Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1986 Apr 1;102(4):1447–1458. doi: 10.1083/jcb.102.4.1447

Actin at receptor-rich domains of isolated acetylcholine receptor clusters

PMCID: PMC2114171  PMID: 3958056

Abstract

Acetylcholine receptor (AChR) clusters of cultured rat myotubes, isolated by extraction with saponin (Bloch, R. J., 1984, J. Cell Biol. 99:984-993), contain a polypeptide that co-electrophoreses with purified muscle actins. A monoclonal antibody against actin reacts in immunoblots with this polypeptide and with purified actins. In indirect immunofluorescence, the antibody stains isolated AChR clusters only at AChR domains, strips of membrane within clusters that are rich in receptor. It also stains the postsynaptic region of the neuromuscular junction of adult rat skeletal muscle. Semiquantitative immunofluorescence analyses show that labeling by antiactin of isolated analyses show that labeling by antiactin of isolated AChR clusters is specific and saturable and that it varies linearly with the amount of AChR in the cluster. Filaments of purified gizzard myosin also bind preferentially at AChR-rich regions, and this binding is inhibited by MgATP. These experiments suggest that actin is associated with AChR- rich regions of receptor clusters. Depletion of actin by extraction of isolated clusters at low ionic strength selectively releases the actin- like polypeptide from the preparation. Simultaneously, AChRs redistribute within the plane of the membrane of the isolated clusters. Similarly, brief digestion with chymotrypsin reduces immunofluorescence staining and causes AChR redistribution. Treatments that deplete AChR from clusters in intact cells also reduce immunofluorescent staining for actin in isolated muscle membrane fragments. Upon reversal of these treatments, cluster reformation occurs in regions of the membrane that also stain for actin. I conclude that actin is associated with AChR domains and that changes in this association are accompanied by changes in the organization of isolated AChR clusters.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

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

  1. 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]
  2. 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]
  3. Bennett V., Stenbuck P. J. Identification and partial purification of ankyrin, the high affinity membrane attachment site for human erythrocyte spectrin. J Biol Chem. 1979 Apr 10;254(7):2533–2541. [PubMed] [Google Scholar]
  4. Betz W., Sakmann B. Effects of proteolytic enzymes on function and structure of frog neuromuscular junctions. J Physiol. 1973 May;230(3):673–688. doi: 10.1113/jphysiol.1973.sp010211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. 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]
  10. 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]
  11. Bretscher A., Weber K. Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures. J Cell Biol. 1980 Jul;86(1):335–340. doi: 10.1083/jcb.86.1.335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Burden S. J., DePalma R. L., Gottesman G. S. Crosslinking of proteins in acetylcholine receptor-rich membranes: association between the beta-subunit and the 43 kd subsynaptic protein. Cell. 1983 Dec;35(3 Pt 2):687–692. doi: 10.1016/0092-8674(83)90101-0. [DOI] [PubMed] [Google Scholar]
  13. Burden S. Identification of an intracellular postsynaptic antigen at the frog neuromuscular junction. J Cell Biol. 1982 Sep;94(3):521–530. doi: 10.1083/jcb.94.3.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  15. Burridge K., Connell L. A new protein of adhesion plaques and ruffling membranes. J Cell Biol. 1983 Aug;97(2):359–367. doi: 10.1083/jcb.97.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Carraway C. A., Jung G., Carraway K. L. Isolation of actin-containing transmembrane complexes from ascites adenocarcinoma sublines having mobile and immobile receptors. Proc Natl Acad Sci U S A. 1983 Jan;80(2):430–434. doi: 10.1073/pnas.80.2.430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Cohen C. M., Foley S. F. Spectrin-dependent and -independent association of F-actin with the erythrocyte membrane. J Cell Biol. 1980 Aug;86(2):694–698. doi: 10.1083/jcb.86.2.694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Craig S. W., Pardo J. V. Gamma actin, spectrin, and intermediate filament proteins colocalize with vinculin at costameres, myofibril-to-sarcolemma attachment sites. Cell Motil. 1983;3(5-6):449–462. doi: 10.1002/cm.970030513. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Fambrough D. M., Hartzell H. C. Acetylcholine receptors: number and distribution at neuromuscular junctions in rat diaphragm. Science. 1972 Apr 14;176(4031):189–191. doi: 10.1126/science.176.4031.189. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Flanagan J., Koch G. L. Cross-linked surface Ig attaches to actin. Nature. 1978 May 25;273(5660):278–281. doi: 10.1038/273278a0. [DOI] [PubMed] [Google Scholar]
  23. Fowler V., Taylor D. L. Spectrin plus band 4.1 cross-link actin. Regulation by micromolar calcium. J Cell Biol. 1980 May;85(2):361–376. doi: 10.1083/jcb.85.2.361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. Gröschel-Stewart U., Schreiber J., Mahlmeister C. Production of specific antibodies to contractile proteins, and their use in immunofluorescence microscopy. I. Antibodies to smooth and striated chicken muscle myosins. Histochemistry. 1976 Feb 26;46(3):229–236. doi: 10.1007/BF02462786. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Hubbard A. L., Ma A. Isolation of rat hepatocyte plasma membranes. II. Identification of membrane-associated cytoskeletal proteins. J Cell Biol. 1983 Jan;96(1):230–239. doi: 10.1083/jcb.96.1.230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kelly P. T., Cotman C. W. Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide. J Cell Biol. 1978 Oct;79(1):173–183. doi: 10.1083/jcb.79.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Koch G. L., Smith M. J. An association between actin and the major histocompatibility antigen H-2. Nature. 1978 May 25;273(5660):274–278. doi: 10.1038/273274a0. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Lazarides E., Burridge K. Alpha-actinin: immunofluorescent localization of a muscle structural protein in nonmuscle cells. Cell. 1975 Nov;6(3):289–298. doi: 10.1016/0092-8674(75)90180-4. [DOI] [PubMed] [Google Scholar]
  34. Lubit B. W. Association of beta-cytoplasmic actin with high concentrations of acetylcholine receptor (AChR) in normal and anti-AChR-treated primary rat muscle cultures. J Histochem Cytochem. 1984 Sep;32(9):973–981. doi: 10.1177/32.9.6379042. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Luna E. J., Fowler V. M., Swanson J., Branton D., Taylor D. L. A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity. J Cell Biol. 1981 Feb;88(2):396–409. doi: 10.1083/jcb.88.2.396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lux S. E. Spectrin-actin membrane skeleton of normal and abnormal red blood cells. Semin Hematol. 1979 Jan;16(1):21–51. [PubMed] [Google Scholar]
  38. Miledi R., Slater C. R. Electrophysiology and electron-microscopy of rat neuromuscular junctions after nerve degeneration. Proc R Soc Lond B Biol Sci. 1968 Feb 27;169(1016):289–306. doi: 10.1098/rspb.1968.0012. [DOI] [PubMed] [Google Scholar]
  39. Nghiêm H. O., Cartaud J., Dubreuil C., Kordeli C., Buttin G., Changeux J. P. Production and characterization of a monoclonal antibody directed against the 43,000-dalton v1 polypeptide from Torpedo marmorata electric organ. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6403–6407. doi: 10.1073/pnas.80.20.6403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  41. Offer G., Moos C., Starr R. A new protein of the thick filaments of vertebrate skeletal myofibrils. Extractions, purification and characterization. J Mol Biol. 1973 Mar 15;74(4):653–676. doi: 10.1016/0022-2836(73)90055-7. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Salpeter M. M. Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. I. The distribution of acetylcholinesterase at motor end plates of a vertebrate twitch muscle. J Cell Biol. 1967 Feb;32(2):379–389. doi: 10.1083/jcb.32.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sanes J. R., Hall Z. W. Antibodies that bind specifically to synaptic sites on muscle fiber basal lamina. J Cell Biol. 1979 Nov;83(2 Pt 1):357–370. doi: 10.1083/jcb.83.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sanes J. R. Laminin, fibronectin, and collagen in synaptic and extrasynaptic portions of muscle fiber basement membrane. J Cell Biol. 1982 May;93(2):442–451. doi: 10.1083/jcb.93.2.442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sealock R. Cytoplasmic surface structure in postsynaptic membranes from electric tissue visualized by tannic-acid-mediated negative contrasting. J Cell Biol. 1982 Feb;92(2):514–522. doi: 10.1083/jcb.92.2.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sealock R., Paschal B., Beckerle M., Burridge K. Talin is a post-synaptic component of the rat neuromuscular junction. Exp Cell Res. 1986 Mar;163(1):143–150. doi: 10.1016/0014-4827(86)90566-5. [DOI] [PubMed] [Google Scholar]
  48. Sealock R. Visualization at the mouse neuromuscular junction of a submembrane structure in common with Torpedo postsynaptic membranes. J Neurosci. 1982 Jul;2(7):918–923. doi: 10.1523/JNEUROSCI.02-07-00918.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Shear C. R., Bloch R. J. Vinculin in subsarcolemmal densities in chicken skeletal muscle: localization and relationship to intracellular and extracellular structures. J Cell Biol. 1985 Jul;101(1):240–256. doi: 10.1083/jcb.101.1.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shear C. R., Sankey S. L., Resneck W. G., Bloch R. J. Heterogeneity of the chicken ALD muscle: evidence for a minor, twitch fiber type. Exp Neurol. 1984 Sep;85(3):506–522. doi: 10.1016/0014-4886(84)90027-x. [DOI] [PubMed] [Google Scholar]
  52. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  53. Steck T. L. The organization of proteins in the human red blood cell membrane. A review. J Cell Biol. 1974 Jul;62(1):1–19. doi: 10.1083/jcb.62.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Strader C. D., Lazarides E., Raftery M. A. The characterization of actin associated with postsynaptic membranes from Torpedo californica. Biochem Biophys Res Commun. 1980 Jan 29;92(2):365–373. doi: 10.1016/0006-291x(80)90342-3. [DOI] [PubMed] [Google Scholar]
  55. Strzelecka-Gołaszewska H., Próchniewicz E., Nowak E., Zmorzyński S., Drabikowski W. Chicken-gizzard actin: polymerization and stability. Eur J Biochem. 1980 Feb;104(1):41–52. doi: 10.1111/j.1432-1033.1980.tb04397.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES