Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1992 Nov 2;119(4):883–892. doi: 10.1083/jcb.119.4.883

Calcium influx into neurons can solely account for cell contact- dependent neurite outgrowth stimulated by transfected L1

PMCID: PMC2289701  PMID: 1429842

Abstract

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human L1 as a culture substrate for rat PC12 cells and rat cerebellar neurons. PC12 cells and cerebellar neurons extended longer neurites on human L1 expressing cells. Neurons isolated from the cerebellum at postnatal day 9 responded equally as well as those isolated at postnatal day 1-4, and this contrasts with the failure of these older neurons to respond to the transfected human neural cell adhesion molecule (NCAM). Human L1-dependent neurite outgrowth could be blocked by antibodies that bound to rat L1 and, additionally, the response could be fully inhibited by pertussis toxin and substantially inhibited by antagonists of L- and N-type calcium channels. Calcium influx into neurons induced by K+ depolarization fully mimics the L1 response. Furthermore, we show that L1- and K+(-)dependent neurite outgrowth can be specifically inhibited by a reduction in extracellular calcium to 0.25 microM, and by pretreatment of cerebellar neurons with the intracellular calcium chelator BAPTA/AM. In contrast, the response was not inhibited by heparin or by removal of polysialic acid from neuronal NCAM both of which substantially inhibit NCAM-dependent neurite outgrowth. These data demonstrate that whereas NCAM and L1 promote neurite outgrowth via activation of a common CAM-specific second messenger pathway in neurons, neuronal responsiveness to NCAM and L1 is not coordinately regulated via posttranslational processing of NCAM. The fact that NCAM- and L1-dependent neurite outgrowth, but not adhesion, are calcium dependent provides further evidence that adhesion per se does not directly contribute to neurite outgrowth.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. Acheson A., Sunshine J. L., Rutishauser U. NCAM polysialic acid can regulate both cell-cell and cell-substrate interactions. J Cell Biol. 1991 Jul;114(1):143–153. doi: 10.1083/jcb.114.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bixby J. L., Harris W. A. Molecular mechanisms of axon growth and guidance. Annu Rev Cell Biol. 1991;7:117–159. doi: 10.1146/annurev.cb.07.110191.001001. [DOI] [PubMed] [Google Scholar]
  3. Bixby J. L., Lilien J., Reichardt L. F. Identification of the major proteins that promote neuronal process outgrowth on Schwann cells in vitro. J Cell Biol. 1988 Jul;107(1):353–361. doi: 10.1083/jcb.107.1.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bixby J. L., Pratt R. S., Lilien J., Reichardt L. F. Neurite outgrowth on muscle cell surfaces involves extracellular matrix receptors as well as Ca2+-dependent and -independent cell adhesion molecules. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2555–2559. doi: 10.1073/pnas.84.8.2555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bixby J. L., Zhang R. Purified N-cadherin is a potent substrate for the rapid induction of neurite outgrowth. J Cell Biol. 1990 Apr;110(4):1253–1260. doi: 10.1083/jcb.110.4.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Campenot R. B., Draker D. D. Growth of sympathetic nerve fibers in culture does not require extracellular calcium. Neuron. 1989 Dec;3(6):733–743. doi: 10.1016/0896-6273(89)90242-0. [DOI] [PubMed] [Google Scholar]
  7. Chang S., Rathjen F. G., Raper J. A. Extension of neurites on axons is impaired by antibodies against specific neural cell surface glycoproteins. J Cell Biol. 1987 Feb;104(2):355–362. doi: 10.1083/jcb.104.2.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cole G. J., Akeson R. Identification of a heparin binding domain of the neural cell adhesion molecule N-CAM using synthetic peptides. Neuron. 1989 Feb;2(2):1157–1165. doi: 10.1016/0896-6273(89)90182-7. [DOI] [PubMed] [Google Scholar]
  9. Cole G. J., Glaser L. A heparin-binding domain from N-CAM is involved in neural cell-substratum adhesion. J Cell Biol. 1986 Feb;102(2):403–412. doi: 10.1083/jcb.102.2.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Daniloff J. K., Levi G., Grumet M., Rieger F., Edelman G. M. Altered expression of neuronal cell adhesion molecules induced by nerve injury and repair. J Cell Biol. 1986 Sep;103(3):929–945. doi: 10.1083/jcb.103.3.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Doherty P., Ashton S. V., Moore S. E., Walsh F. S. Morphoregulatory activities of NCAM and N-cadherin can be accounted for by G protein-dependent activation of L- and N-type neuronal Ca2+ channels. Cell. 1991 Oct 4;67(1):21–33. doi: 10.1016/0092-8674(91)90569-k. [DOI] [PubMed] [Google Scholar]
  12. Doherty P., Barton C. H., Dickson G., Seaton P., Rowett L. H., Moore S. E., Gower H. J., Walsh F. S. Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms. J Cell Biol. 1989 Aug;109(2):789–798. doi: 10.1083/jcb.109.2.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Doherty P., Cohen J., Walsh F. S. Neurite outgrowth in response to transfected N-CAM changes during development and is modulated by polysialic acid. Neuron. 1990 Aug;5(2):209–219. doi: 10.1016/0896-6273(90)90310-c. [DOI] [PubMed] [Google Scholar]
  14. Doherty P., Fruns M., Seaton P., Dickson G., Barton C. H., Sears T. A., Walsh F. S. A threshold effect of the major isoforms of NCAM on neurite outgrowth. Nature. 1990 Feb 1;343(6257):464–466. doi: 10.1038/343464a0. [DOI] [PubMed] [Google Scholar]
  15. Doherty P., Moolenaar C. E., Ashton S. V., Michalides R. J., Walsh F. S. The VASE exon downregulates the neurite growth-promoting activity of NCAM 140. Nature. 1992 Apr 30;356(6372):791–793. doi: 10.1038/356791a0. [DOI] [PubMed] [Google Scholar]
  16. Doherty P., Rimon G., Mann D. A., Walsh F. S. Alternative splicing of the cytoplasmic domain of neural cell adhesion molecule alters its ability to act as a substrate for neurite outgrowth. J Neurochem. 1992 Jun;58(6):2338–2341. doi: 10.1111/j.1471-4159.1992.tb10984.x. [DOI] [PubMed] [Google Scholar]
  17. Doherty P., Rowett L. H., Moore S. E., Mann D. A., Walsh F. S. Neurite outgrowth in response to transfected N-CAM and N-cadherin reveals fundamental differences in neuronal responsiveness to CAMs. Neuron. 1991 Feb;6(2):247–258. doi: 10.1016/0896-6273(91)90360-c. [DOI] [PubMed] [Google Scholar]
  18. Doherty P., Skaper S. D., Moore S. E., Leon A., Walsh F. S. A developmentally regulated switch in neuronal responsiveness to NCAM and N-cadherin in the rat hippocampus. Development. 1992 Jul;115(3):885–892. doi: 10.1242/dev.115.3.885. [DOI] [PubMed] [Google Scholar]
  19. Doherty P., Walsh F. S. Neurite guidance molecules. Curr Opin Cell Biol. 1989 Dec;1(6):1102–1106. doi: 10.1016/s0955-0674(89)80057-2. [DOI] [PubMed] [Google Scholar]
  20. Fischer G., Künemund V., Schachner M. Neurite outgrowth patterns in cerebellar microexplant cultures are affected by antibodies to the cell surface glycoprotein L1. J Neurosci. 1986 Feb;6(2):605–612. doi: 10.1523/JNEUROSCI.06-02-00605.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hlavin M. L., Lemmon V. Molecular structure and functional testing of human L1CAM: an interspecies comparison. Genomics. 1991 Oct;11(2):416–423. doi: 10.1016/0888-7543(91)90150-d. [DOI] [PubMed] [Google Scholar]
  22. Kadmon G., Kowitz A., Altevogt P., Schachner M. Functional cooperation between the neural adhesion molecules L1 and N-CAM is carbohydrate dependent. J Cell Biol. 1990 Jan;110(1):209–218. doi: 10.1083/jcb.110.1.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kadmon G., Kowitz A., Altevogt P., Schachner M. The neural cell adhesion molecule N-CAM enhances L1-dependent cell-cell interactions. J Cell Biol. 1990 Jan;110(1):193–208. doi: 10.1083/jcb.110.1.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kater S. B., Mills L. R. Regulation of growth cone behavior by calcium. J Neurosci. 1991 Apr;11(4):891–899. doi: 10.1523/JNEUROSCI.11-04-00891.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Koike T., Martin D. P., Johnson E. M., Jr Role of Ca2+ channels in the ability of membrane depolarization to prevent neuronal death induced by trophic-factor deprivation: evidence that levels of internal Ca2+ determine nerve growth factor dependence of sympathetic ganglion cells. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6421–6425. doi: 10.1073/pnas.86.16.6421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kuhn T. B., Stoeckli E. T., Condrau M. A., Rathjen F. G., Sonderegger P. Neurite outgrowth on immobilized axonin-1 is mediated by a heterophilic interaction with L1(G4). J Cell Biol. 1991 Nov;115(4):1113–1126. doi: 10.1083/jcb.115.4.1113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Landmesser L., Dahm L., Tang J. C., Rutishauser U. Polysialic acid as a regulator of intramuscular nerve branching during embryonic development. Neuron. 1990 May;4(5):655–667. doi: 10.1016/0896-6273(90)90193-j. [DOI] [PubMed] [Google Scholar]
  28. Lemmon V., Farr K. L., Lagenaur C. L1-mediated axon outgrowth occurs via a homophilic binding mechanism. Neuron. 1989 Jun;2(6):1597–1603. doi: 10.1016/0896-6273(89)90048-2. [DOI] [PubMed] [Google Scholar]
  29. Lindner J., Rathjen F. G., Schachner M. L1 mono- and polyclonal antibodies modify cell migration in early postnatal mouse cerebellum. 1983 Sep 29-Oct 5Nature. 305(5933):427–430. doi: 10.1038/305427a0. [DOI] [PubMed] [Google Scholar]
  30. Lumsden A., Cohen J. Axon guidance in the vertebrate central nervous system. Curr Opin Genet Dev. 1991 Aug;1(2):230–235. doi: 10.1016/s0959-437x(05)80075-9. [DOI] [PubMed] [Google Scholar]
  31. Mann D. A., Doherty P., Walsh F. S. Increased intracellular cyclic AMP differentially modulates nerve growth factor induction of three neuronal recognition molecules involved in neurite outgrowth. J Neurochem. 1989 Nov;53(5):1581–1588. doi: 10.1111/j.1471-4159.1989.tb08555.x. [DOI] [PubMed] [Google Scholar]
  32. Martini R., Schachner M. Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and myelin-associated glycoprotein) in regenerating adult mouse sciatic nerve. J Cell Biol. 1988 May;106(5):1735–1746. doi: 10.1083/jcb.106.5.1735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Matsunaga M., Hatta K., Nagafuchi A., Takeichi M. Guidance of optic nerve fibres by N-cadherin adhesion molecules. Nature. 1988 Jul 7;334(6177):62–64. doi: 10.1038/334062a0. [DOI] [PubMed] [Google Scholar]
  34. McGuire J. C., Greene L. A., Furano A. V. NGF stimulates incorporation of fucose or glucosamine into an external glycoprotein in cultured rat PC12 pheochromocytoma cells. Cell. 1978 Oct;15(2):357–365. doi: 10.1016/0092-8674(78)90004-1. [DOI] [PubMed] [Google Scholar]
  35. Miura M., Asou H., Kobayashi M., Uyemura K. Functional expression of a full-length cDNA coding for rat neural cell adhesion molecule L1 mediates homophilic intercellular adhesion and migration of cerebellar neurons. J Biol Chem. 1992 May 25;267(15):10752–10758. [PubMed] [Google Scholar]
  36. Miura M., Kobayashi M., Asou H., Uyemura K. Molecular cloning of cDNA encoding the rat neural cell adhesion molecule L1. Two L1 isoforms in the cytoplasmic region are produced by differential splicing. FEBS Lett. 1991 Sep 2;289(1):91–95. doi: 10.1016/0014-5793(91)80915-p. [DOI] [PubMed] [Google Scholar]
  37. Moore S. E., Thompson J., Kirkness V., Dickson J. G., Walsh F. S. Skeletal muscle neural cell adhesion molecule (N-CAM): changes in protein and mRNA species during myogenesis of muscle cell lines. J Cell Biol. 1987 Sep;105(3):1377–1386. doi: 10.1083/jcb.105.3.1377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Moos M., Tacke R., Scherer H., Teplow D., Früh K., Schachner M. Neural adhesion molecule L1 as a member of the immunoglobulin superfamily with binding domains similar to fibronectin. Nature. 1988 Aug 25;334(6184):701–703. doi: 10.1038/334701a0. [DOI] [PubMed] [Google Scholar]
  39. Morgenstern J. P., Land H. A series of mammalian expression vectors and characterisation of their expression of a reporter gene in stably and transiently transfected cells. Nucleic Acids Res. 1990 Feb 25;18(4):1068–1068. doi: 10.1093/nar/18.4.1068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mujoo K., Spiro R. C., Reisfeld R. A. Characterization of a unique glycoprotein antigen expressed on the surface of human neuroblastoma cells. J Biol Chem. 1986 Aug 5;261(22):10299–10305. [PubMed] [Google Scholar]
  41. Nagafuchi A., Takeichi M. Cell binding function of E-cadherin is regulated by the cytoplasmic domain. EMBO J. 1988 Dec 1;7(12):3679–3684. doi: 10.1002/j.1460-2075.1988.tb03249.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Neugebauer K. M., Tomaselli K. J., Lilien J., Reichardt L. F. N-cadherin, NCAM, and integrins promote retinal neurite outgrowth on astrocytes in vitro. J Cell Biol. 1988 Sep;107(3):1177–1187. doi: 10.1083/jcb.107.3.1177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rathjen F. G. A neurite outgrowth-promoting molecule in developing fiber tracts. Trends Neurosci. 1988 May;11(5):183–184. doi: 10.1016/0166-2236(88)90116-6. [DOI] [PubMed] [Google Scholar]
  44. Rathjen F. G., Schachner M. Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion. EMBO J. 1984 Jan;3(1):1–10. doi: 10.1002/j.1460-2075.1984.tb01753.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Reber B. F., Reuter H. Dependence of cytosolic calcium in differentiating rat pheochromocytoma cells on calcium channels and intracellular stores. J Physiol. 1991 Apr;435:145–162. doi: 10.1113/jphysiol.1991.sp018502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Reichardt L. F., Tomaselli K. J. Extracellular matrix molecules and their receptors: functions in neural development. Annu Rev Neurosci. 1991;14:531–570. doi: 10.1146/annurev.ne.14.030191.002531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Reid R. A., Hemperly J. J. Variants of human L1 cell adhesion molecule arise through alternate splicing of RNA. J Mol Neurosci. 1992;3(3):127–135. doi: 10.1007/BF02919404. [DOI] [PubMed] [Google Scholar]
  48. Rutishauser U., Acheson A., Hall A. K., Mann D. M., Sunshine J. The neural cell adhesion molecule (NCAM) as a regulator of cell-cell interactions. Science. 1988 Apr 1;240(4848):53–57. doi: 10.1126/science.3281256. [DOI] [PubMed] [Google Scholar]
  49. Saffell J. L., Walsh F. S., Doherty P. Direct activation of second messenger pathways mimics cell adhesion molecule-dependent neurite outgrowth. J Cell Biol. 1992 Aug;118(3):663–670. doi: 10.1083/jcb.118.3.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Seilheimer B., Schachner M. Studies of adhesion molecules mediating interactions between cells of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann cells in culture. J Cell Biol. 1988 Jul;107(1):341–351. doi: 10.1083/jcb.107.1.341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Silver R. A., Lamb A. G., Bolsover S. R. Calcium hotspots caused by L-channel clustering promote morphological changes in neuronal growth cones. Nature. 1990 Feb 22;343(6260):751–754. doi: 10.1038/343751a0. [DOI] [PubMed] [Google Scholar]
  52. Simon Horst, Klinz Stephan, Fahrig Thomas, Schachner Melitta. Molecular Association of the Neural Adhesion Molecules L1 and N-CAM in the Surface Membrane of Neuroblastoma Cells is Shown by Chemical Cross-linking. Eur J Neurosci. 1991 Jul;3(7):634–640. doi: 10.1111/j.1460-9568.1991.tb00849.x. [DOI] [PubMed] [Google Scholar]
  53. Singer S. J. Intercellular communication and cell-cell adhesion. Science. 1992 Mar 27;255(5052):1671–1677. doi: 10.1126/science.1313187. [DOI] [PubMed] [Google Scholar]
  54. Takeichi M. Cadherin cell adhesion receptors as a morphogenetic regulator. Science. 1991 Mar 22;251(5000):1451–1455. doi: 10.1126/science.2006419. [DOI] [PubMed] [Google Scholar]
  55. Wolff J. M., Frank R., Mujoo K., Spiro R. C., Reisfeld R. A., Rathjen F. G. A human brain glycoprotein related to the mouse cell adhesion molecule L1. J Biol Chem. 1988 Aug 25;263(24):11943–11947. [PubMed] [Google Scholar]

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

RESOURCES