Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Oct 2;115(2):365–379. doi: 10.1083/jcb.115.2.365

Microtubule polymer assembly and transport during axonal elongation

PMCID: PMC2289149  PMID: 1717484

Abstract

As axons elongate, tubulin, which is synthesized in the cell body, must be transported and assembled into new structures in the axon. The mechanism of transport and the location of assembly are presently unknown. We report here on the use of tubulin tagged with a photoactivatable fluorescent group to investigate these issues. Photoactivatable tubulin, microinjected into frog embryos at the two- cell stage, is incorporated into microtubules in neurons obtained from explants of the neural tube. When activated by light, a fluorescent mark is made on the microtubules in the axon, and transport and turnover can be visualized directly. We find that microtubules are generated in or near the cell body and continually transported distally as a coherent phase of polymer during axon elongation. This vectorial polymer movement was observed at all levels on the axon, even in the absence of axonal elongation. Measurements of the rate of polymer translocation at various places in the axon suggest that new polymer is formed by intercalary assembly along the axon and assembly at the growth cone in addition to transport of polymer from the cell body. Finally, polymer movement near the growth cone appeared to respond in a characteristic manner to growth cone behavior, while polymer proximally in the axon moved more consistently. These results suggest that microtubule translocation is the principal means of tubulin transport and that translocation plays an important role in generating new axon structure at the growth cone.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Baas P. W., Black M. M. Individual microtubules in the axon consist of domains that differ in both composition and stability. J Cell Biol. 1990 Aug;111(2):495–509. doi: 10.1083/jcb.111.2.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bamburg J. R., Bray D., Chapman K. Assembly of microtubules at the tip of growing axons. Nature. 1986 Jun 19;321(6072):788–790. doi: 10.1038/321788a0. [DOI] [PubMed] [Google Scholar]
  3. Bamburg J. R. The axonal cytoskeleton: stationary or moving matrix? Trends Neurosci. 1988 Jun;11(6):248–249. doi: 10.1016/0166-2236(88)90101-4. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Black M. M., Lasek R. J. Slow components of axonal transport: two cytoskeletal networks. J Cell Biol. 1980 Aug;86(2):616–623. doi: 10.1083/jcb.86.2.616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brady S. T., Lasek R. J., Allen R. D. Fast axonal transport in extruded axoplasm from squid giant axon. Science. 1982 Dec 10;218(4577):1129–1131. doi: 10.1126/science.6183745. [DOI] [PubMed] [Google Scholar]
  7. Brady S. T., Lasek R. J., Allen R. D. Video microscopy of fast axonal transport in extruded axoplasm: a new model for study of molecular mechanisms. Cell Motil. 1985;5(2):81–101. doi: 10.1002/cm.970050203. [DOI] [PubMed] [Google Scholar]
  8. Bray D., Bunge M. B. Serial analysis of microtubules in cultured rat sensory axons. J Neurocytol. 1981 Aug;10(4):589–605. doi: 10.1007/BF01262592. [DOI] [PubMed] [Google Scholar]
  9. Fath K. R., Lasek R. J. Two classes of actin microfilaments are associated with the inner cytoskeleton of axons. J Cell Biol. 1988 Aug;107(2):613–621. doi: 10.1083/jcb.107.2.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gilson C. A., Ackland N., Burnside B. Regulation of reactivated elongation in lysed cell models of teleost retinal cones by cAMP and calcium. J Cell Biol. 1986 Mar;102(3):1047–1059. doi: 10.1083/jcb.102.3.1047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gorbsky G. J., Borisy G. G. Microtubules of the kinetochore fiber turn over in metaphase but not in anaphase. J Cell Biol. 1989 Aug;109(2):653–662. doi: 10.1083/jcb.109.2.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gorbsky G. J., Sammak P. J., Borisy G. G. Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends. J Cell Biol. 1987 Jan;104(1):9–18. doi: 10.1083/jcb.104.1.9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harris W. A., Holt C. E., Smith T. A., Gallenson N. Growth cones of developing retinal cells in vivo, on culture surfaces, and in collagen matrices. J Neurosci Res. 1985;13(1-2):101–122. doi: 10.1002/jnr.490130108. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Hirokawa N., Hisanaga S., Shiomura Y. MAP2 is a component of crossbridges between microtubules and neurofilaments in the neuronal cytoskeleton: quick-freeze, deep-etch immunoelectron microscopy and reconstitution studies. J Neurosci. 1988 Aug;8(8):2769–2779. doi: 10.1523/JNEUROSCI.08-08-02769.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Hyman A., Drechsel D., Kellogg D., Salser S., Sawin K., Steffen P., Wordeman L., Mitchison T. Preparation of modified tubulins. Methods Enzymol. 1991;196:478–485. doi: 10.1016/0076-6879(91)96041-o. [DOI] [PubMed] [Google Scholar]
  19. Keith C. H., Blane K. Sites of tubulin polymerization in PC12 cells. J Neurochem. 1990 Apr;54(4):1258–1268. doi: 10.1111/j.1471-4159.1990.tb01957.x. [DOI] [PubMed] [Google Scholar]
  20. Keith C. H. Slow transport of tubulin in the neurites of differentiated PC12 cells. Science. 1987 Jan 16;235(4786):337–339. doi: 10.1126/science.2432662. [DOI] [PubMed] [Google Scholar]
  21. Lang I., Scholz M., Peters R. Molecular mobility and nucleocytoplasmic flux in hepatoma cells. J Cell Biol. 1986 Apr;102(4):1183–1190. doi: 10.1083/jcb.102.4.1183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lasek R. J., Garner J. A., Brady S. T. Axonal transport of the cytoplasmic matrix. J Cell Biol. 1984 Jul;99(1 Pt 2):212s–221s. doi: 10.1083/jcb.99.1.212s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Letourneau P. C. Analysis of microtubule number and length in cytoskeletons of cultured chick sensory neurons. J Neurosci. 1982 Jun;2(6):806–814. doi: 10.1523/JNEUROSCI.02-06-00806.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Lim S. S., Sammak P. J., Borisy G. G. Progressive and spatially differentiated stability of microtubules in developing neuronal cells. J Cell Biol. 1989 Jul;109(1):253–263. doi: 10.1083/jcb.109.1.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Luby-Phelps K., Taylor D. L., Lanni F. Probing the structure of cytoplasm. J Cell Biol. 1986 Jun;102(6):2015–2022. doi: 10.1083/jcb.102.6.2015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Mitchison T., Kirschner M. Cytoskeletal dynamics and nerve growth. Neuron. 1988 Nov;1(9):761–772. doi: 10.1016/0896-6273(88)90124-9. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Paschal B. M., Shpetner H. S., Vallee R. B. MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties. J Cell Biol. 1987 Sep;105(3):1273–1282. doi: 10.1083/jcb.105.3.1273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Paschal B. M., Vallee R. B. Retrograde transport by the microtubule-associated protein MAP 1C. Nature. 1987 Nov 12;330(6144):181–183. doi: 10.1038/330181a0. [DOI] [PubMed] [Google Scholar]
  34. Sawin K. E., Mitchison T. J. Poleward microtubule flux mitotic spindles assembled in vitro. J Cell Biol. 1991 Mar;112(5):941–954. doi: 10.1083/jcb.112.5.941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Schroer T. A., Sheetz M. P. Functions of microtubule-based motors. Annu Rev Physiol. 1991;53:629–652. doi: 10.1146/annurev.ph.53.030191.003213. [DOI] [PubMed] [Google Scholar]
  37. Schulze E., Kirschner M. Microtubule dynamics in interphase cells. J Cell Biol. 1986 Mar;102(3):1020–1031. doi: 10.1083/jcb.102.3.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shpetner H. S., Vallee R. B. Identification of dynamin, a novel mechanochemical enzyme that mediates interactions between microtubules. Cell. 1989 Nov 3;59(3):421–432. doi: 10.1016/0092-8674(89)90027-5. [DOI] [PubMed] [Google Scholar]
  39. Tanaka E. M., Kirschner M. W. Microtubule behavior in the growth cones of living neurons during axon elongation. J Cell Biol. 1991 Oct;115(2):345–363. doi: 10.1083/jcb.115.2.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Vale R. D. Intracellular transport using microtubule-based motors. Annu Rev Cell Biol. 1987;3:347–378. doi: 10.1146/annurev.cb.03.110187.002023. [DOI] [PubMed] [Google Scholar]
  41. Warren R. H., Brunside B. Microtubules in cone myoid elongation in the teleost retina. J Cell Biol. 1978 Jul;78(1):247–259. doi: 10.1083/jcb.78.1.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Weisenberg R. C., Flynn J., Gao B. C., Awodi S. Microtubule gelation-contraction in vitro and its relationship to component a of slow axonal transport. Cell Motil Cytoskeleton. 1988;10(1-2):331–340. doi: 10.1002/cm.970100137. [DOI] [PubMed] [Google Scholar]
  43. Weisenberg R. C., Flynn J., Gao B. C., Awodi S., Skee F., Goodman S. R., Riederer B. M. Microtubule gelation-contraction: essential components and relation to slow axonal transport. Science. 1987 Nov 20;238(4830):1119–1122. doi: 10.1126/science.2446388. [DOI] [PubMed] [Google Scholar]

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

RESOURCES