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. 1984 Jul 1;99(1 Pt 1):148–154. doi: 10.1083/jcb.99.1.148

Role of the cytoskeleton in the formation, stabilization, and removal of acetylcholine receptor clusters in cultured muscle cells

PMCID: PMC2275607  PMID: 6539781

Abstract

We have examined the effects of microtubule- and microfilament- disrupting drugs on the stability, formation, and removal of acetylcholine (ACh) receptors and ACh receptor clusters on the surface of aneurally cultured chick embryonic myotubes. (a) In muscle cell cultures, cytochalasin D (0.2 microgram/ml) or B (2.0 micrograms/ml) causes the dispersal of 50-60% of the existing clusters over a 24-h period (visualized with rhodamine-conjugated alpha-bungarotoxin); Colcemid (0.5 micrograms/ml) has no affect on these clusters. The total number of cell surface ACh receptors does not decline during this period (measured by [125I]alpha-bungarotoxin binding) in the presence of either drug. (b) When cells are treated with biotinylated alpha- bungarotoxin and fluorescent avidin, ACh receptors are cross-linked and rapidly internalized (Axelrod, D., 1980, Proc. Natl. Acad. Sci. USA., 77: 4823-4827). Within 6 h, I have found that 0-15% of the existing large clusters remain. Cytochalasin D or B had no effect on this removal of clusters; however, Colcemid completely prevented the removal of clusters from the cell surface. (c) Addition of chick brain extract to chick myotubes causes an increase in the synthesis and clustering of ACh receptors (Jessell et al., 1979, Proc. Natl. Acad. Sci. USA. 76: 5397-5401). Cytochalasin D caused a slight increase in the number of receptors synthesized in the presence of brain extract whereas Colcemid had no effect on the synthesis and insertion of new receptors into the plasma membrane induced by the brain extract. However, both drugs prevented the increase in the number of receptor clusters. These results are consistent with the hypothesis that receptor clusters are stabilized by actin-containing filaments, but that the movement of receptors in the plane of the membrane requires Colcemid-sensitive microtubules.

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Selected References

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  1. Anderson M. J., Cohen M. W. Fluorescent staining of acetylcholine receptors in vertebrate skeletal muscle. J Physiol. 1974 Mar;237(2):385–400. doi: 10.1113/jphysiol.1974.sp010487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aronson J., Inoué S. Reversal by light of the action of N-methyl N-desacetyl colchicine on mitosis. J Cell Biol. 1970 May;45(2):470–477. doi: 10.1083/jcb.45.2.470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Axelrod D. Crosslinkage and visualization of acetylcholine receptors on myotubes with biotinylated alpha-bungarotoxin and fluorescent avidin. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4823–4827. doi: 10.1073/pnas.77.8.4823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Axelrod D., Ravdin P. M., Podleski T. R. Control of acetylcholine receptor mobility and distribution in cultured muscle membranes. A fluorescence study. Biochim Biophys Acta. 1978 Jul 20;511(1):23–38. doi: 10.1016/0005-2736(78)90062-7. [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. Barak L. S., Yocum R. R., Nothnagel E. A., Webb W. W. Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin. Proc Natl Acad Sci U S A. 1980 Feb;77(2):980–984. doi: 10.1073/pnas.77.2.980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ben-Ze'ev A., Duerr A., Solomon F., Penman S. The outer boundary of the cytoskeleton: a lamina derived from plasma membrane proteins. Cell. 1979 Aug;17(4):859–865. doi: 10.1016/0092-8674(79)90326-x. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. Bursztajn S., Fischbach G. D. Evidence that coated vesicles transport acetylcholine receptors to the surface membrane of chick myotubes. J Cell Biol. 1984 Feb;98(2):498–506. doi: 10.1083/jcb.98.2.498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Connolly J. A., Kalnins V. I., Cleveland D. W., Kirschner M. W. Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence. J Cell Biol. 1978 Mar;76(3):781–786. doi: 10.1083/jcb.76.3.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Connolly J. A., St John P. A., Fischbach G. D. Extracts of electric lobe and electric organ from Torpedo californica increase the total number as well as the number of aggregates of chick myotube acetylcholine receptors. J Neurosci. 1982 Sep;2(9):1207–1213. doi: 10.1523/JNEUROSCI.02-09-01207.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Croop J., Toyama Y., Dlugosz A. A., Holtzer H. Selective effects of phorbol 12-myristate 13-acetate on myofibrils and 10-nm filaments. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5273–5277. doi: 10.1073/pnas.77.9.5273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Edwards C., Frisch H. L. A model for the localization of acetylcholine receptors at the muscle endplate. J Neurobiol. 1976 Jul;7(4):377–381. doi: 10.1002/neu.480070409. [DOI] [PubMed] [Google Scholar]
  18. Fischbach G. D., Cohen S. A. The distribution of acetylcholine sensitivity over uninnervated and innervated muscle fibers grown in cell culture. Dev Biol. 1973 Mar;31(1):147–162. doi: 10.1016/0012-1606(73)90326-6. [DOI] [PubMed] [Google Scholar]
  19. Fischbach G. D. Synapse formation between dissociated nerve and muscle cells in low density cell cultures. Dev Biol. 1972 Jun;28(2):407–429. doi: 10.1016/0012-1606(72)90023-1. [DOI] [PubMed] [Google Scholar]
  20. Frank E., Fischbach G. D. ACh receptors accumulate at newly formed nerve-muscle synapses in vitro. Soc Gen Physiol Ser. 1977;32:285–291. [PubMed] [Google Scholar]
  21. Garrels J. I., Gibson W. Identification and characterization of multiple forms of actin. Cell. 1976 Dec;9(4 Pt 2):793–805. doi: 10.1016/0092-8674(76)90142-2. [DOI] [PubMed] [Google Scholar]
  22. Geiger B. A 130K protein from chicken gizzard: its localization at the termini of microfilament bundles in cultured chicken cells. Cell. 1979 Sep;18(1):193–205. doi: 10.1016/0092-8674(79)90368-4. [DOI] [PubMed] [Google Scholar]
  23. Geiger B., Tokuyasu K. T., Dutton A. H., Singer S. J. Vinculin, an intracellular protein localized at specialized sites where microfilament bundles terminate at cell membranes. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4127–4131. doi: 10.1073/pnas.77.7.4127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hall Z. W., Lubit B. W., Schwartz J. H. Cytoplasmic actin in postsynaptic structures at the neuromuscular junction. J Cell Biol. 1981 Sep;90(3):789–792. doi: 10.1083/jcb.90.3.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jessell T. M., Siegel R. E., Fischbach G. D. Induction of acetylcholine receptors on cultured skeletal muscle by a factor extracted from brain and spinal cord. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5397–5401. doi: 10.1073/pnas.76.10.5397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lubit B. W., Schwartz J. H. An antiactin antibody that distinguishes between cytoplasmic and skeletal muscle actins. J Cell Biol. 1980 Sep;86(3):891–897. doi: 10.1083/jcb.86.3.891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Osborn M., Franke W. W., Weber K. Visualization of a system of filaments 7-10 nm thick in cultured cells of an epithelioid line (Pt K2) by immunofluorescence microscopy. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2490–2494. doi: 10.1073/pnas.74.6.2490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Osborn M., Weber K. The display of microtubules in transformed cells. Cell. 1977 Nov;12(3):561–571. doi: 10.1016/0092-8674(77)90257-4. [DOI] [PubMed] [Google Scholar]
  29. Pardo J. V., Pittenger M. F., Craig S. W. Subcellular sorting of isoactins: selective association of gamma actin with skeletal muscle mitochondria. Cell. 1983 Apr;32(4):1093–1103. doi: 10.1016/0092-8674(83)90293-3. [DOI] [PubMed] [Google Scholar]
  30. Peng H. B., Cheng P. C., Luther P. W. Formation of ACh receptor clusters induced by positively charged latex beads. Nature. 1981 Aug 27;292(5826):831–834. doi: 10.1038/292831a0. [DOI] [PubMed] [Google Scholar]
  31. Peng H. B. Cytoskeletal organization of the presynaptic nerve terminal and the acetylcholine receptor cluster in cell cultures. J Cell Biol. 1983 Aug;97(2):489–498. doi: 10.1083/jcb.97.2.489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Poo M. Rapid lateral diffusion of functional A Ch receptors in embryonic muscle cell membrane. Nature. 1982 Jan 28;295(5847):332–334. doi: 10.1038/295332a0. [DOI] [PubMed] [Google Scholar]
  33. Prives J., Fulton A. B., Penman S., Daniels M. P., Christian C. N. Interaction of the cytoskeletal framework with acetylcholine receptor on th surface of embryonic muscle cells in culture. J Cell Biol. 1982 Jan;92(1):231–236. doi: 10.1083/jcb.92.1.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stya M., Axelrod D. Diffusely distributed acetylcholine receptors can participate in cluster formation on cultured rat myotubes. Proc Natl Acad Sci U S A. 1983 Jan;80(2):449–453. doi: 10.1073/pnas.80.2.449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Tank D. W., Wu E. S., Webb W. W. Enhanced molecular diffusibility in muscle membrane blebs: release of lateral constraints. J Cell Biol. 1982 Jan;92(1):207–212. doi: 10.1083/jcb.92.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Toyama Y., Forry-Schaudies S., Hoffman B., Holtzer H. Effects of taxol and Colcemid on myofibrillogenesis. Proc Natl Acad Sci U S A. 1982 Nov;79(21):6556–6560. doi: 10.1073/pnas.79.21.6556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Whalen R. G., Butler-Browne G. S., Gros F. Protein synthesis and actin heterogeneity in calf muscle cells in culture. Proc Natl Acad Sci U S A. 1976 Jun;73(6):2018–2022. doi: 10.1073/pnas.73.6.2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Young S. H., Poo M. M. Rapid lateral diffusion of extrajunctional acetylcholine receptors in the developing muscle membrane of Xenopus tadpole. J Neurosci. 1983 Jan;3(1):225–231. doi: 10.1523/JNEUROSCI.03-01-00225.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]

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