Striated muscle tropomyosin-enriched microfilaments ... - Springer Link

.Journal of Muscle Research and Cell Motility 11, 191-202 (1990)

Striated muscle tropomyosin-enriched microfilaments of developing muscles of chicken embryos SEU-MEI W A N G ~, SEU-HWA W A N G x, JENNY LI-CHUN LIN2 and JIM JUNG-CHING LINz*

IDepartment of Anatomy, College of Medicine, National Taiwan University, Taipei, Taiwan 2Department of Biology, University of Iowa, Iowa City, Iowa 52242, USA Received 1 July 1989, revised 14 October 1989; accepted 18 October 1989

Summary The striated muscle tropomyosin-enrichedmicrofilamentswere isolated from developing muscles in ovo by the previously described method with a monoclonal antibody against striated muscle isoforms of tropomyosin (Lin & Lin, 1986). Twodimensional gel analysis of the isolated microfilamentsfrom developing heart, thigh and breast muscles revealed the coexistence of non-muscle isoforms of tropomyosin and actin throughout all stages of embryogenesis. A small but significantamount of skeletal muscle isoforms (~, fl) of tropomyosins and their phosphorylated forms was detected in the microfilamentsisolated from hearts of 6--15-day-old embryos. These skeletal isoforms of tropomyosins disappeared after this stage of embryogenesis. In addition, we also detected both embryonic and adult isoforms of troponin T in early developing hearts. In developing thigh and breast muscles, the presence of non-muscle tropomyosin isoforms 2, 3a and 3b in the isolated microfilamentswas apparent. The contents of tropomyosin isoform 2 were decreased with development and this non-muscle isoform completely disappeared at the 15th day of embryogenesis. On the other hand, the non-muscle tropomyosin isoforms 3a and 3b were present throughout all stages of development. Double-label immunofluorescencemicroscopy with monoclonal CH1 (anti-striated muscle isoforms of tropomyosin) and CG]/6 (anti-non-muscle isoforms of tropomyosin) on the isolated, glycerinated skeletal and cardiac muscle cells of 10-day-old or 13-day-old embryos confirmed the colocalizationof muscle and non-muscle isoforms of tropomyosins within the same cells. These results suggest that different isoforms of actin and tropomyosin can assemble into a class of microfilaments (i.e. striated muscle tropomyosin-enrichedmicrofilaments)in ovo, which may transform into the thin filaments of mature muscle cells.

Introduction The expression of contractile proteins in developing muscle cells is generally switched from non-muscle isoforms to muscle isoforms (Fischman, 1970; Caplan et al., 1983; Carmon et al., 1978; Devlin & Emerson, 1978; Garrels & Gibson, 1978; Montarras et al., 1981, 1982; Patterson & Strohman, 1972). The myofibrillogenesis has been well studied on cultured myotubes and cardiomyocytes by electron microscopy (Shimada, 1971; Shimada et al., 1967; Fischman, 1971; Peng et al., 1981) and by immunofluorescence microscopy (Dlugosz et al.; 1984; Wang et al., 1988). The results from these studies demonstrate that at the earliest stage of development, individual nascent myofibrils appear to be formed from pre-existing stress fibre-like structures (SFLS) and at the later stage, all SFLS are replaced by mature myofibrils (Peng et al., 1981; Dlugosz et al., 1984; Antin et al., 1986). Moreover, recent studies on cardiac myofibrillogenesis strongly support that new myofibrils are directly derived from SFLS (Wang et al., 1988). How do the microfilaments of SFLS transform into the thin filaments of myofibrils in ovo? Will the transformation involve a reorganization of contractile *Author to whomcorrespondenceshouldbe addressed. 0142-4319/90 $03.00 + .I2 9 I990 Chapman and Hall Ltd.

proteins on the microfilaments? Using a monoclonal CL2 antibody against striated muscle tropomyosin, we have developed a method to isolate a set of striated muscle tropomyosin-enriched microfilaments from cultured skeletal muscle cells and have demonstrated a significant amount of non-muscle isoforms of actin and tropomyosin present in the isolated microfilaments of differentiating muscle cells (Lin & Lin, 1986). The isolated microfilaments possesses the characteristics of both microfilaments and thin filaments. Therefore, different isoforms of actin and tropomyosin can assemble into a set of filaments which eventually become the thin filaments of muscle cells. Such an approach has not previously been carried out on in ovo myofibrillogenesis. In the present study, we have used the same approach to isolate the striated muscle tropomyosin-enriched microfilaments from skeletal and cardiac muscles of chicken embryos at various stages of development. Biochemical characterization of these isolated microfilaments revealed a coexistence of non-muscle and muscle isoforms of actin and tropomyosin in the earlier developmental samples. As

192 development progressed, the non-muscle isoforms decreased substantially. A transient expression of skeletal tropomyosin isoforms (a, d) and their phosphorylated forms was detected in the heart at the earlier stage of development. Furthermore, cardiac troponin T appeared to switch from embryonic isoform to adult isoform during heart development. The colocalization of different isoforms of tropomyosin in the same cells was further demonstrated by double-label immunofluorescence on the isolated, glycerinated, embryonic muscle cells with isoform-specific monclonal antibodies.

W A N G , W A N G , LIN and LIN gave rise to either a halo zone around the tropomyosin or a white spot.

Isolation of embryonic cardiac and skeletal muscle cells Heart and thigh muscles of 10-I3-day-old chicken embryos were minced and glycerinated at 4 ~ overnight with buffer containing 50% glycerol, 75 mM KC1, 10 mM Tris pH 7.0, 2 mM EGTA, 2 mM MgC12. The muscle fragments were gently homogenized to dissociate cardiomyocytes and myotubes from the tissues. The isolated cells were allowed to attach to the albumin-coated slides before staining with antibodies.

Immunofluorescence microscopy Materials and methods

Monoclonal anti-tropomyosin antibodies The preparation and characterization of anti-tropomyosin monoclonal antibodies, CH1, CL2 and CGfl6 were reported previously (Lin et al., 1985a). Antibodies CH1 and CL2 specifically recognize the striated muscle isoforms of tropomyosins, whereas CGfl6 recognizes smooth muscle and non-muscle isoforms of tropomyosins. The class of CH1 and CGfl6 antibodies has been previously determined as IgG~ and IgM, respectively. Thus they are suitable for double-label indirect immunofluorescence microscopy.

Isolation of striated muscle tropomyosin-enriched microfilaments We have previously shown that monoclonal CL2 antibody appeared to be able to specifically immunoprecipitate the skeletal tropomyosin-enriched microfilaments from myotubes, but not from fibroblasts (Linet al, 1985a; Lin & Lin, 1986). Therefore, CL2 antibody was used here to isolate the striated muscle tropomyosin-enriched microfilaments from embryonic heart, thigh and breast muscles. Chicken muscles at different stages of development were dissected, minced and briefly treated with Triton/glycerol solution (0.05% Triton X-100, 0.1 M Pipes, 5 mM MgC12 0.2 mM EGTA, 4 M glycerol). After homogenization and centrifugation at 12 800g for 15 rain, a 1/10 volume of CL2 antibody in ascites fluid was added to the resulting supematant. The mixture was incubated for 1 h at room temperature. The antibody-induced aggregates of microfilaments were collected by centrifugation at 12 800g for 15 rain and washed twice with phosphate buffered saline (PBS) containing 5 mM MgC12 and 0.2 mM EGTA. The resulting pellets (i.e. striated muscle tropomyosin-enriched microfilaments) were dissolved in gel sample buffer for one- and twodimensional gel analysis.

One- and two-dimensional gel electrophoresis One-dimensional SDS PAGE was carried out as described by Laemmli (1970) with a low concentration of bisacrylamide (Blattler et al., 1972). Two-dimensional (2D) gel electrophoresis was performed by a modification (Linet al, 1985b) of O'Farrell's method (1975) with pH 4-6 ampholines. The Coomassie Bluestained gels (after pictures had been taken for the record) were then processed for silver stain according to the method of Merrill and co-workers (1981). In most cases, the Coomassie Blue dye interferred with the silver staining of tropomyosin and

The isolated, glycerinated muscle cells were fixed in 4% paraformaldhyde in 0.I M phosphate buffer, pH 7.2, for I0 rain. After washing with PBS, cells were treated with cold methanol for 10 min and then rinsed in PBS. For single-label immunofluorescence, cells were reacted with CL2 antibody and then fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (heavy and light chains). For double-label immunofluorescence, cells were reacted with a mixture of CH1 (IgG~ class) and CGfl6 (IgM class). After washing with PBS, cells were further incubated with FITC-conjugated goat anti-mouse ~ chainspecific IgG and tetramethylrhodamine isothiocyanate (TRITC)conjugated goat anti-mouse bt chain-specific IgG. After extensive washing in PBS, cells were mounted in 2% propylgallate, 80% glycerol in 0.05 M carbonate buffer (Giloh & Sedat, 1982). Observations were made using a Leitz Orthoplan microscope equipped for epifluorescence illumination.

Results As reported previously, monoclonal CL2 antibody reacted specifically with cardiac muscle tropomyosin, 0cand fl-isoforms of thigh and leg muscle tropomyosin and breast muscle tropomyosin, but not with gizzard tropomyosin or chick embryo fibroblast (CEF) tropomyosin (Lin & Lin, 1986). We have further shown that CL2 antibody was able to selectively isolate a class of skeletal muscle tropomyosin-enrichefl microfilaments from cultured myotubes but not the fibroblastic tropomyosinenriched microfilaments from CEF cells (Lin & Lin, 1986). Furthermore, we have previously shown that during microfilament isolation, the filaments do not exchange their components with exogeneous molecules, such as Factin (Matsumura et al., 1983). This specificity allows us to use this study for the isolation of the striated muscle tropomyosin-enriched microfilaments from heart, thigh and breast muscles of developing embryos. Characterization of the isolated microfilaments from developing muscle cells might reveal the assembly of different isoforms of contractile proteins during myogenesis. The striated muscle tropomyosin-enrichefl microfilaments (CL2-MF) isolated from &day-old embryonic hearts contained major protein components of 0r d- and y-actin as well as cardiac muscle tropomyosin (H) and its phosphorylated form (PH), as detected on Coomassie Blue-stained 2D gel (Fig. 1A). Several minor protein spots

Striated muscle thin filament development

193

Fig. 1. Two-dimensional gel analysis of the isolated striated tropomyosin-enriched microfilaments from hearts of 6-day-old (A and

B), 9-day-old (C and D) and 11-day-old (E and F) chicken embryos. Coomassie Blue-stained (A, C and E) and silver-stained (B, D and F) protein patterns are shown with the acidic end to the left. HC and LC indicate the positions of heavy and light chains of monoclonal antibody CL2. P~, P/J and PH refer to the phosphorylated forms of skeletal muscle 0r (~), ]~-tropomyosin (/J) and cardiac muscle tropomyosin (H), respectively; 3a and 3b are the chick embryo fibrolast (CEF) tropomyosin isoforms. TnT and E are adult and embryonic isoforms, respectively, of cardiac troponin T.

could be detected by silver staining of the same gel iFig. 1B). Among them, the skeletal muscle isoforms (~ and ]/) of tropomyosins and their phosphorylated forms, as well as the non-muscle tropomyosin isoform 3a and 3b were identified by their comigration in 2D gels with purified

tropomyosin isoforms (Linet al., 1985b). Although the cardiac and skeletal ~-tropomyosins have been shown to be identical in rabbit (Lewis & Smillie, 1980), these two isoforms in chicken apparently exhibit electrophoretic and immunological differences (Hayashi et al., 1979; Linet al.,

194 1985b). This may suggest that in chicken the cardiac atropomyosin gene differs from the skeletal a-tropomyosin gene. In fact, recent studies on avian tropomyosin genes have provided some basis for this idea (Lindquester et al., 1989). At least two spots of each of the embryonic (E) and adult isoforms of cardiac troponin T (TnT) were identified by immunoblots of 2D gel with monoclonal antibody against troponin T and their comigration with purified adult chicken cardiac troponin T (Jin & Lin, 1988). These isoelectric variants of embryonic and adult cardiac troponin T might be derived from post-translational modifications or from the aggregation of troponin T during isoelectric focusing. By using cDNA and genomic cloning, Cooper and Ordahl (1985) have reported an embryonic and an adult isoform from chicken hearts, which are generated from the same gene by alternative splicing. At the earliest developmental stage examined, the aactin was already the major isoform in heart muscles, although a significant amount of non-muscle isoforms of r- and 7-actin could be detected by Coomassie Blue staining of the gels. The relative amount of phosphorylated form of cardiac tropomyosin (PH) found in the isolated CL2-MF appeared to be greater than cardiac tropomyosin (H). These two forms of cardiac tropomyosin reached about an equal amount in the microfilaments isolated from 13-day-old embryonic heart (Fig. 2A). The function of phosphorylated tropomyosin in embryonic muscles remains to be determined. By using highly sensitive silver staining, not only non-muscle tropomyosin isoform (3a and 3b) but also skeletal muscle tropomyosin isoforms (a, Pa, r, Pfl) could be demonstrated (Fig. 1B). As development progressed, the non-muscle isoforms of tropomyosin 3a and 3b, like r- and ~-actin, were persistently present in the isolated CL2-MF (Figs 1 and 2). Suprisingly, the skeletal muscle isoforms of tropomyosins were also found in the isolated CL2-MF from 6-15-day-old embryonic hearts. Moreover, the embryonic isoform of cardiac troponin T was present in the isolated microfilaments of embryonic hearts at all stages of development. The adult isoform of cardiac troponin T began to accumulate and assemble into the isolated microfilaments of hearts at the 9-day-old embryo stage. It has been shown that both isoforms are derived from a single cardiac troponin T gene via developmentally regulated alternative splicing (Cooper & Ordahl, 1985). Therefore, the cardiac troponin T isoform switching in chicken appears to occur very early in development. Recently, we have found that the switching was not complete within a week after hatching (Jin et al., 1989). The detection of both isoforms in the isolated microfilaments suggests that both proteins are capable of associating with actin filaments. Whether they have slightly altered function or not remains to be determined. No striated muscle tropomyosin-enriched microfilaments (CL2-MF) were obtained from the isolation of thigh and breast muscles of chicken embryos before 8-day in ovo

WANG, WANG, LIN and LIN development. This suggests that the development of cardiac muscle is more advanced than that of skeletal muscles. Figure 3 shows the 2D gel analysis of the isolated CL2-MF from 10-20-day-old embryonic thigh muscles. In contrast to heart development, the a-actin did not become the major actin isoform before 15-day in ovo development (Fig. 3D). The amount of phosphorylated and fl-tropomyosin found in the isolated CL2-MF appeared to be greater than or equal to the unphosphorylated a- and fl-tropomyosins before 15-day in ovo development. This result is consistent with the previous report by Montarras and co-workers (1982). Non-muscle tropomyosins identified were isoforms 2, 3a and 3b. Like /J- and ?-actin, non-muscle tropomyosin 3a and 3b were coexistent with muscle isoforms throughout development. On the other hand, the non-muscle tropomyosin 2 disappeared from the isolated CL2-MF of thigh muscles at 18-day in ovo development (Fig. 3E). The 2D gel analysis of the CL2-MF isolated from breast muscles of developing embryos (Fig. 4) showed a similar trend with that for thigh muscles. The a-actin was the major actin isoform after 15-day in ovo development (data not shown). As in the embryonic thigh muscles, the fl-tropomyosin and its phosphorylated form were predominant to the ~-tropomyosin and its phosphorylated form before 18-day in ovo development (Fig.4C). Nonmuscle tropomyosin isoforms 3a and 3b were detected in the CL2-MF throughout the embryogenesis stages. As development proceeded, the non-muscle tropomyosin 2 also disappeared at 18-day in ovo development (Fig. 4C). The CL2 antibody gave the typical I band staining in the isolated glycerinated thigh muscle cell (Fig. 5A) and cardiac muscle cell (Fig. 5B). The staining pattern was observed in the muscle cells isolated from 9-20-day-old embryos. These results suggest that the striated muscle tropomyosin-enriched microfilaments isolated here were actually the precursors for sarcomeric thin filaments. In order to confirm the coexistence of muscle and nonmuscle isoforms of tropomyosin in the isolated CL2-MF, double-label immunofluorescence was performed on the isolated, glycerinated cardiac muscle cells (Fig. 5C-F) and thigh muscle cells (Fig. 6A-D) of 10-13-day-old embryos. Most of the isolated muscle cells did contain both muscle and non-muscle isoforms of tropomyosin. Figure 5C and E demonstrated a periodic staining with anti-muscle tropomyosin CH1 antibody on the isolated, glycerinated cardiac muscle cells. On the same cells, a fibrillar or more diffuse staining was obtained with anti-non-muscle tropomyosin CG/J6 antibody (Fig. 5D and F). The same results were also obtained on the isolated, glycerinated thigh muscle cells (Fig. 6). Immature thigh muscle cells contained some non-striated fibrillar staining structures with CGfl6 antibody (Fig. 6B), which did not stain with CH1 antibody (Fig. 6A). However, the majority of fibrillar structures were doubly-stained with both CH1 and CGfl6. A periodic staining by both antibodies on mature myofi-


195

Fig. 2. Two-dimensional gel analysis of the isolated striated tropomyosin-enriched microfilaments from hearts of 13-day-old (A and B), 15-day-old (C and D) and 18-day-old (E and F) chicken embryos. Coomassie Blue-stained (A, C and E) and silver-stained (B, D and F) protein patterns are shown with the acidic end to the left. HC and LC indicate the positions of heavy and light chains of monoclonal antibody CL2. P0r P/J and PH refer to the phosphorylated forms of skeletal muscle 0r (0r fi-tropomyosin (fi) and cardiac muscle tropomyosin (H), respectively; 3a and 3b are CEF tropomyosin isoforms. TnT and E are adult and embryonic isoforms, respectively, of cardiac troponin T. brils was frequently observed in most of the isolated thigh muscles (Fig. 6C and D). In many cases, the staining intensity of CG]/6 antibody was weaker compared with that of CH1 antibody.

Discussion

We have shown that a class of striated muscle tropomyosin-enriched microfilaments could be isolated from

196

W A N G , W A N G , LIN and LIN

Fig. 3. Two-dimensional gel analysis of the isolated striated tropomyosin-enriched microfilaments from thigh muscles of chicken embryos at different stages of development. (A) 10-day-old; (B) 12-day-old; (C) 13-day-old; (D) 15-day-old; (E) 18-day-old; (F) 20day-old. Coomasie Blue-stained protein patterns are shown with the acidic end to the left. HC and LC indicate the positions of heavy and light chains of monoclonal CL2 antibody. P~ and P~ refer to the phosphorylated forms of skeletal muscle ~- and/~-tropomyosin, respectively; 2, 3a and 3b are the CEF tropomyosin isoforms.


197

Fig. 4. Two-dimensional gel analysis of the isolated striated tropomyosin-enriched microfilaments from breast muscle of chicken embryos at different stages of development. (A) 10-day-old; (B) 13-day-old; (C) IS-day-old; (D) 20-day-old. Coomassie Blue-stained protein patterns are shown here with the acidic end to the left. HC and LC indicate the positions of heavy and light chains of monoclonal CL2 antibody. P~ and Pfl refer to the phosphorylated forms of skeletal muscle ~- and fl-tropomyosin, respectively; 2, 3a and 3b are the CEF tropomyosin isoforms.

developing skeletal muscles and hearts by a monoclonal CL2 antibody. This class of microfilaments may represent the precursor of the thin filaments of adult muscles. These microfilaments were composed of muscle and non-muscle isoforrns of actin and tropomyosin throughout all the developmental stages examined. As the skeletal muscle cells matured, one of the non-muscle isoforms (isoform 2) of tropomyosin decreased and disappeared between 15-18-day-old in ovo development. This time course appeared to be coincident with the disappearance of skeletal tropomyosin isoforms in the developing cardiac muscles. The coexistence of muscle and non-muscle tropomyosin in the same cardiac or skeletal muscle cells was further confirmed by double-label immunofluorescence. These results are in good agreement with our previous finding on in vitro culture myogenic cells (Lin & Lin, 1986). Therefore, we concluded that non-musde isoforms of actin and tropomyosin as well as their muscle

isoforms were incorporated into a set of striated tropomyosin-enriched microfilaments and at least one nonmuscle tropomyosin isoform (isoform 2) was gradually decreased and disappeared upon the maturation of thin filaments. Using double-label immunofluorescence with both ~actin-specific and 7-actin-specific antibodies, Otey and coworkers (1988) have demonstrated that both muscle and non-muscle actins were colocalized in the same myotubes and in the same striations of mature myofibrils. However, at the electron microscopical level, they have detected a quantitative but not qualitative difference in distribution of muscle and non-muscle actin isoforms in differentiating myogenic cells. In the present study, we have demonstrated the persistent presence of non-muscle tropomyosin isoforms 3a and 3b (low M r isoforms) along with muscle tropomyosin in the isolated CL2-MF of developing muscle cells. These low M r isoforms of non-muscle

198

W A N G , W A N G , LIN and LIN

Fig. 5. Immunofluorescence analysis of tropomyosin isosforrns in the isolated, glycerinated muscle cells. Indirect Immunofluores cence with monoclonal CL2 antibody was performed on the glycerinated skeletal muscle cells (A) of 13-day old embryo and cardiac muscle cells (B) of X0-day-old chicken embryos. It is clear that muscle isoforms of tropomyosin recognized by CL2 antibody are already organized in a typical I-band striation in both types of muscle cells at this stage of embryogenesis. Double-label immunofluorescence with CH1 antibody against striated muscle isoforms of tropomyosin (C and E) and CGfl6 antibody against nonmuscle isoforms of tropomyosin (D and F) was performed on the isolated, glycerinated cardiac muscle cells of 10-day-old (C and D) and 13-day-old (E and F) chicken embryos. Coexistence of both muscle and non-muscle forms of tropomyosin with the same developing cardiac cell is evident. Bar in A, B and D, 20 #m; Bar in F, 10/2m.


199

Fig. 6. Double-.label immunofluorescence analysis of tropomyosin isoforms in the isolated, glycerinated skeletal muscle cells~ Muscle cells from thighs of I0 day-old (A and B) and 13-day old (C, D) chicken embryos were processed for indirect immunofluorescence with CHI antibody against striated muscle isoforms of tropomyosin (A and C) and CGfi0 antibody against non-muscle isoforms of tropomyosin (B and D). In immature muscle cell, the striated muscle isoforms of tropomyosin (A) are continuously distributed along the microfilament bundles which also contain non-muscle isoforms of cropomyosins. However, cells at this stage of development also contain some microfilament bundles, which only have non-muscle tropomyosin isoforms. In more mature muscle cells, the striated muscle tropomyosin isoforms are organized into a typical I band appearance. The non-muscle tropomyosin isoforms are more diffusely distributed along the I band and other cytoplasmic area.

tropomyosin have been previously shown to localize at both stress fibres and cortical ruffles of CEF cells (Lin et aI., 1988). Together with non-muscle fl- and 7-actin, they may constitute a set of functionally distinct microfilaments in differentiating muscle cells. However, the present approach would not allow us to distinguish this class of microfilaments from the isolated CL2-MF. Previous studies on cultured muscle cells and cells recovering from ethyl methanesulphonate treatment have led to the conclusion that early assembly of myofilaments occurs in the intimate association with a single preexisting SFLS. The SFLS were proposed to act as templates or scaffolds for nascent myofibrils and to disappear

after the myofibrils formed (Dlugosz et al., 1984; Antin et aL, 1986). Our results do not seem to support this idea of two separate filament systems but suggest that there is a gradual transformation of one filament system with muscle isoforms replacing non-muscle isoforms. The recent study on cultured cardiac myocytes has further demonstrated that presumptive SFLS also contain 0~-, fl, and 7actins, as well as muscle tropomyosin (Wang et a]., 1988). Occasionally, some of the cultured immature cardiac myocytes with no myofibrils are stained only with antimuscle tropomyosin antibody but not with anti-nonmuscle tropomyosin antibody. This class of cells is not observed in the present study. However, cultured cardiac

200

WANG, WANG, LIN and LIN

myocytes with sarcomeric staining patterns by antimuscle tropomyosin antibody are also stained with anti-nonmuscle tropomyosin antibody (Greaser et al., 1989). This result is in good agreement with the present study (Fig. 5C-E). Therefore, our results are also consistent with the idea that myofibrils may form directly by the growth of nascent myofibrils without any SFLS templates (Sanger et al., 1986; Mittal et al., 1987). The molecular mechanism for this transformation may involve a complex protein isoform transition. For example, in thigh and breast muscles, we have observed a decreasing amount and finally disappearance of nonmuscle tropomyosin isoform 2, as development progresses. In developing heart muscles, a transient expression of embryonic isoform of cardiac troponin T and skeletal muscle isoforms of tropomyosin was found to associate with the isolated microfilaments. These muscle proteins disappear at the same developmental stages as that for the disappearance of non-muscle tropomyosin 2 in skeletal muscles. Morover, we have repeatedly found a higher content of phosphorylated muscle tropomyosin in the striated tropomyosin-enriched microfilaments isolated from earlier developing muscle cells, as compared with unphosphorylated muscle tropomyosin. This suggests that during embryogenesis, the conversion of microfilaments into striated tropomyosin-enriched microfilaments is accompanied with phosphorylation of muscle tropomyosin. Whether this phosphorylation occurs before or after muscle tropomyosin assembly on microfilaments remains unclear. In a preliminary experiment with pulsechase labelling of cultured myotubes, we have found that the striated tropomyosin-enriched microfilaments from 10 rain pulse-labelled myotubes do not contain phosphorylated forms of newly synthesized tropomyosin. After 10 min chase, the phosphorylated tropomyosins begin to increase in the isolated microfilaments. It takes 60 min to reach plateau. These results may suggest that the phosphorylation of tropomyosin occurs on the microfilaments. The function of phosphorylated tropomyosin in developing muscles remains unclear. However, the site of phosphorylation in vitro and in vivo appears to be identical, which locate at Ser-283 residue in skeletal muscle tropomyosin (Mak et al., 1978; Montgomery & Mak, 1984). This region has been shown to be involved in several key functions of tropomyosin; interaction of tropomyosin with troponin T (Brisson et al., 1986), cooperative binding of tropomyosin to actin filaments (Mak & Smillie,

1981) and perhaps cooperative binding of myosin subfragment 1to regulated F-actin (Greene & Eienberg, 1980). Therefore, the phosphorylation of muscle tropomyosins in earlier embryos may enhance the head-to-tail polymerization of tropomyosin dimers, which then strengthens the binding to actin filaments. The competition between muscle and non-muscle tropomyosin for binding to microfilaments may well be a part of the transformation mechanism. In his regard, Heeley and co-workers (1989) have recently demonstrated that the phosphorylated tropomyosin has substantially higher viscosities at low ionic strengths than the unphosphorylated protein. This suggests a greater tendency for the phosphorylated tropomyosin to form head-to-tail polymerization. However, the F-actin binding abilties between the phosphorylated and unphosphorylated forms do not seem to differ significantly. Cardiac troponin T isoform switching during development has been observed in chichen hearts by cDNA cloning and mRNA analysis (Cooper & Ordahl, 1985). In this study, we have clearly shown that two isoforms of cardiac troponin T exist in the isolated microfilaments of embryonic hearts. This suggests that both isoforms are assembled on the actin microfilaments during early development. The embryonic isoform persists throughout embryogenesis and disappears within a week after hatching (Jin et al., 1989). Recently, a troponin T isoform switching was also observed in developing rat hearts (Jin & Lin, 1988). However the time course of troponin T isoform switching is different between chicken and rat. In rat heart, the switching occurs after birth and switching is complete within 2 weeks after birth. This difference in time course for troponin T switching may simply reflect the species difference. The troponin T isoforms may play a slightly altered function in response to a change in functional demand for developing muscle cells.

Acknowledgements This work was supported in part by grant (NSC76-0412B002-107) from the National Science Council, Republic of China to Seu-Mei Wang and by grants from the National Institutes of Health (HD18577, GM40580), and the Muscular Dystrophy Association to Jim J.-C. Lin. Dr. J.J.C. Lin is a recipient of a Pew Scholarship in Biomedical Sciences from the Pew Memorial Trust.

References ANTIN, P. B., TOKUNAKA, S., NACHMIAS, V. T. & HOLTZER, H.

(1986) Role of stress fiber-like structures in assembling nascent myofibrils in myoblasts recovering from exposure to ethyl methanesulfonate.]. Cell Biol. 102, 1464-79. BLATTLER, D. P., GARNER, F., VAN SLYKE, K. & BRADLEY, A. (1972)

Quantitative electrophoresis in polyacrylamide gels of 2-40%. ]. Chromatogr. 64, 147-55.

BRISSON, J. R., GROLOSINSKA, K., SMILLIE, L. B. & SYKES, B. D. (1986) I n t e r a c t i o n o f t r o p o m y o s i n a n d t r o p o n i n T: A p r o t o n n u c l e a r m a g n e t i c r e s o n a n c e s t u d y . Biochemistry 25, 4548--55. CAPLAN, A. I., FISZMAN, M. Y. & EPPENBERGER, H. M. (1983)

Molecular and cell isoforms during development. Science 221, 921-7.

Striated muscle thin filament development (1978) Synthesis of tropomyosin in myogenic cultures and in RNA-directed cell free system: qualitative changes in the polypeptides. Cell 14, 393-401. COOPER, T. A. & ORDAHL, C. P. (1985) A single cardiac troponin T gene generates embryonic and adult isoforms via developmentally regulated alternate splicing. ]. Biol. Chem. 260, 11140-8. DEVLIN, R. B. & EMERSON, C. P. JR. (1978) Coordinate regulation of contractile protein synthesis during myoblast differentiation. Cell 13, 599--611. CARMON, Y., NEWMAN, S. & YAFFE, D.

DLUGOSZ, A. A., ANTIN, P. B., NACHMIAS, V. T. & HOLTZER, H.

(1984) The relationship between stress fiber-like structures and nascent myofibrils in cultured cardiac myocytes. ]. Cell Biol. 99, 2268-78. FISCHMAN,D. A. (1970) The synthesis and assembly of myofibrils in embryonic muscles. Curr. Top. Dev. BioL 5, 235-80. FISCHMAN,D. A. (1971) The fine structure of muscle differentiation in monolayer culture. In Research in Muscle Development and the Muscle Spindle (edited by BANKER, B., PRYZYBYLSKI, R., VAN DER MEULEN, J. & VICTOR, M.).

pp 163-75. New York: Elsevier. (1978) Identification and characterization of multiple forms of actin. Cell 9, 793-805. GILOH, H. & SEDAT, J. W. (1982) Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugated by n-propyl gallate. Science 217, 1252-5.

GARRELS, J. I. & GIBSON, W.

GREASER, M. L., HANDEL, S. E., WANG, S-M., SCHULTZ, E.,

(1989) Assembly of titin, myosin, actin and tropomyosin into myofibrils in cultured chick cardiomyocytes. In Cellular and Molecular Biology of Muscle Development (edited by KEDES, L. H. & STOCKDALE,F. E.), pp. 247--57. New York: Alan R. Liss. GREENE, L. E. & EISENBERG, E. (1980) Cooperative binding of myosin subfragment-1 to the actin-troponin-tropomyosin complex. Proc. Natl. Acad. Sci. USA 77, 2616-20. HAYASHI, J., ISHIMODA, T. & HIRABAYASHI, T. (1979) On the heterogeneity and organ specificity of chicken tropomyosins. ]. Biochem. 81, 1487-95. BULINSKI, J. C., LIN, J. J.-C & LESSARD, J. L.

HEELEY, D. H., WATSON, M. H., MAK A. S., DUBORD, P. & SMILLIE,

L. B. (1989) Effect of phosphorylation on the interaction and functional properties of rabbit striated muscle ~tropomyosin. ]. Biol. Chem. 264, 2424-30. JIN, J.-P & LIN, J. J.-C (1988) Rapid purification of mammalian cardiac troponin T and its isoform switching in rat hearts during development. ]. Biol. Chem. 263, 7309-15. JIN, J.-P., LIN, J. L.-C & LIN, J. J.-C (1989) Troponin T isoform switching during heart development. Ann. N. Y. Acad. Sci. (in press). LAEMMLI,U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227, 680-5. LEWIS, W. G. & SMILLIE, L. B. (1980) The amino acid sequence of rabbit cardiac tropomyosin. ]. Biol. Chem. 255, 6854-9. LIN, J. J.-C., CHOU, C. S. & LIN, J. L.-C. (1985a) Monoclonal antibodies against chicken tropomyosin isoforms: production, characterization and application. Hybridoma 4, 223-42.

201 (1988) Differential localization of tropomyosin isoforms in cultured nonmuscle cells, d]. Cell Biol. 107, 563-72. LIN, J. J.-C., HELFMAN, D. M., HUGHES, S. H. & CHOU, C.-S. (1985b) Tropomyosin isoforms in chicken embryo fibroblasts: Purification, characterization, and changes in Rous sarcoma virus-transformed cells. J. Cell Biol. 100, 692-703. LIN, J. J.-C. & LIN, J. L.-C (1986) Assembly of different isoforms of actin and tropomyosin into the skeletal tropomyosinenriched microfilaments during differentiation of muscle cells in vitro. J. Cell Biol. 103, 2173-83. LIN, J. J.-C., HEGMAN, T. E., & LIN, J. L..-C

LINDQUESTER, G. J., FLACH, J. E., FLEENOR, D. E., HICKMAN, K. H. &

DEVLIN,R. B. (1989) Avain tropomyosin gene expression. Nucl. Acid. Res. 17, 2099-118. MAK, A. S. & SMILLIE, L. B., (1981) Structural interpretation of the two-site binding of troponin on the muscle thin filament. J. Mol. Biol. 149, 541-50. MAK, A. S., SMILLIE, L. B. & BARANY, M. (1978) Specific phosphorylation at serine-283 of a tropomyosin from frog skeletal and rabbit skeletal and cardiac muscle. Proc. Natl. Acad. Sci. USA 75, 3588-92. MATSUMURA, F., YAMASHIRO-MATSUMARA, S. & LIN, J. J.-C

(1983) Isolation and characterization of tropomyosincontaining microfilaments from cultured cells. ]. Biol. Chem. 258, 6636-44. MERRIL, C. R., GOLDMAN, D., SEDMAN, S. A. & EBERT, M. H.

(1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211, 1437-8. MITTAL, B., SANGER, J. M. & SANGER, J. W. (1987) Visualization of myosin in living cells. J. Cell Biol. 105, 1753-60. MONTARRAS, D., FISZMAN, M. Y., & GROS, F. (1981) Characterization of the tropomyosin present in various chick embryo muscle types and in muscle cells differentiated in vitro. J. Biol Chem. 256, 4081--6. MONTARRAS, D., FISZMAN, M. Y. & GRAS, F. (1982) Changes in tropomyosin during development of chick embryonic skeletal muscles in vivo and during differentiation of chick muscle cells in vitro. ]. Biol. Chem. 257, 545-8. MONTGOMERY, K & MAK, A. S. (1984) In vitro phosphorylation of tropomyosin by a kinase from chicken embryo. ]. Biol. Chem. 259, 5555-60. O'FARRELL,P. H. (1975) High resolution two-dimensional electrophoresis of proteins. ]. Biol. Chem. 250, 40O7-21. OTEY, C. A., KALNOSKI, M. H. & BULINSKI, I. C. (1988) Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle. Cell Motil. Cytoskel. 9, 337-48. PATTERSON, B. & STROHMAN, R. C. (1972) Myosin synthesis in cultures of differentiating chick embryo skeletal muscle. Dev. Biol. 29, 113-8. PENG, H. B., WOLOSEWICK, I. J. & CHENG, P.-C (1981) The development of myobrils in cultured muscle cells: A whole mount and thin-section electron microscopic study. Dev. Biol. 88, 121-6. SANGER, J. M., MITTAL, B., POCHAPIN, M. B. & SANGER, J. W.

(1986) Myofibrillogenesis in living cells microinjected with fluorescently labeled ~-actinin. ]. Cell Biol. 102, 2053-66.

202 SHIMADA, Y., FISHMAN, D. A. & MOSCONA, A. A. (1967) The fine structure of embryonic chick skeletal muscle cells differentiation in vitro. J. Cell Biol. 35, 445-3. SHIMADA, Y (1971) Electron microscope observations on the fusion of chick myoblasts in vitro. ]. Cell Biol. 48, 128-42.

WANG, WANG, LIN and LIN WANG, S.-M., GREASER,M. L., SCHULTZ, E., BULINSKI,J. C., LIN, J. J.-C & LESSARD,J. L. (1988) Studies on cardiac myofibrillogenesis with antibodies to titin, tropomyosin and myosin. J. Cell Biol. 107, 1075-83.