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. 1984 Apr 1;98(4):1523–1536. doi: 10.1083/jcb.98.4.1523

Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton

PMCID: PMC2113240  PMID: 6425303

Abstract

Neurofilaments in the axons of mammalian spinal cord neurons are extensively cross-linked; consequently, the filaments and their cross- bridges compose a three-dimensional lattice. We have used antibody decoration in situ combined with tissue preparation by the quick- freeze, deep-etch technique to locate three neurofilament polypeptides (195, 145, and 73 Kd) within this lattice. When antibodies against each polypeptide were incubated with detergent-extracted, formaldehyde-fixed samples of rabbit spinal cord, each antibody assumed a characteristic distribution: anti-73-Kd decorated the neurofilament core uniformly, but not the cross-bridges; anti-145-Kd also decorated the core, but less uniformly; sometimes the anti-145-Kd antibodies were located over the bases of cross-bridges. In contrast, anti-195-Kd primarily decorated the cross-bridges between the neurofilaments. These observations show that the 73-Kd polypeptide is a component of the central core of neurofilaments, and that the 195-Kd polypeptide is a component of the inter-neurofilamentous cross-bridges. It is consistent with this conclusion that we found few cross-bridges between neurofilaments in the optic nerves of neonatal rabbits during a developmental period when the ratio of 195 to 73 or 145-Kd polypeptides is much lower than in adults. The ratio of 195-Kd polypeptide to the other two neurofilament polypeptides also appeared much lower in the cell bodies and dendrites than in axons of adult spinal cord neurons, when the dispositions of the three polypeptides were studied by immunofluorescence experiments. The cell bodies apparently contain neurofilaments composed primarily of 145- and 73-Kd polypeptides, because we observed antibody decoration of individual neurofilaments in the cell bodies with anti-73- and -145-Kd, but not with anti-195-Kd. We conclude that the 195-Kd polypeptide participates in a cross-linking function, and that this function is, at least in certain neurons, most prevalent in the mature axon.

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Selected References

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  1. Bloom G. S., Vallee R. B. Association of microtubule-associated protein 2 (MAP 2) with microtubules and intermediate filaments in cultured brain cells. J Cell Biol. 1983 Jun;96(6):1523–1531. doi: 10.1083/jcb.96.6.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ellisman M. H., Porter K. R. Microtrabecular structure of the axoplasmic matrix: visualization of cross-linking structures and their distribution. J Cell Biol. 1980 Nov;87(2 Pt 1):464–479. doi: 10.1083/jcb.87.2.464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Geisler N., Kaufmann E., Weber K. Proteinchemical characterization of three structurally distinct domains along the protofilament unit of desmin 10 nm filaments. Cell. 1982 Aug;30(1):277–286. doi: 10.1016/0092-8674(82)90033-2. [DOI] [PubMed] [Google Scholar]
  4. Geisler N., Weber K. Self-assembly in Vitro of the 68,000 molecular weight component of the mammalian neurofilament triplet proteins into intermediate-sized filaments. J Mol Biol. 1981 Sep 25;151(3):565–571. doi: 10.1016/0022-2836(81)90011-5. [DOI] [PubMed] [Google Scholar]
  5. Granger B. L., Lazarides E. Structural associations of synemin and vimentin filaments in avian erythrocytes revealed by immunoelectron microscopy. Cell. 1982 Aug;30(1):263–275. doi: 10.1016/0092-8674(82)90032-0. [DOI] [PubMed] [Google Scholar]
  6. Griffith L. M., Pollard T. D. Evidence for actin filament-microtubule interaction mediated by microtubule-associated proteins. J Cell Biol. 1978 Sep;78(3):958–965. doi: 10.1083/jcb.78.3.958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Heuser J. E. Procedure for freeze-drying molecules adsorbed to mica flakes. J Mol Biol. 1983 Sep 5;169(1):155–195. doi: 10.1016/s0022-2836(83)80179-x. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Hirokawa N., Cheney R. E., Willard M. Location of a protein of the fodrin-spectrin-TW260/240 family in the mouse intestinal brush border. Cell. 1983 Mar;32(3):953–965. doi: 10.1016/0092-8674(83)90080-6. [DOI] [PubMed] [Google Scholar]
  10. Hirokawa N. Cross-linker system between neurofilaments, microtubules, and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method. J Cell Biol. 1982 Jul;94(1):129–142. doi: 10.1083/jcb.94.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hirokawa N., Heuser J. E. Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells. J Cell Biol. 1981 Nov;91(2 Pt 1):399–409. doi: 10.1083/jcb.91.2.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hoffman P. N., Lasek R. J. The slow component of axonal transport. Identification of major structural polypeptides of the axon and their generality among mammalian neurons. J Cell Biol. 1975 Aug;66(2):351–366. doi: 10.1083/jcb.66.2.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Leterrier J. F., Liem R. K., Shelanski M. L. Interactions between neurofilaments and microtubule-associated proteins: a possible mechanism for intraorganellar bridging. J Cell Biol. 1982 Dec;95(3):982–986. doi: 10.1083/jcb.95.3.982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Mage M. G. Preparation of Fab fragments from IgGs of different animal species. Methods Enzymol. 1980;70(A):142–150. doi: 10.1016/s0076-6879(80)70045-9. [DOI] [PubMed] [Google Scholar]
  16. Matus A., Bernhardt R., Hugh-Jones T. High molecular weight microtubule-associated proteins are preferentially associated with dendritic microtubules in brain. Proc Natl Acad Sci U S A. 1981 May;78(5):3010–3014. doi: 10.1073/pnas.78.5.3010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Metuzals J., Montpetit V., Clapin D. F. Organization of the neurofilamentous network. Cell Tissue Res. 1981;214(3):455–482. doi: 10.1007/BF00233488. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Moon H. M., Wisniewski T., Merz P., De Martini J., Wisniewski H. M. Partial purification of neurofilament subunits from bovine brains and studies on neurofilament assembly. J Cell Biol. 1981 Jun;89(3):560–567. doi: 10.1083/jcb.89.3.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Peters A., Vaughn J. E. Microtubules and filaments in the axons and astrocytes of early postnatal rat optic nerves. J Cell Biol. 1967 Jan;32(1):113–119. doi: 10.1083/jcb.32.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schiff P. B., Fant J., Horwitz S. B. Promotion of microtubule assembly in vitro by taxol. Nature. 1979 Feb 22;277(5698):665–667. doi: 10.1038/277665a0. [DOI] [PubMed] [Google Scholar]
  22. Schlaepfer W. W. Studies on the isolation and substructure of mammalian neurofilaments. J Ultrastruct Res. 1977 Nov;61(2):149–157. doi: 10.1016/s0022-5320(77)80081-6. [DOI] [PubMed] [Google Scholar]
  23. Schnapp B. J., Reese T. S. Cytoplasmic structure in rapid-frozen axons. J Cell Biol. 1982 Sep;94(3):667–669. doi: 10.1083/jcb.94.3.667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sharp G. A., Shaw G., Weber K. Immunoelectronmicroscopical localization of the three neurofilament triplet proteins along neurofilaments of cultured dorsal root ganglion neurones. Exp Cell Res. 1982 Feb;137(2):403–413. doi: 10.1016/0014-4827(82)90042-8. [DOI] [PubMed] [Google Scholar]
  25. Shaw G., Osborn M., Weber K. An immunofluorescence microscopical study of the neurofilament triplet proteins, vimentin and glial fibrillary acidic protein within the adult rat brain. Eur J Cell Biol. 1981 Dec;26(1):68–82. [PubMed] [Google Scholar]
  26. Shaw G., Weber K. Differential expression of neurofilament triplet proteins in brain development. Nature. 1982 Jul 15;298(5871):277–279. doi: 10.1038/298277a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Smith D. S., Järlfors U., Cameron B. F. Morphological evidence for the participation of microtubules in axonal transport. Ann N Y Acad Sci. 1975 Jun 30;253:472–506. doi: 10.1111/j.1749-6632.1975.tb19223.x. [DOI] [PubMed] [Google Scholar]
  28. Sternberger L. A., Sternberger N. H. Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. Proc Natl Acad Sci U S A. 1983 Oct;80(19):6126–6130. doi: 10.1073/pnas.80.19.6126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tsukita S., Usukura J., Tsukita S., Ishikawa H. The cytoskeleton in myelinated axons: a freeze-etch replica study. Neuroscience. 1982;7(9):2135–2147. doi: 10.1016/0306-4522(82)90125-7. [DOI] [PubMed] [Google Scholar]
  30. Willard M. B. Genetically determined protein polymorphism in the rabbit nervous system. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3641–3645. doi: 10.1073/pnas.73.10.3641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Willard M., Simon C. Antibody decoration of neurofilaments. J Cell Biol. 1981 May;89(2):198–205. doi: 10.1083/jcb.89.2.198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Willard M., Simon C. Modulations of neurofilament axonal transport during the development of rabbit retinal ganglion cells. Cell. 1983 Dec;35(2 Pt 1):551–559. doi: 10.1016/0092-8674(83)90189-7. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Zackroff R. V., Idler W. W., Steinert P. M., Goldman R. D. In vitro reconstitution of intermediate filaments form mammalian neurofilament triplet polypeptides. Proc Natl Acad Sci U S A. 1982 Feb;79(3):754–757. doi: 10.1073/pnas.79.3.754. [DOI] [PMC free article] [PubMed] [Google Scholar]

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