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MUSCLE CELL BIOLOGY AND CELL MOTILITY
Department of Physiology, School of Medicine, University of Maryland at Baltimore, Baltimore, Maryland
Submitted 1 September 2005 ; accepted in final form 2 October 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-actinin and Z disk epitopes of titin. Z disks and M bands can assemble independently at spatially distant sites. As sarcomerogenesis proceeds, these structures interdigitate to produce a more mature organization. Fast-twitch muscle myosin accumulates in the myoplasm and assembles into A bands only after Z disks and M bands assume their typical interdigitated striations. The periodicities of M bands remain constant at 
1.8 µm throughout sarcomerogenesis, whereas distances between Z disks increase from 1.1 µm in early sarcomeres to 1.8 µm in more mature structures. Our findings indicate for the first time that primitive M bands self-assemble independently of Z disks, that obscurin is a component of these primitive M bands in skeletal muscle cells, and that A bands assemble only after M bands and Z disks integrate into maturing sarcomeres.
obscurin; titin; myosin; myomesin; -actinin; A band
Two models of sarcomerogenesis have been proposed that invoke interactions of developing thin and thick filaments with other elements of the cytoskeleton. Holtzer and colleagues (38) suggested that during early myofibrillogenesis, thin and thick filaments assemble independently on stress fiber-like structures (SFLS). SFLS develop into nonstriated myofibrils (NSMF), which progress to nascent striated myofibrils that in turn develop into fully mature, striated myofibrils (SMF). During the transition of NSMF to SMF, adjacent strands of thin and thick filaments align initially at cell borders and subsequently throughout the cytoplasm. A key feature of this model, therefore, is that the earliest precursors of mature thin and thick filaments form independently in the myoplasm of developing muscle, but that later stages of development proceed along common filaments. Sanger and colleagues (34) postulated an alternative model that evokes three distinct structures during myocyte development: premyofibrils, nascent myofibrils, and mature myofibrils. Premyofibrils contain transitory arrays of I-Z-I complexes consisting of sarcomeric actin occupying primitive I bands that are attached to precursors of Z disks, termed Z bodies, that are enriched in -actinin and interact with miniature A bands composed of nonmuscle myosin II. Premyofibrils progress to nascent myofibrils, which develop into mature myofibrils with the concurrent replacement of nonmuscle myosin II by muscle myosin II (37). A key feature of this model, therefore, is that the precursors of thin and thick filaments form along the same structures, which together develop into mature sarcomeres.
Although the two proposed models are distinct, they are similar in that structural proteins are essential to the proper assembly and incorporation of actin and myosin into mature myofibrils (13, 28, 34, 35, 38, 43). Thus, during the initial assembly of myofibrils, Z bodies composed of -actinin, the NH2-terminal region of titin, nebulin, and T-cap/telethonin contribute to the polarized organization of thin actin filaments to form I-Z-I brushes that become incorporated into forming sarcomeres (17, 19, 20, 24, 29, 31, 33). Likewise, proteins of the M band, including myomesin, M protein, and the COOH-terminal region of titin and obscurin, play a key role in the integration of myosin thick filaments into periodic A bands (14, 15, 18, 25, 43, 46, 48). The close association of the giant protein titin (3–4 MDa) with both thin and thick filaments, and its presence at both primitive Z disks and M bands, has led to the idea that it serves as a molecular blueprint for the coordinated assembly of the key elements of sarcomeres during early myofibrillogenesis (17, 18, 27, 41, 42). The central role postulated for titin is consistent with each of the current models of sarcomerogenesis summarized above.
We have studied the role of obscurin in the assembly of sarcomeres. Together with nebulin and titin, obscurin (800 kDa) is the third giant structural protein associated with the contractile apparatus that is expressed in vertebrate striated muscle. Like titin, it is a multidomain protein composed of tandem adhesion modules and signaling domains (36, 49). Unlike titin and nebulin, which are integral components of sarcomeres, obscurin closely surrounds the myofibrils at the level of the M band and the Z disk (26). This unique distribution allows it to bind to a small isoform of ankyrin-1 in the network sarcoplasmic reticulum (SR), suggesting that it contributes to the regular alignment of the network SR with the M bands and Z disks of each sarcomere (4, 26). Obscurin also associates with the contractile apparatus through its binding to titin, sarcomeric myosin, and myosin binding protein C slow (5, 25, 49). Overexpression of the COOH terminus of obscurin in primary myotubes disrupts the assembly of sarcomeric myosin, suggesting that it also is essential for the organization and regular assembly of A bands (25). During myofibrillogenesis in cultured neonatal rat cardiomyocytes, obscurin associates primarily with developing M bands and, at a later time and to a lesser extent, with Z disks (8, 49). In whole mount preparations of embryonic chicken hearts, however, obscurin is detected initially at the developing Z disk and only later at the M band, with a concomitant loss of Z disk staining (49).
Herein we analyze the distribution of obscurin during the de novo assembly of myofibrils in the mouse myogenic cell line C2C12. Our results indicate that, consistent with earlier results in neonatal cardiomyocytes (5, 8, 49), obscurin associates first with the developing M band and only later with the more mature Z disk. We report, however, that primordial M band structures consisting of M band epitopes of titin, myomesin, and obscurin assemble before the appearance of the heavy chain of fast sarcomeric myosin and nearly concurrently with primitive Z disk structures composed of -actinin, Z disk epitopes of titin, and actin filaments. Remarkably, at this early stage of myofibrillogenesis, primitive M bands and Z disks can assemble independently in spatially segregated regions of the myoplasm. Only at later stages of development, when M bands and Z disks interdigitate, as observed in mature sarcomeres, does the fast isoform of muscle myosin begin to assemble into A bands. Our studies suggest that models of de novo myofibrillogenesis may need to be modified to account for the ability of Z disks and M bands to form independently before the assembly of mature sarcomeres.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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80% confluence 24–48 h later and were subcultured onto sterile glass coverslips in 35-mm-diameter petri dishes. Cultures were maintained in growth medium until they reached 60–70% confluence and subsequently were switched to differentiation medium (DMEM containing 5% horse serum; GIBCO-BRL/Invitrogen), which was replaced every 24 h. Samples were processed for immunofluorescence labeling at selected time points as indicated in RESULTS.
Primary antibodies.
The following primary antibodies were used: rabbit anti-obscurin (generated against the COOH terminus of the protein, 3 µg/ml; Ref. 26), rabbit anti-titin-x112-x113 (labels at the Z line, 3 µg/ml; a gift from Dr. C. C. Gregorio; Ref. 20), rabbit anti-titin-M-x246 (specific for the M band portion of titin, 3 µg/ml; a gift from Dr. S. Labeit; Ref. 10), mouse anti-myosin II (clone MY-32, 1:400 dilution; Sigma, St. Louis, MO), mouse anti--actinin (1:400 dilution; Sigma), mouse anti-myomesin-B4 (1:1 dilution; a gift from Drs. J.-C. Perriard and E. Ehler; Ref. 21), mouse anti-titin-N2A (raised against I band epitopes of titin, 3 µg/ml; a gift from Dr. C. C. Gregorio; Refs. 9 and 30). We used phalloidin coupled to Alexa Fluor 488 (1:200 dilution; Molecular Probes, Eugene, OR) to label actin.
Immunofluorescence staining and confocal microscopy. Cultures of C2C12 myotubes were rinsed with PBS and fixed with 2% paraformaldehyde for 15 min at room temperature (RT). Subsequently, cells were permeabilized with 0.1% Triton X-100 for 10 min at RT, rinsed with PBS, and incubated in PBS, 0.1% BSA, and 10 mM NaN3 (PBS-BSA) for 1–2 h at RT before being immunolabeled with primary antibodies. Samples were counterstained with goat anti-rabbit-Alexa Fluor 568 and goat anti-mouse Alexa Fluor 488 or goat-anti-mouse Alexa Fluor 633 (1:200 dilution; Molecular Probes) for 1 h at RT. Cells were washed extensively with PBS-BSA, mounted with Vectashield (Vector Laboratories, Burlingame, CA), and analyzed using a confocal laser scanning microscope (model 410; Zeiss, Tarrytown, NY) equipped with a x63 magnification, 1.4 numerical aperture lens objective. Additional details are provided elsewhere (24).
Measurements of Z disk and M band periodicity.
C2C12 cells induced to differentiate for 24, 48, 72, and 96 h were labeled with antibodies to the Z disk protein, -actinin, and titin epitopes located at the M band. Digital images were obtained under confocal optics as described above. Distances between neighboring Z disks and between neighboring M bands were measured using Image software (Scion, Frederick, MD) and analyzed using StatView software (SAS Institute, Cary, NC).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Obscurin and the early formation of M bands and Z disks. C2C12 myotubes, induced to differentiate for 24 h, were immunolabeled with antibodies against the COOH terminus of obscurin (26) and major M band and Z disk proteins. Obscurin was abundant in the cytoplasm, where it was diffusely distributed or aggregated (Fig. 1, A and B). Fast-twitch sarcomeric myosin, the appearance of which has been linked to advanced myogenic differentiation in C2C12 cells (11), was not detected at this stage (Fig. 1A'). Embryonic myosin and slow-twitch myosin, which are the predominant isoforms early in myofibrillogenesis, were distributed diffusely in the myoplasm as previously reported (11, 37, 39). By contrast, myomesin, a bona fide M band protein, was readily detected in the aggregates containing obscurin (Fig. 1, B and B'). Where obscurin appeared to be continuous (Fig. 1B), however, myomesin showed some periodicity (Fig. 1B'), suggesting that myomesin assembles into primitive M band-like structures before obscurin.
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20% of cultured cells that were still at the myoblast stage, however, titin epitopes at the M band assumed either a diffuse cytoplasmic distribution or were concentrated along filaments with occasional periodicity (Fig. 1D). By contrast, myomesin was either not detected or barely detected along the same filaments in primitive striations (Fig. 1D'). Comparisons of obscurin and the M band epitopes of titin produced similar results (data not shown). Thus the appearance of the M band epitopes of titin and their organization along filaments is likely to occur slightly before myomesin, which is in turn likely to accumulate and become organized into periodic structures before obscurin. Fast-twitch muscle myosin was not present at significant levels in any of the myotubes differentiated for 24 h (see, e.g., Fig. 1A'). Soon after the formation of myotubes, therefore, several components of the M band sequentially concentrated along the same filaments, well before the appearance of fast-twitch sarcomeric myosin.
When we examined components of Z disks in early myotubes, we detected -actinin in some myotubes that failed to express NH2-terminal, Z disk epitopes of titin (data not shown). When both proteins were abundantly expressed, however, they assumed a periodic macular distribution (Fig. 2, A and A''), consistent with the presence of Z bodies. The Z disk epitopes of titin and -actinin colocalized in many such structures (Fig. 2, A and A''), but not all. -Actinin was present in some structures that lacked the Z disk epitopes of titin (Fig. 2, A and A'') and in which both proteins were present, and -actinin sometimes was organized more extensively than titin (Fig. 2, A and A''). At early stages of sarcomere formation, therefore, -actinin is detected in Z bodies before the NH2 terminus of titin.
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-actinin and obscurin (Fig. 2, B and B'') and -actinin and M band epitopes of titin (Fig. 2, C and C''). -Actinin was present at Z bodies in some myotubes that failed to show obscurin or M band epitopes of titin at primitive M bands (data not shown), suggesting that it was expressed and organized earlier. In developing myotubes in which both -actinin and obscurin were amply expressed, however, -actinin was organized in primitive striations along premyofibrils, which tended to appear preferentially near the periphery of the cell (Fig. 2B'), whereas obscurin was detected along nonstriated fibrils in the center of the cell (Fig. 2B) and also diffusely in the cytoplasm (Fig. 2B), where it did not codistribute with -actinin (Fig. 2B''). Furthermore, -actinin and the M band epitopes of titin were organized either continuously along filaments or in primitive, short striations along distinct myofibrils (Fig. 2, C and C'). -Actinin was spatially segregated from the M band region of titin in early C2C12 myotubes (Fig. 2C'') as reported above for obscurin. These results suggest that Z disk and M band components can assemble independently in spatially distinct fibrils of myotubes differentiated for 24 h.
We next asked whether, like Z bodies, the early M bands that form independently in C2C12 myotubes are also associated with actin filaments. Using antibodies against -actinin and to M band epitopes of titin and fluorescent phalloidin to label filamentous actin, we found that M bands (Fig. 2D) that formed without intervening Z disks (Fig. 2D') did indeed assemble along actin filaments (Fig. 2D'') and that these filaments were distinct from those associated with primitive Z disks (Fig. 2D'''). Figure 2E shows an additional example of primitive M bands and Z disks forming along distinct actin filaments. The large, closed arrowheads point to adjacent bundles of actin filaments, in green. The small, open arrowheads point to either M band epitopes of titin or -actinin, shown in light blue and orange, respectively, to indicate their colocalization with actin but not with each other. Our results suggest that during the initial stages of myofibril assembly (i.e., first 24 h after induction of differentiation), M band and Z disk proteins assemble concurrently but independently along discrete actin filaments.
We performed double-immunolabeling experiments with antibodies against epitopes of titin located at the Z disk, I band, and M band to learn how titin incorporates into developing sarcomeres (Fig. 3). In myotubes differentiated for 24 h, the Z disk and M band epitopes of titin were distributed either continuously along developing fibrils (Fig. 3, A and B) or in short striations spaced closely together (Fig. 3 A and B). Antibodies to the I band region of titin labeled the same structures, although weakly and in a more continuous fashion, even when Z disk and M band epitopes were organized periodically (Fig. 3, A' and B', respectively). This result may be explained by the presence of auxiliary proteins bound to the I band region of titin that inhibits binding of antibodies. Alternatively, each end of titin may anchor to its respective structure independently, allowing most of the molecule to remain flexible enough to fold over itself. As a result, epitopes in neighboring molecules would not be aligned laterally.
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We next compared the proteins of M bands to those of Z disks (Fig. 5). In most myotubes, -actinin and Z disk epitopes of titin organized into short, periodic Z bands along nascent myofibrils (Fig. 5, A and A''), with obscurin midway between them, at presumptive M bands (Fig. 5, B and B''). M band epitopes of titin and -actinin were similarly organized into alternating striations throughout the cytoplasm (Fig. 5, C and C''). Thus, at 48 h of differentiation, myofibrils were incorporated into interdigitating striations of M band and Z disk components, some of which had been spatially segregated at 24 h (Fig. 2).
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-actinin and M band epitopes of titin concentrated in alternating striations (Fig. 7, C and C''). Similar degrees of organization were observed for the three different titin epitopes examined at 72 h postdifferentiation (Fig. 7, D and E), suggesting that by that time, the I band epitopes of titin were as accessible to antibodies and as regularly organized as its Z disk and M band epitopes or, alternatively, that titin had become fully extended.
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-actinin were present in regularly spaced striations at the level of the M band (Fig. 8B') and the Z disk (Fig. 8C'), respectively, and titin was well organized not only at Z disks and M bands but also at I bands (Fig. 8, B–E).
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-actinin, and between developing M bands, which were detected using antibodies to the M band epitopes of titin (Fig. 9). We made at least 75 different measurements in at least 20 cells studied in a total of eight independent experiments to obtain the data for each time point. At 24 h of differentiation, -actinin showed a periodicity of 1.1 µm. This value increased to 1.8 µm at 48 h and reached 2 µm by 96 h. By contrast, titin epitopes at the M band showed a periodicity of 1.7 µm at 24 h, which gradually increased to 2 µm by 96 h. These findings suggest that during early sarcomerogenesis, M bands already show a periodicity that approximates that of mature sarcomeres, whereas Z disks require an extra day to reach the same periodicity. These results are consistent with our observation that the earliest M bands and Z disks in C2C12 myotubes at 24 h of differentiation assemble independently.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Obscurin in myofibrillogenesis. Obscurin is expressed early in myofibrillogenesis, when it can be distributed diffusely in the myoplasm or concentrated in primordial M bands, which are defined by the presence of myomesin and M band epitopes of titin. The absence of obscurin from some of these early structures suggests that it may be incorporated into M bands after titin and myomesin. Alternatively, obscurin may be incorporated into the earliest M bands but can be difficult to detect because of its presence in small amounts or because of steric hindrance of its COOH terminus, which was recognized by the antibodies we used. If obscurin arrives at the primitive M band after the COOH terminus of titin and myomesin, however, it could help to explain their topography in adult myofibers, because both titin and myomesin are integral M-band components (32), whereas obscurin surrounds M bands (26). Thus obscurin may wrap around and stabilize a core complex of titin and myomesin shortly after they assemble at developing M bands.
During the next 24 h of differentiation, obscurin, together with myomesin and titin, becomes more extensively organized into periodic M bands along bundles of actin filaments that begin to accumulate adult skeletal isoforms of myosin. C2C12 cells express developmental and slow-twitch myosin during early stages of differentiation (11, 37, 39). The expression of developmental myosin is rapidly downregulated as the heavy chain of adult fast myosin accumulates in the myoplasm along with other myogenic proteins, including -actinin-3 and components of the dystrophin-associated protein complex (11). Our studies focused on the temporospatial expression of adult fast-twitch sarcomeric myosin using an antibody (clone My32) that specifically recognizes it rather than a generic antibody that detects all different myosin isoforms, because the time of appearance and degree of organization of adult fast-twitch myosin are indicative of the level of myogenic maturity of developing C2C12 myotubes (11).
Obscurin at M bands associates only with fast-twitch myosin in regular A bands between 72 and 96 h of differentiation, when it also associates with Z disks. Because obscurin is subjected to extensive alternative splicing (49), we cannot rule out the possibility that different isoforms of obscurin sharing the COOH-terminal epitope are present around M bands and Z disks, where they may play distinct roles. Evidence suggests, however, that obscurin plays an important role in the assembly of A bands, but not of Z disks, both in the rat (25) and in Caenorhabditis elegans, in which it is known as unc-89 (7).
Obscurin's delayed association with assembled Z disks also suggests that it may facilitate later events in myofibrillogenesis, such as the periodic alignment of the SR around each sarcomere via its direct association with small ankyrin-1 (4, 26) or through the lateral alignment and fusion of mature myofibrils into larger bundles. Consistent with the latter scenario, Borisov et al. (8) demonstrated that obscurin in cardiocytes was organized in striations at the level of the Z disk when desmin was still diffusely distributed.
Titin in myofibrillogenesis. Epitopes in the Z disk and M band regions of titin start to assume a periodic distribution early in sarcomerogenesis after only 24 h of differentiation (e.g., Fig. 3). By contrast, epitopes in the I band region of titin assume clear sarcomeric periodicity only 24–48 h later. These observations are consistent with an earlier study that monitored the temporospatial integration of different titin epitopes into fibrils as they formed. Antibodies to titin that recognized epitopes at or close to the Z line showed a periodic pattern earlier than antibodies that bound to epitopes at the A-I junction or within the A band, which showed a continuous staining pattern (16). Our observations, however, are in disagreement with a previous study that suggested that COOH-terminal epitopes of titin become organized into periodic M bands slightly later than titin's NH2-terminal epitopes incorporate into Z disks in embryonic chicken heart rudiments (14). A possible explanation for this discrepancy may be the poor sensitivity of the MAbs used in the previous study compared with the PAbs used in our present experiments. Alternatively, by examining myotubes 24 h after we induced differentiation, we may have failed to detect the earliest stages in the assembly of titin into Z disks. We think this possibility is unlikely, however, because C2C12 muscle cells differentiated for 24 h showed less mature as well as more mature structures, and we detected periodic labeling by antibodies to the M band epitopes of titin just as reliably as we detected periodic labeling by antibodies to its Z disk region (e.g., Fig. 3).
Therefore, our present study indicates that the NH2- and COOH-terminal ends of titin are each targeted to their appropriate structures in developing sarcomeres at nearly the same time of differentiation. At this stage, however, the middle of the titin molecule is harder to detect in periodic structures, perhaps because it is still folded or masked by auxiliary proteins, rendering epitopes located in the I band region relatively inaccessible to immunolabeling. This hypothesis is compatible with an earlier suggestion that titin in early sarcomeres is not yet fully extended between Z disks and M bands (41, 44, 45). Also in agreement with this hypothesis is that when we did observe punctate labeling for I band epitopes of titin at this stage of differentiation, it overlapped extensively with labeling for either Z disk or M band epitopes (Fig. 3). This observation in turn suggests that an individual titin molecule in early sarcomeres may be well organized at nascent Z disks or M bands, but not at both simultaneously. Alternatively, a single titin molecule may stretch laterally between assembling Z bodies or M bands in adjacent myofibrils. Either of these interpretations of our results is consistent with our finding that M bands can assemble independently of Z disks. The ability of titin to serve as a template for the assembly of sarcomeric components may therefore develop later as its NH2 terminus becomes anchored at Z disks and its COOH terminus simultaneously becomes anchored at M bands.
Early M bands. We found that M band structures first appear in developing C2C12 myotubes almost simultaneously with primitive Z disks and that they can assemble independently. Although some of our results are consistent with previous reports that Z lines appear before M bands (35, 37), the presence of nascent Z disks and M bands forming independently in the same myotubes suggests that the time differential is likely to be small.
It might be argued that the ability of M bands to form independently of Z disks is particular to C2C12 cells, which are transformed and adapted for the conditions of tissue culture. We think that this hypothesis is unlikely for two reasons. First, we have made similar observations in primary cultures of rat myotubes (our unpublished results). Second, our findings are consistent with previous reports with mutant or otherwise altered muscle cells in invertebrates. Although invertebrate M bands and associated A bands likely assemble by mechanisms different from those in vertebrates, they, too, can form in the absence of Z disks. Preferential disruption of thin filaments with the persistence of thick filaments organized into A bands has been demonstrated in the body wall muscle of C. elegans mutants (47). A similar independence has been shown in muscles of Drosophila mutants deficient in sarcomeric actin, which assembles A bands with ultrastructurally normal M bands in the absence of intervening Z disks (6). Other conditions, too, lead to the presence of M bands without associated Z disks in vertebrate muscle. For example, cardiocytes entering mitosis retain M bands for a longer time than they do Z disks (2). Skeletal myotubes treated with taxol to stabilize microtubules assemble perfectly normal A bands with central H zones (and presumably normal M bands) in the absence of Z disks and I bands (40). Moreover, our results agree with previous observations that have suggested that I-Z-I brushes and thick myosin filaments assemble as separate units during myofibrillogenesis (16, 22, 23) and provide strong evidence for the concurrent, yet independent, assembly of primitive Z disks and M bands along distinct actin filaments.
The appearance of spatially segregated M bands early in the process of differentiation of C2C12 cells suggests that these structures may play a more important role than previously thought in early myofibrillogenesis. Measurements of the distances between neighboring M bands during sarcomerogenesis show that they assume a periodicity typical of mature sarcomeres earlier than neighboring Z disks do (Fig. 9). This finding raises the possibility that in addition to the key role played by nearly mature M bands in the assembly of A bands late in the formation of sarcomeres (14, 43, 46), nascent M bands may help to define the spacing of developing sarcomeres at the early stages of myofibrillogenesis. Experiments to test this hypothesis are in progress.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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) or M bands (
) were determined from confocal images of immunolabeled C2C12 myotubes at selected times (i.e., 24, 48, 72, and 96 h) after initiation of differentiation. A total of 76, 120, 167, and 366 measurements were made in cultures at 24, 48, 72, and 96 h, respectively, from a total of 35–45 cells in 8 independent experiments to determine the distances between neighboring M bands. Similarly, a total of 158, 106, 96, and 75 measurements were made in cultures at 24, 48, 72, and 96 h, respectively, from a total of 35–40 cells in 8 independent experiments to determine the distances between neighboring Z disks. The results show that primordial M bands had a periodicity of