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Journal of Cell Science 112, 4101-4112 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS0651
Initiation and maturation of I-Z-I bodies in the growth tips of transfected myotubes K. Ojima1,*, Z. X. Lin1,2,*, Z. Q. Zhang1,2,*, T. Hijikata3, S. Holtzer1, S. Labeit4, H. L. Sweeney1 and H. Holtzer1,‡ 1Department
of Physiology and Cell and Developmental Biology, The School of Medicine, University of Pennsylvania, Philadelphia, PA l9l04, USA 2Department of Cell Biology, Beijing Institute for Cancer Research, Beijing Medical University, Beijing l00034, China 3Department of Anatomy, The School of Medicine, Gunma University, Maebashi, Japan 4Department for Anesthesiology and Intensive Operative Care, Klinikum, Mannheim, Germany *These three authors contributed equally to this report ‡Author for correspondence (e-mail:
[email protected])
Accepted 25 August; published on WWW 3 November 1999
SUMMARY While over a dozen I-Z-I proteins are expressed in postmitotic myoblasts and myotubes it is unclear how, when, or where these first assemble into transitory I-Z-I bodies (thin filament/Z-band precursors) and, a short time later, into definitive I-Z-I bands. By double-staining the growth tips of transfected myotubes expressing (a) MYCtagged s-α-actinins (MYC/s-α-actinins) or (b) green fluorescent protein-tagged titin cap (GFP/T-cap) with antibodies against MYC and I-Z-I band proteins, we found that the de novo assembly of I-Z-I bodies and their maturation into I-Z-I bands involved relatively concurrent, cooperative binding and reconfiguration of, at a minimum, 5 integral Z-band molecules. These included s-α-actinin, nebulin, titin, T-cap and α-actin. Resolution of the ~1.0 µm
α-actin/nebulin/tropomyosin/troponin polarized thin filament complexes occurred subsequent to the maturation of Z-bands into a dense tetragonal configuration. Of α-actinin particular interest is finding that mutant MYC/s-α peptides (a) lacking spectrin-like repeats 1-4, or consisting of spectrin-like repeats 1-4 only, as well as (b) mutants/fragments lacking titin or α-actin binding sites, were promptly and exclusively incorporated into de novo assembling I-Z-I bodies and definitive I-Z-I bands as was exogenous full length MYC/s-α-actinin or GFP/T-cap.
INTRODUCTION
myoblasts and myotubes (Antin et al., 1986; Lin et al., 1994; Komiyama et al., 1994; Moncman and Wang, 1996; Shimada et al., 1996; Holtzer et al., 1997; Mayans et al., 1998) resemble the metastable cytoskeletal stress fibers in non-muscle cells (Lazarides and Burridge, 1975; Verkhovsky et al., 1995; Cramer et al., 1997; Hall, 1998). Disparate observations, ranging from binding studies in cell free systems through high resolution immunoelectron microscopy of specific epitopes, have demonstrated linkages in mature Z-bands between s-α-actinin, and (a) the C-terminal of nebulin (Wang and Wright, 1988; Labeit and Kolmerer, 1995a; Wang et al., 1996; Millevoi et al., 1998) and (b) the N terminus of titin (Wang et al., 1996; Maruyama et al., 1986; Fürst et al., 1988; Labeit and Kolmerer, 1995b; Sebestyen et al., 1995; Sorimachi et al., 1997; Young et al., 1998). Recently telethonin, or titin-cap (T-cap), a 19 kDa protein, has also been shown to be an integral molecule in mature Z-bands. T-cap binds to the titin Z1-Z2 domains (Mues et al., 1998; Gregorio et al., 1998) at the periphery of mature, tetragonal Z-bands (Morris et al., 1990; Schroeter et al., 1996). Despite this consensus, there are divergent views regarding
Much is known about how in cell free systems two proteins interact by way of specific binding sites. Similarly, much is known regarding how, in in vitro systems, monomers of myofibrillar proteins, especially actin and MHC, self-assemble into structures bearing some resemblance to native thick and thin filaments characteristic of mature striated myofibrils (SMFs). Less is known about the in vivo mechanisms which, during development, control the formation of complex structures such as the relatively invariant I-Z-I bands, structures that are highly conserved in SMFs of all vertebrates (FranziniArmstrong and Fischman, 1996). This report focuses on the reconfigurations undergone by precursor I-Z-I bodies as they transform into definitive I-Z-I bands in maturing postmitotic myoblasts and myotubes. It centers on the changing temporospatial relationships of s-αactinin, a major component of all Z-bands, relative to the other major I-Z-I band proteins, namely nebulin, titin, T-cap, α-actin, tropomyosin and troponin-I. Morphologically, the longitudinally oriented, transitory I-Z-I bodies in skeletal
Key words: Myofibrillogenesis, MYC-tagged s-α-actinin, GFPtagged T-cap, Titin, Nebulin, α-Actin
4102 K. Ojima and others the changing relationships among the nascent I-Z-I monomers as they assemble into definitive I-Z-I bands. Fürst et al. (1989), Fulton and Isaacs (1991), Trinick (1994), and van der Ven et al. (1993) have suggested that titin is polymerized into long homopolymers earlier than all other myofibrillar proteins. Komiyama et al. (1990) reported that nebulin and α-actinin were detected before titin, whereas Moncman and Wang (1996) reported that actin and titin accumulated before nebulin. Others have reported that the assembly of most myofibrillar proteins into sarcomeric structures occurs more or less concomitantly within both ‘new-born’ postmitotic, mononuleated myoblasts and myotubes (Holtzer et al., 1957, 1997; Hill et al., 1986; Lin et al., 1994; Komiyama et al., 1994; van der Ven and Fürst, 1997; Mues et al., 1998). To address some of these issues, we have used a highly sensitive procedure for tracking minute quantities of nascent s-α-actinin. Schultheiss et al. (1992) and Lin et al. (1998) reported that in transfected myotubes, expressed MYC/s-αactinins are readily followed by staining with anti-MYC. Full length and several mutant MYC/s-α-actinins were incorporated exclusively into the definitive Z-bands in mature SMFs. The high signal to background ratio using anti-MYC to localize MYC/s-α-actinins relative to other myofibrillar proteins is superior to conventional doublestained fluorescent images (see Lin et al., 1998, and Figures therein). This transfection protocol, along with myotubes expressing GFP/T-cap, has been used in this report to follow changes in the I-Z-I bodies as they transform into definitive I-Z-I-bands. In addition this transition is followed in the growth tips, rather than in the shafts, of early myotubes. Protrusive growth tips with giant ruffled membranes cap both ends of the shafts of all myotubes (Lin et al., 1987). Growth tips regulate two inter-related activities; (1) the elongation of anisodiametric myotubes, and (2) the de novo initiation and maturation of new sarcomeres. SMFs in myotubes increase in length by the addition of newly assembled sarcomeres that are continuously annealed to the distal ends of myofibrils that had been assembled in the shaft earlier (Holtzer et al., 1973; Franzini-Armstrong and Fischman, 1996). Flattened growth tips are more favorable for studying the localization of nascent I-Z-I proteins than are the myotube shafts for, as determined in the confocal microscope, they measure 1.0-2.5 µm in the z-axis, whereas the shaft of the same myotube can exceed 14 µm. Our findings are consistent with a model that postulates that the initiation of I-Z-I bodies involves the relatively concurrent and cooperative interaction of, at a minimum, 5 integral Z-band proteins, s-α-actinin, the C-terminal of nebulin, the N-terminal of titin, T-cap and cardiac- and/or sarcomeric α-actin (c- or s-α-actin). No one of these proteins self-assembles into long homopolymers prior to that of the others. While the purpose for constructing a series of αactinin truncations was to attempt to delineate specific protein-protein interactions, and to effect targeted disruptions of I-Z-I assembly events, such disruptions or dominant negatives, were not observed. Intriguingly MYC/s-α-actinin mutants lacking domains which, from in vitro binding studies would have been predicted to be assembly incompetent, were promptly and exclusively targeted to all precursor I-Z-I bodies and definitive I-Z-I bands.
MATERIALS AND METHODS Cell culture Primary cultures of myogenic cells were obtained from day 11 chick embryonic pectoral muscle (Lin et al., 1987, 1998). Cells were plated onto collagen-coated Aclar coverslips (Pro-Plastics, Linden, NJ) at an initial density of 4.5×105 cells per 35 mm culture dish. Most observations were made on 70 hour and 80 hour cultures. Plasmids The construction of the MYC/s-α-actinin cDNAs (Fig. 1) and their introduction into the vector pcDNA3 (Invitrogen) have been described in detail (Lin et al., 1998). The molecular mass of the expressed MYCtagged constructs was confirmed by western blots of total extracts from transfected and untransfected cultures and by immunocytochemistry (Schultheiss et al., 1992; Hijikata et al., 1997; Lin et al., 1998). The construction and properties of the GFP/T-cap cDNA are detailed by Gregorio et al. (1998). Briefly a PCR fragment containing the entire T-cap open reading frame was generated and introduced into a pEGFP-C1 (Clontech) or a pCMV/MYC construct. 24 hour cultures, containing over 98% mononuclated cells, were transfected with 2 µg DNA per dish for 12 hours, using standard calcium phosphate precipitation methods (Lin et al., 1998). Antibodies (A) To localize the expressed MYC-tagged peptides, a mAb to the MYC epitope (clone 9E10; a generous gift from Dr S. Monroe, MRC, Cambridge, UK) was used (1:10) in most experiments. In occasional experiments rabbit polyclonals (1:200; Upstate Biotechnology, Lake Placid, NY) were used. (B) Endogenous s-α-actinin was localized with a mAb (1:100; clone 9A2B8) and a rabbit affinity-purified polyclonal (1:100; Lin et al., 1987; Lu et al., 1992; Hijikata et al., 1997). These antibodies stain all I-Z-I structures; they do not stain any cytoskeletal structures in non-muscle cells. (C) mAbs against cardiac α-actin (undiluted; clone Acl-20-4.2) and against skeletal α-actin (1:400; clone 5C5) were purchased from American Research Products (Belmont, MA) and Sigma (St Louis, MO), respectively: they do not stain actin isoforms in non-muscle cells. (D) An affinity purified antibody against troponin I, which binds specifically to I-bands, was used (1:1000; a generous gift from Dr S. Hitchcock-Gregorgi, School of Medicine and Dentistry, Piscataway, NJ). (E) Tropomyosin was localized with a rabbit affinity-purified antibody (1:1000; Organon Teknika Co; Durham, NC) which stains I-bands; it does not stain cytoskeletal structures in non-muscle cells (Lin et al., 1998). (F) Titin epitopes within the Z-band were localized with anti-Z1-Z2 (1:50; Gregorio et al., 1998) and with anti-titin T20 (undiluted; a generous gift from Dr K. Weber, Max Planck Institute, Göttingen, Germany). (G) Titin T-cap was localized with anti-T-cap (1:50) as described by Gregorio et al. (1998). (H) Anti-nebulin SH3 (undiluted) and antinebulin M177-M181 (1:50) were used to localize the C-terminal of nebulin (Millevoi et al., 1998). An mAb anti-nebulin (1:50; NB2) was purchased from Sigma. (I) Desmin was localized as described by Schultheiss et al. (1991). (J) Anti-MIR (1:100) was used as described by Linke et al. (1999). Antibody staining Staining has been described in detail by Hijikata et al. (1997) and Lin et al. (1998). Dishes were treated with blocking buffer (BB) (2% BSA in PBS) for 30 minutes before incubation with primary antibodies that had been diluted with BB. Staining for both c- and s-α-actin followed procedures recommended by suppliers. All secondary antibodies (Jackson Immuno Research Labs, West Grove, PA) were affinitypurified goat IgGs conjugated with rhodamine (Rho-), Texas Red (TR-) or fluorescein (FITC-). Some dishes were stained with Rho- or FITC-phalloidin (3.3 µM, Molecular Probes Inc., Eugene, OR). Bleed-through fluoresence, or false-positives, were minimized by running duplicate series. In one series, each of the two primary
Precursor I-Z-I bodies into I-Z-I bands 4103 antibodies was stained with either a Rho- or a FITC-conjugated secondary. In the second series, the same primary antibodies were stained in the opposite fashion. For confocal mincroscopy, Texas Red was used rather than rhodamine. Nuclei were detected with DAPI (4, 6-diamidino-2-phenylindole dihydrochloride; Polyscience, Warrington, PA). Specimens were mounted in 60% glycerol in PBS containing 2.5% DABCO (1, 4-diazabicyclo (2, 2, 2) octane; Sigma).
transfected myotubes in 80 hour cultures fall into 2 overlapping categories. Roughly 40% are still immature, displaying 0.2-0.8 µm MYC/I-Z-I bodies in their shafts and growth tips. The remaining ~60%, however, now display numerous SMFs in their shafts, each positive for definitive, periodic ~0.1 µm wide MYC/Z-bands, ~1.0 µm wide I-bands and 1.6 µm wide A-bands. In mature myotubes irregularly-shaped MYC/I-Z-I bodies are confined largely to elongating growth tips. In brief, within hours most disordered MYC/I-Z-I bodies in the shafts of 70 hour myotubes transform into ordered SMFs with typical 0.1 µm wide MYC/Z-bands, whereas irregularly-shaped 0.20.8 µm MYC/I-Z-I bodies continue to be assembled in their growth tips. Many myotubes in 80 hour cultures contract spontaneously. Lin et al. (1998) reported that, as anticipated from in vitro binding studies, MYC/FL (Fig. 1) was differentially incorporated into mature periodic Z-bands in SMFs in day 5-8 myotubes. Unexpectedly, however, so were mutants lacking actin binding sites (MYC/A−; Fig. 1) or fragments consisting of actin binding sites only (MYC/A+; Fig. 1). The staining patterns of MYC/FL, MYC/A− and MYC/A+ were indistinguishable from that of endogenous s-α-actinin. These mutant MYC/s-αactinin peptides did not act as dominant-negatives in early maturing myotubes. Fig. 2B-C′′ confirm and extend these
Microscopy Cells were examined by a Leica DM Model IRB fluoresence microscope, using filter sets which were selected for Rho-, FITC- or DAPI. Photographs were taken with either 63×1.6 (NA 1.4) or 100× (NA 1.3) oil immersion objectives and with 400 ASA film (T-MAX; Eastman Kodak Co., Rochester, NY). The continuous assembly and addition of de novo assembled ~2.0 µm wide sarcomeres in the elongating growth tips to the distal ends of the earlier assembled SMFs in the shaft was monitored on an inverted Zeiss microscope with phase-contrast objectives and a DAGE-72 camera interfaced with a Panasonic video recorder. During the 10 hour observation period the cultures were kept in a 37°C, CO2-gassed incubator adapted to the microscope stage. A laser scanning confocal microscope (LSM 510; Carl Zeiss, Inc., Germany) was used. Conventional EM sections and EM sections decorated with HMM were prepared and examined in a Type H-800 (Hitachi Ltd, Tokyo, Japan) as described by Ishikawa et al. (1969); Toyama et al. (1982) and Lin et al. (1998). Z-bands, which measure ~0.1 µm in width in EM sections, measure ~0.3 µm in the fluoresence microscope after staining with antibodies to MYC/FL MYC-tagged s-α-actinin peptides, or T20 (Lu et (1-897 aa) al., 1992; Lin et al., 1998). To avoid confusion in the text, the size of fluorescent Z-bands was taken as 0.l µm and all other fluorescent objects were MYC/4Spr+ calibrated accordingly.
MYC 1
Distribution of nascent MYC/s-αactinin peptides in the shaft of immature 70 hour and mature 80 hour transfected myotubes In transfected 70-80 hour cultures, 30-60% of the hundreds of myotubes/dish were positive for anti-MYC. If there were differences between untransfected myotubes and those expressing (a) the first 5 MYC/s-α-actinin peptides shown in Fig. 1 or (b) the GFP/Tcap or MYC/T-cap peptides, then such differences have escaped our detection. The two exceptions in Fig. 1, MYC/EFT+ and MYC/T+, will be described by the Discussion. Immature transfected 70 hour myotubes display numerous, longitudinally-oriented, 0.2-0.8 µm MYC/I-Z-I bodies both in their shafts (Fig. 2A,A′) and growth tips (see below). They have yet to assemble any SMFs with mature ~0.1 µm wide MYC/Z-bands, ~1.0 µm wide I-bands or 1.6 µm wide Abands. 70 hour untransfected or transfected myotubes do not contract spontaneously. In contrast, owing to asynchronous rates of fusion, maturation and elongation, the
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RESULTS
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Fig. 1. Maps of the MYC/s-α-actinins. MYC/FL is the full length s-α-actinin (aa 1-897); MYC/4Spr+ consists of spectrin-like repeats 1-4 only (lacking aa l-254 and 723-897); MYC/4Spr− lacks spectrin like repeats 1-4 (aa 255-722); MYC/A+ consists of aa 1-254 including the ABD; MYC/A− lacks aa 1-254 including ABD; MYC/EFT+ lacks aa 1-719; MYC/T+ lacks aa 1-804. The functional domains were mapped on the basis of the data of Blanchard et al. (1989), Hemmings et al. (1992), McGough et al. (1994), Flood et al. (1995), Sorimachi et al. (1997), Young et al. (1998) and Chan et al. (1998).
4104 K. Ojima and others Fig. 2. (A) anti-MYC, (A′) anti-nebulin (NB2), and (A′′) DAPI. Triple-stained, transfected (MYC/FL) 70 hour culture illustrating the abundance and longitudinal alignment of the I-Z-I bodies in the shaft of an immature myotube. Despite the absence of morphological Z-, I- and A-bands, the major myofibrillar proteins, e.g. s-αactinin, s-α-actin, tropomyosin, troponin I, titin, nebulin, MHC and MLC− are present as non-SMFs in such immature myotubes (Lin et al., 1994). Note the colocalization of the 2 antibodies. (A′′) Distribution of myogenic and nonmyogenic nuclei in this microscopic field. (B) antiMYC, (B′) anti-tropomyosin, and (B′′) DAPI. Triplestained, transfected (MYC/FL) 80 hour culture illustrating the SMFs in the shafts of 3 relatively mature myotubes. Arrows point to a transfected myotube. Asterisks mark 2 untransfected, out-of-focus myotubes. Morphologically the recently assembled ~0.1 µm wide MYC/Z-bands, as well as the tropomyosin-positive I-bands, are identical to those in similarly stained adult SMFs. (C) Anti-MYC, (C′) anti-troponin-I, and (C′′) DAPI. Triple-stained, transfected (MYC/A+) 80 hour culture. Arrow points to the shaft of a transfected myotube. Asterisks mark 2 untransfected myotubes. Despite lacking spectrin-like repeats and known titin binding sites, the targeting and incorporation of MYC/A+ were indistinguishable from that of MYC/FL (for details see Lin et al., 1998). (D) anti-MYC, (D′) Rho-phalloidin and (D′′) DAPI. Triple-stained, transfected (MYC/4Spr−) 80 hour culture. Immature and mature myotubes displaying definitive MYC/Z-bands and MYC/I-Z-I bodies (left and right asterisks). The short thin filament complexes in these myotubes (D′) have yet to be organized into laterally aligned ~1.0 µm I-bands (see below). At this stage of maturation and at this magnification their overlapping arrangement in the z-axis results in what appears to be continuous staining with Rho-phalloidin. Myotubes expressing MYC/4Spr+ are indistinguishable from those expressing MYC/4Spr− (see below). Bars, 10 µm.
findings to nascent MYC/Z-bands in the shafts of myotubes in 80 hour cultures. The localization of anti-MYC and antitropomyosin in these transfected and untransfected myotubes are shown in Fig. 2B and B′, and of anti-MYC and anti-troponin-I in Fig. 2C and C′. Similar images of ~0.1 µm periodic MYC/Zbands and ~1.0 µm wide I-bands are observed after doublestaining with antibodies to MYC and c-or s-α-actin (see below). In vitro binding studies have led to the conclusion that the binding/bundling properties of s-α-actinin, including its incorporation into Z-bands in adult SMFs, depends on the 4 spectrin-like repeats self-assembling into anti-parallel homodimers (Baron et al., 1987; Imamura et al., 1988; Blanchard et al., 1989; Kahana and Gratzer, 1991; Meyer and Aebi, 1990; McGough et al., 1994; Flood et al., 1995; Chan et al., 1998). To determine whether in vivo dimerization of spectrin-like repeats played an obligatory role in targeting and/or incorporation of s-α-actinin into Z-bands, cultures were transfected with (a) MYC/4Spr+ or (b) MYC/4Spr− (Fig. 1). Contrary to expectations based on in vitro binding studies that these fragments would be assembly-incompetent, both were
selectively targeted to 0.2-0.8 µm I-Z-I bodies in immature, and to ~0.1 µm Z-bands in mature, myotubes (Fig. 2D, and 2D′). Furthermore neither of these fragments acted as dominant negatives, nor were they cytotoxic even in day 6 myotubes. Whether they will display more subtle deleterious affects in older myotubes is being investigated. Similarity of z-bands in the SMFs in 80 hour myotubes and in adult SMFs It was important to determine whether there were differences in morphology or molecular composition in mature Z-bands in 80 hour myotubes and those in definitive Z-bands in adult SMFs. Two antibodies have identified distinct epitopes of nebulin within the Z-band of adult SMFs (Labeit and Kolmerer, 1995a; Millevoi et al., 1998). One is an SH3 domain at the nebulin C-terminal. The second, N-terminal to the SH3 domain, is a region of nebulin repeats M177-M181. Fig. 3A and 3A′ demonstrate that the antibody to the nebulin-SH3 domain stains the newly assembled ~0.1 µm wide MYC/Zbands as it does the Z-bands in adult SMFs. The coincidence
Precursor I-Z-I bodies into I-Z-I bands 4105 Fig. 3. (A) anti-MYC, (A′) anti-nebulin SH3, and (A′′) DAPI. Triple-stained myotube expressing MYC/4Spr+ in an 80 hour culture. The localization of antibodies to nebulin-SH3 in nascent Zbands is identical to their localization in mature Z-bands in adult SMFs. Arrows point to lateral short, definitive SMFs common in cultured myotubes. (B) anti-MYC, (B′), anti-titin Z1-Z2, and (B′′) DAPI. Triplestained myotube expressing MYC/4Spr−. The strict colocalization of these 2 antibodies to Z-bands in MYC/4Spr− transfected myotubes also obtains in myotubes expressing MYC/FL, MYC/A+, or MYC/4Spr+. (C) Triplestained preparation of a GFP/T-cap transfected myotube. The GFP is localized in the fluorescein channel (C). As the endogenous avian T-cap does not stain with human anti-T-cap only the incorporated exogenous human T-cap is visualized (C′; rhodamine channel). Two nontransfected myotubes in this the microscopic field are not visible. Arrows point to 2 of the 3 nuclei within this expressing myotube (C′′). (D) anti-MYC, and (D′) anti-desmin. High magnification, double-stained, MYC/A+ transfected 80 hour culture illustrating mature MYC/Z-bands in the shaft and MYC/I-Z-I bodies in the growth tip of adjacent myotubes (arrows). In the confocal microscope the shaft measures ~14 µm in the z-axis, the growth tip ~1.0-2.0 µm. Bars, 10 µm.
of MYC/s-α-actinin structures with the C-terminal epitopes of nebulin extends to SMFs so fine as to be barely detectable under the light microscope (arrows in Fig. 3A-3A′). Preparations double-stained with anti-MYC and anti-nebulin M177-M181 demonstrate similarly precise costaining of mature MYC/Z-bands in newly assembled SMFs (see below). Labeit and Kolmerer (1995b), Gregorio et al. (1998), and Mues et al. (1998) have described two distinct epitopes which are also buried within the ~0.1 µm wide Z-bands in adult SMFs. One is against the first 200 aa of titin, termed Z1-Z2. The second is against T-cap, a newly identified 19 kDa protein that binds to the N-terminal titin residues Z1-Z2. Fig. 3B and B′ illustrate that Z-bands in myotubes expressing MYC/4Spr− that had matured during the previous <10 hours bind antibodies to titin Z1-Z2 in the same manner as do Z-bands in adult SMFs. It will be interesting to estimate the number of the different kinds of integral proteins packed into the minute, but definitive Z-band indicated in Fig. 3B and B′ (arrows). The localization of T-cap to mature Z-bands in 80 hour myotubes is shown not by expressing MYC/s-α-actinin, but by transfecting myotubes with GFP/T-cap cDNA. The expressed GFP/T-cap is directly visualized in the fluorescein channel (Fig. 3C), localization of anti-T-cap in the rhodamine channel (Fig.
3C′). It is to be emphasized that anti-T-cap was prepared against human T-cap and does not stain chick Z-bands (unpublished data). Accordingly its selective localization in myotubes transfected with human T-cap must mark a conserved epitope and that the human protein can be incorporated into Z-bands in birds as well as in humans. Selective Z-band staining was also observed when myotubes transfected with MYC/T-cap were decorated with anti-MYC (not shown). In brief, morphologically and in molecular composition mature Z-bands in myotubes in 80 hour cultures share many properties with Z-bands in adult SMFs. Transition of I-Z-I bodies into I-Z-I bands in elongating growth tips of maturing myotubes Overviews illustrating the major structural differences between I-Z-I bodies and I-Z-I bands in the light and electron microscopes are shown in Fig. 3D and D′ and Fig. 4. The inset in Fig. 4B is a section through a growth tip decorated with heavy meromyosin, demonstrating that the oppositely polarized thin filament complexes insert by their barbed ends into the electron opaque cores of the I-Z-I bodies as they do in mature I-Z-I bands (Ishikawa et al., 1969). The paucity and relatively random distribution of thick filaments in this region
4106 K. Ojima and others
A Fig. 4. EM sections through the shaft (A) and the growth tip (B) of different myotubes from the same transfected (MYC/4Spr−) 80 hour culture. In conventional EM sections transfected cells cannot be distinguished from untransfected cells. (A) The relatively mature arrangement of the major myofibrillar proteins in the recently assembled I-Z-I bands, including interdigitation with laterally aggregated 1.6 µm thick filaments. (B) Although it is not possible to measure the length of the individual thin filaments that extend from the electron-opaque core of each IZ-I body (short arrows), variations in their size and superimposition in the z-axis, is evident.These images correlate well with the micrographs in Fig. 3D and D′. Note too the scattered nascent thick filaments (long arrows) that assemble independently of the I-Z-I bodies (for details regarding thick filament assembly, see Holtzer et al., 1997). Bars, 0.5 µm.
B
is consistent with the notion that in early stages of myofibril assembly the initiation of I-Z-I body assembly is relatively independent of the assembly of thick filament complexes (Holtzer et al., 1997). The addition of de novo assembled ~2.0 µm wide sarcomeres to the distal ends of SMFs in the shaft of living 80 hour myotubes was followed by time-lapse video microscopy (Lin et al., 1994). In 9 different growth tips 5-15 nascent sarcomeres were added per 10 hours. Virtually 100% of these were confined to the ventral surface of the elongating growth tips. In contrast, the dorsal surface rapidly extended and retracted sizable lamellipodia (see micrographs below). In morphology these de novo assembled sarcomeres were indistinguishable from those assembled earlier in the shafts of untransfected myotubes. As the average 70-80 hour myotube doubles, or even triples, in length in day 3-4 cultures, over half of the mature sarcomeres in older myotubes must have been initially assembled in elongating growth tips. Clearly the striking reconfigurations of the I-Z-I bodies into definitive I-Z-I bands are readily followed in the growth tips of the elongating myotubes and at most this transition requires an hour or two. Tight temporo-spatial coupling in elongating growth tips of s-α-actinin peptides with nebulin, titin and Tcap If initiation and/or stabilization of I-Z-I bodies requires relatively concurrent and cooperative interactions between (a) s-α-actinin,
(b) nebulin, and (c) titin, then, without exception each irregularly-shaped MYC/s-α-actinin structure in the growth tips of transfected myotubes should costain precisely with antibodies against these integral Z-band proteins. If T-cap is also involved in the initiation of I-Z-I bodies, then GFP/T-cap or MYC/T-cap should likewise precisely colocalize with other integral Z-band proteins. Antibodies to C-terminal nebulin SH3 and to nebulin modules M177-M181 show this colocalization to all MYC/I-ZI bodies (Fig. 5A and A′, B and B′). Nebulin-SH3 or nebulin M177-M181 bodies negative for MYC/s-α-actinin have not been observed. Conversely MYC/s-α-actinin bodies negative for nebulin-SH3 or M177-M181 have not been observed. The invariable linkage between MYC/s-α-actinin and nebulin is further underscored in transiently misaligned I-Z-I bodies (Fig. 5B and B′; see below). Arrays of misaligned I-Z-I bodies are assembled in roughly 40% of the growth tips in all myotubes, untransfected or transfected. Importantly, comparably misaligned mature I-Z-I bands are not observed in the shafts of untransfected or transfected myotubes in 80 hour cultures. We suggest that despite their normal appearance and molecular composition, misoriented I-Z-I bodies are probably degraded before they are transformed into I-Z-I bands (see Discussion). I-Z-I bodies have been described in ‘new-born’, postmitotic, mononucleated myoblasts that emerged from their terminal mitosis l0-l5 hours earlier (Lin et al., 1994). As shown in Fig. 5C and C′, newly assembled, barely resolvable I-Z-I bodies in postmitotic mononucleated myoblasts display linkages
Precursor I-Z-I bodies into I-Z-I bands 4107 Fig. 5. (A) anti-MYC, (A′) anti-nebulin SH3. Doublestained micrographs of a MYC/A− growth tip. They illustrate the precise colocalization of each MYCpositive structure with each nebulin SH3-positive structure. Two types of costaining structures are evident: (1) linearly aligned MYC/I-Z-I bodies, and (2) incipient MYC/Z-bands of variable widths (double arrows). The laterally flattened edges of the myotube rich in MYC/I-Z-I bodies (asterisks) measure ~1.0 µm in the z-axis. The anti-nebulin SH3 stains the nuclei in myotubes weakly. (B) anti-MYC, (B′) anti-nebulin M177-181. Double-stained micrographs of a growth tip and an immature shaft expressing MYC/4Spr−. Both the longitudinally and short-lived transversely oriented I-Z-I bodies (arrows), precisely costain with anti-MYC and anti-nebulin M177-181. The growth tip is likely to be in the process of fusing with the immature, myotube at upperright. Asterisks marks a nebulin positive fibroblast nucleus. (C) anti-MYC, (C′) anti-nebulin SH3. Doublestained micrograph from a MYC/FL transfected culture illustrating the precise codistribution of MYC/FL and nebulin SH3 in (a) Z-bands of mature SMFs, and (b) early emerging I-Z-I bodies of a postmitotic, mononucleated myoblast (arrows; see Lin et al., 1994). Note nuclear staining in the myoblast. (D) anti-MYC, (D′) anti-titin Z1-Z2. Double-stained preparation of a MYC/A+ transfected culture. Both the transfected growth tip and the postmitotic mononucleated, myoblast (arrows) have assembled numerous MYC/I-Z-I bodies that costain with anti-titin Z1-Z2. Bars, 10 µm.
between MYC/s-α-actinin and nebulin SH3. Linkage between s-α-actinin and nebulin M177-M181 bodies have also been detected in postmitotic myoblasts (not shown). If there is an obligatory requirement for the N-terminal of titin or T-cap for (a) the initiation of I-Z-I bodies and (b) their subsequent maturation into I-Z-I bands, then double-staining any transfected myotube should result in the precise costaining of every MYC/s-α-actinin structure with both anti-titin Z1-Z2 and anti-T-cap. As illustrated in Fig. 5D and D′ each MYC/ IZ-I body precisely costained with anti-titin Z1-Z2. Similar images were obtained by double-staining with anti-MYC and anti-titin T20 (not shown). Note too in Fig. 5D and D′, that the
elongated, postmitotic, mononucleated myoblast displays titin Z1-Z2-positive I-Z-I bodies similar to those in growth tips or immature shafts. Anti-MIR, however, which localizes along titin at the A-I junction does not colocalize with MYC/s-αactinin at the most distal region of the growth tip. It localizes more proximally as a doublet associated with emergent MHCpositive A-bands. Comparable differential accessibility of titin epitopes in situ has been reported by Schultheiss et al. (1990) using anti-T20 vs anti-T1 relative to the disassembly/assembly of myofibrils in cultured cardiomyocytes. GFP/T-cap expressed in transfected mature myotubes and growth tips also colocalized to all anti-Z1-Z2 positive
4108 K. Ojima and others Fig. 6. (A) GFP/T-cap (fluorescein channel) and (A′) anti-Z1-Z2 (rhodamine channel). Double-stained preparation revealing codistribution of expressed GFP/T-cap and anti-Z1-Z2. The most distal transfected growth tip in Fig. 6A′ is slightly out of focus in order to visualize the 2 near-by non-transfected myotubes (asterisks). (B) AntiMYC and (B′) antitropomyosin. Micrographs of a double-stained MYC/FL transfected growth tip showing the distribution of periodic MYC-structures that appear to be embedded in broad filaments that stain continuously with anti-tropomyosin. The MYCpositive structures fall into two over-lapping classes: (1) distal MYC/I-Z-I bodies, and (2) more proximal incipient wide MYC/Z-bands (arrows). The mature ~0.1 µm MYC/Z-bands and ~1.0 µm wide I-bands lie still more proximal in the shaft. (C) Anti-MYC and (C′) antitroponin I. Double-stained micrographs of a MYC/4Spr− expressing growth tip illustrating a ‘disoriented’ subset of I-Z-I bodies (arrows). Even in misaligned MYC/I-Z-I bodies the invariable tight linkage between mutant MYC/s-α-actinin peptides and troponin-I is maintained. Note staining of incipient striations with anti-MYC vs continuous staining with anti-troponin I. (D) anti-nebulin M177-181 and (D′) Rho-phalloidin. Doublestained preparation of a MYC/FL transfected growth tip illustrating that incipient M177181 positive Z-bands in the shaft are part of continuously staining, longitudinally and/or transversely oriented, F-actin filament complexes. Note too, the double-stained ectopic aggregates (arrows). Ectopic streaks appear in ~10% of both untransfected and transfected myotubes. Bars, 10 µm.
structures (Fig. 6A and A′). No titin Z1-Z2 body was negative for GFP/T-cap: no GFP/T-cap-positive body was negative for Z1-Z2. Identical results were obtained staining MYC/T-cap transfected cells with anti-MYC (not shown). In summary 4 proteins integral to the tetragonal configuration of Z-bands in adult SMFs and mature myotubes, namely s-α-actinin, the C-terminal 50 kDa of nebulin, the extreme 20 kDa N-terminal of titin and T-cap, are already present in the disordered de novo assembled 0.2-0.8 µm I-Z-I
precursors. Titin, nebulin or T-cap did not assemble into microscopically detectable homopolymers prior to complexing with other integral Z-band proteins. Temporo-spatial coupling of I-Z-I bodies with tropomyosin, troponin-I and α-actin in growth tips There are similarities and differences in the relationships of the proteins that constitute the thin filament complexes as the I-ZI bodies mature. First we describe the similarities in behavior
Precursor I-Z-I bodies into I-Z-I bands 4109 Fig. 7. (A) anti-MYC, and (A′) anti-c-α-actin. Double-stained, MYC/A+ transfected growth tip illustrating the proximal/distal gradient in maturation of MYC/I-Z-I bodies into MYC/Z-bands (arrows at left). In the relatively thick proximal region the longitudinally, over-lapping thin filament complexes stain continuously with anti-c-αactin. An out-of-focus, modestsized (~8.0 µm in the z-axis) cα-actin giant ruffled membrane is indicated by an asterisk. (B) anti-nebulin M177-181 and (B′) anti-c-α-actin. Doublestained cells illustrating the variable morphologies of elongating growth tips. The most distal~40 µm of the larger growth tip (asterisks) positive for c-α-actin lacks M177-181. The giant ruffled membranes associated with the growth tips are positive for c-α-actin but negative for titin, nebulin and T-cap. They characterize~20% of all growth tips, transfected or untransfected. Note what appear to be exocytosed c-α-actin droplets (arrows). These droplets are also positive for s-α-actinin, titin and nebulin (for details see Lin et al., 1989). Bars, 10 µm.
of tropomyosin, troponin-I, and c- or s-α-actin relative to maturing I-Z-I bodies, then the differences. The common staining pattern of the I-band proteins in elongating growth tips is illustrated in Fig. 6B-7B′. In the light microscope the MYC/peptides appear distally as irregularlyshaped, punctate bodies within what appears to be continuously staining thin filament complexes. (Note this in contrast to that observed in thin EM sections in Fig. 4.) These thin filament complexes are positive for tropomyosin (Fig. 6B′) troponin-I (Fig. 6C′), Rhodamine-phalloidin (Fig. 6D′) and cα-actin (Fig. 7A′). A distal-proximal gradient marks the progressive reconfigurations of the MYC/I-Z-I bodies first into broad incipient MYC/Z-bands and then narrow, mature 0.1 µm MYC/Z-bands. But even in the region of incipient and mature MYC/Z-bands the short, over-lapping thin filament complexes (Fig. 4B) stain continuously. It has long been recognized that during the transition from non-striated to striated myofibrils both the Z- and A-bands display the mature ~2.0 µm Z-band periodicity and 1.6 µm width, respectively, well before the Iband filaments display their ~1.0 µm length (Shimada and Obinata, 1977; Antin et al., 1986; Lin et al., 1994). Fig. 6B-D′ also illustrate transient arrays of misaligned I-Z-I bodies that must either be realigned or turned over by unknown editing mechanisms before maturing into I-Z-I bands. The differences between the distribution of tropomyosin, and troponin-I, on the one hand, and of rhodamine-phalloidin and c- and/or s-α-actin on the other, are as follows: (1) In no instance have tropomyosin or troponin-I extended distally beyond MYC/s-α-actinin bodies. In 8 out of 50 growth tips, however, MYC/A+ bodies extended up to 5 µm beyond the most distal tropomyosin or troponin-I positive bodies (not shown). Failure to detect tropomyosin or troponin-I in this
small sub-set of I-Z-I bodies may mean that (a) these molecules are not obligatory for the initial initiation of the I-Z-I bodies, or (b) their low fluoresence is due to unavailability or density of epitopes, titer of antibodies, etc. (2) In contrast, in 20 out of 50 growth tips c- or s-α-actin positive outgrowths extended up to 50 µm beyond the most distal MYC/A+ bodies (compare Fig. 7A and A′ with B and B′). Rapidly elongating c- or s-αactin-positive growth tips often terminate in bizarre ruffled membranes that may measure up to 15 µm in the z-axis (Fig. 7B′). Further details regarding the down regulation of β- and γ-actin in these giant growth tips, as well as the respective roles of c- and s-α-actin isoforms in (a) the protrusive activity responsible for elongation, (b) the initiation and maturation of polarized ~1.0 µm long thin filament complexes, and (c) the assembly of the subsarcolemmal α-actin meshwork, will be described elsewhere. DISCUSSION The two inter-related salient findings in this report are: (1) MYC/s-α-actinin mutants/fragments which lack (a) known actin or titin binding sites (b) spectrin-like repeats, as well as (c) peptides consisting of spectrin-like repeats only, are selectively incorporated into normal I-Z-I bodies and I-Z-I bands, and (2) the de novo assembly of the precursor I-Z-I bodies and their maturation into definitive I-Z-I bands involves a tight temporospatial coupling of, at least, 5 integral Z-band proteins, s-α-actinin, nebulin, titin, T-cap and c- or s- α-actin. Given the morphological and molecular complexity of both I-Z-I bodies and I-Z-I bands, it is surprising and difficult to understand how MYC-tagged fragments of s-α-actinin that
4110 K. Ojima and others were intended to be assembly-incompetent (extrapolating from their known in vitro binding properties), in fact were selectively incorporated into both structures. Originally we expected that MYC/s-α-actinin mutants lacking N-terminal actin, or C-terminal titin binding sites would act as dominantnegatives, that they would form amorphous intracellular precipitates, subvert the assembly of normal s-α-actinin structures, and probably be cytotoxic (contrast findings of Schultheiss et al., 1991 with those of Hijikata et al., 1997 and Lin et al., 1998). Similarly, given the view that the anti-parallel homodimerization of spectrin-like repeats accounts for the actin binding/cross linking properties of s-α-actinin, we expected MYC/4Spr− and MYC/4Spr+ to have comparable deleterious affects on myofibrillogenesis. This did not happen. Interestingly, while this work was in progress Young et al. (1998) reported that at least one site of interaction between titin and s-α-actinin is also dimerization-independent. Nevertheless, the possibility remains that MYC/s-α-actinins lacking known binding sites dimerize with endogenous s-α-actinin and, as a component of this heterodimer in a piggy-back fashion incorporated into I-Z-I structures. Hijikata et al. (1997) directly addressed this issue of heterodimers in PtK2 cells. By doubletransfecting PtK2 cells with MYC/FL and MYC/A− they demonstrated that these expressed peptides did not interact with each other, nor were the MYC/A− peptides incorporated into any of the α-actinin structures of the cell (e.g. dense bodies, adhesion plaques, etc.) by dimerizing with the endogenous wild-type α-actinin. On the other hand, Chan et al. (1998), using chemical cross-linkers on extracts from mass cultures of double-transfected COS cells, detected dimerization of expressed s-α-actinin-2 and s-α-actinin-3 in SDS-gels. Additional experiments examining single cells should resolve this challenging issue. In contrast to the peptides discussed above, others such as MYC/EFT+ and MYC/T+ in Fig. 1, were not selectively incorporated into I-Z-I bodies or I-Z-I bands but accumulated diffusely throughout both the sarcoplasm and all nuclei (data not shown). These mutants were so cytotoxic that few myotubes survived beyond 80 hours. Similarly some expressed MYC/nebulin C-terminal fragments were not selectively incorporated into I-Z-I bodies and I-Z-I bands. These fragments accumulated diffusely throughout the entire myotube, including nuclei, and were cytotoxic to varying degrees (Ojima et al., 1999). Pending more information regarding (a) the mechanisms regulating the relationship between incorporation, normal turn-over and competition with endogenous molecules and (b) the presence in I-Z-I structures of other known and unknown molecules (e.g. PIP2, Fukami et al., 1992; assemblases, Liu et al., 1997; see also Goll et al., 1991), further speculations as to how some expressed Z-band peptides are incorporated selectively whereas others accumulate diffusely throughout the myotubes and are cytotoxic, would be premature. At no stage in the assembly of I-Z-I structures was there evidence that monomers of α-actinin, nebulin, titin, T-cap and c- and/or s-α-actin self-assembled into microscopically-stable homopolymers. When first detected, these proteins were components of supramolecular complexes. Long homopolymers of nebulin or titin did not prefigure the subsequent polymerization of α-actin into polarized ~1.0 µm long thin filament complexes. Whether titin or nebulin function
as molecular rulers for thick and thin filaments (Trinick, 1994; Wang et al., 1996), or whether they play a role in mediating the interdigitation of preformed I-Z-I complexes and polarized 1.6 µm thick filament complexes (Holtzer et al., 1997), they do so, not as long homopolymers, but as part of already heteropolymeric, elongating structures. These findings raise the issue of whether homopolymers of any myofibrillar proteins ever self-assemble in vivo, as they clearly do in vitro. Probably one of the major functions of the numerous actin capping, severing, bundling, etc. proteins in myotubes is to preclude the self-assembly of α-actin into long homopolymers, as well as blocking premature interactions with thick filament complexes (Fig. 4). Comparable regulatory proteins may also preclude inappropriate self-assembly of other myofibrillar proteins as well (Holtzer et al., 1997). The data in this report, particularly Fig. 3D and D′ and Fig. 4, are not readily reconciled with the hypothesis that ‘minisarcomeres’ are intermediates in the assembly of SMFs (Sanger et al., 1989; Rhee et al., 1994). There are no stages in the transition of irregularly-shaped I-Z-I bodies into I-Z-I bands where closely packed, tetragonal Z-bands progressively separate (rather then reconfigure) to acquire their ~2.0 µm periodicity by the interposition of gradually lengthening thick filaments. Also the failure of anti-MIR to stain I-Z-I bodies would question the notion of ‘mini-sarcomeres’ bounded by mature Z-bands. Additionally, despite difficulties, due to the plane of sectioning, in accurately measuring the irregularly oriented thick filaments in Fig. 4, we estimate that they approximate in length normal mature thick filaments. Importantly, they are positive for both s-MHC and MLCs (data not shown). In brief, even myosin, although capable of selfassembly into filaments of various lengths in vitro, most likely requires MLCs to achieve its invariant in vivo length of 1.6 µm and probably involves myomesin and/or C-protein for lateral alignment into A-bands in vivo (Lin et al., 1994). Whether single nascent thick filament complexes are also titin positive remains to be determined (see Holtzer et al., 1997). The fact that the truncated s-α-actinin peptides integrated into both electron-opaque I-Z-I bodies and I-Z-I bands is consistent with their binding by multiple side-by-side interactions with the endogenous s-α-actinin and therefore may not be a reliable indicator of how s-α-actinin behaves during normal I-Z-I assembly. Consistent with this notion was the failure to find exogenous Z-band peptides that were not also positive for their endogenous counterparts. MYC- or GFP-Zband peptides were always components of chimeric structures consisting of endogenous and exogenous molecules, suggesting that the latter by themselves did not initiate the assembly of I-Z-I bodies. On the other hand, in Drosophila, both MHC isoforms and IFM-specific tagged Act88F actin expressed in null muscles lacking endogenous MHC and Act88F, respectively, assembled morphologically normal thick and thin filament complexes (Wells et al., 1996; Brault et al., 1999). It will be of interest to determine whether peptides of MHC or α-actin as radically truncated as those of s-α-actinin, titin or nebulin act as dominant negatives or incorporate into thick or thin filament complexes. It is our impression that tropomyosin and troponin I monomers may not be required for initiation of I-Z-I bodies, but shortly later are required for stabilization and/or reconfiguration. However, as these interpretations were based
Precursor I-Z-I bodies into I-Z-I bands 4111 on fluorescent images beyond the resolution, and at the limit of sensitivity, of the fluorescence microscope they must be confirmed by immuno-electron microscopy. On the other hand there is additional evidence that c-α-actin is as central to the assembly of I-Z-I bodies as is titin. K. Ojima et al. (unpublished data) have found that in both transfected and untransfected growth tips antibodies to titin Z1-Z2 and tropomodulin, a pointed end actin capping protein (Littlefield and Fowler, 1998) also colocalized in I-Z-I bodies. These findings are consistent with the view that while in normal development the initial involvement of α-actin in I-Z-I body initiation probably requires concurrent interactions with s-αactinin, nebulin, titin, T-cap, and perhaps tropomodulin, its elongation into polarized ~1.0 µm thin filaments probably requires subsequent interactions with tropomyosin and troponin-I. One of the major limitations in this study is defining ‘relatively concurrent’ interactions of the 5 or more integral Z-band proteins versus a multi-step temporal sequence in binding monomers. A paradigm for this issue is the kinetics of assembly of adhesion plaques and subsequent centrifugal elongation of their attached stress fibers in non-muscle cells. Integrin clustering induced by beads coated with antibodies or fibronectin, followed by ligand occupancy, results in the local and relatively concurrent accumulation of integrin, tensin, FAK and talin, rapidly followed by vinculin, α-actinin and Factin. These interactions involve conformational changes driven by nucleoside triphosphate hydrolysis (Yamada and Miyamoto, 1995; Lewis and Schwartz, 1996). Comparable kinetics may mediate the initiation and subsequent conformational changes undergone by integral Z-band proteins as they condense from disordered I-Z-I bodies into semi-crystalline Z-bands. Sebestyen et al. (1995), Gautel et al. (1993), describe titin phosphorylation sites for serine-proline kinases localized within the M- and Z-bands, respectively, whereas Mayans et al. (1998) describe an autoregulated titin kinase at the C-terminal. Furthermore, the phosphorylation mediated by cdc2 kinase and recombinant ERK1 kinase is higher in extracts from embryonic muscle as compared with those from mature muscle. With the increasingly detailed characterization of the I-Z-I body to I-Z-I band transition, probing the role(s) in real time of such signal transduction pathways as the nebulin SH3/titin kinases should prove interesting. Equally intriguing is whether the initiation and maturation of I-Z-I bodies into I-Z-I bands can occur in the absence of any one of the integral Z-band proteins. This work was supported by Grants 5-P01-HL15835, HL59470 and AR 32147 from the National Institutes of Health, from the Muscular Dystrophy Association, and by DFG (La6815-I). We are indebted to Dr J. Murray for many helpful suggestions.
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