Abstract
Dystrophin is localized, in normal muscle fibers, on the cytoplasmic surface of the sarcolemma. The function of this protein is not known but, according to its structure and intracellular distribution, it seems likely that dystrophin interacts with other cytoskeletal proteins to form a complex linkage between myofibrils, sarcolemma and extracellular matrix. To evaluate the possibility that dystrophin deficiency induces, per se, a disarray in the cytoskeleton, we studied three components of this structure in muscle fibers of the dystrophic mdx mouse in a phase preceding the onset of necrosis. Vinculin, abundant in sarcolemmal structures called costameres, desmin, the principal component of intermediate filaments and nebulin, constituent of the socalled “third filament” within the sarcomere, were stained with the indirect immunofluorescence technique in cryostat sections. The same monoclonal antibodies were used in Western blots of proteins extracted from the same muscles. No difference was observed in the distribution or in the relative abundance of the three proteins, comparing muscles from 18 days-old mdx and control mice. Our results indicate that the lack of dystrophin does not induce, per se, alterations in the structures linking the sarcolemma to the contractile apparatus. It is likely that the structural damage in dystrophin-less muscle fibers is initially confined to limited portions of the plasma membrane. These focal lesions, impairing intracellular calcium homeostasis, can lead to muscle fiber necrosis.
Similar content being viewed by others
References
Alloisio N, Morle L, Bachir D, Guetarni D, Colonna P, Delaunay J (1985) Red cell sialoglycoprotein β in homozygous and heterozygous 4.1(-) hereditary elliptocytosis. Biochim Biophys Acta 816: 57–62
Arahata K, Ishiura S, Ishiguro T, Tsukahara T, Suhara Y, Eguchi C, Ishihara T, Nonaka I, Ozawa E, Sugita H (1983) Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide. Nature 333: 861–866
Bakker AJ, Head SI, Williams DA, Stephenson DG (1993) Ca2+ levels in myotubes grown from the skeletal muscle of dystrophic (mdx) and normal mice. J Physiol (Lond) 460: 1–13
Belkin AM, Oraatsky OI, Glukhova MA, Koteliansky VE (1988) Immunolocalization of meta-vinculin in human smooth and cardiac muscles. J Cell Biol 107: 545–553
Bonilla E, Miranda AF, Prelle A, Salviati G, Betto R, Zeviani M, Schon EA, DiMauro S, Rowland LP (1988) Immunocytochemical study of nebulin in Duchenne muscular dystrophy. Neurology 38: 1600–1603
Bulfield G, Siller WG, Wight PAL, Moore KJ (1984) X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci USA 81: 1189–1192
Campbell KP, Kahl SD (1989) Association of dystrophin and an integral membrane glycoprotein. Nature 338: 259–262
Carpenter S, Karpati G, Zubrzycka-Gaarn EE, Bulman DE, Ray PN, Worton RG (1990) Dystrophin is localized to the plasma membrane of human skeletal muscle fibers by electron-microscopic cytochemical study. Muscle Nerve 13: 376–380
Cullen MJ, Jaros E (1988) Ultrastructure of the skeletal muscle in the X chromosome-linked dystrophic (mdx) mouse. Acta Neuropathol 77: 69–81
Cullen MJ, Fulthorpe JJ, Harris JB (1992) The distribution of desmin and titin in normal and dystrophic human muscle. Acta Neuropathol 83: 158–169
Dickson G, Azad A, Morris GE, Simon H, Noursadeghi M, Walsh FS (1992) Colocalization and molecular association of dystrophin with laminin at the surface of mouse and human myotubes. J Cell Sci 103: 1223–1233
Dunn JF, Bannister N, Kemp GJ, Publicover SJ (1993) Sodium is elevated in mdx muscles: ionic interactions in dystrophic cells. J Neurol Sci 114: 76–80
Ervasti JM, Campbell KP (1991) Membrane organization of the dystrophin-glycoprotein complex. Cell 66: 1121–1131
Ervasti JM, Campbell KP (1993) A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J Cell Biol 122: 809–823
Franco A, Lansman JB (1990) Calcium entry through stretchinactivated ion channels in mdx myotubes. Nature 344: 670–673
Fürst D, Nave R, Osborn M, Weber K, Bardosi A, Arcidiacono N, Ferro M, Romano V, Romeo G (1987) Nebulin and titin expression in Duchenne muscular dystrophy appears normal. FEBS Lett 224: 49–53
Glukhova MA, Kabakov AE, Belkin AM, Frid MG, Ornatsky OI, Zhidkova NI, Koteliansky VE (1986) Metavinculin distribution in adult human tissues and cultured cells. FEBS Lett 207: 139–141
Granger BL, Lazarides E (1979) Desmin and vimentin coexist at the periphery of the myofibril Z disc. Cell 18: 1053–1063
Hoffman EP, Brown RH Jr, Kunkel LM (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51: 919–928
Hoffman EP, Fischbeck KH, Brown RH, Johnson M, Medori R, Loike JD, Harris JB, Waterston R, Brooke M, Specht L, Kupsky W, Chamberlain J, Caskey CT, Shapiro F, Kunkel LM (1988) Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy. N Engl J Med 318: 1363–1368
Hutter OF, Burton FL, Bovell DL (1991) Mechanical properties of normal and mdx mouse sarcolemma: bearing on function of dystrophin. J Muscle Res Cell Motil 12: 585–589
Ibraghimov-Beskrovnaya O, Ervasti JM, Leveille CJ, Slaughter CA, Sernett SW, Campbell KP (1992) Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature 355: 696–702
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685
Law DJ, Tidball JG (1993) Dystrophin deficiency is associated with myotendinous junction defects in prenecrotic and fully regenerated skeletal muscle. am J Pathol 142: 1513–1523
Massa R, Karpati G, Weller B, Carpenter S, Shoubridge E (1991) Quantitative studies of dystrophin-positive fibers in mdx muscles. Neurology 41 [Suppl 1]: 136
Masuda T, Fujimaki N, Ozawa E, Ishikawa H (1992) Confocal laser microscopy of dystrophin localization in guinea pig skeletal muscle fibers. J Cell Biol 119: 543–548
Matsumura K, Shimizu T, Nonaka I, Mannen T (1989) Immunochemical study of connectin (titin) in neuromuscular diseases using a monoclonal antibody: connectin is degraded extensively in Duchenne muscular dystrophy. J Neurol Sci 93: 147–156
Minetti C, Tanji K, Bonilla E (1992) Immunologic study of vinculin in Duchenne muscular dystrophy. Neurology 42: 1751–1754
Moggio M, Fagiolari G, Prelle A, Gallanti A, Sciacco M, Scarlato G (1992) Lack of anionic phospholipid calcium binding sites in Duchenne muscular dystrophy. Muscle Nerve 15: 325–331
Ohlendieck K, Campbell KP (1991) Dystrophin-associated proteins are greatly reduced in skeletal muscle from mdx mice. J Cell Biol 115: 1685–1694
Ohlendieck K, Ervasti JM, Snook JB, Campbell KP (1991) Dystrophin-glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma. J Cell Biol 112: 125–148
Ohlendieck K, Matsumura K, Ionasescu V, Towbin JA, Bosch EP, Weinstein SL, Sernett SW, Campbell KP (1993) Duchenne muscular dystrophy: deficiency of dystrophin-associated proteins in the sarcolemma. Neurology 43: 795–800
Pardo JV, D'Angelo Siliciano J, Craig SW (1983) A vinculin-containing cortical lattice in skeletal muscle: transverse lattice elements “costameres” mark sites of attachment between myofibrils and sarcolemma. Proc Natl Acad Sci USA 80: 1008–1012
Pearson J, Sabarra A (1974) A method for obtaining longitudinal cryostat sections of living muscle without contraction artifacts. Stain Technol 49: 143–146
Petrof BJ, Shrager JB, Stedman HH, Kelly AM, Sweeney HL (1993) Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc Natl Acad Sci USA 90: 3710–3714
Porter GA, Dmytrenko GM, Winkelmann JC, Bloch RJ (1992) Dystrophin colocalizes with β-spectrin in distinct subsarcolemmal domains in mammalian skeletal muscle. J Cell Biol 117: 997–1005
Reddy PA, Anandavalli TE, Anandaraj MPJS (1986) Calcium activated neutral proteases (milli- and micro-CANP) and endogenous CANP inhibitor of muscle in Duchenne muscular dystrophy. Clin Chim Acta 160: 281–288
Salviati G, Betto R, Ceoldo S, Biasia E, Bonilla E, Miranda AF, DiMauro S (1989) Cell fractionation studies indicate that dystrophin is a protein of surface membranes of skeletal muscle. Biochem J 258: 837–841
Shear CR, Bloch RJ (1985) Vinculin in subsarcolemmal densities in chicken skeletal muscle: localization and relationship to intracellular and extracellular structures. J Cell Biol 101: 240–256
Sicinski P, Geng Y, Ryder-Cook AS, Barnard EA, Darlison MG, Barnard PJ (1989) The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science 244: 1578–1580
Small JV, Fürst DO, Thornell LE (1992) The cytoskeletal lattice of muscle cells. Eur J Biochem 208: 559–572
Spencer MJ, Tidball JG (1992) Calpain concentration is elevated although net calcium-dependent proteolysis is suppressed in dystrophin-deficient muscle. Exp Cell Res 203: 107–114
Stedman HH, Sweeney HL, Shrager JB, Maguire HC, Panettieri RA, Petrof B, Narusawa M, Leferovich JM, Sladky JT, Kelly AM (1991) The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature 352: 536–539
Straub V, Bittner RE, Léger JJ, Voit T (1992) Direct visualization of the dystrophin network on skeletal muscle fiber membrane. J Cell Biol 119: 1183–1191
Terracio L, Simpson DG, Hilenski L, Carver W, Decker RS, Vinson N, Borg TK (1990) Distribution of vinculin in the Z-disk of striated muscle: analysis by laser scanning confocal microscopy. J Cell Physiol 145: 78–87
Tokuyasu KT, Dutton AH, Singer SJ (1983) Immunoelectron microscopic studies of desmin (skeletin) localization and intermediate filament organization in chicken skeletal muscle. J Cell Biol 96: 1727–1735
Torres LFB, Duchen LW (1987) The mutant mdx: inherited myopathy in the mouse. Brain 110: 269–299
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354
Turner PR, Westwood T, Regen CM, Steinhardt RA (1988) Increased protein degradation results from elevated free calcium levels found in muscle mdx mice. Nature 335: 735–738
Turner PR, Fong P, Denetclaw WF, Steinhardt RA (1991) Increased calcium influx in dystrophic muscle. J Cell Biol 115: 1701–1712
Voit T, Patel K, Dunn MJ, Dubowitz V, Strong PN (1989) Distribution of dystrophin, nebulin and Ricinus communis I (RCA I)-binding glycoprotein in tissues of normal and mdx mice. J Neurol. Sci 89: 199–211
Wakayama Y, Shibuya S (1991) Gold-labelled dystrophin molecule in muscle plasmalemma of mdx control mice as seen by electron microscopy of deep etching replica. Acta Neuropathol 82: 178–184
Wang K, Wright J (1988) Architecture of the sarcomere matrix of skeletal muscle: immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin filaments anchored at the Z line. J Cell Biol 107: 2199–2212
Weller B, Karpati G, Carpenter S (1990) Dystrophin-deficient mdx muscle fibers are preferentially vulnerable to necrosis induced by experimental lengthening contractions. J Neurol Sci 100: 9–13
Wood DS, Zeviani M, Prelle A, Bonilla E, Salviati G, Miranda AF, DiMauro S, Rowland LP (1987) Is nebulin the defective gene product in Duchenne muscular dystrophy? N Engl J Med 316: 107–108
Zubrzycka-Gaarn EE, Bulman DE, Karpati G, Burghes AHM, Belfall B, Klamut HJ, Talbot J, Hodges RS, Ray PN, Worton RG (1988) The Duchenne muscular dystrophy gene product is localized in sarcolemma of human skeletal muscle. Nature 333: 466–469
Author information
Authors and Affiliations
Additional information
Supported by grants from Telethon, Italy to R. Massa and to L. Castellani. G. Silvestri is the recipient of a Telethon post-graduate fellowship
Rights and permissions
About this article
Cite this article
Massa, R., Castellani, L., Silvestri, G. et al. Dystrophin is not essential for the integrity of the cytoskeleton. Acta Neuropathol 87, 377–384 (1994). https://doi.org/10.1007/BF00313607
Received:
Revised:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00313607