Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Apr 2;121(2):375–386. doi: 10.1083/jcb.121.2.375

Dynamics of the neuronal intermediate filaments

PMCID: PMC2200102  PMID: 8468352

Abstract

We have analyzed the dynamics of neuronal intermediate filaments in living neurons by using the method of photobleaching of fluorescently- labeled neurofilament L protein and immunoelectron microscopy of incorporation sites of biotinylated neurofilament L protein. Low-light- level imaging and photobleaching of growing axons of mouse sensory neurons did not affect the rate of either axonal growth or the addition of intermediate filament structures at the axon terminal, suggesting that any perturbations caused by these optical methods would be minimal. After laser photobleaching, recovery of fluorescence did occur slowly with a recovery half-time of 40 min. Furthermore, we observed a more rapid fluorescence recovery in growing axons than in quiescent ones, indicating a growth-dependent regulation of the turnover rate. Incorporation sites of biotin-labeled neurofilament L protein were localized as numerous discrete sites along the axon, and they slowly elongated to become continuous arrays 24 h after injection. Collectively, these results indicate that neuronal intermediate filaments in growing axons turn over within the small area of the axoplasm possibly by the mechanism of lateral and segmental incorporation of new subunits.

Full Text

The Full Text of this article is available as a PDF (3.5 MB).

Selected References

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

  1. Albers K., Fuchs E. The expression of mutant epidermal keratin cDNAs transfected in simple epithelial and squamous cell carcinoma lines. J Cell Biol. 1987 Aug;105(2):791–806. doi: 10.1083/jcb.105.2.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Angelides K. J., Smith K. E., Takeda M. Assembly and exchange of intermediate filament proteins of neurons: neurofilaments are dynamic structures. J Cell Biol. 1989 Apr;108(4):1495–1506. doi: 10.1083/jcb.108.4.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black M. M., Keyser P., Sobel E. Interval between the synthesis and assembly of cytoskeletal proteins in cultured neurons. J Neurosci. 1986 Apr;6(4):1004–1012. doi: 10.1523/JNEUROSCI.06-04-01004.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chin S. S., Liem R. K. Expression of rat neurofilament proteins NF-L and NF-M in transfected non-neuronal cells. Eur J Cell Biol. 1989 Dec;50(2):475–490. [PubMed] [Google Scholar]
  5. Geisler N., Plessmann U., Weber K. The complete amino acid sequence of the major mammalian neurofilament protein (NF-L). FEBS Lett. 1985 Mar 25;182(2):475–478. doi: 10.1016/0014-5793(85)80357-4. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Glass J. D., Griffin J. W. Neurofilament redistribution in transected nerves: evidence for bidirectional transport of neurofilaments. J Neurosci. 1991 Oct;11(10):3146–3154. doi: 10.1523/JNEUROSCI.11-10-03146.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gorbsky G. J., Sammak P. J., Borisy G. G. Microtubule dynamics and chromosome motion visualized in living anaphase cells. J Cell Biol. 1988 Apr;106(4):1185–1192. doi: 10.1083/jcb.106.4.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guan R. J., Hall F. L., Cohlberg J. A. Proline-directed protein kinase (p34cdc2/p58cyclin A) phosphorylates bovine neurofilaments. J Neurochem. 1992 Apr;58(4):1365–1371. doi: 10.1111/j.1471-4159.1992.tb11351.x. [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., 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]
  12. Hisanaga S., Gonda Y., Inagaki M., Ikai A., Hirokawa N. Effects of phosphorylation of the neurofilament L protein on filamentous structures. Cell Regul. 1990 Jan;1(2):237–248. doi: 10.1091/mbc.1.2.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hisanaga S., Hirokawa N. Molecular architecture of the neurofilament. II. Reassembly process of neurofilament L protein in vitro. J Mol Biol. 1990 Feb 20;211(4):871–882. doi: 10.1016/0022-2836(90)90080-6. [DOI] [PubMed] [Google Scholar]
  14. Hisanaga S., Hirokawa N. Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing. J Mol Biol. 1988 Jul 20;202(2):297–305. doi: 10.1016/0022-2836(88)90459-7. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Hollenbeck P. J. The transport and assembly of the axonal cytoskeleton. J Cell Biol. 1989 Feb;108(2):223–227. doi: 10.1083/jcb.108.2.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lasek R. J. Polymer sliding in axons. J Cell Sci Suppl. 1986;5:161–179. doi: 10.1242/jcs.1986.supplement_5.10. [DOI] [PubMed] [Google Scholar]
  18. Lawson S. N., Harper A. A., Harper E. I., Garson J. A., Anderton B. H. A monoclonal antibody against neurofilament protein specifically labels a subpopulation of rat sensory neurones. J Comp Neurol. 1984 Sep 10;228(2):263–272. doi: 10.1002/cne.902280211. [DOI] [PubMed] [Google Scholar]
  19. Lewis S. A., Cowan N. J. Genetics, evolution, and expression of the 68,000-mol-wt neurofilament protein: isolation of a cloned cDNA probe. J Cell Biol. 1985 Mar;100(3):843–850. doi: 10.1083/jcb.100.3.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lim S. S., Edson K. J., Letourneau P. C., Borisy G. G. A test of microtubule translocation during neurite elongation. J Cell Biol. 1990 Jul;111(1):123–130. doi: 10.1083/jcb.111.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lu X., Lane E. B. Retrovirus-mediated transgenic keratin expression in cultured fibroblasts: specific domain functions in keratin stabilization and filament formation. Cell. 1990 Aug 24;62(4):681–696. doi: 10.1016/0092-8674(90)90114-t. [DOI] [PubMed] [Google Scholar]
  22. McCormick M. B., Coulombe P. A., Fuchs E. Sorting out IF networks: consequences of domain swapping on IF recognition and assembly. J Cell Biol. 1991 Jun;113(5):1111–1124. doi: 10.1083/jcb.113.5.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miller R. K., Vikstrom K., Goldman R. D. Keratin incorporation into intermediate filament networks is a rapid process. J Cell Biol. 1991 May;113(4):843–855. doi: 10.1083/jcb.113.4.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mitchison T. J. Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence. J Cell Biol. 1989 Aug;109(2):637–652. doi: 10.1083/jcb.109.2.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Monteiro M. J., Cleveland D. W. Expression of NF-L and NF-M in fibroblasts reveals coassembly of neurofilament and vimentin subunits. J Cell Biol. 1989 Feb;108(2):579–593. doi: 10.1083/jcb.108.2.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Nakamura Y., Takeda M., Angelides K. J., Tada K., Hariguchi S., Nishimura T. Assembly, disassembly, and exchange of glial fibrillary acidic protein. Glia. 1991;4(1):101–110. doi: 10.1002/glia.440040112. [DOI] [PubMed] [Google Scholar]
  28. Ngai J., Coleman T. R., Lazarides E. Localization of newly synthesized vimentin subunits reveals a novel mechanism of intermediate filament assembly. Cell. 1990 Feb 9;60(3):415–427. doi: 10.1016/0092-8674(90)90593-4. [DOI] [PubMed] [Google Scholar]
  29. Nixon R. A., Lewis S. E. Differential turnover of phosphate groups on neurofilament subunits in mammalian neurons in vivo. J Biol Chem. 1986 Dec 15;261(35):16298–16301. [PubMed] [Google Scholar]
  30. Nixon R. A., Sihag R. K. Neurofilament phosphorylation: a new look at regulation and function. Trends Neurosci. 1991 Nov;14(11):501–506. doi: 10.1016/0166-2236(91)90062-y. [DOI] [PubMed] [Google Scholar]
  31. Nixon R. A. Slow axonal transport. Curr Opin Cell Biol. 1992 Feb;4(1):8–14. doi: 10.1016/0955-0674(92)90052-e. [DOI] [PubMed] [Google Scholar]
  32. Okabe S., Hirokawa N. Actin dynamics in growth cones. J Neurosci. 1991 Jul;11(7):1918–1929. doi: 10.1523/JNEUROSCI.11-07-01918.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Okabe S., Hirokawa N. Axonal transport. Curr Opin Cell Biol. 1989 Feb;1(1):91–97. doi: 10.1016/s0955-0674(89)80043-2. [DOI] [PubMed] [Google Scholar]
  34. Okabe S., Hirokawa N. Differential behavior of photoactivated microtubules in growing axons of mouse and frog neurons. J Cell Biol. 1992 Apr;117(1):105–120. doi: 10.1083/jcb.117.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Okabe S., Hirokawa N. Microtubule dynamics in nerve cells: analysis using microinjection of biotinylated tubulin into PC12 cells. J Cell Biol. 1988 Aug;107(2):651–664. doi: 10.1083/jcb.107.2.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Okabe S., Hirokawa N. Turnover of fluorescently labelled tubulin and actin in the axon. Nature. 1990 Feb 1;343(6257):479–482. doi: 10.1038/343479a0. [DOI] [PubMed] [Google Scholar]
  37. Pachter J. S., Liem R. K. The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve. Dev Biol. 1984 May;103(1):200–210. doi: 10.1016/0012-1606(84)90021-6. [DOI] [PubMed] [Google Scholar]
  38. Parysek L. M., Goldman R. D. Distribution of a novel 57 kDa intermediate filament (IF) protein in the nervous system. J Neurosci. 1988 Feb;8(2):555–563. doi: 10.1523/JNEUROSCI.08-02-00555.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Reinsch S. S., Mitchison T. J., Kirschner M. Microtubule polymer assembly and transport during axonal elongation. J Cell Biol. 1991 Oct;115(2):365–379. doi: 10.1083/jcb.115.2.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schlaepfer W. W., Lynch R. G. Immunofluorescence studies of neurofilaments in the rat and human peripheral and central nervous system. J Cell Biol. 1977 Jul;74(1):241–250. doi: 10.1083/jcb.74.1.241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Shaw G., Weber K. The distribution of the neurofilament triplet proteins within individual neurones. Exp Cell Res. 1981 Nov;136(1):119–125. doi: 10.1016/0014-4827(81)90043-4. [DOI] [PubMed] [Google Scholar]
  43. Sihag R. K., Nixon R. A. Phosphorylation of the amino-terminal head domain of the middle molecular mass 145-kDa subunit of neurofilaments. Evidence for regulation by second messenger-dependent protein kinases. J Biol Chem. 1990 Mar 5;265(7):4166–4171. [PubMed] [Google Scholar]
  44. Soellner P., Quinlan R. A., Franke W. W. Identification of a distinct soluble subunit of an intermediate filament protein: tetrameric vimentin from living cells. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7929–7933. doi: 10.1073/pnas.82.23.7929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Steinert P. M., Steven A. C., Roop D. R. The molecular biology of intermediate filaments. Cell. 1985 Sep;42(2):411–420. doi: 10.1016/0092-8674(85)90098-4. [DOI] [PubMed] [Google Scholar]
  46. Tsien R. Y. Fluorescent indicators of ion concentrations. Methods Cell Biol. 1989;30:127–156. doi: 10.1016/s0091-679x(08)60978-4. [DOI] [PubMed] [Google Scholar]
  47. Vigers G. P., Coue M., McIntosh J. R. Fluorescent microtubules break up under illumination. J Cell Biol. 1988 Sep;107(3):1011–1024. doi: 10.1083/jcb.107.3.1011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Vikstrom K. L., Borisy G. G., Goldman R. D. Dynamic aspects of intermediate filament networks in BHK-21 cells. Proc Natl Acad Sci U S A. 1989 Jan;86(2):549–553. doi: 10.1073/pnas.86.2.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wong P. C., Cleveland D. W. Characterization of dominant and recessive assembly-defective mutations in mouse neurofilament NF-M. J Cell Biol. 1990 Nov;111(5 Pt 1):1987–2003. doi: 10.1083/jcb.111.5.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES