Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1986 Mar 1;102(3):748–761. doi: 10.1083/jcb.102.3.748

Evidence for a polarity in the distribution of proteins from the cytoskeleton in Torpedo marmorata electrocytes

PMCID: PMC2114141  PMID: 2936752

Abstract

The subcellular distribution of the 43,000-D protein (43 kD or v1) and of some major cytoskeletal proteins was investigated in Torpedo marmorata electrocytes by immunocytochemical methods (immunofluorescence and immunogold at the electron microscope level) on frozen-fixed sections and homogenates of electric tissue. A monoclonal antibody directed against the 43-kD protein (Nghiem, H. O., J. Cartaud, C. Dubreuil, C. Kordeli, G. Buttin, and J. P. Changeux, 1983, Proc. Natl. Acad. Sci. USA, 80:6403-6407), selectively labeled the postsynaptic membrane on its cytoplasmic face. Staining by anti-actin and anti-desmin antibodies appeared evenly distributed within the cytoplasm: anti-desmin antibodies being associated with the network of intermediate-sized filaments that spans the electrocyte, and anti-actin antibodies making scattered clusters throughout the cytoplasm without preferential labeling of the postsynaptic membrane. On the other hand, a dense coating by anti-actin antibodies became apparent on the postsynaptic membrane in homogenates of electric tissue pointing to the possible artifactual redistribution of a soluble cytoplasmic actin pool. Anti-fodrin and anti-ankyrin antibodies selectively labeled the non-innervated membrane of the cell. F actin was also detected in this membrane. Filamin and vinculin, two actin-binding proteins recently localized at the rat neuromuscular junction (Bloch, R. J., and Z. W. Hall, 1983, J. Cell Biol., 97:217-223), were detected in the electrocyte by the immunoblot technique but not by immunocytochemistry. The data are interpreted in terms of the functional polarity of the electrocyte and of the selective interaction of the cytoskeleton with the innervated and non-innervated domains of the plasma membrane.

Full Text

The Full Text of this article is available as a PDF (2.2 MB).

Selected References

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

  1. Barrantes F. J., Mieskes G., Wallimann T. Creatine kinase activity in the Torpedo electrocyte and in the nonreceptor, peripheral v proteins from acetylcholine receptor-rich membranes. Proc Natl Acad Sci U S A. 1983 Sep;80(17):5440–5444. doi: 10.1073/pnas.80.17.5440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barrantes F. J., Neugebauer D. C., Zingsheim H. P. Peptide extraction by alkaline treatment is accompanied by rearrangement of the membrane-bound acetylcholine receptor from Torpedo marmorata. FEBS Lett. 1980 Mar 24;112(1):73–78. doi: 10.1016/0014-5793(80)80131-1. [DOI] [PubMed] [Google Scholar]
  3. Bartholdi M., Barrantes F. J., Jovin T. M. Rotational molecular dynamics of the membrane-bound acetylcholine receptor revealed by phosphorescence spectroscopy. Eur J Biochem. 1981 Nov;120(2):389–397. doi: 10.1111/j.1432-1033.1981.tb05716.x. [DOI] [PubMed] [Google Scholar]
  4. Bennett V., Davis J. Erythrocyte ankyrin: immunoreactive analogues are associated with mitotic structures in cultured cells and with microtubules in brain. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7550–7554. doi: 10.1073/pnas.78.12.7550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bennett V. Purification of an active proteolytic fragment of the membrane attachment site for human erythrocyte spectrin. J Biol Chem. 1978 Apr 10;253(7):2292–2299. [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Bloom F. E., Barrnett R. J. Fine structural localization of acetylcholinesterase in electroplaque of the electric eel. J Cell Biol. 1966 Jun;29(3):475–495. doi: 10.1083/jcb.29.3.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bourgeois J. -P., Ryter A., Menez A., Fromageot P., Boquet P., Changeux J. -P. Localization of the cholinergic receptor protein in Electrophorus electroplax by high resolution autoradiography. FEBS Lett. 1972 Sep 1;25(1):127–133. doi: 10.1016/0014-5793(72)80469-1. [DOI] [PubMed] [Google Scholar]
  10. Bourgeois J. P., Popot J. L., Ryter A., Changeux J. P. Quantitative studies on the localization of the cholinergic receptor protein in the normal and denervated electroplaque from Electrophorus electricus. J Cell Biol. 1978 Oct;79(1):200–216. doi: 10.1083/jcb.79.1.200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Burden S. J., Sargent P. B., McMahan U. J. Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve. J Cell Biol. 1979 Aug;82(2):412–425. doi: 10.1083/jcb.82.2.412. [DOI] [PMC free article] [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. Cartaud J., Sobel A., Rousselet A., Devaux P. F., Changeux J. P. Consequences of alkaline treatment for the ultrastructure of the acetylcholine-receptor-rich membranes from Torpedo marmorata electric organ. J Cell Biol. 1981 Aug;90(2):418–426. doi: 10.1083/jcb.90.2.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Changeux J. P. The acetylcholine receptor: an "allosteric" membrane protein. Harvey Lect. 1979 1980;75:85–254. [PubMed] [Google Scholar]
  16. Coudrier E., Reggio H., Louvard D. Characterization of an integral membrane glycoprotein associated with the microfilaments of pig intestinal microvilli. EMBO J. 1983;2(3):469–475. doi: 10.1002/j.1460-2075.1983.tb01446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dellagi K., Brouet J. C., Perreau J., Paulin D. Human monoclonal IgM with autoantibody activity against intermediate filaments. Proc Natl Acad Sci U S A. 1982 Jan;79(2):446–450. doi: 10.1073/pnas.79.2.446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fifková E., Delay R. J. Cytoplasmic actin in neuronal processes as a possible mediator of synaptic plasticity. J Cell Biol. 1982 Oct;95(1):345–350. doi: 10.1083/jcb.95.1.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Froehner S. C., Gulbrandsen V., Hyman C., Jeng A. Y., Neubig R. R., Cohen J. B. Immunofluorescence localization at the mammalian neuromuscular junction of the Mr 43,000 protein of Torpedo postsynaptic membranes. Proc Natl Acad Sci U S A. 1981 Aug;78(8):5230–5234. doi: 10.1073/pnas.78.8.5230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Giraudat J., Devillers-Thiery A., Perriard J. C., Changeux J. P. Complete nucleotide sequence of Torpedo marmorata mRNA coding for the 43,000-dalton nu 2 protein: muscle-specific creatine kinase. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7313–7317. doi: 10.1073/pnas.81.23.7313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Glenney J. R., Jr, Glenney P. Fodrin is the general spectrin-like protein found in most cells whereas spectrin and the TW protein have a restricted distribution. Cell. 1983 Sep;34(2):503–512. doi: 10.1016/0092-8674(83)90383-5. [DOI] [PubMed] [Google Scholar]
  24. Gysin R., Yost B., Flanagan S. D. Immunochemical and molecular differentiation of 43 000 molecular weight proteins associated with Torpedo neuroelectrocyte synapses. Biochemistry. 1983 Dec 6;22(25):5781–5789. doi: 10.1021/bi00294a016. [DOI] [PubMed] [Google Scholar]
  25. Hamilton S. L., McLaughlin M., Karlin A. Formation of disulfide-linked oligomers of acetylcholine receptor in membrane from torpedo electric tissue. Biochemistry. 1979 Jan 9;18(1):155–163. doi: 10.1021/bi00568a024. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. Klymkowsky M. W., Heuser J. E., Stroud R. M. Protease effects on the structure of acetylcholine receptor membranes from Torpedo californica. J Cell Biol. 1980 Jun;85(3):823–838. doi: 10.1083/jcb.85.3.823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. LUFT J. H. The fine structure of electric tissue. Exp Cell Res. 1958;14(Suppl 5):168–182. [PubMed] [Google Scholar]
  31. 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]
  32. Lazarides E., Nelson W. J. Expression of spectrin in nonerythroid cells. Cell. 1982 Dec;31(3 Pt 2):505–508. doi: 10.1016/0092-8674(82)90306-3. [DOI] [PubMed] [Google Scholar]
  33. Levine J., Willard M. Fodrin: axonally transported polypeptides associated with the internal periphery of many cells. J Cell Biol. 1981 Sep;90(3):631–642. doi: 10.1083/jcb.90.3.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lo M. M., Garland P. B., Lamprecht J., Barnard E. A. Rotational mobility of the membrane-bound acetylcholine receptor of Torpedo electric organ measured by phosphorescence depolarisation. FEBS Lett. 1980 Mar 10;111(2):407–412. doi: 10.1016/0014-5793(80)80838-6. [DOI] [PubMed] [Google Scholar]
  35. Matus A., Ackermann M., Pehling G., Byers H. R., Fujiwara K. High actin concentrations in brain dendritic spines and postsynaptic densities. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7590–7594. doi: 10.1073/pnas.79.23.7590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Maupin-Szamier P., Pollard T. D. Actin filament destruction by osmium tetroxide. J Cell Biol. 1978 Jun;77(3):837–852. doi: 10.1083/jcb.77.3.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Neubig R. R., Krodel E. K., Boyd N. D., Cohen J. B. Acetylcholine and local anesthetic binding to Torpedo nicotinic postsynaptic membranes after removal of nonreceptor peptides. Proc Natl Acad Sci U S A. 1979 Feb;76(2):690–694. doi: 10.1073/pnas.76.2.690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. Porter S., Froehner S. C. Interaction of the 43K protein with components of Torpedo postsynaptic membranes. Biochemistry. 1985 Jan 15;24(2):425–432. doi: 10.1021/bi00323a028. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Rosenbluth J. Synaptic membrane structure in Torpedo electric organ. J Neurocytol. 1975 Dec;4(6):697–712. doi: 10.1007/BF01181631. [DOI] [PubMed] [Google Scholar]
  45. Rousselet A., Cartaud J., Devaux P. F., Changeux J. P. The rotational diffusion of the acetylcholine receptor in Torpeda marmorata membrane fragments studied with a spin-labelled alpha-toxin: importance of the 43 000 protein(s). EMBO J. 1982;1(4):439–445. doi: 10.1002/j.1460-2075.1982.tb01188.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rousselet A., Cartaud J., Devaux P. F. Importance des interactions protéine-protéine dans les maintien de la structure des fragments excitables de l'organe électrique de Torpedo marmorata. C R Seances Acad Sci D. 1979 Sep 24;289(5):461–463. [PubMed] [Google Scholar]
  47. SCHOFFENIELS E. Ion movements studied with single isolated electroplax. Ann N Y Acad Sci. 1959 Aug 28;81:285–306. doi: 10.1111/j.1749-6632.1959.tb49314.x. [DOI] [PubMed] [Google Scholar]
  48. Saitoh T., Wennogle L. P., Changeux J. P. Factors regulating the susceptibility of the acetylcholine receptor protein to heat inactivation. FEBS Lett. 1979 Dec 15;108(2):489–494. doi: 10.1016/0014-5793(79)80595-5. [DOI] [PubMed] [Google Scholar]
  49. Sanes J. R., Chiu A. Y. The basal lamina of the neuromuscular junction. Cold Spring Harb Symp Quant Biol. 1983;48(Pt 2):667–678. doi: 10.1101/sqb.1983.048.01.070. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. 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]
  52. 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]
  53. 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]
  54. Singer S. J., Ash J. F., Bourguignon L. Y., Heggeness M. H., Louvard D. Transmembrane interactions and the mechanisms of transport of proteins across membranes. J Supramol Struct. 1978;9(3):373–389. doi: 10.1002/jss.400090308. [DOI] [PubMed] [Google Scholar]
  55. Sobel A., Heidmann T., Hofler J., Changeux J. P. Distinct protein components from Torpedo marmorata membranes carry the acetylcholine receptor site and the binding site for local anesthetics and histrionicotoxin. Proc Natl Acad Sci U S A. 1978 Jan;75(1):510–514. doi: 10.1073/pnas.75.1.510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sobel A., Weber M., Changeux J. P. Large-scale purification of the acetylcholine-receptor protein in its membrane-bound and detergent-extracted forms from Torpedo marmorata electric organ. Eur J Biochem. 1977 Oct 17;80(1):215–224. doi: 10.1111/j.1432-1033.1977.tb11874.x. [DOI] [PubMed] [Google Scholar]
  57. St John P. A., Froehner S. C., Goodenough D. A., Cohen J. B. Nicotinic postsynaptic membranes from Torpedo: sidedness, permeability to macromolecules, and topography of major polypeptides. J Cell Biol. 1982 Feb;92(2):333–342. doi: 10.1083/jcb.92.2.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. 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]
  59. 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]
  60. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Tyler J. M., Hargreaves W. R., Branton D. Purification of two spectrin-binding proteins: biochemical and electron microscopic evidence for site-specific reassociation between spectrin and bands 2.1 and 4.1. Proc Natl Acad Sci U S A. 1979 Oct;76(10):5192–5196. doi: 10.1073/pnas.76.10.5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Walker J. H., Boustead C. M., Witzemann V. The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein. EMBO J. 1984 Oct;3(10):2287–2290. doi: 10.1002/j.1460-2075.1984.tb02127.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wennogle L. P., Changeux J. P. Transmembrane orientation of proteins present in acetylcholine receptor-rich membranes from Torpedo marmorata studied by selective proteolysis. Eur J Biochem. 1980 May;106(2):381–393. doi: 10.1111/j.1432-1033.1980.tb04584.x. [DOI] [PubMed] [Google Scholar]
  64. Wieland T., Miura T., Seeliger A. Analogs of phalloidin. D-Abu2-Lys7-phalloin, an F-actin binding analog, its rhodamine conjugate (RLP) a novel fluorescent F-actin-probe, and D-Ala2-Leu7-phalloin, an inert peptide. Int J Pept Protein Res. 1983 Jan;21(1):3–10. [PubMed] [Google Scholar]

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

RESOURCES