Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1987 Jan 1;104(1):97–108. doi: 10.1083/jcb.104.1.97

Disruption and reformation of the acetylcholine receptor clusters of cultured rat myotubes occur in two distinct stages

PMCID: PMC2117041  PMID: 3793764

Abstract

We have examined the redistribution of acetylcholine receptor (AChR) intramembrane particles (IMPs) when AChR clusters of cultured rat myotubes are experimentally disrupted and allowed to reform. In control myotubes, the AChR IMPs are evenly distributed within the AChR domains of cluster membrane. Shortly after addition of azide to disrupt clusters, IMPs become unevenly scattered, with some microaggregation. After longer treatment, IMPs are depleted from AChR domains with no further change in IMP distribution. Contact domains of clusters are relatively poor in IMPs both before and after cluster dispersal. Upon visualization with fluorescent alpha-bungarotoxin, some AChR in azide- treated samples appear as small, bright spots. These spots do not correspond to microaggregates seen in freeze-fracture replicas, and probably represent receptors that have been internalized. The internalization rate is insufficient to account completely for the loss of IMPs from clusters, however. During reformation of AChR clusters upon removal of azide, IMP concentration in receptor domains increases. At early stages of reformation, IMPs appear in small groups containing compact microaggregates. At later times, AChR domains enlarge and IMPs within them assume the evenly spaced distribution characteristic of control clusters. These observations suggest that the disruption of clusters is accompanied by mobilization of AChR from a fixed array, allowing AChR IMPs to diffuse away from the clusters, to form microaggregates, and to become internalized. Cluster reformation appears to be the reverse of this process. Our results are thus consistent with a two-step model for AChR clustering, in which the concentration of IMPs into a small membrane region precedes their rearrangement into evenly spaced sites.

Full Text

The Full Text of this article is available as a PDF (5.6 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. Axelrod D. Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol. 1981 Apr;89(1):141–145. doi: 10.1083/jcb.89.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Bevan S., Steinbach J. H. The distribution of alpha-bungarotoxin binding sites of mammalian skeletal muscle developing in vivo. J Physiol. 1977 May;267(1):195–213. doi: 10.1113/jphysiol.1977.sp011808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bloch R. J. Acetylcholine receptor clustering in rat myotubes: requirement for CA2+ and effects of drugs which depolymerize microtubules. J Neurosci. 1983 Dec;3(12):2670–2680. doi: 10.1523/JNEUROSCI.03-12-02670.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bloch R. J. Actin at receptor-rich domains of isolated acetylcholine receptor clusters. J Cell Biol. 1986 Apr;102(4):1447–1458. doi: 10.1083/jcb.102.4.1447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bloch R. J. Dispersal and reformation of acetylcholine receptor clusters of cultured rat myotubes treated with inhibitors of energy metabolism. J Cell Biol. 1979 Sep;82(3):626–643. doi: 10.1083/jcb.82.3.626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bloch R. J., Geiger B. The localization of acetylcholine receptor clusters in areas of cell-substrate contact in cultures of rat myotubes. Cell. 1980 Aug;21(1):25–35. doi: 10.1016/0092-8674(80)90111-7. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Bloch R. J. Loss of acetylcholine receptor clusters induced by treatment of cultured rat myotubes with carbachol. J Neurosci. 1986 Mar;6(3):691–700. doi: 10.1523/JNEUROSCI.06-03-00691.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bloch R. J., Steinbach J. H. Reversible loss of acetylcholine receptor clusters at the developing rat neuromuscular junction. Dev Biol. 1981 Jan 30;81(2):386–391. doi: 10.1016/0012-1606(81)90305-5. [DOI] [PubMed] [Google Scholar]
  13. Burden S. Development of the neuromuscular junction in the chick embryo: the number, distribution, and stability of acetylcholine receptors. Dev Biol. 1977 Jun;57(2):317–329. doi: 10.1016/0012-1606(77)90218-4. [DOI] [PubMed] [Google Scholar]
  14. Bursztajn S., Berman S. A., McManaman J. L., Watson M. L. Insertion and internalization of acetylcholine receptors at clustered and diffuse domains on cultured myotubes. J Cell Biol. 1985 Jul;101(1):104–111. doi: 10.1083/jcb.101.1.104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Bursztajn S., McManaman J. L., Appel S. H. Organization of acetylcholine receptor clusters in cultured rat myotubes is calcium dependent. J Cell Biol. 1984 Feb;98(2):507–517. doi: 10.1083/jcb.98.2.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cohen S. A., Pumplin D. W. Clusters of intramembrane particles associated with binding sites for alpha-bungarotoxin in cultured chick myotubes. J Cell Biol. 1979 Aug;82(2):494–516. doi: 10.1083/jcb.82.2.494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. DIAMOND J., MILEDI R. A study of foetal and new-born rat muscle fibres. J Physiol. 1962 Aug;162:393–408. doi: 10.1113/jphysiol.1962.sp006941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fambrough D. M. Control of acetylcholine receptors in skeletal muscle. Physiol Rev. 1979 Jan;59(1):165–227. doi: 10.1152/physrev.1979.59.1.165. [DOI] [PubMed] [Google Scholar]
  19. Frank E., Fischbach G. D. Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses. J Cell Biol. 1979 Oct;83(1):143–158. doi: 10.1083/jcb.83.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goldstein J. L., Anderson R. G., Brown M. S. Coated pits, coated vesicles, and receptor-mediated endocytosis. Nature. 1979 Jun 21;279(5715):679–685. doi: 10.1038/279679a0. [DOI] [PubMed] [Google Scholar]
  21. Harris A. J., Kuffler S. W., Dennis M. J. Differential chemosensitivity of synaptic and extrasynaptic areas on the neuronal surface membrane in parasympathetic neurons of the frog, tested by microapplication of acetylcholine. Proc R Soc Lond B Biol Sci. 1971 Apr 27;177(1049):541–553. doi: 10.1098/rspb.1971.0046. [DOI] [PubMed] [Google Scholar]
  22. Land B. R., Podleski T. R., Salpeter E. E., Salpeter M. M. Acetylcholine receptor distribution on myotubes in culture correlated to acetylcholine sensitivity. J Physiol. 1977 Jul;269(1):155–176. doi: 10.1113/jphysiol.1977.sp011897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Matthews-Bellinger J. A., Salpeter M. M. Fine structural distribution of acetylcholine receptors at developing mouse neuromuscular junctions. J Neurosci. 1983 Mar;3(3):644–657. doi: 10.1523/JNEUROSCI.03-03-00644.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Muller J., Kachadorian W. A., DiScala V. A. Evidence that ADH-stimulated intramembrane particle aggregates are transferred from cytoplasmic to luminal membranes in toad bladder epithelial cells. J Cell Biol. 1980 Apr;85(1):83–95. doi: 10.1083/jcb.85.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Owicki J. C., McConnell H. M. Lateral diffusion in inhomogeneous membranes. Model membranes containing cholesterol. Biophys J. 1980 Jun;30(3):383–397. doi: 10.1016/S0006-3495(80)85103-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Petit V. A., Edidin M. Lateral phase separation of lipids in plasma membranes: effect of temperature on the mobility of membrane antigens. Science. 1974 Jun 14;184(4142):1183–1185. doi: 10.1126/science.184.4142.1183. [DOI] [PubMed] [Google Scholar]
  27. Pumplin D. W., Bloch R. J. Lipid domains of acetylcholine receptor clusters detected with saponin and filipin. J Cell Biol. 1983 Oct;97(4):1043–1054. doi: 10.1083/jcb.97.4.1043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pumplin D. W., Drachman D. B. Myasthenic patients' IgG causes redistribution of acetylcholine receptors: freeze-fracture studies. J Neurosci. 1983 Mar;3(3):576–584. doi: 10.1523/JNEUROSCI.03-03-00576.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pumplin D. W., Fambrough D. M. Turnover of acetylcholine receptors in skeletal muscle. Annu Rev Physiol. 1982;44:319–335. doi: 10.1146/annurev.ph.44.030182.001535. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Role L. W., Matossian V. R., O'Brien R. J., Fischbach G. D. On the mechanism of acetylcholine receptor accumulation at newly formed synapses on chick myotubes. J Neurosci. 1985 Aug;5(8):2197–2204. doi: 10.1523/JNEUROSCI.05-08-02197.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Salpeter M. M., Spanton S., Holley K., Podleski T. R. Brain extract causes acetylcholine receptor redistribution which mimics some early events at developing neuromuscular junctions. J Cell Biol. 1982 May;93(2):417–425. doi: 10.1083/jcb.93.2.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Saxton M. J. Lateral diffusion in an archipelago. Effects of impermeable patches on diffusion in a cell membrane. Biophys J. 1982 Aug;39(2):165–173. doi: 10.1016/S0006-3495(82)84504-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schreiner G. F., Unanue E. R. Membrane and cytoplasmic changes in B lymphocytes induced by ligand-surface immunoglobulin interaction. Adv Immunol. 1976;24:37–165. doi: 10.1016/s0065-2776(08)60329-6. [DOI] [PubMed] [Google Scholar]
  35. Slater C. R. Postnatal maturation of nerve-muscle junctions in hindlimb muscles of the mouse. Dev Biol. 1982 Nov;94(1):11–22. doi: 10.1016/0012-1606(82)90063-x. [DOI] [PubMed] [Google Scholar]
  36. Stya M., Axelrod D. Mobility and detergent extractability of acetylcholine receptors on cultured rat myotubes: a correlation. J Cell Biol. 1983 Jul;97(1):48–51. doi: 10.1083/jcb.97.1.48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Triller A., Cluzeaud F., Pfeiffer F., Betz H., Korn H. Distribution of glycine receptors at central synapses: an immunoelectron microscopy study. J Cell Biol. 1985 Aug;101(2):683–688. doi: 10.1083/jcb.101.2.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wade J. B., Stetson D. L., Lewis S. A. ADH action: evidence for a membrane shuttle mechanism. Ann N Y Acad Sci. 1981;372:106–117. doi: 10.1111/j.1749-6632.1981.tb15464.x. [DOI] [PubMed] [Google Scholar]
  39. Yee A. G., Fischbach G. D., Karnovsky M. J. Clusters of intramembranous particles on cultured myotubes at sites that are highly sensitive to acetylcholine. Proc Natl Acad Sci U S A. 1978 Jun;75(6):3004–3008. doi: 10.1073/pnas.75.6.3004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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