiation. Although the synthesis of most of the proteins in this class decreases by 3-6 fold, the synthesis of two proteins (2 and 5) decreases to rates which are barely detectable in cultures of muscle fibers. Two proteins in this class (p and y) migrate on gels at the positions of nonmuscle actins (Whalen et al., 1976; Storti and Rich, 1976). Their synthesis decreases only slightly during myoblast differentiation, and they are some of the proteins most actively synthesized by both myoblasts and differentiated fibers. C/ass V Seven proteins (8, 13, 18, 22, 24, 25 and 26) are synthesized at rates which fluctuate during myoblast differentiation (data not shown). For example, protein 8 is synthesized at a high rate in dividing myoblasts, at a low rate during the period of myoblast fusion (12-30 hr in l-10 CM medium) and then at a high rate again in differentiated muscle fibers. The other proteins in this class also show irregular changes in synthesis during myoblast differentiation. The analysis of synthesis of these thirty proteins shows that very few if any of these proteins undergo changes in synthesis identical to those observed for the contractile proteins. From these results, we conclude that contractile protein synthesis follows a unique pattern of regulation during myoblast differentiation. Discussion The results of this study show that the synthesis of myosin heavy chain, two myosin light chains, two subunits of troponin and two subunits of tropomyosin is initiated simultaneously at the time of myoblast fusion. The synthesis of these proteins then increases with similar kinetics to high rates which are virtually identical. Both the time of activation and the rates of synthesis of at least seven contractile proteins are therefore highly coordinated during myoblast differentiation.

Coordinate

Synthesis

of Contractile

Proteins

607

r

-: -I I \ 0 Figure

5. Kinetics

of Synthesis

of 30 Muscle

Proteins

during

Myoblast

IO HOURS

20 30 AFTER

40 MEDIUM

50 60 CHANGE

1

7‘0

Differentiation

The synthesis of thirty muscle cell proteins was quantitated by densitometry of the same autoradiograms used for the analysis of contractile protein synthesis in Figure 3. Their positions on the two-dimensional gels and their numerical designations are shown in Figure 1. These proteins were placed into classes according to their kinetics of synthesis. (A) Proteins whose synthesis is initiated at fusion: 11(A), 14(U), 28( 0), 29(A), 30(O). (B) Proteins which are made in dividing myoblasts but whose synthesis increases during myoblast differentiation: 3(A), 6(A), 7(B), 9(O), D,(O), Ds( 0). Note that spot 7 is found at a slightly more acidic position on autoradiograms after fusion (see Figures 1A and lB), suggesting that this spot may represent more than a single protein component. (C) Proteins whose synthesis remains relatively constant throughout myoblast differentiation: l(W), 4(a), 17(O), 19(A), 20( 0). (D) Proteins whose synthesis decreases during myoblast differentiation: P(U), y(O), 2(A), 5(A), 15(O), 16(O). The ordinate for the p and y actins is on the right.

Cell 608

We do not detect the synthesis of any of the contractile proteins, except possibly the musclespecific actin (a), in cultures of dividing myoblasts. This result is consistent with those of our previous studies, in which we quantitated the cellular rates of myosin heavy chain synthesis during myoblast differentiation using a sensitive immunoprecipitation assay and measuring incorporation of amino acids into MHC and specific activities of intracellular amino acid pools (Emerson and Beckner, 1975; Emerson, 1977). Myosin heavy chain synthesis was undetected in cultures of dividing myoblasts even though rates of synthesis as low as 20 molecules per nucleus per min could have been detected. In this study, we show that following myoblast fusion, the rates of synthesis of the other contractile proteins increase at least 500 fold. The extent of this increase was determined from measurements of 35S-methionine incorporation into individual proteins, and incorporation at different stages of myoblast differentiation was normalized to a constant amount of radioactivity incorporated into total protein at each stage. This normalization assures that the observed increase in contractile protein synthesis during myoblast differentiation reflects a differential activation of synthesis rather than an overall increase in the rate of protein synthesis in muscle cells or changes in amino acid pools or cell number. In our previous studies, we have examined these parameters and have shown that the synthesis of myosin heavy chain increases at least 2000 fold from fewer than 20 molecules per nucleus per min in dividing myoblasts to 40,000 molecules per nucleus per min in muscle fibers (Emerson, 1977). The differential increase in synthesis of MHC and the other contractile proteins demonstrated in this study must therefore reflect large increases in the cellular rates of synthesis of all the contractile proteins. The magnitude of this increase suggests that dividing myoblasts do not synthesize contractile proteins and that their synthesis is activated de novo at the time of myoblast fusion. Other studies of contractile protein synthesis during myoblast differentiation in cultures of chick and calf muscle have indicated that myosin heavy chain, as well as muscle actin and tropomyosin, are synthesized by cultures of dividing myoblasts. After fusion, their synthesis increases by as little as 5-10 fold (Paterson and Strohman, 1972; Whalen et al., 1976). The primary chick and calf muscle cultures used in these other studies, however, had a high background (approximately 10%) of differentiated muscle cells even at the earliest stages of culture. In contrast, secondary cultures of quail muscle cells initially consist virtually exclusively of rapidly dividing myoblasts and are uncontaminated (~0.5%) by post-mitotic or fused muscle

cells or by other cell types. The homogeneity of the myoblasts in quail cultures has been established by 3H-thymidine autoradiography, clonal analysis, and cytological measurements of multinucleated and antimyosin immunofluorescent staining cells (Buckley and Konigsberg, 1974; Emerson, 1977; Devlin and Konigsberg, 1977). The presence of differentiated muscle cells in chick and calf myoblast cultures seems the most reasonable explanation for the apparent high levels of contractile protein synthesis and the rather small activation following the onset of fusion reported in these previous studies. The synthesis of muscle actin (a) also increases at the time of myoblast fusion. The rate of muscle actin synthesis, however, is 3 times that of the other contractile proteins and continues to increase in newly formed fibers after the other contractile proteins attain their maximum rates. These quantitative and kinetic differences indicate that muscle actin synthesis is regulated by somewhat different processes than the other contractile proteins. Our data, however, do not explain whether myoblasts synthesize muscle-specific actin at very low rates, and we do not exclude the possibility that actin synthesis is activated coordinately with the other contractile proteins, but then synthesized at higher rates as a result of factors unrelated to this primary activation, such as a greater stability or translational efficiency of actin mRNA in newly formed muscle fibers. There are also probably post-translational mechanisms which regulate the assembly of contractile proteins into myofibrils. We show that all contractile proteins except actin are synthesized at the same molar rates in cultures of cross-striated muscle fibers, even though it is known that they are not present in the same molar ratios in myofibrils of functional muscle (Potter, 1974). Presumably, there is either differential turnover or free pools of unassembled contractile proteins in muscle (Morkin et al., 1973). Of all the contractile proteins examined in this study, myosin light chain LC3 is the only protein in adult breast muscle whose synthesis is not activated during myoblast differentiation in cell culture. The absence of LC3 synthesis has previously been reported in studies of cultured rat muscle (Yablonka and Yaffe, 1976), although rabbit embryonic muscle contains small amounts of LC3 (Streter, Balint and Gergely, 1975). These results indicate that LC3 is synthesized later during muscle development and is therefore regulated differently from the other contractile proteins. In this regard, it is of some interest to note that with the exception of LC3, all the contractile proteins in adult quail breast muscle are synthesized by embryonic breast muscle cells in culture, and proteins from adult

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and cultured embryonic muscle co-migrate exactly on two-dimensional gels. This indicates that the contractile proteins expressed in cultures of embryonic muscle are structurally very similar, if not identical, to the proteins in adult muscle. Consequently, there does not appear to be a switch in synthesis during development from embryonic to adult forms of the contractile proteins. There could, of course, be structural differences to subtle to be detected by two-dimensional gel electrophoresis, and there are probably differences in the proportions of some of the contractile protein subunits during the transition from fetal to adult stages of development, as has been proposed for tropomyosin (Roy, Potter, and Sarkar, 1974). For example, in our hands, tropomyosin purified from adult quail breast muscle consists of at least 4 times as much TM2 as TM1, even though both subunits are synthesized at equal rates by cultures of crossstriated muscle fibers. In addition to the contractile proteins, other proteins also undergo changes in synthesis during myoblast differentiation. The synthesis of some of these proteins is initiated at the time of myoblast fusion (class I proteins) and therefore may be regulated coordinately with the contractile proteins. Although we cannot unequivocably identify any of these proteins at this time, it would not be surprising if they were other muscle-specific proteins (for example, a-actinin, creatine phosphokinase). Most of the thirty proteins examined, however, undergo changes in synthesis which can readily be distinguished both kinetically and quantitatively from the contractile proteins. In fact, some proteins (numbers 2, 5) which are actively synthesized by dividing myoblasts cease being synthesized after myoblast fusion. These results therefore indicate that regulatory processes other than activation of contractile protein synthesis also occur during myoblast differentiation. The activation of contractile protein synthesis is temporally correlated with the time of myoblast fusion. In previous studies, we have shown that fusion is not a prerequisite for contractile protein synthesis-that is, complete activation of synthesis can occur in mononucleated muscle cells under conditions which promote the cessation of myoblast cell division (Emerson and Beckner, 1975; Emerson, 1977). The synthesis of contractile proteins therefore appears to be initiated by mechanisms which respond to events occuring during the protraction of the G1 phase of the myoblast cell division cycle. A molecular basis for this activation, however, is not known. One basic question which remains unresolved is whether this activation is regulated by transcriptional or translational control mechanisms. Previous studies of myosin heavy

chain and actin mRNAs in cultured and embryonic muscle have suggested a variety of mechanisms for regulating contractile protein synthesis during muscle development. It has been proposed that the amount of MHC mRNA (Strohman et al., 1977) and actin mRNA (Paterson, Roberts and Yaffe, 1974) present in muscle cells determines the levels of MHC and actin synthesis-that is, transcription of these mRNAs regulates the synthesis of MHC and actin. On the other hand, it has also suggested that MHC mRNA and actin mRNA are present in the form of RNP particles in dividing myoblasts, and that post-transcriptional processes are responsible for the activation of MHC and actin synthesis (Heywood, Kennedy and Bester, 1973; Bag and Sarkar, 1975, 1976). Finally, other studies suggest that MHC mRNA is synthesized by dividing myoblasts, but is unstable until shortly before fusion when this mRNA becomes much more stable (Buckingham et al., 1974; Buckingham, Cohen and Gros, 1976). It is clear that a comprehensive study of the synthesis, processing and stabilities of the contractile protein mRNAs in relation to their translational activities in differentiating muscle cells will be required to resolve unequivocally the question of transcriptional or translational control of contractile protein synthesis. Sequence-specific as well as translational assays for the contractile protein mRNAs will almost certainly be required to undertake such experiments. The demonstration in this study, however, that the contractile proteins are synthesized coordinately during myoblast differentiation raises the exciting possibility that a detailed understanding of the molecular basis of this regulation will provide unique insights into the mechanisms which coordinate gene expression during cellular differentiation. Experimental

Procedures

Muscle Cell Culture Secondary cultures of myoblasts were prepared from breast muscle of IO day embryos of Japanese quail, Coturnix coturnix (Konigsberg, 1971). Cultures were inoculated with 15,000 cells per 60 mm gelatin-coated dish and initially maintained at 36.5”C in a 4% Cot atmosphere in a growth medium consisting of 15% selected horse serum and 10% chick embryo extract in Eagle’s MEM supplemented with Eagle’s nonessential amino acids, penicillin, streptomycin and fungizone. Cultures were refed with growth medium after 24 hr. After 48 hr, cultures consist of a rapidly dividing population of myoblasts uncontaminated byfibroblasts or differentiated muscle cells (Buckley and Konigsberg, 1974). At this time, growth medium was removed and replaced with I-10 CM medium, which consists of 1% chick embryo extract, 10% horse serum and MEM supplemented with the same components present in growth medium (Emerson and Beckner, 1975; Emerson, 1977). In addition to having reduced concentrations of embryo extract and horse serum, l-10 CM medium was conditioned by exposure for 48 hr to cultures of dividing myoblasts (Konigsberg, 1971). As a result of this conditioning process, l-10 CM medium does not support continued myoblast cell

Cell 610

division, but promotes fusion and the rapid initiation of myosin synthesis (Emerson, 1977). Cultures in l-10 CM medium were labeled for 2 hr with 80 &i/ ml of ?S-methionine (450 Ci/mmole) at various times during myoblast differentiation, as specified in the text. Each plate was then washed 3 times in saline G and the cells were extracted with 0.2 ml of isoelectric focusing buffer [0.2% SDS, 2% NP-40, 5% mercaptoethanol, 0.8% ampholyte (ph 4-6), 0.8% ampholyte (pH 5-7), 0.4% ampholyte (ph 3-lo), 9.5 M urea]. Samples were stored at -80°C. Incorporation of Y-methionine into protein was measured by incubating 2 ~1 aliquots of cell extracts in 1 ml of 0.3 N NaOH for 20 min at room temperature and then in 15% TCA for 20 min at 4°C. TCA precipitates were collected on celotate filters (Millipore Corporation), solubilized with NCS (Amersham-Searle) and counted in a Beckman LS-230 scintillation counter. Purification of Muscle proteins Contractile proteins were purified from the breast muscle of adult quail. Tropomyosin, troponin and actin were purified from an ether powder of muscle according to the procedure of Spudich and Watt (1971). Three rounds of isoelectric precipitation and ammonium sulfate fractionation were required to purify both tropomyosin and troponin. Pure actin was recovered from the O40% ammonium sulfate fraction obtained during tropomyosin purification. On SDS-acrylamide gels, the tropomyosin separated into two bands with molecular weights of 36,000 (TM1) and 34,000 daltons (TM2). The troponin was resolved into three bands with molecular weights of 40,000 (TNJ, 21,000 (TN3 and 18,000 daltons (TN,). Actin migrated as a single band with a molecular weight of 43,000 daltons. Myosin heavy chain and light chains were purified by a modification of the procedure of Baril, Love and Herrmann (1966). Glycerinated muscle was subjected to repeated rounds of ionic precipitation, and the extracted myosin was purified by ultracentrifugation, chromatography on DEAE-cellulose and ammonium sulfate fractionation. Myosin heavy chain migrated as a single band with a molecular weight of 200,000 daltons on SDS/ urea acrylamide gels. Three light chains were present with molecular weights of 25,000 (LC1), 18,000 (LCZ) and 16,000 daltons (L&). The region of the gel corresponding to the heavy chain was excised, and the myosin heavy chain was extracted and used to raise antibodies in rabbits (Emerson, 1977). Two-Dimensional Gel Electrophoresis Purified contractile proteins and 3sS-labeled cell extracts were analyzed by electrophoresis on two-dimensional gels according to the procedure of O’Farrell (1975). Briefly, samples were first subjected to electrophoresis on isoelectric focusing gels (2.5 mm in diameter) containing either a pH gradient of 4-7 or 3-10. A pH gradient of 4-7 was optimal for resolving the different forms of actin and the tropomyosin subunits. Isoelectric focusing gels were equilibrated for 30 min at room temperature in 10 ml of SDS buffer [8 M urea, 1.5% SDS, 5% mercaptoethanol, 0.06 M Tris (pH 8.3)]. TCA precipitation of the SDS buffer after this incubation showed that ~8% of the radioactive protein was eluted from the gels during this procedure. Equilibrated gels were then placed directly on top of a 2.25 mm thick SDS slab gel containing a lo-15% gradient of acrylamide. Isoelectric focusing gels were not embedded in agarose since the diameter of these gels was slightly wider than the width of the SDS slab gel, thus forming an air-tight seal when pushed down onto the SDS gel. The buffers used were the same as those described by Laemmli (1970). The gel was subjected to electrophoresis for 10 hr at 100 V, stained with Amido black and destained in 7.5% acetic acid/40% methanol. Gels were dried for autoradiography by heating under vaccum. In some experiments, gels were prepared for fluorography according to the procedures of Laskey and Mills (1975). Autoradiograms were prepared by exposure of dried gels for

100-300 hr to Kodak Medical X-ray film, which was then developed using Kodak X-ray developer. Fluorograms were prepared by exposure at -70°C of PPO-impregnated gels to Kodak RP Royal X-Omat film which was preflashed to an optical density of 0.15 (Laksey and Mills, 1975). Both the exposure time and the development times of autoradiograms and fluorograms were carefully controlled to permit quantitative comparisons of gels containing labeled proteins from different stages of myoblast differentiation. To quantitate optical density at specific positions on autoradiograms and fluorograms, the developed films were scanned with a Photoscan System P-1000 Densitometer (Optronits International, Inc.) using a 100 p path length. These data were processed using a computer which graphically displayed the otpical densities at 100 p* intervals over the scanned region of the autoradiogram. The total optical density of a specific spot on an autoradiogram was then determined by summing the optical densities within the spot and subtracting background optical densities, which were determined by scanning regions adjacent to the spot. This photoscanning system records optical densities linearly up to 3 OD units, and we demonstrated that the optical density at specific spots on an autoradiogram is a linear function of the time of exposure of the X-ray film to the dried gel. Analysis of 35S radioactivity in specific regions of the gels by scintillation counting (see below) confirmed that optical densities determined using this photoscan system are proportional to the radioactivity in these gels over the range of these measurements. The use of autoradiography and fluorography for quantitation of radioactivity in acrylamide gels has been well documented (Fairbanks et al., 1965: Bonner and Laskey, 1974; Laskey and Mills, 1975). The photoscan system permits high resolution scanning of X-ray film, making possible quantitation of radioactivity in closely resolved proteins. For some experiments, radioactivity in specific proteins resolved on two-dimensional gels was determined by scintillation counting. Regions of dried gels corresponding to stained proteins or spots on autoradiograms were excised, hydrated and then incubated with 0.5 ml of NCS for 2 hr at 55°C. 35S radioactivity was determined by scintillation counting in a toluene-based fluor. Counting effeciency for 35S was 90%. This procedure, although quantitative, has a more limited resolving power than the photoscan system, since it is difficult to excise accurately closely adjacent proteins from the dried gels. lmmunoprecipitation of Myosin Heavy Chain 35S-methionine incorporation into myosin heavy chain was measured by immunoprecipitation of the heavy chain from cell extracts using an antibody specific to myosin heavy chain (Emerson, 1977). Aliquots from cell extracts were added to antibody buffer [0.3 M NaCl, 1% Triton X-100, 1% deoxycholate, 0.03 M Tris (pH 7.6), 1 mg/ml BSA, 10 pg/ml myosin carrier]. Ammonium sulfate-fractionated antimyosin antiserum prepared by immunization of rabbits with purified quail myosin was added at concentrations which were shown to give quantitative precipitation of myosin heavy chain. After incubation at 4°C overnight, immunoprecipitates were collected by centrifugation through 1% sucrose in antibody buffer and washed 3 times with antibody buffer. The pellet was then suspended in SDS buffer and electrophoresed on 5% SDS/urea acrylamide gels. Regions of the gel corresponding to mysoin heavy chain were excised, and the amount of radioactivity present in mysoin heavy chain was determined by scintillation counting. Acknowledgments This research was supported by grants from the National Institute of Child Health and Human Development and the Muscular Dystrophy Association, and a Career Development Award to C.P.E. We thank Shari Avenius for her excellent technical assistance and Drs. Robert Grainger, Suzanne Emerson and Irwin

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Konigsberg for criticism during the preparation of this manuscript. We also wish to thank Dr. Robert Kretsinger for the use of his computer program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

October

7, 1977;

revised

January

9, 1978

(troponin

I). Biochem.

J. 737, 145-154.

Heywood, S. M., Kennedy, D. S. and Bester, A. J. (1973). Stored myosin messenger in embryonic chick muscle. FEBS Letters 53, 69-72. Izant, J. G. and Lazarides, E. (1977). Invariance and heterogeneity in the major structural and regulatory proteins of chick muscle cells revealed by two-dimensional gel electrophoresis. Proc. Nat. Acad. Sci. USA 74, 1450-1454. Konigsberg, I. R. (1971). Diffusion-mediated fusion. Dev. Biol.26, 133-152.

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expression during differentiaCold Spring Harbor Symp.