Regulation of neurofilament interactionsin vitro by natural and synthetic polypeptides sharing Lys-Ser-Pro sequences with the heavy neurofilament subunit NF-H: Neurofilament crossbridging by antiparallel sidearm overlapping

  • J. P. Gou
  • T. Gotow
  • P. A. Janmey
  • J. F. Leterrier
Cellular Engineering


Neurofilaments are organised into parallel bundles in axons through cross-bridges formed by lateral projections of neurofilament subunits. Pure neurofilaments form gels in vitro, consisting of interconnected parallel arrays of filaments regulated by the phosphorylation level of neurofilament subunits. Neurofilament-associated polypeptides sharing phosphorylated epitopes with the repetitive lysine-serine-proline (Lys-Ser-Pro) motifs of the neurofilament heavy subunit sidearm are characterised: they regulate in vitro the neurofilament gelation kenetics in a concentration-and phosphorylation-dependent manner. Studies with synthetic peptides show that interactions between neurofilaments involve both acid and base amino acid residues of neurofilament sidearms and demonstrate the opposite effects of peptides containing either one (inhibition) or two (activation) Lys-Ser-Pro motifs. Electron microscopy reveals an organised network of native neurofilament sidearms, regulated by the phosphorylation level of neurofilament subunits, suggesting a structural transition between intra- and inter-neurofilament sidearm interactions. These results favour the hypothesis of a mechanism of neurofilament crossbridging through the variable antiparallel overlapping of the phosphorylable Lys-Ser-Pro domains of neurofilament sidearms from adjacent filaments, following an equilibrium regulated by neurofilament-associated proteins, bivalent cations and the phosphorylation level of Lys-Ser-Pro motifs from both neurofilament sidearms and neurofilament-associated proteins.


Neurofilaments Sidearms Crossbridging Antiparallel interactions Phosphorylation Associated Proteins Lysine-serine-proline motifs 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Anderton, B. H., Breinburg D., Downes, M. J., Green, P. J., Tomlinson, B. E., Ulrich, J., Wood, J. N. andKahn, J. (1982): ‘Monoclonal antibodies show that neurofibrillary tangles and neurofilaments share antigenic determinants,’Nature,298, pp. 84–86CrossRefGoogle Scholar
  2. Arima, K., Murayama, S., Mukoyama, M. andInose, T. (1992): ‘Immunocytochemical and ultrastructural studies of neuronal and oligodentroglial cytoplasmic inclusions in multiple system atrophy. 1 Neuronal cytoplasmic inclusions,’Acta Neuropathol. Berl.,83, pp. 453–460CrossRefGoogle Scholar
  3. Brown, A. andLasek, R. J. (1993): ‘Neurofilaments move apart freely when released from the circumferential constraint of the axonal plasma membrane,’Cell Motil. Cytoskel.,26, pp. 313–324CrossRefGoogle Scholar
  4. Brown, A. andLasek, R. J. (1995): ‘Polylysine cross-links axoplasmic neurofilaments into tight bundles,’Cell Motil. Cytoskel.,31, pp. 9–21CrossRefGoogle Scholar
  5. Carden, M. J., Schlaepfer, W. W. andLee, V. M.-Y. (1985): ‘The structure, biochemical properties and immunogenicity of neurofilament peripheral regioins are determined by phosphorylation state,’J. Biol. Chem.,260, pp. 9805–9817Google Scholar
  6. Ching, G. Y. andLiem, R. K. H. (1993): ‘Assembly of type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments,’J. Cell. Biol.,122, pp. 1232–1335CrossRefGoogle Scholar
  7. Chou, P. Y. andFasman, G. D. (1978): ‘Prediction of the secondary structure of proteins from their amino acid sequence,’Adv. Enzymol.,47, pp. 45–148Google Scholar
  8. Cleveland, D. W., Fisher, S. G., Kirschner, M. W. andLaemmli, U. K. (1977): ‘Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis,’J. Biol. Chem.,252, pp. 1102–1106Google Scholar
  9. Coleman, M. P. andAnderton, B. H. (1990): ‘Phosphate-dependent monoclonal antibodies to neurofilaments and Alzheimer neurofibrillary tangles recognize a synthetic phosphopeptide,’N. Neurochem.,54, pp. 1548–1555CrossRefGoogle Scholar
  10. Dautigny, A., Pham-dinh, D., Roussel, C., Felix, J. M., Nussbaum, J. L. andJolles, P. (1988): ‘The large neurofilament subunit (NF-H) of the rat: cDNA cloning andin situ detection,’Biochem. Biophys. Res. Commun.,154, pp. 1099–1106CrossRefGoogle Scholar
  11. Eyer, J. andLeterrier, J. F. (1988): ‘Influence of the phosphorylation state of neurofilament proteins on the interactions between purified filamentsin vitro,’Biochem. J.,252, pp. 655–660Google Scholar
  12. Eyer, J., Mclean, W. G. andLeterrier, J. F. (1989): ‘Effect of a single dose of ββ′-iminodipropionitrilein vivo on the properties of neurofilamentsin vitro: comparison with the effect of iminodipropionitrile added directly to neurofilamentsin vitro,’J. Neurochem.,52, pp. 1759–1765CrossRefGoogle Scholar
  13. Eyer, J. andPeterson, A. (1994): ‘Neurofilament-deficient axons and perikaryal aggregates in viable transgenic mice expressing a neurofilament-β-Galactosidase fusion protein,’Neuron,12, pp. 389–405CrossRefGoogle Scholar
  14. Figlewicz, D. A., Krizus, A., Martinoli, M. G., Meininger, V., Dib, G. A., Rouleau, M. andJulien, J. P. (1994): ‘Variants in the heavy neurofilament subunit are associated with the development of amyotrophic lateral sclerosis,’Hum. Mol. Gen.3, pp. 1757–1761Google Scholar
  15. Fliegner, K. H. andLiem, R. K. H. (1991): ‘Cellular and molecular biology of neuronal intermediate filaments,’Int. Rev. Cytol.,131, pp. 109–167CrossRefGoogle Scholar
  16. Fuchs, E. andWeber, K. (1994): ‘Intermediate filaments: structure, dynamics, function and disease,’Annu. Rev. Biochem.,63, pp. 345–382Google Scholar
  17. Galloay, P. G., Mulvihill, P. andPerry, G. (1992): ‘Filaments of Lewy bodies contain insoluble cytoskeletal elements,’Am. J. Pathol.,14, pp. 809–822Google Scholar
  18. Gotow, T., Takeda, M., Tanaka, T. andHashimoto, P. H. (1992): ‘Macromolecular structure of reassembled neurofilaments as revealed by the quick-freeze deep-etch mica method: Difference between NF-M and NF-H subunits in their ability to form cross-bridges,’Eur. J. Cell. Biol.,58, pp. 331–345Google Scholar
  19. Gotow, T. andTanaka, J. (1994): ‘Phosphorylation of neurofilament H subunit as related to arrangement of neurofilaments,’J Neurosci. Res.,37, pp. 691–713CrossRefGoogle Scholar
  20. Gotow, T., Tanaka, T., Nakamura, Y. andTakeda, M. (1994): ‘Dephosphorylation of the largest neurofilament subunit protein influences the structure of crossbridges in reassembled neurofilaments,’J. Cell. Sci.,107, pp. 1949–1957Google Scholar
  21. Gou, J. P., Eyer, J. andLeterrier, J. F. (1995): ‘Progressive hyperphosphorylation of neurofilament heavy subunits with aging: Possible involvement in the mechanism of neurofilament accumulation,’Biochem. Biophys. Res. Comm.,215, pp. 368–376CrossRefGoogle Scholar
  22. Guiroy, D. C., Shankar, S. K., Gibbs Jr, C. J.,Messenheimer, J. A., Das, S. andGajdusek, D. C. (1989): ‘Neuronal degeneration and neurofilament accumulation in the trigeminal ganglia in Creutzfeld-Jakob disease,’Ann. Neurol.,25, pp. 102–106CrossRefGoogle Scholar
  23. Hedreen, J. C. andKoliatsos, V. E. (1994): ‘Phosphorylated neurofilaments in neuronal pericarya and dendrites in human brain following axonal damage,’J. Neuropathol. Exp. Neurol.,53, pp. 663–671Google Scholar
  24. Heins, S., Wong, P. C., Müller, S., Goldie, K., Cleveland, D. W. andAebi, U. (1993): ‘The rod domain of NF-L determines neurofilament architecture whereas the end domains specify filament assembly and network formation,’J. Cell. Biol.,123, pp. 1517–1533CrossRefGoogle Scholar
  25. Hirokawa, N. (1982): ‘Cross-linker system between neurofilaments, microtubules and membranous organelles in frog axons revealed by the quick-freeze, deep-etching method,’J. Cell. Biol.,94, pp. 129–142CrossRefGoogle Scholar
  26. Hirokawa, N., Glicksman, M. A. andWillard, M. B. (1984): ‘Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton,’J. Cell. Biol.,98, pp. 1523–1536CrossRefGoogle Scholar
  27. Hirokawa, N., Hisanaga, S. andShiomura, Y. (1988): ‘MAP2 is a component of crossbridges between microtubules and neurofilamentsin vivo andin vitro. Quick-freeze, deep etch immunoelectron microscopy and reconstitution studies,’J. Neurosci.,8, pp. 2769–2779Google Scholar
  28. Hisanaga, S. andHirokawa, N. (1988): ‘Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing,’J. Mol. Biol.,202, pp. 297–305CrossRefGoogle Scholar
  29. Hisanaga, S. andHirokawa, N. (1989): ‘The effect of dephosphorylation on the structure of projections of neurofilaments,’J. Neurosci.,9, pp. 959–966Google Scholar
  30. Hisanaga, S., Matsuoka, Y., Nishizawa, K., Saito, T., Inagaki, M. andHirokawa, N. (1994): ‘Phosphorylation of native and reassembled neurofilaments composed of NF-L, NF-M and NF-H by the catalytic subunit of cAMP-dependent protein kinase,’Mol. Biol. Cell.,5, pp. 161–172Google Scholar
  31. Inagaki, M., Matsudoka, Y., Tsujimura, K., Ando, S., Tokui, T., Tahahashi, T. andInagaki, N. (1996): ‘Dynamic properties of intermediate filaments: regulation by phosphorylation,’Bioessays,18, pp. 481–487CrossRefGoogle Scholar
  32. Janmey, P. A., Euteneuer, U., Traub, P. andSchliwa, M. (1991): ‘Viscoelastic properties of vimentin compared with other filamentous biopolymer networks,’J. Cell. Biol.,113, pp. 1155–1160CrossRefGoogle Scholar
  33. Janmey, P. A., Hvidt, S., Kas, J., Lerche, D., Maggs, A., Sackmann, E., Schliwa, M. andStossel, T. P. (1994): ‘The mechanical properties of actin gels. Elastic modulus and filament motion,’J. Biol. Chem.,269, pp. 32503–32513Google Scholar
  34. Julien, J.-P. andMushinsky, W. E. (1983): ‘The distribution of phosphorylation sites among identified proteolytic fragments of mammalian neurofilaments,’J. Biol. Chem.,258, pp. 4019–4025Google Scholar
  35. Julien, J.-P., Smoluk, G. D. andMushinsky, W. E. (1983): ‘Characteristics of the protein kinase activity associated with rat neurofilament preparations,’Biochim. Biophys. Acta,755, pp. 25–31Google Scholar
  36. Klee, M. K., Xu, Z., Wong, P. C. andCleveland, D. W. (1993): ‘Neurofilaments are obligate heteropolymersin vivo,’J. Cell. Biol.,122, pp. 1337–1350CrossRefGoogle Scholar
  37. Kyte, J. andDoolittle, R. F. (1982): ‘A simple method for displaying the hydropathic character of a protein,’J. Molecular Biol.,157, pp. 105–132CrossRefGoogle Scholar
  38. Laemmli, U. K. (1970): ‘Cleavage of structural proteins during the assembly of the head of bacteriophage T4,’Nature,227, pp. 680–685CrossRefGoogle Scholar
  39. Langui, D., Probst, A., Anderton, B. H., Brion, J. P. andUlrich, J. (1990): ‘Aluminium-induced tangles in cultured rat neurones. Enhanced effect of aluminium by addition of maltol,’Acta Neuropathol.,80, pp. 649–655CrossRefGoogle Scholar
  40. Leterrier, J. F., Liem, R. K. H. andShelanski, M. L. (1982): ‘Interactions between neurofilaments and microtubule-associated proteins: a possible mechanism for intraorganellar bridging,’J. Cell. Biol.,95, pp. 982–986CrossRefGoogle Scholar
  41. Leterrier, J. F. andEyer, J. (1987): ‘Properties of highly viscous gels formed by neurofilamentsin vitro: a possible consequence of a specific inter-filament cross-bridging,’Biochem. J.,245, pp. 93–101Google Scholar
  42. Leterrier, J. F., Eyer, J., Weiss D. G. andLinden, M. (1991): ‘In vitro studies of the physical interactions between neurofilaments, microtubules and mitochondria isolated from the central nervous system,’inPaillotin, G. (Ed.): ‘The living cell in four dimensions.’Am. Inst. Phys. Conf. Proc.,226, Gif sur Yvette, France, pp. 91–105Google Scholar
  43. Leterrier, J. F., Langui, D., Probst, A. andUlrich, J. (1992): ‘A molecular mechanism for the induction of neurofilament bundling by aluminium ions,’J. Neurochem.,58, pp. 2060–2070CrossRefGoogle Scholar
  44. Leterrier, J. F., Langui, D., Probst, A. andUlrich, J. (1993): ‘A molecular mechanism of aluminium neurotoxicity (Matters arising: reply),’J. Neurochem.,60, pp. 385–387CrossRefGoogle Scholar
  45. Leterrier, J. F., Hartwig, J., Kas, J., Vegners, R. andJanmey, P. A. (1996): ‘Mechanical effects of neurofilament crossbridges: modulation by phosphorylation, lipids, and interactions with Factin,’J. Biol. Chem.,271, pp. 15687–15694CrossRefGoogle Scholar
  46. Lieberburg, I., Spinner, N., Snyder, S., Anderson, J., Goldgaber, D., Smulwitz, M., Carroll, Z., Emanuel, B. S., Breitner, J. andRubin, L. (1989): ‘Cloning of a cDNA encoding the rat high molecular weight neurofilament peptide (NF-H): Developmental and tissue expression in the rat and mapping of its human homologue to chromosomes 1 and 22,’Proc. Natl. Acad. Sci. USA,86, pp. 2463–2467CrossRefGoogle Scholar
  47. Lowry, O. H., Rosebrough, N. J., Farr, A. L. andRandall, R. J. (1951): ‘Protein measurement using the Folin phenol reagent,’J. Biol. Chem.,193, pp. 265–275Google Scholar
  48. MacLean-Fletcher, S. D. andPollard, T. D. (1980): ‘Viscosimetric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors,’J. Cell. Biol.,85, pp. 414–428CrossRefGoogle Scholar
  49. Mata, M., Kupina, M. andFink, D. J. (1992): ‘Phosphorylation-dependent neurofilament epitopes are reduced at the node of Ranvier,’J. Neurocytol.,21, pp. 199–210CrossRefGoogle Scholar
  50. Nagakawa, T., Chen, J., Zhang, Z., Kanai, Y. andHirokawa, N. (1995): ‘Two distinct functions of the carboxy-terminal tail domain of NF-M upon neurofilament assembly: cross-bridge formation and longitudinal elongation of filaments,’J. Cell. Biol.,129, pp. 411–429CrossRefGoogle Scholar
  51. Napolitano, E. W., Chin, S. S., Colman, D. R. andLiem, R. K. H. (1987): ‘Complete amino acid sequence andin vitro expression of rat NF-M, the middle molecular weight neurofilament protein,’J. Neurosci.,7, pp. 2590–2599Google Scholar
  52. Nixon, R. A. (1992): ‘Slow axonal transport,’Curr. Opin. Cell Biol.,4, pp. 8–14CrossRefGoogle Scholar
  53. Nixon, R. A. (1993): ‘The regulation of neurofilament protein dynamics by phosphorylation: Clues to neurofibrillary pathobiology,’Brain Pathol.,3, pp. 29–38Google Scholar
  54. Nixon, R. A., Paskevich, P. A., Sihag, R. K. andThayer, C. Y. (1994): ‘Phosphorylation on carboxyl terminus domains of neurofilament proteins in retinal ganglion cell neuronsin vivo: Influence on regional neurofilament accumulation, interneurofilament spacing, and axon caliber,’J. Cell. Biol.,126, pp. 1031–1046CrossRefGoogle Scholar
  55. Okabe, S., Miyasaka, H. andHirokawa, N. (1993): ‘Dynamics of the neuronal intermediate filament,’J. Cell. Biol.,121, pp. 375–386CrossRefGoogle Scholar
  56. Papasozomenos, S. C., Binder, L. I., Bender, P. K. andPayne, M. R. (1985): ‘Microtubule-associated protein 2 within axon of spinal motor neurons: association with microtubules and neurofilament in normal and β,β′-iminodipropionotile treated axons,’J. Cell Biol.,100, pp. 74–85CrossRefGoogle Scholar
  57. Probst, A., Langui, D., Lautenschlager, C., Ulrich, J., Brion, J. P. andAnderton, B. H. (1988): ‘Progressive supranuclear palsy: extensive neuropil threads in addition to neurofibrillary tangles. Very similar antigenicity of subcortical neuronal pathology in progressive nuclear palsy and Alzheimer’s disease,’Acta Neuropathol. Berl.,77, pp. 61–68CrossRefGoogle Scholar
  58. Schagger, H. andvon Jagow, G. (1987): ‘Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100kDa,’Anal. Biochem.,166, pp. 368–379CrossRefGoogle Scholar
  59. Shaw, G. andHou, Z. (1990): ‘Bundling and cross-linking of intermediate filaments of the nervous system,’J. Neurosci. Res.,25, pp. 561–568CrossRefGoogle Scholar
  60. Shaw, G. (1991): ‘Neurofilaments proteins,’inBurgoyne, R. D. (Ed.), ‘The neuronal cytoskeleton’ (Wiley-Liss, New York) pp. 186–213Google Scholar
  61. Shea, T. B. andBeermann, M. L. (1994a): ‘Respective roles of neurofilaments, microtubules, MAP1B, and tau in neurite out-growth and stabilization,’Mol. Biol. Cell.,5, pp. 863–875Google Scholar
  62. Shea, T. B. andBeermann, M. L. (1994b): ‘Multiple interactions of aluminium with neurofilament subunits: regulation by phosphate-dependent interactions between C-terminal extensions of the high and middle molecular weight subunits,’J. Neurosci. Res.,38, pp. 160–166CrossRefGoogle Scholar
  63. Shetty, K. T., Link, W. T. andPant, H. C. (1993): ‘Cdc2-like kinase from rat spinal cord specificity phosphorylates KSPXK motifs in neurofilament proteins. Isolation and characterization,’Proc. Natl. Acad. Sci. USA,90, pp. 6844–6848CrossRefGoogle Scholar
  64. Shetty, V. K., Link, W. T., Jaffe, H., Wang, J. andPant, H. C. (1995): ‘Neuronal cyclin-dependent kinase-5 phosphorylation sites in neurofilament protein (NF-H) are dephosphorylated by protein phosphatase 2a,’J. Neurochem.,64, pp. 2681–2690Google Scholar
  65. Smith, M. A., Rudnicka-Nawrot, Richey, P. L., Praprotnik, D., Mulvihill, P., Miller, C. A., Sayre, L. M. andPerry, G. (1995): ‘Carbonyl-related posttranslational modification of neurofilament protein in the neurofibrillary pathology of Alzheimer’s disease,’J. Neurochem.,64, pp. 2660–2666CrossRefGoogle Scholar
  66. Sobue, S., Hashizume, Y. Yasuda, T., Mukai, E., Kumagai, T., Mitsuma, T. andTrojanowski, J. Q. (1990): ‘Phosphorylated high molecular weight neurofilament protein in lower motor neurons in amyotrophic lateral sclerosis and other neurodegenerative diseases involving ventral horn cells,’Acta Neuropathol.,79, pp. 402–408CrossRefGoogle Scholar
  67. Takahashi, M., Amin, N., Grant, P. andPant, H. C. (1995): ‘P13sucl associates with a cdc2-like kinase in a multimeric cytoskeletal complex in squid axoplams,’J. Neurosci.,15, pp. 6222–6229Google Scholar
  68. Toru-Delbauffe, D. andPierre, M. (1983): ‘A rat brain protein kinase phosphorylating specifically neurofilaments,’FEBS Lett.,162, pp. 230–234CrossRefGoogle Scholar
  69. Toru-Delfauffe, D., Pierre, M., Osty, J., Chantoux, F. andFrancon, J. (1986): ‘Properties of neurofilament protein kinase,’Biochem. J.,235, pp. 283–289Google Scholar
  70. Towbin, H., Staehelin, T. andGordon, J. (1979): ‘Electrophoresis transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications,’Proc. Natl. Acad. Sci. USA,76, pp. 4350–4354CrossRefGoogle Scholar
  71. Tyler, J. M. andBranton, D. (1980): ‘Rotary shadowing of extended molecules dried from glycerol,’J. Ultrastruct. Res.,71, pp. 95–102CrossRefGoogle Scholar
  72. Vickers, J. C., Delacourte, A. andMorrison, J. H. (1992): ‘Progressive transformation of the cytoskeleton associated with normal aging andAlzheimer’s disease,’Brain Res.,594, pp. 273–278CrossRefGoogle Scholar
  73. Vickers, J. C., Riederer, B. M., Marugg, R. A., Buee-Scherrer, V., Buee, L., Delacourte, A. andMorrison, J. H. (1994): ‘Alterations in neurofilament protein immunoreactivity in human hippocampal neurons related to normal aging and Alzheimer’s disease,’Neurosci.,62, pp. 1–43CrossRefGoogle Scholar
  74. Willard, M. andSimon, C. (1981): ‘Antibody decoration of neurofilaments,’J. Cell Biol.,89, pp. 198–205CrossRefGoogle Scholar
  75. Wood, J. N. andAnderton, B. H. (1981): ‘Monoclonal antibodies to mammalian neurofilaments,’Biosci. Rep.,1, pp. 263–268CrossRefGoogle Scholar

Copyright information

© IFMBE 1998

Authors and Affiliations

  • J. P. Gou
    • 1
  • T. Gotow
    • 2
  • P. A. Janmey
    • 3
  • J. F. Leterrier
    • 1
  1. 1.U298 InsermCHRUAngersFrance
  2. 2.Department of Anatomy I & Cell BiologyOsaka University Medical SchoolOsakaJapan
  3. 3.Department of Medicine, Brigham & Women’s HospitalHarvard Medical SchoolBostonUSA

Personalised recommendations