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. 1991 Jun 1;113(5):1133–1144. doi: 10.1083/jcb.113.5.1133

Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle

PMCID: PMC2289019  PMID: 2040646

Abstract

Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between Torpedo dystrophin and the previously described 270/300-kD Torpedo postsynaptic protein. Dystrophin was found in both AChR-rich and AChR-poor regions of the innervated face of the electroplaque. Immunogold experiments showed that AChR and dystrophin were closely intermingled in the AChR domains. In contrast, dystrophin appeared to be absent from many or all AChR-rich domains of the rat neuromuscular junction and of AChR clusters in cultured muscle (Xenopus laevis). It was present, however, in the immediately surrounding membrane (deep regions of the junctional folds, membrane domains interdigitating with and surrounding AChR domains within clusters). These results suggest that dystrophin may have a role in organization of AChR in electric tissue. Dystrophin is not, however, an obligatory component of AChR domains in muscle and, at the neuromuscular junction, its roles may be more related to organization of the junctional folds.

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Selected References

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  1. Anderson M. J., Cohen M. W., Zorychta E. Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells. J Physiol. 1977 Jul;268(3):731–756. doi: 10.1113/jphysiol.1977.sp011879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson M. J., Fambrough D. M. Aggregates of acetylcholine receptors are associated with plaques of a basal lamina heparan sulfate proteoglycan on the surface of skeletal muscle fibers. J Cell Biol. 1983 Nov;97(5 Pt 1):1396–1411. doi: 10.1083/jcb.97.5.1396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Anderson M. J. Nerve-induced remodeling of muscle basal lamina during synaptogenesis. J Cell Biol. 1986 Mar;102(3):863–877. doi: 10.1083/jcb.102.3.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Appleyard S. T., Dunn M. J., Dubowitz V., Scott M. L., Pittman S. J., Shotton D. M. Monoclonal antibodies detect a spectrin-like protein in normal and dystrophic human skeletal muscle. Proc Natl Acad Sci U S A. 1984 Feb;81(3):776–780. doi: 10.1073/pnas.81.3.776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bloch R. J., Hall Z. W. Cytoskeletal components of the vertebrate neuromuscular junction: vinculin, alpha-actinin, and filamin. J Cell Biol. 1983 Jul;97(1):217–223. doi: 10.1083/jcb.97.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bloch R. J. Isolation of acetylcholine receptor clusters in substrate-associated material from cultured rat myotubes using saponin. J Cell Biol. 1984 Sep;99(3):984–993. doi: 10.1083/jcb.99.3.984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bloch R. J., Morrow J. S. An unusual beta-spectrin associated with clustered acetylcholine receptors. J Cell Biol. 1989 Feb;108(2):481–493. doi: 10.1083/jcb.108.2.481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bloch R. J., Pumplin D. W. Molecular events in synaptogenesis: nerve-muscle adhesion and postsynaptic differentiation. Am J Physiol. 1988 Mar;254(3 Pt 1):C345–C364. doi: 10.1152/ajpcell.1988.254.3.C345. [DOI] [PubMed] [Google Scholar]
  9. Bridgman P. C., Carr C., Pedersen S. E., Cohen J. B. Visualization of the cytoplasmic surface of Torpedo postsynaptic membranes by freeze-etch and immunoelectron microscopy. J Cell Biol. 1987 Oct;105(4):1829–1846. doi: 10.1083/jcb.105.4.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Buckle V. J., Guenet J. L., Simon-Chazottes D., Love D. R., Davies K. E. Localisation of a dystrophin-related autosomal gene to 6q24 in man, and to mouse chromosome 10 in the region of the dystrophia muscularis (dy) locus. Hum Genet. 1990 Aug;85(3):324–326. doi: 10.1007/BF00206755. [DOI] [PubMed] [Google Scholar]
  11. Byers T. J., Husain-Chishti A., Dubreuil R. R., Branton D., Goldstein L. S. Sequence similarity of the amino-terminal domain of Drosophila beta spectrin to alpha actinin and dystrophin. J Cell Biol. 1989 Oct;109(4 Pt 1):1633–1641. doi: 10.1083/jcb.109.4.1633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Carr C., Fischbach G. D., Cohen J. B. A novel 87,000-Mr protein associated with acetylcholine receptors in Torpedo electric organ and vertebrate skeletal muscle. J Cell Biol. 1989 Oct;109(4 Pt 1):1753–1764. doi: 10.1083/jcb.109.4.1753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chang H. W., Bock E., Bonilla E. Dystrophin in electric organ of Torpedo californica homologous to that in human muscle. J Biol Chem. 1989 Dec 15;264(35):20831–20834. [PubMed] [Google Scholar]
  14. Chelly J., Kaplan J. C., Maire P., Gautron S., Kahn A. Transcription of the dystrophin gene in human muscle and non-muscle tissue. Nature. 1988 Jun 30;333(6176):858–860. doi: 10.1038/333858a0. [DOI] [PubMed] [Google Scholar]
  15. Chen Q., Sealock R., Peng H. B. A protein homologous to the Torpedo postsynaptic 58K protein is present at the myotendinous junction. J Cell Biol. 1990 Jun;110(6):2061–2071. doi: 10.1083/jcb.110.6.2061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cullen M. J., Walsh J., Nicholson L. V., Harris J. B. Ultrastructural localization of dystrophin in human muscle by using gold immunolabelling. Proc R Soc Lond B Biol Sci. 1990 May 22;240(1297):197–210. doi: 10.1098/rspb.1990.0034. [DOI] [PubMed] [Google Scholar]
  17. Ervasti J. M., Ohlendieck K., Kahl S. D., Gaver M. G., Campbell K. P. Deficiency of a glycoprotein component of the dystrophin complex in dystrophic muscle. Nature. 1990 May 24;345(6273):315–319. doi: 10.1038/345315a0. [DOI] [PubMed] [Google Scholar]
  18. Fardeau M., Tomé F. M., Collin H., Augier N., Pons F., Léger J., Léger J. Présence d'une protéine de type dystrophine au niveau de la jonction neuromusculaire dans la dystrophie musculaire de Duchenne et la souris mutante "mdx". C R Acad Sci III. 1990;311(5):197–204. [PubMed] [Google Scholar]
  19. Fertuck H. C., Salpeter M. M. Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after 125I-alpha-bungarotoxin binding at mouse neuromuscular junctions. J Cell Biol. 1976 Apr;69(1):144–158. doi: 10.1083/jcb.69.1.144. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fong P. Y., Turner P. R., Denetclaw W. F., Steinhardt R. A. Increased activity of calcium leak channels in myotubes of Duchenne human and mdx mouse origin. Science. 1990 Nov 2;250(4981):673–676. doi: 10.1126/science.2173137. [DOI] [PubMed] [Google Scholar]
  21. Fox G. Q., Richardson G. P. The developmental morphology of Torpedo marmorata: electric organ--myogenic phase. J Comp Neurol. 1978 Jun 1;179(3):677–697. doi: 10.1002/cne.901790313. [DOI] [PubMed] [Google Scholar]
  22. Franco A., Jr, Lansman J. B. Calcium entry through stretch-inactivated ion channels in mdx myotubes. Nature. 1990 Apr 12;344(6267):670–673. doi: 10.1038/344670a0. [DOI] [PubMed] [Google Scholar]
  23. Froehner S. C., Douville K., Klink S., Culp W. J. Monoclonal antibodies to cytoplasmic domains of the acetylcholine receptor. J Biol Chem. 1983 Jun 10;258(11):7112–7120. [PubMed] [Google Scholar]
  24. Froehner S. C., Luetje C. W., Scotland P. B., Patrick J. The postsynaptic 43K protein clusters muscle nicotinic acetylcholine receptors in Xenopus oocytes. Neuron. 1990 Oct;5(4):403–410. doi: 10.1016/0896-6273(90)90079-u. [DOI] [PubMed] [Google Scholar]
  25. Froehner S. C., Murnane A. A., Tobler M., Peng H. B., Sealock R. A postsynaptic Mr 58,000 (58K) protein concentrated at acetylcholine receptor-rich sites in Torpedo electroplaques and skeletal muscle. J Cell Biol. 1987 Jun;104(6):1633–1646. doi: 10.1083/jcb.104.6.1633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Froehner S. C. Peripheral proteins of postsynaptic membranes from Torpedo electric organ identified with monoclonal antibodies. J Cell Biol. 1984 Jul;99(1 Pt 1):88–96. doi: 10.1083/jcb.99.1.88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Giloh H., Sedat J. W. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science. 1982 Sep 24;217(4566):1252–1255. doi: 10.1126/science.7112126. [DOI] [PubMed] [Google Scholar]
  28. Hammonds R. G., Jr Protein sequence of DMD gene is related to actin-binding domain of alpha-actinin. Cell. 1987 Oct 9;51(1):1–1. doi: 10.1016/0092-8674(87)90002-x. [DOI] [PubMed] [Google Scholar]
  29. Heuser J. E., Salpeter S. R. Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane. J Cell Biol. 1979 Jul;82(1):150–173. doi: 10.1083/jcb.82.1.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hirokawa N., Heuser J. E. Internal and external differentiations of the postsynaptic membrane at the neuromuscular junction. J Neurocytol. 1982 Jun;11(3):487–510. doi: 10.1007/BF01257990. [DOI] [PubMed] [Google Scholar]
  31. Hoffman E. P., Brown R. H., Jr, Kunkel L. M. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 1987 Dec 24;51(6):919–928. doi: 10.1016/0092-8674(87)90579-4. [DOI] [PubMed] [Google Scholar]
  32. Hoffman E. P., Kunkel L. M. Dystrophin abnormalities in Duchenne/Becker muscular dystrophy. Neuron. 1989 Jan;2(1):1019–1029. doi: 10.1016/0896-6273(89)90226-2. [DOI] [PubMed] [Google Scholar]
  33. Hoffman E. P., Watkins S. C., Slayter H. S., Kunkel L. M. Detection of a specific isoform of alpha-actinin with antisera directed against dystrophin. J Cell Biol. 1989 Feb;108(2):503–510. doi: 10.1083/jcb.108.2.503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Jasmin B. J., Cartaud A., Ludosky M. A., Changeux J. P., Cartaud J. Asymmetric distribution of dystrophin in developing and adult Torpedo marmorata electrocyte: evidence for its association with the acetylcholine receptor-rich membrane. Proc Natl Acad Sci U S A. 1990 May;87(10):3938–3941. doi: 10.1073/pnas.87.10.3938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Khurana T. S., Hoffman E. P., Kunkel L. M. Identification of a chromosome 6-encoded dystrophin-related protein. J Biol Chem. 1990 Oct 5;265(28):16717–16720. [PubMed] [Google Scholar]
  36. Kingsley R. E., Cole N. L. Preparation of cultured mammalian cells for transmission and scanning electron microscopy using Aclar film. J Electron Microsc Tech. 1988 Sep;10(1):77–85. doi: 10.1002/jemt.1060100110. [DOI] [PubMed] [Google Scholar]
  37. Koenig M., Monaco A. P., Kunkel L. M. The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. Cell. 1988 Apr 22;53(2):219–228. doi: 10.1016/0092-8674(88)90383-2. [DOI] [PubMed] [Google Scholar]
  38. Kordeli E., Cartaud J., Nghiêm H. O., Pradel L. A., Dubreuil C., Paulin D., Changeux J. P. Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes. J Cell Biol. 1986 Mar;102(3):748–761. doi: 10.1083/jcb.102.3.748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kramarcy N. R., Sealock R. Dystrophin as a focal adhesion protein. Collocalization with talin and the Mr 48,000 sarcolemmal protein in cultured Xenopus muscle. FEBS Lett. 1990 Nov 12;274(1-2):171–174. doi: 10.1016/0014-5793(90)81356-s. [DOI] [PubMed] [Google Scholar]
  40. LaRochelle W. J., Froehner S. C. Determination of the tissue distributions and relative concentrations of the postsynaptic 43-kDa protein and the acetylcholine receptor in Torpedo. J Biol Chem. 1986 Apr 25;261(12):5270–5274. [PubMed] [Google Scholar]
  41. MAYOR H. D., HAMPTON J. C., ROSARIO B. A simple method for removing the resin from epoxy-embedded tissue. J Biophys Biochem Cytol. 1961 Apr;9:909–910. doi: 10.1083/jcb.9.4.909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Mandel J. L. Dystrophin. The gene and its product. Nature. 1989 Jun 22;339(6226):584–586. doi: 10.1038/339584a0. [DOI] [PubMed] [Google Scholar]
  43. Miranda A. F., Bonilla E., Martucci G., Moraes C. T., Hays A. P., Dimauro S. Immunocytochemical study of dystrophin in muscle cultures from patients with Duchenne muscular dystrophy and unaffected control patients. Am J Pathol. 1988 Sep;132(3):410–416. [PMC free article] [PubMed] [Google Scholar]
  44. Mitra A. K., McCarthy M. P., Stroud R. M. Three-dimensional structure of the nicotinic acetylcholine receptor and location of the major associated 43-kD cytoskeletal protein, determined at 22 A by low dose electron microscopy and x-ray diffraction to 12.5 A. J Cell Biol. 1989 Aug;109(2):755–774. doi: 10.1083/jcb.109.2.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Mongini T., Ghigo D., Doriguzzi C., Bussolino F., Pescarmona G., Pollo B., Schiffer D., Bosia A. Free cytoplasmic Ca++ at rest and after cholinergic stimulus is increased in cultured muscle cells from Duchenne muscular dystrophy patients. Neurology. 1988 Mar;38(3):476–480. doi: 10.1212/wnl.38.3.476. [DOI] [PubMed] [Google Scholar]
  46. Morton M. E., Froehner S. C. The alpha 1 and alpha 2 polypeptides of the dihydropyridine-sensitive calcium channel differ in developmental expression and tissue distribution. Neuron. 1989 May;2(5):1499–1506. doi: 10.1016/0896-6273(89)90196-7. [DOI] [PubMed] [Google Scholar]
  47. Peng H. B., Froehner S. C. Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells. J Cell Biol. 1985 May;100(5):1698–1705. doi: 10.1083/jcb.100.5.1698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Peng H. B., Nakajima Y. Membrane particle aggregates in innervated and noninnervated cultures of Xenopus embryonic muscle cells. Proc Natl Acad Sci U S A. 1978 Jan;75(1):500–504. doi: 10.1073/pnas.75.1.500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Porter S., Froehner S. C. Characterization and localization of the Mr = 43,000 proteins associated with acetylcholine receptor-rich membranes. J Biol Chem. 1983 Aug 25;258(16):10034–10040. [PubMed] [Google Scholar]
  50. Pumplin D. W. Acetylcholine receptor clusters of rat myotubes have at least three domains with distinctive cytoskeletal and membranous components. J Cell Biol. 1989 Aug;109(2):739–753. doi: 10.1083/jcb.109.2.739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Ravdin P., Axelrod D. Fluorescent tetramethyl rhodamine derivatives of alpha-bungarotoxin: preparation, separation, and characterization. Anal Biochem. 1977 Jun;80(2):585–592. doi: 10.1016/0003-2697(77)90682-0. [DOI] [PubMed] [Google Scholar]
  52. Rowland L. P. Biochemistry of muscle membranes in Duchenne muscular dystrophy. Muscle Nerve. 1980 Jan-Feb;3(1):3–20. doi: 10.1002/mus.880030103. [DOI] [PubMed] [Google Scholar]
  53. Sahr K. E., Laurila P., Kotula L., Scarpa A. L., Coupal E., Leto T. L., Linnenbach A. J., Winkelmann J. C., Speicher D. W., Marchesi V. T. The complete cDNA and polypeptide sequences of human erythroid alpha-spectrin. J Biol Chem. 1990 Mar 15;265(8):4434–4443. [PubMed] [Google Scholar]
  54. Sakakibara H., Engel A. G., Lambert E. H. Duchenne dystrophy: ultrastructural localization of the acetylcholine receptor and intracellular microelectrode studies of neuromuscular transmission. Neurology. 1977 Aug;27(8):741–745. doi: 10.1212/wnl.27.8.741. [DOI] [PubMed] [Google Scholar]
  55. Sealock R. Identification of regions of high acetylcholine receptor density in tannic acid-fixed postsynaptic membranes from electric tissue. Brain Res. 1980 Oct 20;199(2):267–281. doi: 10.1016/0006-8993(80)90689-7. [DOI] [PubMed] [Google Scholar]
  56. Sealock R., Kavookjian A. Postsynaptic distribution of acetylcholine receptors in electroplax of the torpedine ray, Narcine brasiliensis. Brain Res. 1980 May 19;190(1):81–93. doi: 10.1016/0006-8993(80)91161-0. [DOI] [PubMed] [Google Scholar]
  57. Sealock R., Wray B. E., Froehner S. C. Ultrastructural localization of the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies. J Cell Biol. 1984 Jun;98(6):2239–2244. doi: 10.1083/jcb.98.6.2239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Sicinski P., Geng Y., Ryder-Cook A. S., Barnard E. A., Darlison M. G., Barnard P. J. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science. 1989 Jun 30;244(4912):1578–1580. doi: 10.1126/science.2662404. [DOI] [PubMed] [Google Scholar]
  59. Sottrup-Jensen L., Stepanik T. M., Kristensen T., Wierzbicki D. M., Jones C. M., Lønblad P. B., Magnusson S., Petersen T. E. Primary structure of human alpha 2-macroglobulin. V. The complete structure. J Biol Chem. 1984 Jul 10;259(13):8318–8327. [PubMed] [Google Scholar]
  60. Torres L. F., Duchen L. W. The mutant mdx: inherited myopathy in the mouse. Morphological studies of nerves, muscles and end-plates. Brain. 1987 Apr;110(Pt 2):269–299. doi: 10.1093/brain/110.2.269. [DOI] [PubMed] [Google Scholar]
  61. Watkins S. C., Hoffman E. P., Slayter H. S., Kunkel L. M. Immunoelectron microscopic localization of dystrophin in myofibres. Nature. 1988 Jun 30;333(6176):863–866. doi: 10.1038/333863a0. [DOI] [PubMed] [Google Scholar]
  62. Winkelmann J. C., Chang J. G., Tse W. T., Scarpa A. L., Marchesi V. T., Forget B. G. Full-length sequence of the cDNA for human erythroid beta-spectrin. J Biol Chem. 1990 Jul 15;265(20):11827–11832. [PubMed] [Google Scholar]
  63. Witkowski J. A. Dystrophin-related muscular dystrophies. J Child Neurol. 1989 Oct;4(4):251–271. doi: 10.1177/088307388900400403. [DOI] [PubMed] [Google Scholar]
  64. Woodruff M. L., Theriot J., Burden S. J. 300-kD subsynaptic protein copurifies with acetylcholine receptor-rich membranes and is concentrated at neuromuscular synapses. J Cell Biol. 1987 Apr;104(4):939–946. doi: 10.1083/jcb.104.4.939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wray B. E., Sealock R. Ultrastructural immunocytochemistry of particulate fractions using polyvinyl chloride microculture wells. J Histochem Cytochem. 1984 Oct;32(10):1117–1120. doi: 10.1177/32.10.6481151. [DOI] [PubMed] [Google Scholar]
  66. Yamaguchi M., Ishiura S., Takano-Ohmuro H., Tsukahara T., Arahata K., Obinata T., Tamiya T., Tsuchiya T., Sugita H. Detection of a fast isoform of C-protein with an antiserum directed against the N-terminal portion of dystrophin. Biochem Biophys Res Commun. 1990 May 31;169(1):57–63. doi: 10.1016/0006-291x(90)91432-r. [DOI] [PubMed] [Google Scholar]

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