Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Nov 1;123(3):653–664. doi: 10.1083/jcb.123.3.653

Regulated tyrosine phosphorylation at the tips of growth cone filopodia

PMCID: PMC2200113  PMID: 7693715

Abstract

Several types of evidence suggest that protein-tyrosine phosphorylation is important during the growth of neuronal processes, but few specific roles, or subcellular localizations suggestive of such roles, have been defined. We report here a localization of tyrosine-phosphorylated protein at the tips of growth cone filopodia. Immunocytochemistry using a mAb to phosphorylated tyrosine residues revealed intense staining of the tips of most filopodia of Aplysia axons growing slowly on a polylysine substrate, but of few filopodia of axons growing rapidly on a substrate coated with Aplysia hemolymph, which has growth-promoting material. Cytochalasin D, which causes F-actin to withdraw rapidly from the growth cone, caused the tyrosine-phosphorylated protein to withdraw rapidly from filopodia, suggesting that the protein associates or interacts with actin filaments. Phosphotyrosine has previously been found concentrated at adherens junctions, where bundles of actin filaments terminate, but video-enhanced contrast-differential interference contrast and confocal interference reflection microscopy demonstrated that the filopodial tips were not adherent to the substrate. Acute application of either hemolymph or inhibitors of protein-tyrosine kinases to neurons on polylysine resulted in a rapid loss of intense staining at filopodial tips concomitant with a lengthening of the filopodia (and their core bundles of actin filaments). These results demonstrate that tyrosine-phosphorylated protein can be concentrated at the barbed ends of actin filaments in a context other than an adherens junction, indicate an association between changes in phosphorylation and filament dynamics, and provide evidence for tyrosine phosphorylation as a signaling mechanism in the filopodium that can respond to environmental cues controlling growth cone dynamics.

Full Text

The Full Text of this article is available as a PDF (4.2 MB).

Selected References

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

  1. Akiyama T., Ogawara H. Use and specificity of genistein as inhibitor of protein-tyrosine kinases. Methods Enzymol. 1991;201:362–370. doi: 10.1016/0076-6879(91)01032-w. [DOI] [PubMed] [Google Scholar]
  2. Atashi J. R., Klinz S. G., Ingraham C. A., Matten W. T., Schachner M., Maness P. F. Neural cell adhesion molecules modulate tyrosine phosphorylation of tubulin in nerve growth cone membranes. Neuron. 1992 May;8(5):831–842. doi: 10.1016/0896-6273(92)90197-l. [DOI] [PubMed] [Google Scholar]
  3. Bandtlow C., Zachleder T., Schwab M. E. Oligodendrocytes arrest neurite growth by contact inhibition. J Neurosci. 1990 Dec;10(12):3837–3848. doi: 10.1523/JNEUROSCI.10-12-03837.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bare D. J., Lauder J. M., Wilkie M. B., Maness P. F. p59fyn in rat brain is localized in developing axonal tracts and subpopulations of adult neurons and glia. Oncogene. 1993 Jun;8(6):1429–1436. [PubMed] [Google Scholar]
  5. Bastiani M. J., Goodman C. S. Neuronal growth cones: specific interactions mediated by filopodial insertion and induction of coated vesicles. Proc Natl Acad Sci U S A. 1984 Mar;81(6):1849–1853. doi: 10.1073/pnas.81.6.1849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bentley D., Caudy M. Pioneer axons lose directed growth after selective killing of guidepost cells. Nature. 1983 Jul 7;304(5921):62–65. doi: 10.1038/304062a0. [DOI] [PubMed] [Google Scholar]
  7. Bentley D., Toroian-Raymond A. Disoriented pathfinding by pioneer neurone growth cones deprived of filopodia by cytochalasin treatment. Nature. 1986 Oct 23;323(6090):712–715. doi: 10.1038/323712a0. [DOI] [PubMed] [Google Scholar]
  8. Bixby J. L., Jhabvala P. Inhibition of tyrosine phosphorylation potentiates substrate-induced neurite growth. J Neurobiol. 1992 Jul;23(5):468–480. doi: 10.1002/neu.480230503. [DOI] [PubMed] [Google Scholar]
  9. Bovolenta P., Mason C. Growth cone morphology varies with position in the developing mouse visual pathway from retina to first targets. J Neurosci. 1987 May;7(5):1447–1460. doi: 10.1523/JNEUROSCI.07-05-01447.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bray D., Chapman K. Analysis of microspike movements on the neuronal growth cone. J Neurosci. 1985 Dec;5(12):3204–3213. doi: 10.1523/JNEUROSCI.05-12-03204.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Burmeister D. W., Rivas R. J., Goldberg D. J. Substrate-bound factors stimulate engorgement of growth cone lamellipodia during neurite elongation. Cell Motil Cytoskeleton. 1991;19(4):255–268. doi: 10.1002/cm.970190404. [DOI] [PubMed] [Google Scholar]
  12. Burridge K., Fath K., Kelly T., Nuckolls G., Turner C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol. 1988;4:487–525. doi: 10.1146/annurev.cb.04.110188.002415. [DOI] [PubMed] [Google Scholar]
  13. Caudy M., Bentley D. Pioneer growth cone steering along a series of neuronal and non-neuronal cues of different affinities. J Neurosci. 1986 Jun;6(6):1781–1795. doi: 10.1523/JNEUROSCI.06-06-01781.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Davenport R. W., Dou P., Rehder V., Kater S. B. A sensory role for neuronal growth cone filopodia. Nature. 1993 Feb 25;361(6414):721–724. doi: 10.1038/361721a0. [DOI] [PubMed] [Google Scholar]
  15. DePasquale J. A., Izzard C. S. Accumulation of talin in nodes at the edge of the lamellipodium and separate incorporation into adhesion plaques at focal contacts in fibroblasts. J Cell Biol. 1991 Jun;113(6):1351–1359. doi: 10.1083/jcb.113.6.1351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Elkins T., Zinn K., McAllister L., Hoffmann F. M., Goodman C. S. Genetic analysis of a Drosophila neural cell adhesion molecule: interaction of fasciclin I and Abelson tyrosine kinase mutations. Cell. 1990 Feb 23;60(4):565–575. doi: 10.1016/0092-8674(90)90660-7. [DOI] [PubMed] [Google Scholar]
  17. Forscher P., Kaczmarek L. K., Buchanan J. A., Smith S. J. Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons. J Neurosci. 1987 Nov;7(11):3600–3611. doi: 10.1523/JNEUROSCI.07-11-03600.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Forscher P., Smith S. J. Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone. J Cell Biol. 1988 Oct;107(4):1505–1516. doi: 10.1083/jcb.107.4.1505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fukazawa H., Li P. M., Yamamoto C., Murakami Y., Mizuno S., Uehara Y. Specific inhibition of cytoplasmic protein tyrosine kinases by herbimycin A in vitro. Biochem Pharmacol. 1991 Oct 9;42(9):1661–1671. doi: 10.1016/0006-2952(91)90500-5. [DOI] [PubMed] [Google Scholar]
  20. Goldberg D. J., Burmeister D. W. Looking into growth cones. Trends Neurosci. 1989 Dec;12(12):503–506. doi: 10.1016/0166-2236(89)90110-0. [DOI] [PubMed] [Google Scholar]
  21. Goldberg D. J., Burmeister D. W. Stages in axon formation: observations of growth of Aplysia axons in culture using video-enhanced contrast-differential interference contrast microscopy. J Cell Biol. 1986 Nov;103(5):1921–1931. doi: 10.1083/jcb.103.5.1921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hammarback J. A., Letourneau P. C. Neurite extension across regions of low cell-substratum adhesivity: implications for the guidepost hypothesis of axonal pathfinding. Dev Biol. 1986 Oct;117(2):655–662. doi: 10.1016/0012-1606(86)90334-9. [DOI] [PubMed] [Google Scholar]
  23. Hariharan I. K., Chuang P. T., Rubin G. M. Cloning and characterization of a receptor-class phosphotyrosine phosphatase gene expressed on central nervous system axons in Drosophila melanogaster. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11266–11270. doi: 10.1073/pnas.88.24.11266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Heidemann S. R., Lamoureux P., Buxbaum R. E. Growth cone behavior and production of traction force. J Cell Biol. 1990 Nov;111(5 Pt 1):1949–1957. doi: 10.1083/jcb.111.5.1949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Howard P. K., Sefton B. M., Firtel R. A. Tyrosine phosphorylation of actin in Dictyostelium associated with cell-shape changes. Science. 1993 Jan 8;259(5092):241–244. doi: 10.1126/science.7678470. [DOI] [PubMed] [Google Scholar]
  26. Izzard C. S. A precursor of the focal contact in cultured fibroblasts. Cell Motil Cytoskeleton. 1988;10(1-2):137–142. doi: 10.1002/cm.970100118. [DOI] [PubMed] [Google Scholar]
  27. Kapfhammer J. P., Raper J. A. Collapse of growth cone structure on contact with specific neurites in culture. J Neurosci. 1987 Jan;7(1):201–212. doi: 10.1523/JNEUROSCI.07-01-00201.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kellie S., Horvath A. R., Elmore M. A. Cytoskeletal targets for oncogenic tyrosine kinases. J Cell Sci. 1991 Jun;99(Pt 2):207–211. doi: 10.1242/jcs.99.2.207. [DOI] [PubMed] [Google Scholar]
  29. Lamballe F., Klein R., Barbacid M. trkC, a new member of the trk family of tyrosine protein kinases, is a receptor for neurotrophin-3. Cell. 1991 Sep 6;66(5):967–979. doi: 10.1016/0092-8674(91)90442-2. [DOI] [PubMed] [Google Scholar]
  30. Letourneau P. C. Differences in the organization of actin in the growth cones compared with the neurites of cultured neurons from chick embryos. J Cell Biol. 1983 Oct;97(4):963–973. doi: 10.1083/jcb.97.4.963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lewis A. K., Bridgman P. C. Nerve growth cone lamellipodia contain two populations of actin filaments that differ in organization and polarity. J Cell Biol. 1992 Dec;119(5):1219–1243. doi: 10.1083/jcb.119.5.1219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Loeb D. M., Maragos J., Martin-Zanca D., Chao M. V., Parada L. F., Greene L. A. The trk proto-oncogene rescues NGF responsiveness in mutant NGF-nonresponsive PC12 cell lines. Cell. 1991 Sep 6;66(5):961–966. doi: 10.1016/0092-8674(91)90441-z. [DOI] [PubMed] [Google Scholar]
  33. Maher P. A. Nerve growth factor induces protein-tyrosine phosphorylation. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6788–6791. doi: 10.1073/pnas.85.18.6788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Maher P. A., Pasquale E. B., Wang J. Y., Singer S. J. Phosphotyrosine-containing proteins are concentrated in focal adhesions and intercellular junctions in normal cells. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6576–6580. doi: 10.1073/pnas.82.19.6576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Maness P. F., Aubry M., Shores C. G., Frame L., Pfenninger K. H. c-src gene product in developing rat brain is enriched in nerve growth cone membranes. Proc Natl Acad Sci U S A. 1988 Jul;85(14):5001–5005. doi: 10.1073/pnas.85.14.5001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Matten W. T., Aubry M., West J., Maness P. F. Tubulin is phosphorylated at tyrosine by pp60c-src in nerve growth cone membranes. J Cell Biol. 1990 Nov;111(5 Pt 1):1959–1970. doi: 10.1083/jcb.111.5.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Miller D. R., Lee G. M., Maness P. F. Increased neurite outgrowth induced by inhibition of protein tyrosine kinase activity in PC12 pheochromocytoma cells. J Neurochem. 1993 Jun;60(6):2134–2144. doi: 10.1111/j.1471-4159.1993.tb03498.x. [DOI] [PubMed] [Google Scholar]
  38. O'Connor T. P., Duerr J. S., Bentley D. Pioneer growth cone steering decisions mediated by single filopodial contacts in situ. J Neurosci. 1990 Dec;10(12):3935–3946. doi: 10.1523/JNEUROSCI.10-12-03935.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. O'Dell T. J., Kandel E. R., Grant S. G. Long-term potentiation in the hippocampus is blocked by tyrosine kinase inhibitors. Nature. 1991 Oct 10;353(6344):558–560. doi: 10.1038/353558a0. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Patterson P. H. On the importance of being inhibited, or saying no to growth cones. Neuron. 1988 Jun;1(4):263–267. doi: 10.1016/0896-6273(88)90074-8. [DOI] [PubMed] [Google Scholar]
  42. Pollard T. D., Cooper J. A. Actin and actin-binding proteins. A critical evaluation of mechanisms and functions. Annu Rev Biochem. 1986;55:987–1035. doi: 10.1146/annurev.bi.55.070186.005011. [DOI] [PubMed] [Google Scholar]
  43. Rivas R. J., Burmeister D. W., Goldberg D. J. Rapid effects of laminin on the growth cone. Neuron. 1992 Jan;8(1):107–115. doi: 10.1016/0896-6273(92)90112-q. [DOI] [PubMed] [Google Scholar]
  44. Schanen-King C., Nel A., Williams L. K., Landreth G. Nerve growth factor stimulates the tyrosine phosphorylation of MAP2 kinase in PC12 cells. Neuron. 1991 Jun;6(6):915–922. doi: 10.1016/0896-6273(91)90232-o. [DOI] [PubMed] [Google Scholar]
  45. Sheetz M. P., Baumrind N. L., Wayne D. B., Pearlman A. L. Concentration of membrane antigens by forward transport and trapping in neuronal growth cones. Cell. 1990 Apr 20;61(2):231–241. doi: 10.1016/0092-8674(90)90804-n. [DOI] [PubMed] [Google Scholar]
  46. Squinto S. P., Stitt T. N., Aldrich T. H., Davis S., Bianco S. M., Radziejewski C., Glass D. J., Masiakowski P., Furth M. E., Valenzuela D. M. trkB encodes a functional receptor for brain-derived neurotrophic factor and neurotrophin-3 but not nerve growth factor. Cell. 1991 May 31;65(5):885–893. doi: 10.1016/0092-8674(91)90395-F. [DOI] [PubMed] [Google Scholar]
  47. Takata K., Singer S. J. Phosphotyrosine-modified proteins are concentrated at the membranes of epithelial and endothelial cells during tissue development in chick embryos. J Cell Biol. 1988 May;106(5):1757–1764. doi: 10.1083/jcb.106.5.1757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tarone G., Cirillo D., Giancotti F. G., Comoglio P. M., Marchisio P. C. Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes. Exp Cell Res. 1985 Jul;159(1):141–157. doi: 10.1016/s0014-4827(85)80044-6. [DOI] [PubMed] [Google Scholar]
  49. Tian S. S., Tsoulfas P., Zinn K. Three receptor-linked protein-tyrosine phosphatases are selectively expressed on central nervous system axons in the Drosophila embryo. Cell. 1991 Nov 15;67(4):675–685. doi: 10.1016/0092-8674(91)90063-5. [DOI] [PubMed] [Google Scholar]
  50. Tosney K. W., Landmesser L. T. Growth cone morphology and trajectory in the lumbosacral region of the chick embryo. J Neurosci. 1985 Sep;5(9):2345–2358. doi: 10.1523/JNEUROSCI.05-09-02345.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tsui H. C., Lankford K. L., Klein W. L. Differentiation of neuronal growth cones: specialization of filopodial tips for adhesive interactions. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8256–8260. doi: 10.1073/pnas.82.23.8256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tsukita S., Oishi K., Akiyama T., Yamanashi Y., Yamamoto T., Tsukita S. Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated. J Cell Biol. 1991 May;113(4):867–879. doi: 10.1083/jcb.113.4.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Vetter M. L., Martin-Zanca D., Parada L. F., Bishop J. M., Kaplan D. R. Nerve growth factor rapidly stimulates tyrosine phosphorylation of phospholipase C-gamma 1 by a kinase activity associated with the product of the trk protooncogene. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5650–5654. doi: 10.1073/pnas.88.13.5650. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES