MICROSCOPY RESEARCH AND TECHNIQUE 48:131–141 (2000) Plasma Membrane Cytoskeleton of Muscle: A Fine Structural Analysis SIMON C. WATKINS,1* MICHAEL J. CULLEN,2 ERIC P. HOFFMAN,3 AND LYNN BILLINGTON1 1Department of Cell Biology and Physiology, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania 15261 of Neuroscience, University of Newcastle upon Tyne, Newcastle upon Tyne, Tyne and Weir, England NE1 7RU 3Department of Human Genetics, National Children’s Medical Center, Washington, DC 20010 2Department KEY WORDS dystrophin; electron microscopy; light microscopy; dystrophin cytoskeleton ABSTRACT The discovery of dystrophin and its definition as the causative molecule in Duchenne Muscular Dystrophy has led to a renewed interest in the molecular structure of the muscle fiber plasma membrane and its association with the extracellular basal lamina. The original identification of dystrophin gave credence to the possibility that the plasma membrane of the muscle fiber may be highly organized and involved in maintaining appropriate homeostasis in this actively contracting cellular system. In this review, we examine the currently known members of the muscle fiber plasma membrane cytoskeleton and the interactions that occur between the different members of this complex using histological, electron microscopic, and confocal methods. From our studies and others cited in this review, it is clear that the dystrophin cytoskeletal complex is not completely understood and component molecules continue to be discovered. Perhaps equally importantly, currently defined molecules (such as alpha-actinin or neuronal nitric oxide synthase) are being recognized as being specifically associated with the complex. What is striking from all of the studies, to date, is that while we are able to identify members of the dystrophin cytoskeletal complex and while we are able to associate mutations of individual molecules with disease(s), we are still unable to truly define the roles of each of the molecules in maintaining the normal physiology of the muscle fiber. Microsc. Res. Tech. 48:131–141, 2000. r 2000 Wiley-Liss, Inc. INTRODUCTION Since the discovery of dystrophin in 1987 and the elucidation of its fundamental role in the pathogenesis of Duchenne muscular dystrophy (DMD) (Hoffman et al., 1987), public interest and clinical research have focused largely on attempts to correct the effects of mutations in the dystrophin gene with drug therapy (e.g., Barton-Davis et al., 1999; DeSilva et al., 1987; Haye and Williams, 1994, 1998; Sansome et al., 1993), with surgical intervention (e.g., Bach and McKeon, 1991; Forst and Forst, 1995, 1999; Smith et al., 1993) and with molecular and cellular therapeutics (e.g., Floyd et al, 1998; Howell et al., 1998; Karpati, 1989; Karpati and Petrof, 1998; Law et al., 1993, 1994; Miller et al., 1987; Tremblay et al., 1993). However, the plasmalemmal cytoskeleton of the skeletal muscle fiber is now recognized to be an extremely complex multimolecular network, and mutations of these so-called dystrophin-associated proteins will give rise to diverse and commonly debilitating myopathies. The first discovery of a dystrophin associated protein was by Campbell and Kahl (1989) who reported the association of dystrophin with an integral membrane glycoprotein soon after the discovery of dystrophin. Since this time, there has been a continuous series of discoveries that demonstrate the complexity of the dystrophin cytoskeleton. In addition to dystrophin, there are at least fourteen proteins that link the cytoplasmic myofibrillar contractile elements to the signal transducing molecules of the extracellular matrix and provide structural support to the sarcolemma (Fig. 1). These proteins include an extracellular r 2000 WILEY-LISS, INC. protein (␣-dystroglycan), seven integral membrane proteins (␤-dystroglycan, sarcospan, and ␣-, ␤-, ␦, ␥-, and ⑀-sarcoglycan, four cytoplasmic proteins (␣-actinin-2 and syntrophin ␣1, ␤1, and ␤2) and two isoforms of a subsarcolemmal protein (␣-dystrobrevin-1, -2). It is certain that together with dystrophin these proteins form a critical membrane cytoskeleton at the muscle fiber plasma membrane, functionally responsible for the structural and possibly signal transduction pathway between the contractile elements of a muscle fiber and its extracellular matrix (Ervasti and Campbell, 1993). Description of the physical inter-relationship(s) between these proteins and with dystrophin, and the possible role(s) of these proteins in the DMD and other myopathies is the focus of this review. DYSTROPHIN-ASSOCIATED PROTEINS Dystroglycan Complex The dystroglycan complex (DGC) was initially purified from digitonin-solubilized rabbit skeletal muscle membranes (Ervasti et al., 1990). Two of the components, ␣- and ␤-dystroglycan (156 and 43 DAG, respectively) are post-translationally spliced products of a single gene and are termed dystroglycans because of their demonstrated, direct interaction with dystrophin (dystro-) and the heavily glycosylated (-glycan) nature of the ␣-form (Ibraghimova-Beskrovnaya et al., 1992). *Correspondence to: Simon C. Watkins Ph.D., BSTS 225, 3500 Terrace St., University of Pittsburgh, Pittsburgh PA 15261. E-mail: [email protected] Received 22 September 1999; accepted in revised form 10 October 1999 132 S.C. WATKINS ET AL. Fig. 1. Schematic representation of dystrophin and the dystrophin-associated proteins. Sequence analysis of rabbit skeletal muscle cDNAs reveals a reading frame of 2,685 bases encoding a precursor polypeptide of 895 amino acids with a predicted molecular weight of 97 kDa (IbraghimovBeskrovnaya et al., 1992). The N-terminal portion of the precursor polypeptide encodes the 56 kDa core protein of ␣-dystroglycan while the C-terminal portion is processed into the mature ␤-dystroglycan. Carbohydrate moieties constitute up to two thirds of the molecular mass of ␣-dystroglycan. Beta-dystroglycan has a large extracellular domain with four potential N-glycosylation sites, a transmembrane domain, and a 120 amino acid cytoplasmic tail. Expression of the dystroglycans is ubiquitous. An early report showed ␤-dystroglycan to be present in brain and lung (Ibraghimova-Beskrovnaya et al., 1992) More recently, a comprehensive immunoblot analysis of monkey tissues showed the molecule to be present in every tissue examined including lung, liver, uterus, aorta, cerebrum, cerebellum, spinal cord, and peripheral nerve (Mizuno et al., 1994). Alpha-dystroglycan has also been found in brain tissue (Gee et al., 1993; Montanaro et al., 1995; Tian et al., 1996) and peripheral nerve fibers (Yamada et al., 1994). Importantly, in these locales, the proteins are generally functioning as laminin receptors. Studies of dystrophin binding properties using protein binding assays show that its linkage to the integral membrane components of the DGC is via ␤-dystroglycan (Suzuki et al., 1994). The dystroglycan binding site on dystrophin is located in a widely extended region encompassing the cysteine-rich domain and the first half of the C-terminal domain. ␤-dystroglycan also binds with utrophin or dystrophin related protein, a homolog of dystrophin via an SH3 domain-binding region in its C-terminus (Jung et al., 1995). Further in vitro localization shows that dystrophin from skeletal muscle and brain binds to the cytoplasmic domain of ␤-dystrogly- can (Jung et al., 1995) and that the binding site is on the second half of hinge 4 and the cysteine-rich domain (amino-acids 3054–3271) of dystrophin. The dystrophin binding site is in the proline-rich section at the extreme end of the C-terminus of ␤-dystroglycan (amino acids 880–895). Alpha-dystroglycan is a heavily glycosylated, mucinlike protein that links the transmembrane ␤-dystroglycan to the extracellular matrix protein, laminin (Smalheiser and Kim, 1995). Laminin, a major component of the basal lamina, binds to ␣-dystroglycan via the G repeats of its ␣2 chain (Talts et al., 1999). Alphadystroglycan is a receptor for agrin (Bowe et al., 1994; Gee et al., 1994; Peng et al., 1998, 1999; Sugiyama et al., 1994), and, thus, may contribute to the clustering of acetylcholine receptors at the neuromuscular junction (Peng et al., 1998). Mycobacterium leprae, the bacterium responsible for leprosy, complexed with laminin utilizes ␣-dystroglycan as a target for entry into peripheral nerve Schwann cells (Rambukkana et al., 1998). The morphological relationships of the dystroglycans and the proteins that they bind to have, until recently, been based almost entirely on (very thorough) biochemical information. Immunofluorescence studies have shown that the dystroglycans, like dystrophin, are localized at the sarcolemma (Durbeej et al., 1998; James et al., 1996; Kawajiri et al., 1996, 1998; Peng et al., 1998), but for precise information on how they are positioned relative to dystrophin, ultrastructural immunolabeling is required. Immunogold labeling of ␣-dystroglycan shows that the gold conjugate is localized to the outer face of the sarcolemma, usually on structures projecting from the surface (Fig. 2a) and when ␣-dystroglycan and dystrophin are double labeled on the same section, dystrophin is found on the proximal side of the membrane frequently opposite dystroglycan labeling sites (Fig. 2a). MICROSCOPY OF THE DYSTROPHIN CYTOSKELETON 133 Fig. 2. a. Immunogold labeling of ␣-dystroglycan (10nm particles). Alpha-dystroglycan localizes to the outer surface of the sarcolemma usually on structures projecting from the surface. b. Double immuno- gold labeling of ␣-dystroglycan (15nm particles) and dystrophin. Dystrophin (10nm particles) localizes to the inner surface of the sarcolemma. (Scale bar ⫽ 100nm). Immunogold labeling of ␤-dystroglycan shows that the gold conjugate lies on the membrane or extremely close to it, on either side of the membrane (Fig. 3a) (Cullen et al., 1998). The primary antibody that was used was raised against 15 of the last 16 amino acids at the C-terminus of ␤-dystroglycan, i.e., at the cytoplasmic end of the molecule. The observation that the gold conjugate is sometimes positioned on the extracellular side of the membrane can probably be attributed to the fact that the immunogold probe can lie up to the length of two antibody molecules distant, i.e., ⬃20 nm, from the epitope that it is labeling. Because the antibody was to the C-terminus and because the C-terminus is involved in binding to dystrophin, it could be predicted that double labeling of ␤-dystroglycan and dystrophin would show close colocalization. Double labeling experiments using antibodies to the C-termini of both molecules show that this is indeed the case (Fig. 3b) and 134 S.C. WATKINS ET AL. Fig. 3. a. Immunogold labeling of ␤-dystroglycan. The gold conjugate lies on or very near to both sides of the sarcolemma. b. Double immunogold labeling of the C-termini of ␤-dystroglycan (10nm particles) and dystrophin (5nm particles). (Scale bar ⫽ 100nm). careful measurement of the distance and orientation of one gold probe with respect to the other shows that the two epitopes must be separated by ⬍15 nm (Cullen et al., 1998). While much new information can be gained by performing immunolabeling on cryosections, there is a restriction in what the technique can achieve because one is viewing a membrane in what is essentially just one linear dimension. If the investigator wishes to view the arrangement of a protein at a membrane face on, in two dimensions, a different technique needs to be employed. The technique of choice is freeze-fracture in which membranes are split down the center of the phospholipid bilayer to expose membrane-associated proteins. There are histological artifacts associated with combining this technique with immunolabeling because of the thawing required for the antibody incubation stages (Abeysekera and Robards, 1995) but preliminary studies examining the relationship between ␤-dystroglycan and dystrophin are encouraging. Figure 4a shows a platinum-carbon replica of an extracellular face of a muscle plasma membrane that was immunogold labeled for ␤-dystroglycan. The 10-nm gold particles are distributed over the membrane in a pattern that can be shown statistically to be nonrandom. When the nearest-neighbor distances between labeling sites from a number of such electronmicrographs are measured, the resulting histogram of repeating ␤-dystroglycan molecules has a clear mode at approximately 140 nm, i.e., at value close to that obtained from cryosections (Fig. 4b). Figure 4c. shows a fracture face that has been double labeled for ␤-dystro- MICROSCOPY OF THE DYSTROPHIN CYTOSKELETON 135 glycan (10 nm gold) and dystrophin C-terminus (5 nm gold). As was shown on the cryosections, these two epitopes colocalize extremely closely. Interestingly, when this procedure was repeated using an antibody to the N-terminus of dystrophin, the colocalization is not seen (S. Stevenson, personal communication). It is instructive to examine to what extent the localization studies carried out are in accord with the models derived from biochemical studies. Examination of a number of the published models shows that ␣-dystroglycan lies in an extracellular position binding to laminin in the extracellular matrix (Matsumura and Campbell, 1994; Ohlendieck, 1996; Ozawa et al., 1995; Winder et al., 1995). The ultrastructural results are entirely in accord with this, both when ␣-dystroglycan is labeled by itself or with another protein. The immunogold labeling of ␤-dystroglycan indicates that it is a transmembrane protein and, moreover, the close proximity to the C-terminus of dystrophin when double labeling is carried out, is in agreement with dystrophin binding at the end of the C-terminus of ␤-dystroglycan. The resolution of freeze fracture experiments is high, but labeling sensitivity is still relatively poor and limits any major conclusions of direct molecular interactions. However, the distribution of nearest-neighbor distances suggest that the dystroglycans, along with other components of the DGC, may be separated by dystrophin molecules. Analogous with spectrin, dystrophin is usually depicted as an anti-parallel dimer with apposing N- and C-termini in close proximity to each other. However, it has been alternatively suggested, on the basis of amino acid sequence analysis, that there is little evidence for dimerization (Chan and Kunkel, 1997; Rybakova and Ervasti, 1997; Winder et al., 1995, 1996) and thus the N-terminus may be positioned at some distance from the C-terminus. The doublelabeling experiments in which the N-terminus of dystrophin and the C-terminus of ␤-dystroglycan were not seen to colocalize, lends support to this interpretation. While the absence of dystrophin is central to the pathogenesis of DMD, the exact effect of this loss on the functional physiology of the muscle cell is still not understood. As the members of the DGC are tightly interlinked in an interdependent fashion, it is possible that the pathological sequelae of mutations in the dystrophin gene may have as much to do with the cellular functions of the associated proteins as with dystrophin itself. It is highly likely the dystroglycans are essential to normal homeostasis (particularly given their ubiquitous distribution) and that any mutations causing complete dystroglycan loss are lethal (Jung et al., 1995). Two studies support this assertion. In one, myotubes derived from cells transfected with an antisense dystroglycan construct show a preferential loss of ␣-dystroglycan and undergo apoptosis in vitro (Mon- Fig. 4. a. Platinum-carbon replica of an extracellular face of a sarcolemma immunogold labeled for ␤-dystroglycan. (Scale bar ⫽ 100nm). b. Nearest-neighbor distances between labeling sites of ␤-dystroglycan have a mode of approximately 140nm. c. Freezefracture immunolabeling of dystrophin (5nm particles with open arrows) and ␤-dystroglycan (10nm particles and closed arrows). (Scale bar ⫽ 100nm). 136 S.C. WATKINS ET AL. tanaro et al., 1999). In another, homozygous mouse embryos with a null allele of the dystroglycan gene exhibit disrupted patterns of laminin and collagen and undergo early death in embryonic development due to the disruption of the Reichert’s membrane (Williamson et al., 1997). Other than a single report that mutations in ␤-dystroglycan cause a limb-girdle muscular dystrophy (LGMD) as demonstrated by immunostaining and immunoblot analysis (Salih et al., 1996), there have been no other demonstrated causative associations between mutations in the dystroglycans and non-lethal myopathies. Sarcoglycan Complex The sarcoglycan complex is comprised of ␣-sarcoglycan (previously termed adhalin and 50 DAG), ␤-sarcoglycan (A3b), ␦-sarcoglycan, and ⑀-sarcoglycan. Alphasarcoglycan and ␥-sarcoglycan have been found to be present only in skeletal and cardiac muscle by immunoblotting (Yamamoto et al., 1994) whereas ␤-, ␦- and ⑀-sarcoglycan are ubiquitously distributed (Ettinger et al., 1997; McNally et al., 1998). The sarcoglycan complex is considered to be associated with the dystrophin axis via a lateral association with the dystroglycan complex, but the exact binding sites between the sarcoglycans and dystroglycans have yet to be identified (Ozawa et al., 1995; Suzuki et al., 1994). The cDNA of ␣-sarcoglycan was cloned and shown to include a region coding for a transmembrane domain (McNally et al., 1994; Roberds et al., 1993) and ⑀-sarcoglycan shares a high structural homology to ␣-sarcoglycan (McNally et al., 1998). Madhaven and Jarrett (1995) found that ␣-sarcoglycan was bound by the cysteine-rich sequences of dystrophin using overlay binding experiments to probe gel blots of purified rabbit muscle DGC. The first immunocytochemical reports of the localization of ␣- and ␥-sarcoglycan appeared quickly after their discovery (Ohlendieck and Campbell, 1991) and have been followed by a large number of papers incorporating immunofluorescence studies, many comparing diseased and normal muscle and some reporting developmental changes (e.g., Di Blasi et al., 1996; Matsumura et al., 1992a,b, 1993a,b; Noguchi et al., 1995; Vater et al., 1995; Yamamoto et al., 1994). Without exception, these papers show that the sarcoglycans are normally present at the sarcolemma of the myofibers. Using an antibody that recognizes an epitope in the extracellular portion of ␣-sarcoglycan, we carried out immunogold labeling on control and DMD biopsy samples and detected the gold probe close to the sarcolemma and mostly on its peripheral side (Cullen et al., 1994). A more recent study, using triple immunogold labeling shows that ␣-sarcoglycan localizes to the outer surface of the sarcolemma and that ␤- and ␦-sarcoglycan localize to the inner surface (Wakayama et al., 1999). A deficiency of ␣-sarcoglycan has been associated with severe childhood autosomal recessive muscular dystrophy (SCARMD), a disease whose symptoms and pathology are very similar to DMD (Ben Hamida et al., 1983). SCARMD was first described in North African (Ben Hamida et al., 1983) and Middle Eastern countries (Salih et al., 1993) but has since been described in European (Fardeau et al., 1993), American (Passos- Bueno et al., 1993a,b, 1999; Vainzof et al., 1999a), Asian (Hayashi et al., 1995; Higuchi et al., 1994, 1997) and Australian families (Jones et al., 1998). Since its first association with ␣-sarcoglycan deficiency (Matsumura et al., 1992a), it has become clear that SCARMD is not a single homogeneous genetic disease, but a group of muscular dystrophies with similar phenotypes caused by damage to several different genes (Carrie et al., 1997; Duclos et al., 1998, Holt and Campbell, 1998; Mizuno et al., 1995; Passos-Bueno et al., 1996; Romero et al., 1995). Linkage analysis reveals that some cases of sarcoglycanopathy (now termed limb girdle muscular dystrophy type 2C) are linked to chromosome 13q12 and display a deficiency of ␥-sarcoglycan; some SCARMD phenotypes are independent of any mechanism involving the primary gene defect (Ozawa et al., 1995). Human patients with mutations in the ␥-sarcoglycan present lower levels of dystrophin, ␣- and ␤-sarcoglycan, and disrupted laminin patterns, thus showing that abnormalities of dystrophin may, in some cases, be a secondary phenomenon (Jones et al., 1998; Vainzof et al., 1999b). Ultrastructural studies in a case of ␥-sarcoglycanopathy (LGMD2C) suggest that loss or reduction of the sarcoglycans results in an instability in the plasma membrane, which becomes abnormally indented and convoluted (Hassoni and Cullen, 1999). A murine model of ␥-sarcoglycan deficiency produces a dystrophic phenotype that does not display a secondary loss of dystrophin or of laminin and dystroglycan (Hack et al., 1998). The mice do show a deficiency of ␤- and ␦-sarcoglycan, thus providing additional evidence that muscular dystrophy can occur in the absence of alterations in dystrophin levels. A form of autosomal recessive muscular dystrophy in the BIO 14.6 hamster, another animal model of sarcoglycanopathy, has been reported to be associated with a selective loss of ␦-sarcoglycan and has now been shown to lack the entire sarcoglycan complex (Mizuno et al., 1995). The primary defect is due to a large deletion in the 5’ region of the gene (Sakamoto et al., 1997). Using intravenous injection of Evans blue dye as an in vivo tracer assay, Straub et al. (1998) found that extensive fiber damage takes place in the skeletal and cardiac muscle of the BIO 14.6 hamster due to the disruption of the sarcolemma. Delta-sarcoglycan gene transfer has recently been shown to restore the sarcoglycan complex and sarcolemmal integrity in hamsters (Crosbie et al., 1999; Greelish et al., 1999; Holt et al., 1998; Li et al., 1999). Loss of ␤-sarcoglycan or ␦-sarcoglycan results in the disruption of the sarcoglycan-sarcospan complex in knockout mice. Beta-sarcoglycan-deficiency leads to progressive muscular dystrophy with extensive degeneration and regeneration (Araishi et al., 1999). Deltasarcoglycan deficiency affects both skeletal and smooth muscle, the latter of which leads to the perturbation of vascular function and cardiomyopathy (Coral-Vazquez et al., 1999) Syntrophin Complex The syntrophins constitute a heterogeneous group of 58-kDa intracellular dystrophin-binding and membrane-associated proteins. A 58-kDa peripheral membrane protein was first identified in the Torpedo electric MICROSCOPY OF THE DYSTROPHIN CYTOSKELETON organ (Froehner et al., 1987) and this has since been placed in the syntrophin family. Syntrophin isolated from rabbit skeletal muscle appears as a triplet by one-dimensional SDS electrophoresis and, when separated by two-dimensional gel electrophoresis, appears as two clusters of 58-kDa proteins with different isoelectric points (pI). One, ␣-syntrophin, is slightly acidic (pI ⫽ 6.4) and the other, ␤-syntrophin, is quite basic (pI ⫽ 9.0) (Yamamoto et al., 1993). From predicted amino acid sequences and calculated isoelectric points, ␤-syntrophin has been shown to have two isoforms: ␤1 (p1 ⫽ 9.0) and ␤2 (pI ⫽ 9.4) (Ahn et al., 1996). Alpha1-syntrophin is located on the inner surface of the sarcolemma of skeletal and cardiac muscle and interacts with the carboxy-terminal region of dystrophin (Ahn et al., 1996; Castello et al., 1996; Suzuki et al., 1995; Yang et al., 1995;). Beta1- and ␤2-syntrophin are found in a variety of tissues including muscle (Ahn et al., 1996). As all three syntrophins bind to dystrophin in vitro, it is likely that their differential localization is regulated by interactions with other proteins. Little is known about the normal function of the syntrophins and their association with neuromuscular disease. The syntrophins are down-regulated in the absence of normal dystrophin and their absence may contribute to the dystrophic phenotype (Peters et al., 1994; Tachi et al., 1997). As shown by immunogoldlabeling electron microscopy, ␣1-syntrophin colocalizes with nitric-oxide synthase (nNOS) at the sarcolemma and may bind nNOS (Wakayama et al., 1997). Until recently, the absence of sarcolemmal nNOS in DMD patients and mdx mice was thought to contribute to myofiber necrosis (Brenman et al., 1995; Chang et al., 1996; Haycock et al., 1996). In nNOS-dystrophin null mdx mice, typical dystrophic pathogenesis takes place in the absence of nitric oxide synthesis (Crosbie et al., 1998). However, in ␣1-syntrophin knockout mice, nNOS is absent at the sarcolemma but the structural and functional properties of the muscles remain relatively normal (Kameya et al., 1999), thus making the role of the syntrophins even more obscure. Sarcospan Sarcospan, formerly known as A5 (Yoshida and Ozawa, 1990), is a 25-kDa multisubunit sarcolemmal complex. Northern blot analysis shows ubiquitous expression of a 4.5-kb transcript of sarcospan but a 6.5-kb transcript is found exclusively in skeletal and cardiac muscle. In skeletal muscle, sarcospan is enriched at neuromuscular junctions and myotendinous junctions (Crosbie et al., 1999). Topology algorithms predict that sarcospan has four transmembrane spanning helices with both N- and C-terminal domains located intracellularly, which render sarcospan structurally and functionally analogous to the tetraspan superfamily of proteins (Crosbie et al., 1997). Tetraspans are thought to facilitate control of cell growth, migration, and adhesion through interactions between transmembrane proteins (Laguaudriere-Gesbert et al, 1997; Maecker et al., 1997; Wice and Gordon, 1995; Wright and Tomlinson, 1994). Whether or not sarcospan provides the same functions is not known. Sarcospan precipitates with the dystrophin-glycoprotein complex in a wheat germ agluttinin-Sepharose ion exchange column (Crosbie et al, 1997) and is dependent 137 upon the presence of the sarcoglycan complex for its expression. In ␦-sarcoglycan deficient BIO14.6 hamsters and in ␣-sarcoglycan null mice, both the sarcoglycan complex and sarcospan are missing from the sarcolemma. Injection of adenovirus encoding ␦-sarcoglycan into BIO 14.6 muscles restores sarcospan as well as the sarcoglycan complex mentioned previously (Crosbie et al., 1999). The role of sarcospan in muscle disease has yet to be established. It is known that the presence of sarcospan is greatly reduced in DMD muscle compared with normal muscle (Crosbie et al., 1997). This reduction may contribute to the dystrophic phenotype in DMD. In addition, Crosbie et al. (1997) propose that mutations in the sarcospan gene cause congenital fibrosis of the extraocular muscles (CFEOM) as the gene encoding sarcospan maps to the same narrow region of chromosome 12 as the disease locus for CFEOM. Alpha-Dystrobrevin Alpha-dystrobrevin is a dystrophin homologue that coprecipitates with the both the acetylcholine receptor complex and the dystrophin glycoprotein complex (Blake et al., 1996; Sadoulet-Puccio et al., 1997). In human skeletal muscle, alternative splicing of the dystrobrevin locus on chromosome 18q12.1–12.2 (Ambrose et al., 1997; Sadoulet-Puccio et al., 1996) produces five isoforms of dystrobrevin, two of which are found in skeletal muscle: full-length ␣-dystrobrevin-1 (84 kDa) and a C-terminus truncated ␣-dystrobrevin-2 (65 kDa). During development, ␣-dystrobrevin-1 is found on the cell surface and acetylcholine-rich receptor areas of the myotube. Following agrin-induced acetylcholine receptor clustering, ␣-dystrobrevin-1 becomes relocalized beneath the sarcolemma and restricted to the synaptic region of the myofibers (Nawrotzki et al., 1998). Alphadystrobrevin-2 is concentrated at the synapse but is also present on the extrasynaptic sarcolemma in mature muscle (Peters et al., 1998). The association of ␣-dystrobrevin with the synaptic region suggests that it may have a role in synapse formation and/or stability (Sadoulet-Puccio et al., 1997). The amount of ␣-dystrobrevin is severely reduced in DMD patients and in patients with LGMD arising from loss of one or all of the sarcoglycans (Metzinger et al., 1997; Puca et al., 1998) as well as in mdx mice (Nawrotzki et al., 1998) and thus may contribute to the dystrophic phenotype. Other than this possibility, no disease has been associated with the loss or mutation of ␣-dystrobrevin. Alpha-Actinin-2 The most recently named component of the dystrophin-glycoprotein complex is ␣-actinin-2 (Hance et al., 1999). The C-terminal region of dystrophin interacts with skeletal muscle ␣-actinin-2 and actin in the yeast two-hybrid system. Alpha-actinin-2 colocalizes with the sarcolemma of tissue culture cells and purified sarcolemma vesicles. Alpha-actinin-2 links overlapping actin filaments every 38 nm at the vertebrate muscle Z-lines (Yamaguchi et al., 1985) Alpha-actinin-2 also binds to the ␤1 subunit of integrin (Otey et al., 1990) thus providing critical links between F-actin, dystrophin, and the transmembrane protein, integrin (Pavalko et 138 S.C. WATKINS ET AL. al., 1990). There are yet no known diseases directly associated with the mutations in the ␣-actinin-2 gene. Conclusions Over the last 12 years, our understanding of the muscle fiber plasma membrane cytoskeleton has grown enormously, from the first identification of the dystrophin molecule, to the recognition that the plasma membrane of an actively and constantly contracting cell is supported by an elaborate complex of cytoskeletal molecules. It is certain, given the selective and debilitating diseases that are associated with each of the molecules, that the functionality of each component of the membrane cytoskeleton is complex. However, it is somewhat surprising that while we have been able to identify many (though probably not all) of the components of the muscle fiber cytoskeleton, we have little idea of the true function of each member of the complex. Since Rowland’s first suggestion of the ‘‘membrane hypothesis’’ of DMD (1981) it was apparent that a failure of maintenance of integrity of the muscle fiber plasma membrane may be central to the pathology of DMD. The recognition that dystrophin was specifically associated with the plasma membrane, gave convenient support to the notion that its function was to protect the muscle fiber from damage during repeated cycles of contraction and relaxation. However, it is clear that the role(s) of the members of the plasma membrane cytoskeleton may go far beyond this simple function. Perhaps the most intriguing feature of mutations of the complex, which suggest functionalities far beyond simply protecting the cell membrane from damage, is the diversity of disease phenotypes caused by mutations of the complex. Selective mutations give rise to DMD, congenital muscular dystrophy, limb girdle muscular dystrophy, or oculo-pharangeal muscular dystrophy. Why should selective mutations cause diverse and selected dystrophies of different muscles? 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