We have recently found that the erythroid ankyrin gene, Ank1, expresses isoforms in mouse skeletal muscle, several of which share COOH-terminal sequence with previously known Ank1 isoforms but have a novel, highly hydrophobic 72–amino acid segment at their NH2 termini. Here, through the use of domainspecific peptide antibodies, we report the presence of the small ankyrins in rat and rabbit skeletal muscle and demonstrate their selective association with the sarcoplasmic reticulum. In frozen sections of rat skeletal muscle, antibodies to the spectrin-binding domain (anti-p65) react only with a 210-kD Ank1 and label the sarcolemma and nuclei, while antibodies to the COOH terminus of the small ankyrin (anti-p6) react with peptides of 20 to 26 kD on immunoblots and decorate the myoplasm in a reticular pattern. Mice homozygous for the normoblastosis mutation (gene symbol nb) are deficient in the 210-kD ankyrin but contain normal levels of the small ankyrins in the myoplasm. In nb/nb skeletal muscle, anti-p65 label is absent from the sarcolemma, whereas anti-p6 label shows the same distribution as in control skeletal muscle. In normal skeletal muscle of the rat, anti-p6 decorates Z lines, as defined by antidesmin distribution, and is also present at M lines where it surrounds the thick myosin filaments. Immunoblots of the proteins isolated with rabbit sarcoplasmic reticulum indicate that the small ankyrins are highly enriched in this fraction. When expressed in transfected HEK 293 cells, the small ankyrins are distributed in a reticular pattern resembling the ER if the NH2-terminal hydrophobic domain is present, but they are uniformly distributed in the cytosol if this domain is absent. These results suggest that the small ankyrins are integral membrane proteins of the sarcoplasmic reticulum. We propose that, unlike the 210-kD form of Ank1, previously localized to the sarcolemma and believed to be a part of the supporting cytoskeleton, the small Ank1 isoforms may stabilize the sarcoplasmic reticulum by linking it to the contractile apparatus.

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 ␤-spectrin. In tibialis anterior muscle from mice lacking desmin due to homologous recombination, most costameres are lost. In myofibers from desmin Ϫ/Ϫ 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 Ϫ/Ϫ muscles express similar levels of cytokeratins. Cytokeratins concentrate at the sarcolemma at all three domains of costameres when the latter are retained in desmin Ϫ/Ϫ muscle and redistribute with ␤-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. INTRODUCTIONDuchenne Muscular Dystrophy and related muscular dystrophies are caused by the mutation or loss of dystrophin and dystrophin-associated proteins (Campbell, 1995;Bonnemann et al., 1996;Straub and Campbell, 1997;Ozawa et al., 1998), respectively, but the functions of these proteins in healthy skeletal muscle are still poorly understood. Dystrophin, which is a member of the spectrin superfamily of membrane skeletal proteins (Davison and Critchley, 1988;Koenig et al., 1988;Dhermy, 1991;Ahn and Kunkel, 1993), accumulates in healthy muscle on the cytoplasmic face of the sarcolemma in linear structures that are oriented both longitudinally and transversely (Masuda et al., 1992;Minetti et al., 1992;Porter et al., 1992;Straub et al., 1992;Williams and Bloch, 1999b). The transverse structures, which lie at the sarcolemma over the Z and M lines of nearby myofibrils, are organized in a rib-like pattern and so are referred to as "costameres" (Pardo et al., 1983a). We also use this term to include the longitudinal elements, which, with the transverse domains, form a lattice-like network that underlies most of the skeletal muscle sarcolemma. All three costameric domains are enriched in dystrophin (Porter et al., 1992). We have found that, in the absence of dystrophin, the longitudinal and M line domains of costameres are more susceptible to disruption in Duchenne muscle and in muscle from the mdx mouse (Porter et al., 1992;Williams and Bloch, 1999b; see also Ehmer et al., 1997), suggesting that dystrophin functions more to stabilize these sarcolemmal domains than the domains that overlie Z lines. These studies also suggest that other structures associated with the sarco...

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. β-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, β-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, β-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 β-spectrin. Utrophin, which is expressed in mdx muscle, also codistributes with β-spectrin at the mutant sarcolemma. By contrast, the distribution of structural and intracellular membrane proteins, including α-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.

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There was an error published in J. Cell Sci. 114, 751-762.In Fig. 1, the authors mistakenly included several panels taken from quadriceps muscle that had been used in a previous publication, rather than original images of the extensor digitorum longus. The corrected figure is shown below.

We used double label immunofluorescence and confocal microscopy to examine the organization of beta-spectrin and dystrophin at the sarcolemma of fast twitch myofibers in the Extensor Digitorum Longus (EDL) of the rat. Both beta-spectrin and dystrophin are concentrated in costameres, a rectilinear sarcolemmal array composed of longitudinal strands and transverse elements overlying Z and M lines. In contrast, intercostameric regions, lying between these linear structures, contain significant levels of dystrophin but little detectable beta-spectrin. The dystrophin-associated proteins, syntrophin and beta-dystroglycan, are also concentrated at costameres but, like dystrophin, are present in intercostameric regions as well. Dystrophin is present at costameres and intercostameric regions in fast twitch muscles of the mouse but is absent from all regions of the sarcolemma in the mdx mouse, which lacks dystrophin. Areas of the sarcolemma near myonuclei also contain dystrophin without beta-spectrin, consistent with the idea that the distribution of dystrophin at the sarcolemma is not dependent on beta-spectrin. We conclude that dystrophin is present under all areas of the sarcolemma. The increased fragility of the sarcolemma in patients with Duchennes muscular dystrophy may be explained in part by the absence of dystrophin not only from costameres, but also from intercostameric regions.

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