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. 1978 Aug 1;78(2):597–621. doi: 10.1083/jcb.78.2.597

Subaxolemmal filamentous network in the giant nerve fiber of the squid (Loligo pealei L.) and its possible role in excitability

PMCID: PMC2110117  PMID: 690181

Abstract

A new technique utilizing the squid giant nerve fiber has been developed which permits direct examination of the inner face of the axolemma by scanning electron microscopy. The axoplasm was removed sequentially in a 15-mm long segment of the fiber by intracellular perfusion with a solution of KF, KCl, Ca++-containing seawater, or with pronase. The action potential of the fibers was monitored during these treatments. After brief prefixation in 1% paraformaldehyde and 1% glutaraldehyde, the perfused segment was opened by a lne could be related to information on the detailed morphology of the cytoplasmic face of the axolemma and the ectoplasm. The results obtained by scanning electron microscopy were further substantiated by transmission electron microscopy of thin sections. In addition, living axons were studied with polarized light during axoplasm removal, and the identification of actin by heavy meromyosin labeling and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was accomplished. These observations demonstrate that a three-dimensional network of interwoven filaments, consisting partly of an actinlike protein, is firmly attached to the axolemma. The axoplasmic face of fibers in which the filaments have been removed partially after perfusion with pronase displays smooth membranous blebs and large profiles which sppose the axolemma. In fibers where the excitability has been suppressed by pronase perfusion, approximately one-third of the inner face of the axolemma in the perfusion zone is free of filaments. It is hypothesized that the attachment of axoplasm filaments to the axolemma may have a role in the maintenance of the normal morphology of the axolemma, and, thus, in some aspect of excitability.

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Selected References

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  1. Armstrong C. M., Bezanilla F., Rojas E. Destruction of sodium conductance inactivation in squid axons perfused with pronase. J Gen Physiol. 1973 Oct;62(4):375–391. doi: 10.1085/jgp.62.4.375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ash J. F., Singer S. J. Concanavalin-A-induced transmembrane linkage of concanavalin A surface receptors to intracellular myosin-containing filaments. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4575–4579. doi: 10.1073/pnas.73.12.4575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BAKER P. F., HODGKIN A. L., SHAW T. I. Replacement of the axoplasm of giant nerve fibres with artificial solutions. J Physiol. 1962 Nov;164:330–354. doi: 10.1113/jphysiol.1962.sp007025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buckley I. K. Three dimensional fine structure of cultured cells: possible implications for subcellular motility. Tissue Cell. 1975;7(1):51–72. doi: 10.1016/s0040-8166(75)80007-3. [DOI] [PubMed] [Google Scholar]
  5. Burton P. R., Kirkland W. L. Actin detected in mouse neuroblastoma cells by binding of heavy meromyosin. Nat New Biol. 1972 Oct 25;239(95):244–246. doi: 10.1038/newbio239244a0. [DOI] [PubMed] [Google Scholar]
  6. COSTERO I., POMERAT C. M. Cultivation of neurons from the adult human cerebral and cerebellar cortes. Am J Anat. 1951 Nov;89(3):405–467. doi: 10.1002/aja.1000890304. [DOI] [PubMed] [Google Scholar]
  7. Chang C. M., Goldman R. D. The localization of actin-like fibers in cultured neuroblastoma cells as revealed by heavy meromyosin binding. J Cell Biol. 1973 Jun;57(3):867–874. doi: 10.1083/jcb.57.3.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clarke M., Schatten G., Mazia D., Spudich J. A. Visualization of actin fibers associated with the cell membrane in amoebae of Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1975 May;72(5):1758–1762. doi: 10.1073/pnas.72.5.1758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Elgsaeter A., Branton D. Intramembrane particle aggregation in erythrocyte ghosts. I. The effects of protein removal. J Cell Biol. 1974 Dec;63(3):1018–1036. doi: 10.1083/jcb.63.3.1018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ellisman M. H., Rash J. E., Staehelin L. A., Porter K. R. Studies of excitable membranes. II. A comparison of specializations at neuromuscular junctions and nonjunctional sarcolemmas of mammalian fast and slow twitch muscle fibers. J Cell Biol. 1976 Mar;68(3):752–774. doi: 10.1083/jcb.68.3.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fine R. E., Bray D. Actin in growing nerve cells. Nat New Biol. 1971 Nov 24;234(47):115–118. doi: 10.1038/newbio234115a0. [DOI] [PubMed] [Google Scholar]
  12. Geren B. B., Schmitt F. O. THE STRUCTURE OF THE SCHWANN CELL AND ITS RELATION TO THE AXON IN CERTAIN INVERTEBRATE NERVE FIBERS. Proc Natl Acad Sci U S A. 1954 Sep;40(9):863–870. doi: 10.1073/pnas.40.9.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gilbert D. S., Newby B. J. Neurofilament disguise, destruction and discipline. Nature. 1975 Aug 14;256(5518):586–589. doi: 10.1038/256586a0. [DOI] [PubMed] [Google Scholar]
  14. HODGKIN A. L., KATZ B. The effect of calcium on the axoplasm of giant nerve fibers. J Exp Biol. 1949 Oct;26(3):292-4, pl. doi: 10.1242/jeb.26.3.292. [DOI] [PubMed] [Google Scholar]
  15. Inoue I., Pant H. C., Tasaki I., Gainer H. Release of proteins from the inner surface of squid axon membrane labeled with tritiated N-ethylmaleimide. J Gen Physiol. 1976 Oct;68(4):385–395. doi: 10.1085/jgp.68.4.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ishikawa H., Bischoff R., Holtzer H. Formation of arrowhead complexes with heavy meromyosin in a variety of cell types. J Cell Biol. 1969 Nov;43(2):312–328. [PMC free article] [PubMed] [Google Scholar]
  17. LOWEY S., COHEN C. Studies on the structure of myosin. J Mol Biol. 1962 Apr;4:293–308. doi: 10.1016/s0022-2836(62)80007-2. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. LeBeux Y. J., Willemot J. An ultrastructural study of the microfilaments in rat brain by means of heavy meromyosin labeling. I. The perikaryon, the dendrites and the axon. Cell Tissue Res. 1975 Jun 27;160(1):1–36. doi: 10.1007/BF00219840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. MOMMAERTS W. F. H. M., PARRISH R. G. Studies on myosin. I. Preparation and criteria of purity. J Biol Chem. 1951 Feb;188(2):545–552. [PubMed] [Google Scholar]
  21. Marchesi V. T., Steers E., Jr Selective solubilization of a protein component of the red cell membrane. Science. 1968 Jan 12;159(3811):203–204. doi: 10.1126/science.159.3811.203. [DOI] [PubMed] [Google Scholar]
  22. Metuzals J. Configuration of a filamentous network in the axoplasm of the squid (Loligo pealii L.) giant nerve fiber. J Cell Biol. 1969 Dec;43(3):480–505. doi: 10.1083/jcb.43.3.480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Metuzals J., Izzard C. S. Spatial patterns of threadlike elements in the axoplasm of the giant nerve fiber of the squid (Loligo pealii L.) as disclosed by differential interference microscopy and by electron microscopy. J Cell Biol. 1969 Dec;43(3):456–479. doi: 10.1083/jcb.43.3.456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Metuzals J., Mushynski W. E. Electron microscope and experimental investigations of the neurofilamentous network in Deiters' neurons. Relationship with the cell surface and nuclear pores. J Cell Biol. 1974 Jun;61(3):701–722. doi: 10.1083/jcb.61.3.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mooseker M. S., Tilney L. G. Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells. J Cell Biol. 1975 Dec;67(3):725–743. doi: 10.1083/jcb.67.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nicolson G. L., Marchesi V. T., Singer S. J. The localization of spectrin on the inner surface of human red blood cell membranes by ferritin-conjugated antibodies. J Cell Biol. 1971 Oct;51(1):265–272. doi: 10.1083/jcb.51.1.265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nicolson G. L. Transmembrane control of the receptors on normal and tumor cells. I. Cytoplasmic influence over surface components. Biochim Biophys Acta. 1976 Apr 13;457(1):57–108. doi: 10.1016/0304-4157(76)90014-9. [DOI] [PubMed] [Google Scholar]
  28. Painter R. G., Sheetz M., Singer S. J. Detection and ultrastructural localization of human smooth muscle myosin-like molecules in human non-muscle cells by specific antibodies. Proc Natl Acad Sci U S A. 1975 Apr;72(4):1359–1363. doi: 10.1073/pnas.72.4.1359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pollard T. D., Korn E. D. Electron microscopic identification of actin associated with isolated amoeba plasma membranes. J Biol Chem. 1973 Jan 25;248(2):448–450. [PubMed] [Google Scholar]
  30. Pollard T. D., Shelton E., Weihing R. R., Korn E. D. Ultrastructural characterization of F-actin isolated from Acanthamoeba castellanii and identification of cytoplasmic filaments as F-actin by reaction with rabbit heavy meromyosin. J Mol Biol. 1970 May 28;50(1):91–97. doi: 10.1016/0022-2836(70)90106-3. [DOI] [PubMed] [Google Scholar]
  31. Pollard T. D., Weihing R. R. Actin and myosin and cell movement. CRC Crit Rev Biochem. 1974 Jan;2(1):1–65. doi: 10.3109/10409237409105443. [DOI] [PubMed] [Google Scholar]
  32. Porter K., Prescott D., Frye J. Changes in surface morphology of Chinese hamster ovary cells during the cell cycle. J Cell Biol. 1973 Jun;57(3):815–836. doi: 10.1083/jcb.57.3.815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. 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]
  34. Reynolds J. A., Trayer H. Solubility of membrane proteins in aqueous media. J Biol Chem. 1971 Dec 10;246(23):7337–7342. [PubMed] [Google Scholar]
  35. Röhlich P. Membrane-associated actin filaments in the cortical cytoplasm of the rat mast cell. Exp Cell Res. 1975 Jul;93(2):293–298. doi: 10.1016/0014-4827(75)90453-x. [DOI] [PubMed] [Google Scholar]
  36. Sato H., Tasaki E., Carbone E., Hallett M. Changes in axon birefringence associated with excitation: implications for the structure of the axon membrane. J Mechanochem Cell Motil. 1973;2(3):209–217. [PubMed] [Google Scholar]
  37. Schekman R., Singer S. J. Clustering and endocytosis of membrane receptors can be induced in mature erythrocytes of neonatal but not adult humans. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4075–4079. doi: 10.1073/pnas.73.11.4075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. See Y. P., Metuzals J. Purification and characterization of squid brain myosin. J Biol Chem. 1976 Dec 10;251(23):7682–7689. [PubMed] [Google Scholar]
  39. Singer S. J. Molecular biology of cellular membranes with applications to immunology. Adv Immunol. 1974;19(0):1–66. doi: 10.1016/s0065-2776(08)60251-5. [DOI] [PubMed] [Google Scholar]
  40. Singer S. J. The molecular organization of membranes. Annu Rev Biochem. 1974;43(0):805–833. doi: 10.1146/annurev.bi.43.070174.004105. [DOI] [PubMed] [Google Scholar]
  41. Spudich J. A. Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. II. Purification, properties, and membrane association of actin from amoebae of Dictyostelium discoideum. J Biol Chem. 1974 Sep 25;249(18):6013–6020. [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Steck T. L., Weinstein R. S., Straus J. H., Wallach D. F. Inside-out red cell membrane vesicles: preparation and purification. Science. 1970 Apr 10;168(3928):255–257. doi: 10.1126/science.168.3928.255. [DOI] [PubMed] [Google Scholar]
  45. Tasaki I., Watanabe A., Hallett M. Properties of squid axon membrane as revealed by a hydrophobic probe, 2-p-toluidinylnaphthalene-6-sulfonate. Proc Natl Acad Sci U S A. 1971 May;68(5):938–941. doi: 10.1073/pnas.68.5.938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vial J. D., Garrido J. Actin-like filaments amd membrane rearrangement in oxyntic cells. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4032–4036. doi: 10.1073/pnas.73.11.4032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Villegas J. Axon-Schwann cell interaction in the squid nerve fibre. J Physiol. 1972 Sep;225(2):275–296. doi: 10.1113/jphysiol.1972.sp009940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  49. Wickus G., Gruenstein E., Robbins P. W., Rich A. Decrease in membrane-associated actin of fibroblasts after transformation by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1975 Feb;72(2):746–749. doi: 10.1073/pnas.72.2.746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Woodrum D. T., Rich S. A., Pollard T. D. Evidence for biased bidirectional polymerization of actin filaments using heavy meromyosin prepared by an improved method. J Cell Biol. 1975 Oct;67(1):231–237. doi: 10.1083/jcb.67.1.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Yamada K. M., Spooner B. S., Wessells N. K. Ultrastructure and function of growth cones and axons of cultured nerve cells. J Cell Biol. 1971 Jun;49(3):614–635. doi: 10.1083/jcb.49.3.614. [DOI] [PMC free article] [PubMed] [Google Scholar]

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