Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1985 Nov 1;101(5):1799–1807. doi: 10.1083/jcb.101.5.1799

Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors

PMCID: PMC2113947  PMID: 2997236

Abstract

Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10- and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

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

  1. Amos L. A. Arrangement of high molecular weight associated proteins on purified mammalian brain microtubules. J Cell Biol. 1977 Mar;72(3):642–654. doi: 10.1083/jcb.72.3.642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ben-Ze'ev A., Farmer S. R., Penman S. Mechanisms of regulating tubulin synthesis in cultured mammalian cells. Cell. 1979 Jun;17(2):319–325. doi: 10.1016/0092-8674(79)90157-0. [DOI] [PubMed] [Google Scholar]
  3. Binder L. I., Frankfurter A., Kim H., Caceres A., Payne M. R., Rebhun L. I. Heterogeneity of microtubule-associated protein 2 during rat brain development. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5613–5617. doi: 10.1073/pnas.81.17.5613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Black M. M., Greene L. A. Changes in the colchicine susceptibility of microtubules associated with neurite outgrowth: studies with nerve growth factor-responsive PC12 pheochromocytoma cells. J Cell Biol. 1982 Nov;95(2 Pt 1):379–386. doi: 10.1083/jcb.95.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blose S. H., Meltzer D. I., Feramisco J. R. 10-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin. J Cell Biol. 1984 Mar;98(3):847–858. doi: 10.1083/jcb.98.3.847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  7. Cleveland D. W., Hwo S. Y., Kirschner M. W. Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly. J Mol Biol. 1977 Oct 25;116(2):227–247. doi: 10.1016/0022-2836(77)90214-5. [DOI] [PubMed] [Google Scholar]
  8. Cleveland D. W., Hwo S. Y., Kirschner M. W. Purification of tau, a microtubule-associated protein that induces assembly of microtubules from purified tubulin. J Mol Biol. 1977 Oct 25;116(2):207–225. doi: 10.1016/0022-2836(77)90213-3. [DOI] [PubMed] [Google Scholar]
  9. Cleveland D. W., Lopata M. A., Sherline P., Kirschner M. W. Unpolymerized tubulin modulates the level of tubulin mRNAs. Cell. 1981 Aug;25(2):537–546. doi: 10.1016/0092-8674(81)90072-6. [DOI] [PubMed] [Google Scholar]
  10. Daniels M. The role of microtubules in the growth and stabilization of nerve fibers. Ann N Y Acad Sci. 1975 Jun 30;253:535–544. doi: 10.1111/j.1749-6632.1975.tb19227.x. [DOI] [PubMed] [Google Scholar]
  11. Drubin D. G., Caput D., Kirschner M. W. Studies on the expression of the microtubule-associated protein, tau, during mouse brain development, with newly isolated complementary DNA probes. J Cell Biol. 1984 Mar;98(3):1090–1097. doi: 10.1083/jcb.98.3.1090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gard D. L., Kirschner M. W. A polymer-dependent increase in phosphorylation of beta-tubulin accompanies differentiation of a mouse neuroblastoma cell line. J Cell Biol. 1985 Mar;100(3):764–774. doi: 10.1083/jcb.100.3.764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garrels J. I., Schubert D. Modulation of protein synthesis by nerve growth factor. J Biol Chem. 1979 Aug 25;254(16):7978–7985. [PubMed] [Google Scholar]
  14. Greene L. A., Burstein D. E., Black M. M. The role of transcription-dependent priming in nerve growth factor promoted neurite outgrowth. Dev Biol. 1982 Jun;91(2):305–316. doi: 10.1016/0012-1606(82)90037-9. [DOI] [PubMed] [Google Scholar]
  15. Greene L. A., Liem R. K., Shelanski M. L. Regulation of a high molecular weight microtubule-associated protein in PC12 cells by nerve growth factor. J Cell Biol. 1983 Jan;96(1):76–83. doi: 10.1083/jcb.96.1.76. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Greene L. A., Tischler A. S. Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2424–2428. doi: 10.1073/pnas.73.7.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gunning P. W., Landreth G. E., Bothwell M. A., Shooter E. M. Differential and synergistic actions of nerve growth factor and cyclic AMP in PC12 cells. J Cell Biol. 1981 May;89(2):240–245. doi: 10.1083/jcb.89.2.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
  19. Heidemann S. R., Joshi H. C., Schechter A., Fletcher J. R., Bothwell M. Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells. J Cell Biol. 1985 Mar;100(3):916–927. doi: 10.1083/jcb.100.3.916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Herzog W., Weber K. Fractionation of brain microtubule-associated proteins. Isolation of two different proteins which stimulate tubulin polymerization in vitro. Eur J Biochem. 1978 Dec 1;92(1):1–8. doi: 10.1111/j.1432-1033.1978.tb12716.x. [DOI] [PubMed] [Google Scholar]
  21. Karsenti E., Kobayashi S., Mitchison T., Kirschner M. Role of the centrosome in organizing the interphase microtubule array: properties of cytoplasts containing or lacking centrosomes. J Cell Biol. 1984 May;98(5):1763–1776. doi: 10.1083/jcb.98.5.1763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Karsenti E., Newport J., Hubble R., Kirschner M. Interconversion of metaphase and interphase microtubule arrays, as studied by the injection of centrosomes and nuclei into Xenopus eggs. J Cell Biol. 1984 May;98(5):1730–1745. doi: 10.1083/jcb.98.5.1730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Keates R. A., Hall R. H. Tubulin requires an accessory protein for self assembly in microtubules. Nature. 1975 Oct 2;257(5525):418–421. doi: 10.1038/257418a0. [DOI] [PubMed] [Google Scholar]
  24. Kuznetsov S. A., Rodionov V. I., Gelfand V. I., Rosenblat V. A. Microtubule-associated protein MAP1 promotes microtubule assembly in vitro. FEBS Lett. 1981 Dec 7;135(2):241–244. doi: 10.1016/0014-5793(81)80791-0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Luckenbill-Edds L., Van Horn C., Greene L. A. Fine structure of initial outgrowth of processes induced in a pheochromocytoma cell line (PC12) by nerve growth factor. J Neurocytol. 1979 Aug;8(4):493–511. doi: 10.1007/BF01214805. [DOI] [PubMed] [Google Scholar]
  27. Mareck A., Fellous A., Francon J., Nunez J. Changes in composition and activity of microtubule-associated proteins during brain development. Nature. 1980 Mar 27;284(5754):353–355. doi: 10.1038/284353a0. [DOI] [PubMed] [Google Scholar]
  28. Marsh L., Letourneau P. C. Growth of neurites without filopodial or lamellipodial activity in the presence of cytochalasin B. J Cell Biol. 1984 Dec;99(6):2041–2047. doi: 10.1083/jcb.99.6.2041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Murphy D. B., Borisy G. G. Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2696–2700. doi: 10.1073/pnas.72.7.2696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Pfeffer S. R., Drubin D. G., Kelly R. B. Identification of three coated vesicle components as alpha- and beta-tubulin linked to a phosphorylated 50,000-dalton polypeptide. J Cell Biol. 1983 Jul;97(1):40–47. doi: 10.1083/jcb.97.1.40. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schnapp B. J., Vale R. D., Sheetz M. P., Reese T. S. Single microtubules from squid axoplasm support bidirectional movement of organelles. Cell. 1985 Feb;40(2):455–462. doi: 10.1016/0092-8674(85)90160-6. [DOI] [PubMed] [Google Scholar]
  33. Schubert D., Heinemann S., Kidokoro Y. Cholinergic metabolism and synapse formation by a rat nerve cell line. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2579–2583. doi: 10.1073/pnas.74.6.2579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Seeds N. W., Gilman A. G., Amano T., Nirenberg M. W. Regulation of axon formation by clonal lines of a neural tumor. Proc Natl Acad Sci U S A. 1970 May;66(1):160–167. doi: 10.1073/pnas.66.1.160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Seeds N. W., Maccioni R. B. Proteins from morphologically differentiated neuroblastoma cells promote tubulin polymerization. J Cell Biol. 1978 Feb;76(2):547–555. doi: 10.1083/jcb.76.2.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Shelton D. L., Reichardt L. F. Expression of the beta-nerve growth factor gene correlates with the density of sympathetic innervation in effector organs. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7951–7955. doi: 10.1073/pnas.81.24.7951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sloboda R. D., Rudolph S. A., Rosenbaum J. L., Greengard P. Cyclic AMP-dependent endogenous phosphorylation of a microtubule-associated protein. Proc Natl Acad Sci U S A. 1975 Jan;72(1):177–181. doi: 10.1073/pnas.72.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smith A. P., Varon S., Shooter E. M. Multiple forms of the nerve growth factor protein and its subunits. Biochemistry. 1968 Sep;7(9):3259–3268. doi: 10.1021/bi00849a032. [DOI] [PubMed] [Google Scholar]
  40. Solomon F., Magendantz M., Salzman A. Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins. Cell. 1979 Oct;18(2):431–438. doi: 10.1016/0092-8674(79)90062-x. [DOI] [PubMed] [Google Scholar]
  41. Vale R. D., Schnapp B. J., Reese T. S., Sheetz M. P. Organelle, bead, and microtubule translocations promoted by soluble factors from the squid giant axon. Cell. 1985 Mar;40(3):559–569. doi: 10.1016/0092-8674(85)90204-1. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Yamada K. M., Spooner B. S., Wessells N. K. Axon growth: roles of microfilaments and microtubules. Proc Natl Acad Sci U S A. 1970 Aug;66(4):1206–1212. doi: 10.1073/pnas.66.4.1206. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES