Synemin may function to directly link muscle cell intermediate filaments to both myofibrillar Z-lines and costameres.

Synemin is a large intermediate filament (IF) protein that has been identified in all types of muscle cells in association with desmin- and/or vimentin-containing IFs. Our previous studies (Bellin, R. M., Sernett, S. W., Becker, B., Ip, W., Huiatt, T. W., and Robson, R. M. (1999) J. Biol. Chem. 274, 29493-29499) demonstrated that synemin forms heteropolymeric IFs with major IF proteins and contains a binding site for the myofibrillar Z-line protein alpha-actinin. By utilizing blot overlay assays, we show herein that synemin also interacts with the costameric protein vinculin. Furthermore, extensive assays utilizing the Gal4 yeast two-hybrid system demonstrate interactions of synemin with desmin and vimentin and additionally define more precisely the protein subdomains involved in the synemin/alpha-actinin and synemin/vinculin interactions. The C-terminal approximately 300-amino acid region of synemin binds to the N-terminal head and central rod domains of alpha-actinin and the approximately 150-amino acid C-terminal tail of vinculin. Overall, these interactions indicate that synemin may anchor IFs to myofibrillar Z-lines via interactions with alpha-actinin and to costameres at the sarcolemma via interactions with vinculin and/or alpha-actinin. These linkages would enable the IFs to directly link all cellular myofibrils and to anchor the peripheral layer of myofibrils to the costameres.     

ALP and MLP distribution during myofibrillogenesis in cultured cardiomyocytes

The Z-line is a multifunctional macromolecular complex that anchors sarcomeric actin filaments, mediates interactions with intermediate filaments and costameres, and recruits signaling molecules. Antiparallel alpha-actinin homodimers, present at Z-lines, cross-link overlapping actin filaments and also bind other cytoskeletal and signaling elements. Two LIM domain containing proteins, alpha-actinin associated LIM protein (ALP) and muscle LIM protein (MLP), interact with alpha-actinin, distribute in vivo to Z-lines or costameres, respectively, and, when absent, are associated with heart disease. Here we describe the behavior of ALP and MLP during myofibrillogenesis in cultured embryonic chick cardiomyocytes. As myofibrils develop, ALP and MLP are observed in distinct distribution patterns in the cell. ALP is coincident with alpha-actinin from the first stage of myofibrillogenesis and co-distributes with alpha-actinin to Z-lines and intercalated discs in mature myofibrils. Interestingly, we also demonstrate using ALP-GFP transfection experiments and an in vitro binding assay that the ALP-alpha-actinin binding interaction is not required to target ALP to the Z-line. In contrast, MLP localization is not co-incident with that of alpha-actinin until late stages of myofibrillogenesis; however, it is present in premyofibrils and nascent myofibrils prior to the incorporation of other costameric components such as vinculin, vimentin, or desmin. Our observations support the view that ALP function is required specifically at actin anchorage sites. The subcellular distribution pattern of MLP during myofibrillogenesis suggests that it functions during differentiation prior to the establishment of costameres.     

Identification of a repeated domain within mammalian alpha-synemin that interacts directly with talin

The type VI intermediate filament (IF) protein synemin is a unique member of the IF protein superfamily. Synemin associates with the major type III IF protein desmin forming heteropolymeric intermediate filaments (IFs) within developed mammalian striated muscle cells. These IFs encircle and link all adjacent myofibrils together at their Z-lines, as well as link the Z-lines of the peripheral layer of cellular myofibrils to the costameres located periodically along and subjacent to the sarcolemma. Costameres are multi-protein assemblies enriched in the cytoskeletal proteins vinculin, alpha-actinin, and talin. We report herein a direct interaction of human alpha-synemin with the cytoskeletal protein talin by protein-protein interaction assays. The 312 amino acid insert (SNTIII) present only within alpha-synemin binds to the rod domain of talin in vitro and co-localizes with talin at focal adhesion sites within mammalian muscle cells. Confocal microscopy studies showed that synemin co-localizes with talin within the costameres of human skeletal muscle cells. Analysis of the primary sequences of human alpha- and beta-synemins revealed that SNTIII is composed of seven tandem repeats, each containing a specific Ser/Thr-X-Arg-His/Gln (S/T-X-R-H/Q) motif. Our results suggest human alpha-synemin plays an essential role in linking the heteropolymeric IFs to adherens-type junctions, such as the costameres within mammalian striated muscle cells, via its interaction with talin, thereby helping provide mechanical integration for the muscle cell cytoskeleton.     

Vinculin is a component of an extensive network of myofibril-sarcolemma attachment regions in cardiac muscle fibers

Immunofluorescent staining of bovine and avian cardiac tissue with affinity-purified antibody to chicken gizzard vinculin reveals two new sites of vinculin reactivity. First, vinculin is organized at the sarcolemma in a striking array of rib-like bands, or costameres. The costameres encircle the cardiac muscle cell perpendicular to the long axis of the fiber and overlie the I bands of the immediately subjacent sarcomeres. The second new site of vinculin reactivity is found in bovine cardiocytes at tubular invaginations of the plasma membrane. The frequency and location of these invaginations correspond to the known frequency and distribution of the transverse tubular system in bovine atrial, ventricular, and Purkinje fibers. We do not detect tubular invaginations that stain with antivinculin in avian cardiocytes and, in fact, a transverse tubular system has not been found in avian cardiac fibers. Apparent lateral Z-line attachments to the sarcolemma and its invaginations have been observed in cardiac muscle by electron microscopy in the same regions where we find vinculin. On the basis of these previous ultrastructural findings and our published evidence for a physical connection between costameres and the underlying myofibrils in skeletal muscle, we interpret the immunofluorescence data of this study to mean that, in cardiac muscle, vinculin is a component of an extensive system of lateral attachment of myofibrils to the plasma membrane and its invaginations.     

Subcellular localization of dystrophin and vinculin in cardiac muscle fibers and fibers of the conduction system of the chicken ventricle

The subcellular localization of dystrophin and vinculin was investigated in cardiac muscle fibers and fibers of the conduction system of the chicken ventricle by immunofluorescence confocal microscopy. In ventricular cardiac muscle fibers, strong staining with antibody against dystrophin appeared as regularly arranged transverse striations at the sarcolemmal surface, and faint but uniform staining was seen in narrow strips between these striations. In fibers of the ventricular conduction system, the sarcolemma was stained uniformly with this antibody, but strong staining was found as regular striations in many areas and as scattered patches in other areas of the sarcolemma. These intensely stained striations and scattered patches of dystrophin were colocalized with those of vinculin. Because dystrophin striations were located at the level of Z bands of the underlying myofibrils, they were regarded as the concentration of this protein at costameres together with vinculin. In fibers of the conduction system, myofibrils were close to the sarcolemma where dystrophin and vinculin assumed a striated pattern, at some distance from the cell membrane where these proteins exhibited a patchy distribution, and distant from the sarcolemma where dystrophin was uniformly distributed. These data suggest that the distribution patterns of dystrophin reflect the degree of association between the sarcolemma and underlying myofibrils.     

Sarcolemmal organization in skeletal muscle lacking desmin: evidence for cytokeratins associated with the membrane skeleton at costameres

The sarcolemma of fast-twitch muscle is organized into "costameres," structures that are oriented transversely, over the Z and M lines of nearby myofibrils, and longitudinally, to form a rectilinear lattice. Here we examine the role of desmin, the major intermediate filament protein of muscle in organizing costameres. In control mouse muscle, desmin is enriched at the sarcolemmal domains that lie over nearby Z lines and that also contain beta-spectrin. In tibialis anterior muscle from mice lacking desmin due to homologous recombination, most costameres are lost. In myofibers from desmin -/- quadriceps, by contrast, most costameric structures are stable. Alternatively, Z line domains may be lost, whereas domains oriented longitudinally or lying over M lines are retained. Experiments with pan-specific antibodies to intermediate filament proteins and to cytokeratins suggest that control and desmin -/- muscles express similar levels of cytokeratins. Cytokeratins concentrate at the sarcolemma at all three domains of costameres when the latter are retained in desmin -/- muscle and redistribute with beta-spectrin at the sarcolemma when costameres are lost. Our results suggest that desmin associates with and selectively stabilizes the Z line domains of costameres, but that cytokeratins associate with all three domains of costameres, even in the absence of desmin.     

The dystrophin complex forms a mechanically strong link between the sarcolemma and costameric actin

The absence of dystrophin complex leads to disorganization of the force-transmitting costameric cytoskeleton and disruption of sarcolemmal membrane integrity in skeletal muscle. However, it has not been determined whether the dystrophin complex can form a mechanically strong bond with any costameric protein. We performed confocal immunofluorescence analysis of isolated sarcolemma that were mechanically peeled from skeletal fibers of mouse hindlimb muscle. A population of gamma-actin filaments was stably associated with sarcolemma isolated from normal muscle and displayed a costameric pattern that precisely overlapped with dystrophin. However, costameric actin was absent from all sarcolemma isolated from dystrophin-deficient mdx mouse muscle even though it was localized to costameres in situ. Vinculin, alpha-actinin, beta-dystroglycan and utrophin were all retained on mdx sarcolemma, indicating that the loss of costameric actin was not due to generalized membrane instability. Our data demonstrate that the dystrophin complex forms a mechanically strong link between the sarcolemma and the costameric cytoskeleton through interaction with gamma-actin filaments. Destabilization of costameric actin filaments may also be an important precursor to the costamere disarray observed in dystrophin-deficient muscle. Finally, these methods will be broadly useful in assessing the mechanical integrity of the membrane cytoskeleton in dystrophic animal models lacking other costameric proteins.     

Role of N-cadherin- and integrin-based costameres in the development of rat cardiomyocytes

Costameres, vinculin-containing structures found in skeletal and cardiac muscle, are thought to anchor the Z-discs of the peripheral myofibrils to the sarcolemma. Several lines of evidence indicate that two different sets of costameres, integrin- and N-cadherin-based, are present in cardiac muscles. In this study, immunoblot analysis was used to study the expression of N-cadherin, alpha-catenin, beta-catenin, vinculin, talin, and laminin in rat cardiac muscles at embryonic days 15 and 19, the day of birth (postnatal day 0), postnatal weeks 1, 2, 3, and 4, and in the adult. Double immunofluorescence microscopy was performed to study the spatial and temporal distribution of these two sets of costameres in rat cardiomyocytes. Costameric staining for N-cadherin, codistributed with beta-catenin, was strong from embryonic day 15 up to postnatal week 2, gradually decreased after postnatal week 3, and was undetectable at postnatal week 4 and in the adult. Confocal microscopy showed that N-cadherin colocalized with alpha-actinin at cortical myofibrils. Double-labeling of beta-catenin and talin indicated the coexistence of N-cadherin/catenin- and integrin/talin-based costameres in rat cardiac muscle. Although beta-catenin and vinculin were co-localized at the costamere of cardiomyocytes from embryonic day 15 to postnatal week 3, staining for beta-catenin or talin was mutually exclusive at all stages examined. These results demonstrate the simultaneous, but mutually exclusive, existence of N-cadherin/catenin- and integrin/talin-based costameres in rat cardiomyocytes between late embryonic stages and postnatal week 3, while only integrin/talin-based costameres were found in adult rats. The N-cadherin/catenin-based costameres in rat cardiac muscles may play a role in myofibrillogenesis similar to that of their counterparts in cultured cardiomyocytes.     

Desmin at myotendinous junctions

Myofibrils are linked to the cell membrane at myotendinous junctions located at the ends of muscle fibers, and at costameres, sites positioned periodically along lateral surfaces of muscle cells. Both of these sites are enriched in proteins that link active components of myofibrils to the cell membrane. Costameres are also enriched in desmin intermediate filaments that link passive components of myofibrils to the lateral surfaces of muscle cells. In this study, the possibility that desmin is also found between the terminal Z-disk of myofibrils and the myotendinous junction membrane is examined by immunocytochemistry and by KI-extraction procedures. Data presented show that desmin is located in the filamentous core of cellular processes at myotendinous junctions at sites 30 nm or more from the membrane. This core lies deep to subsarcolemmal material previously shown to contain talin, vinculin, and dystrophin. The distance from desmin to the membrane suggests desmin does not interact directly with membrane proteins at the junction. Immunoblots and indirect immunofluorescence of junctional regions of muscle compared to nonjunctional regions show no apparent enrichment of desmin at junctional sites, although vinculin, another costameric and junctional component, is significantly enriched at junctional regions. These findings show that passive elements of myofibrils may be continuous from myotendinous junctions of muscle origin to insertion via desmin filaments located between terminal Z-disks and the junctional membrane. This can provide a system in parallel to that involving thin filaments, vinculin, and talin for linking myofibrils to the cell membrane at myotendinous junctions.     

Primary longitudinal adhesion structures: plectin-containing precursors of costameres in differentiating human skeletal muscle cells

Plectin is a high molecular mass protein (ca 530 kDa) that binds actin, intermediate filaments, and microtubules. Mutations of the human plectin gene cause epidermolysis bullosa simplex with muscular dystrophy. In mature human skeletal muscle, plectin is localized between neighboring myofibrils and between myofibrils and the sarcolemma, both at the level of Z-discs. In the present study we have analyzed plectin expression patterns with emphasis on its sarcolemmal localization during human skeletal muscle differentiation in vitro. In myoblasts plectin showed a cytoplasmic intermediate filament-like distribution, whereas in myotubes plectin is also found at the level of the sarcolemma. In particular, in early myotubes a specific plectin isoform colocalizes with the costameric proteins vinculin and beta1D integrin in longitudinally orientated structures which increased in number and longitudinal extension upon further maturation. In mature myotubes processes perpendicular to the parallel system of longitudinal structures became apparent. Subsequent to the occurrence of spontaneous myofibrillar contractions, the number of longitudinal streaks decreased, and plectin and other costameric proteins were found in an orderly cross-striated sarcolemmal lattice overlying myofibrillar Z-discs. Our study demonstrates that plectin is preassembled together with vinculin and beta1D integrin into primary longitudinal adhesion structures. After the occurrence of spontaneous contractions, these structures reorient and mature costameres are assembled.     

Plectin tethers desmin intermediate filaments onto subsarcolemmal dense plaques containing dystrophin and vinculin

Plectin is a versatile cytoskeletal linker protein that preferentially localizes at interfaces between intermediate filaments and the plasma membrane in muscle, epithelial cells, and other tissues. Its deficiency causes muscular dystrophy with epidermolysis bullosa simplex. To better understand the functional roles of plectin beneath the sarcolemma of skeletal muscles and to gain some insights into the underlying mechanism of plectin-deficient muscular dystrophy, we studied in vivo structural and molecular relationships of plectin to subsarcolemmal cytoskeletal components, such as desmin, dystrophin, and vinculin, in rat skeletal muscles. Immunogold electron microscopy revealed that plectin fine threads tethered desmin intermediate filaments onto subsarcolemmal dense plaques overlying Z-lines and I-bands. These dense plaques were found to contain dystrophin and vinculin, and thus may be the structural basis of costameres. The in vivo association of plectin with desmin, (meta-)vinculin, dystrophin, and actin was demonstrated by immunoprecipitation experiments. Treatment of plectin immunoprecipitates with gelsolin reduced actin, dystrophin, and (meta-)vinculin but not desmin, implicating that subsarcolemmal actin could partly mediate the interaction between plectin and dystrophin or (meta-)vinculin. Altogether, our data suggest that plectin, along with desmin intermediate filaments, might serve a vital structural role in the stabilization of the subsarcolemmal cytoskeleton.     

Trypanosoma cruzi infection disrupts vinculin costameres in cardiomyocytes

Chagas' disease cardiomyopathy is an important manifestation of Trypanosoma cruzi infection, leading to cardiac dysfunction and serious arrhythmias. We have here investigated by indirect immunofluorescence assay the distribution of vinculin, a focal adhesion protein with a major role in the transmission of contraction force, during the T. cruzi-cardiomyocyte infection in vitro and in vivo. No change in vinculin distribution was observed after 24 h of infection, where control and T. cruzi-infected cardiomyocytes displayed vinculin localized at costameres and intercalated discs. On the other hand, a clear disruption of vinculin costameric distribution was noted after 72 h of infection. A significant reduction in the levels of vinculin expression was observed at all times of infection. In murine experimental Chagas' disease, alteration in the vinculin distribution was also detected in the infected myocardium, with no costameric staining in infected myocytes and irregular alignment of intercalated discs in cardiac fibers. These data suggest that the disruption of costameric vinculin distribution and the enlargement of interstitial space due to inflammatory infiltration may contribute to the reduction of transmission of cardiac contraction force, leading to alterations in the heart function in Chagas' disease.     

Contractile activity and cell-cell contact regulate myofibrillar organization in cultured cardiac myocytes

Adult feline ventricular myocytes cultured on a laminin-coated substratum reestablish intercellular junctions, yet disassemble their myofibrils. Immunofluorescence microscopy reveals that these non- beating heart cells lack vinculin-positive focal adhesions; moreover, intercellular junctions are also devoid of vinculin. When these quiescent myocytes are stimulated to contract with the beta-adrenergic agonist, isoproterenol, extensive vinculin-positive focal adhesions and intercellular junctions emerge. If solitary myocytes are stimulated to beat, an elaborate series of vinculin-positive focal adhesions develop which appear to parallel the reassembly of myofibrils. In cultures where neighboring myocytes reestablish cell-cell contact, myofibrils appear to reassemble from the fascia adherens rather than focal contacts. Activation of beating is accompanied by a significant reduction in the rate of total and cytoskeletal protein synthesis; in fact, myofibrillar reassembly, redevelopment of focal adhesions and fascia adherens junctions require no protein synthesis for at least 24 h, implying the existence of an assembly competent pool of cytoskeletal proteins. Maturation of the fasciae adherens and the appearance of vinculin within Z-line/costameres, does require de novo synthesis of new cytoskeletal proteins. Changes in cytoskeletal protein turnover appear dependent on beta agonist-induced cAMP production, but myofibrillar reassembly is a cAMP-independent event. Such observations suggest that mechanical forces, in the guise of contractile activity, regulate vinculin distribution and myofibrillar order in cultured adult feline heart cells.     

Integrin on developing and adult skeletal muscle

Avian integrin is a complex of integral membrane glycoproteins that appears to function as a dual receptors for both intracellular cytoskeletal and extracellular matrix components. Antibodies were raised against this complex and used to (1) immunolocalize integrin on cryosections of developing and adult muscle tissue and on developing myotube cultures in vitro and (2) immunoaffinity purify integrin from various fiber-type specific muscles. Integrin localization was compared with that of its putative cytoskeletal-associated and extracellular matrix ligands, talin and vinculin and fibronectin and laminin, respectively. The goal was to identify putative sites of interaction between the muscle sarcolemma and the cytoskeleton and the extracellular matrix and to reveal any differences in the molecular composition at these sites. Integrin's distribution on the sarcolemma of early (Day 12) embryonic limb muscle was random and punctate. On late embryonic (Days 17-19) limb muscle tissue its distribution was generally uniform but with occasional increased densities at specific sites along the sarcolemma. Posthatch (greater than 3 weeks) fast twitch muscle showed a highly regionalized distribution. These regions of integrin concentration coincided with densities of acetylcholine receptors, revealed by TRITC alpha-bungarotoxin labeling, and regions of muscle-tendon interaction, identified by morphological criteria. Tissue culture studies also demonstrated integrin densities at analogous sites in vitro, e.g., acetylcholine receptor clusters and sites at which myofibrils terminate at the sarcolemma. These integrin-rich sites were also shown to be Triton X-100 insoluble and therefore presumably are linked to the cytoskeleton or extracellular matrix. The localization of integrin on developing and adult muscle tissue was compared with that of fibronectin, laminin, vinculin, and talin using double, immunofluorescently labeled cryosections. In general, integrin did not colocalize exclusively with any one of its putative ligands. In the embryo, discrete densities of both talin and vinculin were observed at the myotendinous junction, whereas integrin immunoreactivity was widely distributed on muscle, vasculature, nerve, and connective tissue with no discernible sites of increased density. Laminin was primarily associated with muscle and nerve whereas fibronectin was prominent on connective tissue. On posthatch tissue, the distributions of talin, vinculin, laminin, and fibronectin were similar to those in the embryo, whereas the distribution of integrin was restricted to specific sites. The distribution of integrin was also examined for fiber-type specific differences on adu     

Extensive but coordinated reorganization of the membrane skeleton in myofibers of dystrophic (mdx) mice

We used immunofluorescence techniques and confocal imaging to study the organization of the membrane skeleton of skeletal muscle fibers of mdx mice, which lack dystrophin. beta-Spectrin is normally found at the sarcolemma in costameres, a rectilinear array of longitudinal strands and elements overlying Z and M lines. However, in the skeletal muscle of mdx mice, beta-spectrin tends to be absent from the sarcolemma over M lines and the longitudinal strands may be disrupted or missing. Other proteins of the membrane and associated cytoskeleton, including syntrophin, beta-dystroglycan, vinculin, and Na,K-ATPase are also concentrated in costameres, in control myofibers, and mdx muscle. They also distribute into the same altered sarcolemmal arrays that contain beta-spectrin. Utrophin, which is expressed in mdx muscle, also codistributes with beta-spectrin at the mutant sarcolemma. By contrast, the distribution of structural and intracellular membrane proteins, including alpha-actinin, the Ca-ATPase and dihydropyridine receptors, is not affected, even at sites close to the sarcolemma. Our results suggest that in myofibers of the mdx mouse, the membrane- associated cytoskeleton, but not the nearby myoplasm, undergoes widespread coordinated changes in organization. These changes may contribute to the fragility of the sarcolemma of dystrophic muscle.     

The involvement of adherens junction components in myofibrillogenesis in cultured cardiac myocytes.

The distribution of adherens junction (AJ) components was investigated in cultured heart myocytes. These cells, derived from either newborn rats or chick embryos, develop elaborate arrays of myofibrils which become extensive and laterally aligned following several days in culture. The Z-disks in these cells, visualized by immunolabeling with antibodies to muscle-specific alpha-actinin, exhibit a characteristic periodicity of about 2 microns and are in register with those of neighboring myofibrils throughout the sarcoplasm. Vinculin, in these cells, associates with intercellular AJ and cell-matrix adhesions. In addition, this protein is detected in periodic bands located along the lateral cell membranes corresponding to "costamers" previously described by Pardo, J.V., Siliciano, J.D. and Craig, S.W. (Proc. Natn. Acad. Sci. USA, 80, 1008). Similarly, N-cadherin, which is predominantly associated with intercellular junctions, is also detected in periodic striations located mainly on the dorsal and lateral cell surfaces. Using computer-aided three-dimensional microscopy confirmed that these vinculin- and N-cadherin-containing structures are located in extrajunctional sites, apparently associated with Z-disks of peripheral myofibrils. Based on these findings an alternative pathway is proposed for the assembly of vinculin and N-cadherin, which is not triggered by adhesive interactions with extracellular surfaces but rather by interactions at the membrane-cytoplasm interphase with the periphery of the pre-assembled myofibrils. Moreover, we present evidence that antibodies to N-cadherin, which are capable of blocking AJ formation in culture, have an inhibitory effect also on the development and alignment of myofibrils. We discuss the functional significance of the "costameric" organization of vinculin and N-cadherin and consider its involvement both in the lateral alignment of neighboring muscle cells and in the stabilization of developing myofibrils.     

Vinculin in relation to stress fibers in spread platelets.

To investigate the function of vinculin in blood platelets, we studied its localization in relation to other cytoskeletal proteins as well as its state of phosphorylation in platelets allowed to spread on fibrinogen-coated surfaces. By 5 minutes after loading the platelets onto the surfaces the 47 and 20 kDa polypeptides became phosphorylated, indicating activation. By 30 minutes, platelets formed small, typical bundles of fibers which stained brilliantly with rhodamine phalloidin. Myosin and tropomyosin, detected with specific antibodies, were localized in periodic arrays along these bundles. By indirect immunofluorescence, a discrete patch of vinculin was observed at each end of every actin-containing bundle. Vinculin phosphorylation was not detected in immunoprecipitates protected against phosphatases. Interference reflection images showed that regions of close binding to the substratum (adhesion plaques) closely matched the vinculin staining sites. Talin appeared diffusely localized. It could be shown to be present in the plaques when platelets were stabilized with ZnCl2 by the method of Geiger and then sonicated to remove some of the surface membrane. Localizations of vinculin and myosin were unaltered by this treatment. Talin phosphorylation or proteolysis could not account for vinculin translocation. We conclude that platelets, in response to an appropriate physiological surface, form typical actin bundles with vinculin at the termination of each bundle, in close relation to adhesion plaques. The signal for this translocation does not appear to depend on phosphorylation of vinculin or on phosphorylation or proteolysis of talin. Our findings support the conclusion that in platelets, as in nucleated cells, vinculin serves as at least part of the connection between bundled actin fibers and the extracellular matrix. Such a connection seems required for platelets' known ability to exert tension on surfaces.     

Irregular costameres represent nitric oxide synthase-1-positive sarcolemma invaginations enriched in contracted skeletal muscle fibres

NADPH diaphorase histochemistry and NOS-1 immunohistochemistry on 60 m thick frozen sections of rat extensor digitorum longus muscles led to the detection of prominent rings clearly encompassing the surface of the muscle fibres. These so far unknown costameres were usually found as doublets flanking a space of about 2 m width. Because these costameric doublets did not appear in regular periods, we designate them irregular costameres to discriminate them from regular ones with a 1 m periodicity overlying Z-discs and M-lines. Irregular costameres were thicker than the regular ones and free of intercostameres. Immunohistochemistry demonstrated that NOS-1 was co-localized with integral -dystroglycan, -sarcoglycan) and peripheral (caveolin-3, dystrophin) members of the enlarged dystrophin complex in the irregular costameres but not with non-sarcolemmal organized proteins (myosin heavy chain, -actinin, desmin and sarcoplasmic reticulum-located Ca²⁺-dependent ATPase-1). Invaginations of the sarcolemma to form irregular costameres were observed. In teased myofibres the sarcolemma between two following irregular costameres was ballooned, while the irregular costameres themselves clamped the fibres together. Finally, the number of detectable irregular costameres was significantly increased in maximally contracted extensor digitorum longus muscles generated by electric stimulation but decreased in mechanically stretched ones. Combining these observations, we hypothesize that irregular costameres belong to a reserve zone for the sarcolemma necessary for the contraction/relaxation cycle in myofibres.     

The cysteine-rich and C-terminal domains of dystrophin are not required for normal costameric localization in the mouse

Dystrophin has a modular structure and is believed to be critical for muscle cell cytoarchitecture by linking the cytoskeleton to the extracellular matrix. The N-terminus binds to actin and two domains at the C-terminus, the cysteine-rich and C-terminal domains, are associated with the sarcolemma indirectly via the dystroglycan complex. We have generated a mutation in mouse embryonic stem (ES) cells which serves to delete the cysteine-rich and C-terminal domains to address directly their role. We show that these two domains are not necessary for normal costameric organization at the sarcolemma in myotubes derived from the mutant cell line. Furthermore sarcolemmal localization is also apparent in mouse chimaeric musclein vivo.     

Interactions of intermediate filament protein synemin with dystrophin and utrophin

Synemin is a unique, very large intermediate filament (IF) protein present in all types of muscle cells, which forms heteropolymeric intermediate filaments (IFs) with the major IF proteins desmin and/or vimentin. We show herein that tissue-purified avian synemin directly interacts with both dystrophin and utrophin, and that specific expressed regions of both of the mammalian (human) synemin isoforms (alpha-synemin and beta-synemin) directly interact with specific expressed domains/regions of the dystrophin and utrophin molecules. Mammalian synemin is also shown to colocalize with dystrophin within muscle cell cultures. These results indicate that synemin is an important IF protein in muscle cells that helps fortify the linkage between the peripheral layer of cellular myofibrils and the costameric regions located along the sarcolemma and the sarcolemma region located within the neuromuscular and myotendinous junctions (NMJs and MTJs).