Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1987 Jul 1;245(1):93–101. doi: 10.1042/bj2450093

Properties of highly viscous gels formed by neurofilaments in vitro. A possible consequence of a specific inter-filament cross-bridging.

J F Leterrier 1, J Eyer 1
PMCID: PMC1148086  PMID: 3663160

Abstract

Neurofilaments freshly isolated from bovine spinal cord form a reversible gel in vitro, consisting of nearly parallel and interlinked filaments organized in bundles. This phenomenon is obtained above a critical neurofilament concentration and is highly sensitive to denaturation. No gelation occurs with neurofilaments reconstituted from urea-solubilized subunits. The velocity of the gelation kinetics, optimum at a slightly acidic pH, is inhibited by low and high ionic strength and activated by millimolar concentrations of Mg2+ and other bivalent cations. No protein other than the purified neurofilament preparation itself (80-95% neurofilament triplet) is necessary for the formation of a gel. However, purified cytoskeletal proteins from microtubules and neurofilaments influence the viscosity of the native preparation. These observations suggest a reticulation in vitro between neurofilaments, dependent upon a fragile conformation of the polymers and possibly mediated through the high-Mr neurofilament subunits (200 kDa and 150 kDa). The significance of these results is discussed with regard to the inter-neurofilament cross-bridging in situ involving the 200 kDa subunit described by Hirokawa, Glicksman & Willard [(1984) J. Cell Biol. 98, 1523-1536].

Full text

PDF
93

Images in this article

Selected References

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

  1. Aamodt E. J., Williams R. C., Jr Microtubule-associated proteins connect microtubules and neurofilaments in vitro. Biochemistry. 1984 Dec 4;23(25):6023–6031. doi: 10.1021/bi00320a019. [DOI] [PubMed] [Google Scholar]
  2. Berkowitz S. A., Katagiri J., Binder H. K., Williams R. C., Jr Separation and characterization of microtubule proteins from calf brain. Biochemistry. 1977 Dec 13;16(25):5610–5617. doi: 10.1021/bi00644a035. [DOI] [PubMed] [Google Scholar]
  3. Brown P. A., Berlin R. D. Packing volume of sedimented microtubules: regulation and potential relationship to an intracellular matrix. J Cell Biol. 1985 Oct;101(4):1492–1500. doi: 10.1083/jcb.101.4.1492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carden M. J., Eagles P. A. Neurofilaments from ox spinal nerves. Isolation, disassembly, reassembly and cross-linking properties. Biochem J. 1983 Nov 1;215(2):227–237. doi: 10.1042/bj2150227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chiu F. C., Norton W. T. Bulk preparation of CNS cytoskeleton and the separation of individual neurofilament proteins by gel filtration: dye-binding characteristics and amino acid compositions. J Neurochem. 1982 Nov;39(5):1252–1260. doi: 10.1111/j.1471-4159.1982.tb12562.x. [DOI] [PubMed] [Google Scholar]
  6. Dahl D., Bignami A. Neurofilament phosphorylation in development. A sign of axonal maturation? Exp Cell Res. 1986 Jan;162(1):220–230. doi: 10.1016/0014-4827(86)90440-4. [DOI] [PubMed] [Google Scholar]
  7. Delacourte A., Filliatreau G., Boutteau F., Biserte G., Schrevel J. Study of the 10-nm-filament fraction isolated during the standard microtubule preparation. Biochem J. 1980 Nov 1;191(2):543–546. doi: 10.1042/bj1910543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Geisler N., Kaufmann E., Fischer S., Plessmann U., Weber K. Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins. EMBO J. 1983;2(8):1295–1302. doi: 10.1002/j.1460-2075.1983.tb01584.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Griffin J. W., Fahnestock K. E., Price D. L., Hoffman P. N. Microtubule-neurofilament segregation produced by beta, beta'-iminodipropionitrile: evidence for the association of fast axonal transport with microtubules. J Neurosci. 1983 Mar;3(3):557–566. doi: 10.1523/JNEUROSCI.03-03-00557.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Heimann R., Shelanski M. L., Liem R. K. Microtubule-associated proteins bind specifically to the 70-kDa neurofilament protein. J Biol Chem. 1985 Oct 5;260(22):12160–12166. [PubMed] [Google Scholar]
  12. 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]
  13. Hirokawa N., Glicksman M. A., Willard M. B. Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton. J Cell Biol. 1984 Apr;98(4):1523–1536. doi: 10.1083/jcb.98.4.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hoffman P. N., Griffin J. W., Gold B. G., Price D. L. Slowing of neurofilament transport and the radial growth of developing nerve fibers. J Neurosci. 1985 Nov;5(11):2920–2929. doi: 10.1523/JNEUROSCI.05-11-02920.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hoffman P. N., Griffin J. W., Price D. L. Control of axonal caliber by neurofilament transport. J Cell Biol. 1984 Aug;99(2):705–714. doi: 10.1083/jcb.99.2.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hoffman P. N., Thompson G. W., Griffin J. W., Price D. L. Changes in neurofilament transport coincide temporally with alterations in the caliber of axons in regenerating motor fibers. J Cell Biol. 1985 Oct;101(4):1332–1340. doi: 10.1083/jcb.101.4.1332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Julien J. P., Mushynski W. E. The distribution of phosphorylation sites among identified proteolytic fragments of mammalian neurofilaments. J Biol Chem. 1983 Mar 25;258(6):4019–4025. [PubMed] [Google Scholar]
  18. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  19. 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]
  20. Lasek R. J., Oblinger M. M., Drake P. F. Molecular biology of neuronal geometry: expression of neurofilament genes influences axonal diameter. Cold Spring Harb Symp Quant Biol. 1983;48(Pt 2):731–744. doi: 10.1101/sqb.1983.048.01.076. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Leterrier J. F., Wong J., Liem R. K., Shelanski M. L. Promotion of microtubule assembly by neurofilament-associated microtubule-associated proteins. J Neurochem. 1984 Nov;43(5):1385–1391. doi: 10.1111/j.1471-4159.1984.tb05398.x. [DOI] [PubMed] [Google Scholar]
  23. Liem R. K., Hutchison S. B. Purification of individual components of the neurofilament triplet: filament assembly from the 70 000-dalton subunit. Biochemistry. 1982 Jun 22;21(13):3221–3226. doi: 10.1021/bi00256a029. [DOI] [PubMed] [Google Scholar]
  24. Luca F. C., Bloom G. S., Vallee R. B. A monoclonal antibody that cross-reacts with phosphorylated epitopes on two microtubule-associated proteins and two neurofilament polypeptides. Proc Natl Acad Sci U S A. 1986 Feb;83(4):1006–1010. doi: 10.1073/pnas.83.4.1006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. MacLean-Fletcher S. D., Pollard T. D. Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors. J Cell Biol. 1980 May;85(2):414–428. doi: 10.1083/jcb.85.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Minami Y., Murofushi H., Sakai H. Interaction of tubulin with neurofilaments: formation of networks by neurofilament-dependent tubulin polymerization. J Biochem. 1982 Sep;92(3):889–898. doi: 10.1093/oxfordjournals.jbchem.a134003. [DOI] [PubMed] [Google Scholar]
  27. Minami Y., Sakai H. Effects of microtubule-associated proteins on network formation by neurofilament-induced polymerization of tubulin. FEBS Lett. 1986 Jan 20;195(1-2):68–72. doi: 10.1016/0014-5793(86)80132-6. [DOI] [PubMed] [Google Scholar]
  28. Minami Y., Sakai H. Network formation by neurofilament-induced polymerization of tubulin: 200K subunit of neurofilament triplet promotes nucleation of tubulin polymerization and enhances microtubule assembly. J Biochem. 1983 Dec;94(6):2023–2033. doi: 10.1093/oxfordjournals.jbchem.a134557. [DOI] [PubMed] [Google Scholar]
  29. Morris J. R., Lasek R. J. Stable polymers of the axonal cytoskeleton: the axoplasmic ghost. J Cell Biol. 1982 Jan;92(1):192–198. doi: 10.1083/jcb.92.1.192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nixon R. A., Logvinenko K. B. Multiple fates of newly synthesized neurofilament proteins: evidence for a stationary neurofilament network distributed nonuniformly along axons of retinal ganglion cell neurons. J Cell Biol. 1986 Feb;102(2):647–659. doi: 10.1083/jcb.102.2.647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Papasozomenos S. C., Yoon M., Crane R., Autilio-Gambetti L., Gambetti P. Redistribution of proteins of fast axonal transport following administration of beta,beta'-iminodipropionitrile: a quantitative autoradiographic study. J Cell Biol. 1982 Nov;95(2 Pt 1):672–675. doi: 10.1083/jcb.95.2.672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vallee R. B., Borisy G. G. Removal of the projections from cytoplasmic microtubules in vitro by digestion with trypsin. J Biol Chem. 1977 Jan 10;252(1):377–382. [PubMed] [Google Scholar]
  36. Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. 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]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES