Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 May 2;129(4):1093–1101. doi: 10.1083/jcb.129.4.1093

Distribution of alpha-dystroglycan during embryonic nerve-muscle synaptogenesis

PMCID: PMC2120479  PMID: 7744958

Abstract

The distribution of alpha-dystroglycan (alpha DG) relative to acetylcholine receptors (AChRs) and neural agrin was examined by immunofluorescent staining with mAb IIH6 in cultures of nerve and muscle cells derived from Xenopus embryos. In Western blots probed with mAb IIH6, alpha DG was evident in membrane extracts of Xenopus muscle but not brain. alpha DG immunofluorescence was present at virtually all synaptic clusters of AChRs and neural agrin. Even microclusters of AChRs and agrin at synapses no older than 1-2 h (the earliest examined) had alpha DG associated with them. alpha DG was also colocalized at the submicrometer level with AChRs at nonsynaptic clusters that have little or no agrin. The number of large (> 4 microns) nonsynaptic clusters of alpha DG, like the number of large nonsynaptic clusters of AChRs, was much lower on innervated than on noninnervated cells. When mAb IIH6 was included in the culture medium, the large nonsynaptic clusters appeared fragmented and less compact, but the accumulation of agrin and AChRs along nerve-muscle contacts was not prevented. It is concluded that during nerve-muscle synaptogenesis, alpha DG undergoes the same nerve- induced changes in distribution as AChRs. We propose a diffusion trap model in which the alpha DG-transmembrane complex participates in the anchoring and recruitment of AChRs and alpha DG during the formation of synaptic as well as nonsynaptic AChR clusters.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson M. J., Cohen M. W. Nerve-induced and spontaneous redistribution of acetylcholine receptors on cultured muscle cells. J Physiol. 1977 Jul;268(3):757–773. doi: 10.1113/jphysiol.1977.sp011880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Anderson M. S., Kunkel L. M. The molecular and biochemical basis of Duchenne muscular dystrophy. Trends Biochem Sci. 1992 Aug;17(8):289–292. doi: 10.1016/0968-0004(92)90437-e. [DOI] [PubMed] [Google Scholar]
  5. Axelrod D., Ravdin P., Koppel D. E., Schlessinger J., Webb W. W., Elson E. L., Podleski T. R. Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4594–4598. doi: 10.1073/pnas.73.12.4594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baker L. P., Peng H. B. Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells. J Cell Biol. 1993 Jan;120(1):185–195. doi: 10.1083/jcb.120.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bayne E. K., Anderson M. J., Fambrough D. M. Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates. J Cell Biol. 1984 Oct;99(4 Pt 1):1486–1501. doi: 10.1083/jcb.99.4.1486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bewick G. S., Nicholson L. V., Young C., O'Donnell E., Slater C. R. Different distributions of dystrophin and related proteins at nerve-muscle junctions. Neuroreport. 1992 Oct;3(10):857–860. doi: 10.1097/00001756-199210000-00009. [DOI] [PubMed] [Google Scholar]
  9. Bowe M. A., Deyst K. A., Leszyk J. D., Fallon J. R. Identification and purification of an agrin receptor from Torpedo postsynaptic membranes: a heteromeric complex related to the dystroglycans. Neuron. 1994 May;12(5):1173–1180. doi: 10.1016/0896-6273(94)90324-7. [DOI] [PubMed] [Google Scholar]
  10. Campanelli J. T., Hoch W., Rupp F., Kreiner T., Scheller R. H. Agrin mediates cell contact-induced acetylcholine receptor clustering. Cell. 1991 Nov 29;67(5):909–916. doi: 10.1016/0092-8674(91)90364-5. [DOI] [PubMed] [Google Scholar]
  11. Campanelli J. T., Roberds S. L., Campbell K. P., Scheller R. H. A role for dystrophin-associated glycoproteins and utrophin in agrin-induced AChR clustering. Cell. 1994 Jun 3;77(5):663–674. doi: 10.1016/0092-8674(94)90051-5. [DOI] [PubMed] [Google Scholar]
  12. Cohen M. W., Godfrey E. W. Early appearance of and neuronal contribution to agrin-like molecules at embryonic frog nerve-muscle synapses formed in culture. J Neurosci. 1992 Aug;12(8):2982–2992. doi: 10.1523/JNEUROSCI.12-08-02982.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cohen M. W., Moody-Corbett F., Godfrey E. W. Neuritic deposition of agrin on culture substrate: implications for nerve-muscle synaptogenesis. J Neurosci. 1994 May;14(5 Pt 2):3293–3303. doi: 10.1523/JNEUROSCI.14-05-03293.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Douville P. J., Harvey W. J., Carbonetto S. Isolation and partial characterization of high affinity laminin receptors in neural cells. J Biol Chem. 1988 Oct 15;263(29):14964–14969. [PubMed] [Google Scholar]
  15. Ervasti J. M., Campbell K. P. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J Cell Biol. 1993 Aug;122(4):809–823. doi: 10.1083/jcb.122.4.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ervasti J. M., Campbell K. P. Dystrophin and the membrane skeleton. Curr Opin Cell Biol. 1993 Feb;5(1):82–87. doi: 10.1016/s0955-0674(05)80012-2. [DOI] [PubMed] [Google Scholar]
  17. Ervasti J. M., Campbell K. P. Membrane organization of the dystrophin-glycoprotein complex. Cell. 1991 Sep 20;66(6):1121–1131. doi: 10.1016/0092-8674(91)90035-w. [DOI] [PubMed] [Google Scholar]
  18. Fallon J. R., Hall Z. W. Building synapses: agrin and dystroglycan stick together. Trends Neurosci. 1994 Nov;17(11):469–473. doi: 10.1016/0166-2236(94)90135-x. [DOI] [PubMed] [Google Scholar]
  19. Ferns M. J., Campanelli J. T., Hoch W., Scheller R. H., Hall Z. The ability of agrin to cluster AChRs depends on alternative splicing and on cell surface proteoglycans. Neuron. 1993 Sep;11(3):491–502. doi: 10.1016/0896-6273(93)90153-i. [DOI] [PubMed] [Google Scholar]
  20. Ferns M., Hoch W., Campanelli J. T., Rupp F., Hall Z. W., Scheller R. H. RNA splicing regulates agrin-mediated acetylcholine receptor clustering activity on cultured myotubes. Neuron. 1992 Jun;8(6):1079–1086. doi: 10.1016/0896-6273(92)90129-2. [DOI] [PubMed] [Google Scholar]
  21. Gee S. H., Blacher R. W., Douville P. J., Provost P. R., Yurchenco P. D., Carbonetto S. Laminin-binding protein 120 from brain is closely related to the dystrophin-associated glycoprotein, dystroglycan, and binds with high affinity to the major heparin binding domain of laminin. J Biol Chem. 1993 Jul 15;268(20):14972–14980. [PubMed] [Google Scholar]
  22. Gee S. H., Montanaro F., Lindenbaum M. H., Carbonetto S. Dystroglycan-alpha, a dystrophin-associated glycoprotein, is a functional agrin receptor. Cell. 1994 Jun 3;77(5):675–686. doi: 10.1016/0092-8674(94)90052-3. [DOI] [PubMed] [Google Scholar]
  23. Godfrey E. W. Comparison of agrin-like proteins from the extracellular matrix of chicken kidney and muscle with neural agrin, a synapse organizing protein. Exp Cell Res. 1991 Jul;195(1):99–109. doi: 10.1016/0014-4827(91)90504-n. [DOI] [PubMed] [Google Scholar]
  24. Godfrey E. W., Nitkin R. M., Wallace B. G., Rubin L. L., McMahan U. J. Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells. J Cell Biol. 1984 Aug;99(2):615–627. doi: 10.1083/jcb.99.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gordon H., Lupa M., Bowen D., Hall Z. A muscle cell variant defective in glycosaminoglycan biosynthesis forms nerve-induced but not spontaneous clusters of the acetylcholine receptor and the 43 kDa protein. J Neurosci. 1993 Feb;13(2):586–595. doi: 10.1523/JNEUROSCI.13-02-00586.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ibraghimov-Beskrovnaya O., Ervasti J. M., Leveille C. J., Slaughter C. A., Sernett S. W., Campbell K. P. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature. 1992 Feb 20;355(6362):696–702. doi: 10.1038/355696a0. [DOI] [PubMed] [Google Scholar]
  27. Kidokoro Y., Brass B., Kuromi H. Concanavalin A prevents acetylcholine receptor redistribution in Xenopus nerve-muscle cultures. J Neurosci. 1986 Jul;6(7):1941–1951. doi: 10.1523/JNEUROSCI.06-07-01941.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kullberg R. W., Lentz T. L., Cohen M. W. Development of the myotomal neuromuscular junction in Xenopus laevis: an electrophysiological and fine-structural study. Dev Biol. 1977 Oct 1;60(1):101–129. doi: 10.1016/0012-1606(77)90113-0. [DOI] [PubMed] [Google Scholar]
  29. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  30. Lindenbaum M. H., Carbonetto S. Muscular dystrophy: dystrophin and partners at the cell surface. Curr Biol. 1993 Feb;3(2):109–111. doi: 10.1016/0960-9822(93)90167-m. [DOI] [PubMed] [Google Scholar]
  31. Matsumura K., Ervasti J. M., Ohlendieck K., Kahl S. D., Campbell K. P. Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle. Nature. 1992 Dec 10;360(6404):588–591. doi: 10.1038/360588a0. [DOI] [PubMed] [Google Scholar]
  32. Moody-Corbett F., Cohen M. W. Influence of nerve on the formation and survival of acetylcholine receptor and cholinesterase patches on embryonic Xenopus muscle cells in culture. J Neurosci. 1982 May;2(5):633–646. doi: 10.1523/JNEUROSCI.02-05-00633.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Naegele J. R., Barnstable C. J. A carbohydrate epitope defined by monoclonal antibody VC1.1 is found on N-CAM and other cell adhesion molecules. Brain Res. 1991 Sep 13;559(1):118–129. doi: 10.1016/0006-8993(91)90294-6. [DOI] [PubMed] [Google Scholar]
  34. Nastuk M. A., Lieth E., Ma J. Y., Cardasis C. A., Moynihan E. B., McKechnie B. A., Fallon J. R. The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering. Neuron. 1991 Nov;7(5):807–818. doi: 10.1016/0896-6273(91)90283-6. [DOI] [PubMed] [Google Scholar]
  35. Nitkin R. M., Smith M. A., Magill C., Fallon J. R., Yao Y. M., Wallace B. G., McMahan U. J. Identification of agrin, a synaptic organizing protein from Torpedo electric organ. J Cell Biol. 1987 Dec;105(6 Pt 1):2471–2478. doi: 10.1083/jcb.105.6.2471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ohlendieck K., Ervasti J. M., Matsumura K., Kahl S. D., Leveille C. J., Campbell K. P. Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle. Neuron. 1991 Sep;7(3):499–508. doi: 10.1016/0896-6273(91)90301-f. [DOI] [PubMed] [Google Scholar]
  37. Ohlendieck K., Ervasti J. M., Snook J. B., Campbell K. P. Dystrophin-glycoprotein complex is highly enriched in isolated skeletal muscle sarcolemma. J Cell Biol. 1991 Jan;112(1):135–148. doi: 10.1083/jcb.112.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Peng H. B., Baker L. P., Dai Z. A role of tyrosine phosphorylation in the formation of acetylcholine receptor clusters induced by electric fields in cultured Xenopus muscle cells. J Cell Biol. 1993 Jan;120(1):197–204. doi: 10.1083/jcb.120.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Phillips W. D., Noakes P. G., Roberds S. L., Campbell K. P., Merlie J. P. Clustering and immobilization of acetylcholine receptors by the 43-kD protein: a possible role for dystrophin-related protein. J Cell Biol. 1993 Nov;123(3):729–740. doi: 10.1083/jcb.123.3.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Reist N. E., Werle M. J., McMahan U. J. Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscular junctions. Neuron. 1992 May;8(5):865–868. doi: 10.1016/0896-6273(92)90200-w. [DOI] [PubMed] [Google Scholar]
  41. Roberds S. L., Leturcq F., Allamand V., Piccolo F., Jeanpierre M., Anderson R. D., Lim L. E., Lee J. C., Tomé F. M., Romero N. B. Missense mutations in the adhalin gene linked to autosomal recessive muscular dystrophy. Cell. 1994 Aug 26;78(4):625–633. doi: 10.1016/0092-8674(94)90527-4. [DOI] [PubMed] [Google Scholar]
  42. Sealock R., Froehner S. C. Dystrophin-associated proteins and synapse formation: is alpha-dystroglycan the agrin receptor? Cell. 1994 Jun 3;77(5):617–619. doi: 10.1016/0092-8674(94)90045-0. [DOI] [PubMed] [Google Scholar]
  43. Sugiyama J., Bowen D. C., Hall Z. W. Dystroglycan binds nerve and muscle agrin. Neuron. 1994 Jul;13(1):103–115. doi: 10.1016/0896-6273(94)90462-6. [DOI] [PubMed] [Google Scholar]
  44. Sunada Y., Bernier S. M., Kozak C. A., Yamada Y., Campbell K. P. Deficiency of merosin in dystrophic dy mice and genetic linkage of laminin M chain gene to dy locus. J Biol Chem. 1994 May 13;269(19):13729–13732. [PubMed] [Google Scholar]
  45. Tomé F. M., Evangelista T., Leclerc A., Sunada Y., Manole E., Estournet B., Barois A., Campbell K. P., Fardeau M. Congenital muscular dystrophy with merosin deficiency. C R Acad Sci III. 1994 Apr;317(4):351–357. [PubMed] [Google Scholar]
  46. Tsim K. W., Ruegg M. A., Escher G., Kröger S., McMahan U. J. cDNA that encodes active agrin. Neuron. 1992 Apr;8(4):677–689. doi: 10.1016/0896-6273(92)90089-v. [DOI] [PubMed] [Google Scholar]
  47. Vogel Z., Christian C. N., Vigny M., Bauer H. C., Sonderegger P., Daniels M. P. Laminin induces acetylcholine receptor aggregation on cultured myotubes and enhances the receptor aggregation activity of a neuronal factor. J Neurosci. 1983 May;3(5):1058–1068. doi: 10.1523/JNEUROSCI.03-05-01058.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wallace B. G., Qu Z., Huganir R. L. Agrin induces phosphorylation of the nicotinic acetylcholine receptor. Neuron. 1991 Jun;6(6):869–878. doi: 10.1016/0896-6273(91)90227-q. [DOI] [PubMed] [Google Scholar]
  49. Wallace B. G. Staurosporine inhibits agrin-induced acetylcholine receptor phosphorylation and aggregation. J Cell Biol. 1994 May;125(3):661–668. doi: 10.1083/jcb.125.3.661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Xu H., Christmas P., Wu X. R., Wewer U. M., Engvall E. Defective muscle basement membrane and lack of M-laminin in the dystrophic dy/dy mouse. Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5572–5576. doi: 10.1073/pnas.91.12.5572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Ziskind-Conhaim L., Geffen I., Hall Z. W. Redistribution of acetylcholine receptors on developing rat myotubes. J Neurosci. 1984 Sep;4(9):2346–2349. doi: 10.1523/JNEUROSCI.04-09-02346.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES