Variations in dystrophin complex in red and white caudal muscles from Torpedo marmorata.

We present an up-to-date study on the nature, at the protein level, of various members of the dystrophin complex at the muscle cell membrane by comparing red and white caudal muscles from Torpedo marmorata. Our investigations involved immunodetection approaches and Western blotting analysis. We determined the presence or absence of different molecules belonging to the dystrophin family complex by analyzing their localization and molecular weight. Specific antibodies directed against dystrophin, i.e., DRP2 alpha-dystrobrevin, beta-dystroglycan, alpha-syntrophin, alpha-, beta-, gamma-, and delta-sarcoglycan, and sarcospan, were used. The immunofluorescence study (confocal microscopy) showed differences in positive immunoreactions at the sarcolemmal membrane in these slow-type and fast-type skeletal muscle fibers. Protein extracts from T. marmorata red and white muscles were analyzed by Western blotting and confirmed the presence of dystrophin and associated proteins at the expected molecular weights. Differences were confirmed by comparative immunoprecipitation analysis of enriched membrane preparations with anti-beta-dystroglycan polyclonal antibody. These experiments revealed clear complex or non-complex formation between members of the dystrophin system, depending on the muscle type analyzed. Differences in the potential function of these various dystrophin complexes in fast or slow muscle fibers are discussed in relation to previous data obtained in corresponding mammalian tissues. (J Histochem Cytochem 49:857-865, 2001)     

Properties of chicken cardiac dystrophin.

We investigated the presence of dystrophin by immunoblot and immunofluorescence analyses, negative staining, rotatory shadowing and immunogold electron microscopy in chicken cardiac muscle. Saponin was found to be better than Triton X-100 for providing a new 'dystrophin-enriched' solution for use in biochemical studies of the molecule. By Western blot analysis, only a 400-kDa band was revealed with polyclonal antibodies directed against a central region (residues 1178-1723) of the dystrophin molecule and no cross-reactions with other proteins or degraded products were observed. Specific cleavage of the dystrophin molecule showed that the central rod-shaped domain corresponded to a resistant 'core'. This structure might rigidify the protein. By immunofluorescence, dystrophin was localized at the periphery of cardiac ventricular cells. The molecule was examined by electron microscopy and found to have variable lengths (140-160 nm for the monomeric from and about 260 +/- 10 nm or more for oligomeric forms). These oligomeric structures are considered to be associated molecules which are only partially overlapped lengthwise. The precise distribution of dystrophin within the cardiac muscle was determined by visualisation of gold particles in immuno-electron microscopy. Gold particles were found on the sarcolemma with no evidence of any association with cytoplasmic structures. The present data provide further details on the cardiac dystrophin molecule and suggest that its capacity of self-association may elasticize the dystrophin dimer.     

The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle

We use a highly specific and sensitive antibody to further characterize the distribution of dystrophin in skeletal, cardiac, and smooth muscle. No evidence for localization other than at the cell surface is apparent in skeletal muscle and no 427-kD dystrophin labeling was detected in sciatic nerve. An elevated concentration of dystrophin appears at the myotendinous junction and the neuromuscular junction, labeling in the latter being more intense specifically in the troughs of the synaptic folds. In cardiac muscle the distribution of dystrophin is limited to the surface plasma membrane but is notably absent from the membrane that overlays adherens junctions of the intercalated disks. In smooth muscle, the plasma membrane labeling is considerably less abundant than in cardiac or skeletal muscle and is found in areas of membrane underlain by membranous vesicles. As in cardiac muscle, smooth muscle dystrophin seems to be excluded from membrane above densities that mark adherens junctions. Dystrophin appears as a doublet on Western blots of skeletal and cardiac muscle, and as a single band of lower abundance in smooth muscle that corresponds most closely in molecular weight to the upper band of the striated muscle doublet. The lower band of the doublet in striated muscle appears to lack a portion of the carboxyl terminus and may represent a dystrophin isoform. Isoform differences and the presence of dystrophin on different specialized membrane surfaces imply multiple functional roles for the dystrophin protein.     

Complementary distributions of vinculin and dystrophin define two distinct sarcolemma domains in smooth muscle

The sarcolemma of the smooth muscle cell displays two alternating structural domains in the electron microscope: densely-staining plaques that correspond to the adherens junctions and intervening uncoated regions which are rich in membrane invaginations, or caveolae. The adherens junctions serve as membrane anchorage sites for the actin cytoskeleton and are typically marked by antibodies to vinculin. We show here by immunofluorescence and immunoelectron microscopy that dystrophin is specifically localized in the caveolae-rich domains of the smooth muscle sarcolemma, together with the caveolae-associated molecule caveolin. Additional labeling experiments revealed that beta 1 integrin and fibronectin are confined to the adherens junctions, as indicated by their codistribution with vinculin and tensin. Laminin, on the other hand, is distributed around the entire cell perimeter. The sarcolemma of the smooth muscle cell is thus divided into two distinct domains, featuring different and mutually exclusive components. This simple bipartite domain organization contrasts with the more complex organization of the skeletal muscle sarcolemma: smooth muscle thus offers itself as a useful system for localizing, among other components, potential interacting partners of dystrophin.     

Prenatal diagnosis of Duchenne muscular dystrophy by fetal muscle biopsy

Summary Prenatal diagnosis and carrier detection for Duchenne muscular dystrophy (DMD) usually can be performed using DNA analysis. When recombination occurs within the DMD gene, or DNA analysis is uninformative, or in pedigrees where it is unclear whether or not the consultand is a carrier, direct examination of muscle by dystrophin analysis may provide the only means of prenatal diagnosis. We present three cases representing each of these molecular genetic diagnostic dilemmas. In each instance, we used sonographically guided fetal muscle biopsy for dystrophin protein analysis to resolve the dilemma. In the first and third cases, the presence of normal dystrophin was shown by immunofluorescence and this was followed by delivery of an unaffected male fetus. In the second case, dystrophin was not found in fetal muscle tissue implying that this fetus was affected. The absence of dystrophin and affected status was confirmed in skeletal and cardiac muscle obtained after pregnancy termination.     

Mechanical function of dystrophin in muscle cells

We have directly measured the contribution of dystrophin to the cortical stiffness of living muscle cells and have demonstrated that lack of dystrophin causes a substantial reduction in stiffness. The inferred molecular structure of dystrophin, its preferential localization underlying the cell surface, and the apparent fragility of muscle cells which lack this protein suggest that dystrophin stabilizes the sarcolemma and protects the myofiber from disruption during contraction. Lacking dystrophin, the muscle cells of persons with Duchenne muscular dystrophy (DMD) are abnormally vulnerable. These facts suggest that muscle cells with dystrophin should be stiffer than similar cells which lack this protein. We have tested this hypothesis by measuring the local stiffness of the membrane skeleton of myotubes cultured from mdx mice and normal controls. Like humans with DMD mdx mice lack dystrophin due to an x-linked mutation and provide a good model for the human disease. Deformability was measured as the resistance to indentation of a small area of the cell surface (to a depth of 1 micron) by a glass probe 1 micron in radius. The stiffness of the membrane skeleton was evaluated as the increment of force (mdyne) per micron of indentation. Normal myotubes with an average stiffness value of 1.23 +/- 0.04 (SE) mdyne/micron were about fourfold stiffer than myotubes cultured from mdx mice (0.34 +/- 0.014 mdyne/micron). We verified by immunofluorescence that both normal and mdx myotubes, which were at a similar developmental stage, expressed sarcomeric myosin, and that dystrophin was detected, diffusely distributed, only in normal, not in mdx myotubes. These results confirm that dystrophin and its associated proteins can reinforce the myotube membrane skeleton by increasing its stiffness and that dystrophin function and, therefore, the efficiency of therapeutic restoration of dystrophin can be assayed through its mechanical effects on muscle cells.     

Proteolytic susceptibility of the central domain in chicken gizzard and skeletal muscle dystrophins.

We investigated proteolytic susceptibility of the central domain in dystrophin molecules from chicken smooth and skeletal muscles. Dystrophin-enriched preparations from both muscles were made as described in Pons et al. (Proc. Natl. Acad. Sci. USA (1990) 87, 7851-7855). These preparations contained other protein components in addition to dystrophin. Three enzymes (Staphylococcus aureus proteinase, chymotrypsin and trypsin) having different proteolytic specificities were used. Time-courses of proteinase degradation were examined by the Western immunoblot technique using a specific polyclonal serum directed against a fragment (residues 1173-1728) of the dystrophin central domain. We observed accumulation of some major proteinase-resistant fragments, in the 110-160 kDa range originating from that central region of the molecule. Cleavage patterns of the smooth and skeletal muscle preparations were quite similar, but molecular weights of the breakdown products differed slightly. Interpretation of the results was based on two predictive structural models of the dystrophin central domain (Koenig and Kunkel (1990) J. Biol. Chem. 265, 4560-4566 and Cross et al. (1990) FEBS Lett. 262, 87-90). Skip residues at the end of repeat 13 (around the 1740th residue of the dystrophin amino acid sequence), as hypothesized in the Cross model, constitute probably the most sensitive site within the dystrophin central domain for any exogenous (or even endogenous) proteinase. Variations observed between dystrophins from skeletal and smooth muscles also suggest that the structures of both dystrophins differ slightly even within the dystrophin central domain. This precise identification of proteinase-resistant dystrophin fragments of variable lengths is a first step towards further physicochemical studies on the very large and rare dystrophin molecule.     

Dystrophin predominantly localizes to the transverse tubule/Z-line regions of single ventricular myocytes and exhibits distinct associations with the membrane

Dystrophin is a high molecular weight protein present at low abundance in skeletal, cardiac and smooth muscle and in trace amounts in brain. In skeletal muscle, dystrophin is uniformly distributed along the inner surface of the plasma membrane. Biochemical fractionation studies have shown that all detectable skeletal muscle dystrophin is tightly associated with a complex of wheat germ agglutinin (WGA)-binding and concanavalin A (Con A) binding sarcolemmal glycoproteins. Absence of dystrophin is the primary biochemical defect in patients with Duchenne muscular dystrophy and leads to segmental necrosis of their skeletal myofibers. Although present in similar amounts in normal cardiac and skeletal muscle, the absence of dystrophin from cardiac muscle has less severe effects on the survival of cardiac cells. We have therefore examined whether there are differences in the properties of cardiac and skeletal dystrophin. We report that in contrast to skeletal muscle, cardiac dystrophin is distributed between distinct pools: a soluble cytoplasmic pool, a membrane-bound pool not associated with WGA-binding glycoproteins and a membrane-bound pool associated with WGA-binding glycoproteins. Cardiac dystrophin was not associated with any Con A binding glycoproteins. Immunohistochemical localization studies in isolated ventricular myocytes reveal a distinct punctate staining pattern for dystrophin, approximating to the level of the transverse tubule/Z-line and contrasting with the uniform sarcolemmal staining reported for skeletal muscle fibers. The distinct properties of cardiac dystrophin suggest unique roles for this protein in cardiac versus skeletal muscle function.Abbreviations Dys Dystrophin - T-tubule Transverse tubule - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - WGA Wheat Germ Agglutinin - Con A Concanavalin A - DHP Dihydropyridine receptor - FITC Fluorescein Isothiocyanate Conjugate - NAG N-Acetyl-D-Glucosamine - NP-40 NONIDET P-40 - PBS Phosphate-Buffered Saline - TBST Tris Buffered Saline-Tween     

Description of a utrophin associated protein complex in lipid raft domains of human artery smooth muscle cells

The dystrophin-associated protein complex (DAPC) is a multimeric complex that links the extracellular matrix to the actin cytoskeleton, and in some cases dystrophin can be substituted by its autosomal homologue utrophin to form the utrophin-associated protein complex (UAPC). Both complexes maintain the stability of plasma membrane during contraction process and play an important role in transmembrane signaling. Mutations in members of the DAPC are associated with muscular dystrophy and dilated cardiomyopathy. In a previous study with human umbilical cord vessels, we observed that utrophin colocalize with caveolin-1 (Cav-1) which proposed the presence of UAPC in the plasma membrane of vascular smooth muscle (VSM). In the current study, we demonstrated by immunofluorescence analysis, co-immunoprecipitation assays, and subcellular fractionation by sucrose gradients, the existence of an UAPC in lipid raft domains of human umbilical artery smooth muscle cells (HUASMC). This complex is constituted by utrophin, β-DG, ε-SG, α-smooth muscle actin, Cav-1, endothelial nitric oxide synthase (eNOS) and cavin-1. It was also observed the presence of dystrophin, utrophin Dp71, β-SG, δ-SG, δ-SG3 and sarcospan in non-lipid raft fractions. Furthermore, the knockdown of α/β-DG was associated with the decrease in both the synthesis of nitric oxide (NO) and the presence of the phosphorylated (active) form of eNOS; and with a reduction in the downstream activation of some cGMP signaling transduction pathway components. Together these results show the presence of an UAPC complex in HUASMC that may participate in the activity regulation of eNOS and in the vascular function.     

Dystrophin and dystrophin-associated protein in muscles and nerves from monkey

Since all organs (i.e. skeletal, cardiac, smooth muscles and sciatic nerve) are never only taken from a single patient, all these tissues were obtained from one cynomolgus monkey, a model closely resembling humans. This work describes an up-to-date reinvestigation of the dystrophin-glycoprotein complex and related molecules in various monkey tissues such those cited above. We used monoclonal and polyclonal antibodies produced in our laboratory, which are directed against dystrophin, utrophin, short-dystrophin products, alpha-dystrobrevin, beta-dystroglycan, alpha-syntrophin, alpha-, beta-, gamma-, delta-, epsilon-sarcoglycan, and sarcospan. For each molecule, we determined their molecular weight and tissue localization. Regardless of the tissue analyzed, at least one dystrophin or utrophin as full-length molecule and one short-dystrophin product or dystrobrevin as proteins belonging to the dystrophin superfamily were found. Beta-dystroglycan, beta and delta sarcoglycans were always detected, while other sarcoglycans varied from all to only three components. Epsilon sarcoglycan appears to be specific to smooth muscle, which is devoid of alpha sarcoglycan. Sarcospan is only absent from sciatic nerve structures. Among the different muscles investigated in this study, short dystrophin products are only present in cardiac muscle. All of these findings are summarized in one table, which highlight in one single animal the variability of the dystrophin-glycoprotein complex components in relation with the organ studied. This statement is important because any attempt to estimate protein restoration needs in each study the knowledge of the expected components that should be considered normal.     

Immunolocalization of caveolin-1 and caveolin-3 in monkey skeletal,cardiac and uterine smooth muscles

Caveolin, a 20-24 kDa integral membrane protein, is a principal component of caveolar domains. Caveolin-1 is expressed predominantly in endothelial cells, fibroblasts, and adipocytes, while the expression of caveolin-3 is confined to muscle cells. However, their localization in various muscles has not been well documented. Using double-immunofluorescence labeling and confocal laser microscopy, we examined the localization of caveolins-1 and 3 in adult monkey skeletal, cardiac and uterine smooth muscles and the co-immunolocalization of these caveolins with dystrophin, which is a product of the Duchenne muscular dystrophy gene. In the skeletal muscle tissue, caveolin-3 was localized along the sarcolemma except for the transverse tubules, and co-immunolocalized with dystrophin, whereas caveolin-1 was absent except in the blood vessels of the muscle tissue. In cardiac muscle cells, caveolins-1 and -3 and dystrophin were co-immunolocalized on the sarcolemma and transverse tubules. In uterine smooth muscle cells, caveolin-1, but not caveolin-3, was co-immunolocalized with dystrophin on the sarcolemma.     

Presence of invertebrate dystrophin-like products in obliquely striated muscle of the leech, Pontobdella muricata (Annelida, Hirudinea)

Dystrophin is a 427-kDa cytoskeletal protein, which occurs in scant amounts in vertebrate muscle and nerve cells. No previous references to dystrophin or associated proteins in invertebrates at the protein level have been found, while two recent studies investigated the presence of genes encoding proteins homologous to dystrophin in sea urchin and other invertebrates such as Drosophila melanogaster. In this study, the possible presence and distribution of dystrophin-like proteins were studied in different invertebrate muscle cell types and species through Western blot analysis and light and electron microscope immunohistochemistry using a panel of antibodies whose specificities have been determined in vertebrates. Crude protein extracts of leech Pontobdella muricata were analysed by Western blotting. The revealed protein band, with 140kDa molecular weight, was related to dystrophin, utrophin or dystrophin-related protein-2 (DRP2) according to the specificities of the antibodies used to detect them. The immunofluorescence study showed positive immunoreactions in obliquely striated muscle of this hyrudinean. The immunoelectron microscopy study confirmed specific immunogold labelling beneath the sarcolemma of muscle cells. We thus assume that this protein is an invertebrate dystrophin-like product that is referred to as IDLp140. The potential functions of this invertebrate dystrophin-like protein in invertebrate muscles are discussed relative to previous data in vertebrate tissues.     

A Sensitive,Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber) were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2–17% and intra-assay precision, CV 2–10%). Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound.     

Dystrophin (Xp21), a New Phenotype Marker of Cultured Rat Aortic Myocytes

Dystropbin is a low-abundance cytoskeletal protein involved in the maintenance of membrane integrity in striated muscle. Very little is known about its role in smooth muscle. Utrophin (a dystropbin-related protein) is an ubiquitous protein whose role is still unclear. Changes in the expression of both proteins (if any) during phenotypic modulation of smooth muscle have not yet been reported. In contrast, modulated expression of heavy-molecular-weight caldesmon (h-CaD), a well-known specific regulatory protein of the contractile apparatus in smooth muscle, is well documented, along with its nonmuscle isoform, low-molecular-weight caldesmon (l-CaD), and other cytoskeletal proteins. We investigated three properties of cultured rat aortic smooth muscle cells: morphology, contractile ability, and expression of dystrophin, utrophin, h-CaD, and l-CaD. Cells were grown either in serum substitute supplemented medium (U-medium), where they reexpressed contractility, or in fetal calf serum-supplemented medium (F-medium), where they did not. It was found that only cultures grown in U-medium continued expressing dystrophin, even during the proliferation phase, contrary to cells grown in F-medium. However, when F-medium was changed for U-medium the cells recovered their contractility and reexpressed dystrophin. Expression of utrophin, h-CaD, and l-CaD was similar in both culture types. Dystrophin was demonstrated to be a true phenotype marker of cultured rat aortic smooth muscle cells, particularly with respect to their actual contractility.     

Monoclonal antibody evidence for structural similarities between the central rod regions of actinin and dystrophin.

A monoclonal antibody, MANDYS141, binds to both dystrophin and actinin on Western blots (SDS-denatured), but only to actinin in frozen sections of human muscle (native conformation). It differs from a polyclonal cross-reacting antiserum in that it binds to several muscle isoforms of actinin (smooth, fast and slow) from man, mouse and chicken and recognises a quite different part of the proposed triple-helical region of dystrophin (amino acids 1750-2248). The results suggest that structural homologies between actinin and dystrophin occur more than once in their central helical regions and provide experimental support for an actinin-like central rod model for dystrophin.     

Increased neointimal thickening in dystrophin-deficient mdx mice

Background

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), encodes a large cytoskeletal protein present in muscle fibers. While dystrophin in skeletal muscle has been extensively studied, the function of dystrophin in vascular smooth muscle is less clear. Here, we have analyzed the role of dystrophin in injury-induced arterial neointima formation.

Methodology/Principal Findings

We detected a down-regulation of dystrophin, dystroglycan and β-sarcoglycan mRNA expression when vascular smooth muscle cells de-differentiate in vitro. To further mimic development of intimal lesions, we performed a collar-induced injury of the carotid artery in the mdx mouse, a model for DMD. As compared with control mice, mdx mice develop larger lesions with increased numbers of proliferating cells. In vitro experiments demonstrate increased migration of vascular smooth muscle cells from mdx mice whereas the rate of proliferation was similar in cells isolated from wild-type and mdx mice.

Conclusions/Significance

These results show that dystrophin deficiency stimulates neointima formation and suggest that expression of dystrophin in vascular smooth muscle cells may protect the artery wall against injury-induced intimal thickening.     

Patterns of dystrophin expression in developing, adult and regenerating tail skeletal muscle of Amphibian urodeles.

The patterns of expression of dystrophin were investigated by indirect immunofluorescence and by immunoblotting in developing, adult and regenerating tail skeletal muscle of newts Pleurodeles waltl and Notophthalmus viridescens. In this study, a monoclonal antibody H-5A3 directed against the C-terminal region (residues 3357-3660) and a polyclonal antibody raised to the central domain (residues 1173-1738) of the chicken skeletal muscle dystrophin were used. Western blot analysis showed that these antibodies recognized a 400 kDa band of dystrophin (and may be of dystrophin-related protein) in the adult muscle tissues and in newt tail regenerates. During skeletal muscle differentiation or epimorphic regeneration (blastema), anti-dystrophin immunoreactivity gradually accumulated over the periphery of the myofibers. Dystrophin and laminin were first and concomitantly observed at the ends of the newly formed myotubes where they were anchored on connective tissue septa or bone processes by dystrophin-rich myotendinous structures. It is noteworthy that neuromuscular junctions, which most probably also contain dystrophin, are established in urodeles near the ends of the myofibers as shown by histochemical localization of AChE activity or fluorescent bungarotoxin detection of AChRs. In the stump transition zone close to the tail amputation level where tissue regeneration of injured muscle fibers took place, dystrophin staining located on the cytoplasmic surface of myofibers progressively disappeared during the dedifferentiation process which seemed to occur during muscle regeneration as suggested by electron microscopy. Furthermore, double labeling experiments using anti-dystrophin and anti-laminin antibodies showed a good correlation between the remodeling processes of the muscle fiber basal lamina and the loss of dystrophin along the sarcolemma of damaged and presumably dedifferentiating muscle cells.     

Dystrophin is tightly associated with the sarcolemma of mammalian skeletal muscle fibers

Sarcolemmal vesicles with right-side-out configuration were prepared from normal fresh human and rabbit skeletal muscle bundles by incubation in 140 mM KCl solution containing collagenase. The vesicles were used to examine the association of dystrophin, the protein product of the Duchenne muscular dystrophy gene, with the sarcolemma. Western blot analysis, indirect immunofluorescence, and immunoperoxidase staining using specific antibodies raised against the N-terminal and the C-terminal domains show that dystrophin remains associated with the membrane of sarcolemmal vesicles. Indirect immunofluorescence microscopy using permeabilized and unpermeabilized vesicles indicated that both the N-terminus and the C-terminus of dystrophin are localized to the cytoplasmic surface of the sarcolemma. These results suggest that dystrophin has much stronger attachment to the surface membrane than it has to the internal domain of skeletal muscle fibers. Sarcolemmal vesicles thus represent a new system for studying the function of dystrophin and the molecular basis of its association with the sarcolemma.     

High-resolution en-face visualization of the cardiomyocyte plasma membrane reveals distinctive distributions of spectrin and dystrophin

The actin-binding proteins, spectrin and dystrophin, are key components of the plasma membrane-associated cytoskeleton of the cardiac muscle cell. From confocal immunofluorescence studies, the distribution of spectrin appears to overlap with that of dystrophin, but the precise functional differentiation, molecular distributions and spatial relationship of these two cytoskeletal systems remain unclear. Freeze-fracture replica immuno-electron microscopy, in parallel with immunofluorescence/confocal microscopy, were applied to examine at high resolution the spatial relationships between the spectrin and dystrophin membrane-associated cytoskeleton systems in cardiac muscle. Application of freeze-fracture replica cytochemistry, with single and double immunogold labeling, permitted simultaneous examination of the organization of spectrin and dystrophin in en-face views of the plasma membrane at high resolution. In contrast to the close spatial relationship previously demonstrated for dystrophin and β-dystroglycan, no association between the gold label marking dystrophin and that marking spectrin was observed. Our freeze-fracture cytochemical results suggest that the two membrane skeletal networks formed by dystrophin and spectrin in cardiac muscle are independently organized, implying that whatever overlap of function (e.g., in structural support to the plasma membrane) may exist between them, the two systems may each have additional distinctive roles.     

Dystrophin colocalizes with beta-spectrin in distinct subsarcolemmal domains in mammalian skeletal muscle

Duchenne's muscular dystrophy (DMD) is caused by the absence or drastic decrease of the structural protein, dystrophin, and is characterized by sarcolemmal lesions in skeletal muscle due to the stress of contraction. Dystrophin has been localized to the sarcolemma, but its organization there is not known. We report immunofluorescence studies which show that dystrophin is concentrated, along with the major muscle isoform of beta-spectrin, in three distinct domains at the sarcolemma: in elements overlying both I bands and M lines, and in occasional strands running along the longitudinal axis of the myofiber. Vinculin, which has previously been found at the sarcolemma overlying the I bands and in longitudinal strands, was present in the same three structures as spectrin and dystrophin. Controls demonstrated that the labeling was intracellular. Comparison to labeling of the lipid bilayer and of the extracellular matrix showed that the labeling for spectrin and dystrophin is associated with the intact sarcolemma and is not a result of processing artifacts. Dystrophin is not required for this lattice-like organization, as similar domains containing spectrin but not dystrophin are present in muscle from the mdx mouse and from humans with Duchenne's muscular dystrophy. We discuss the possibility that dystrophin and spectrin, along with vinculin, may function to link the contractile apparatus to the sarcolemma of normal skeletal muscle.