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. 1994 Dec 1;127(5):1461–1475. doi: 10.1083/jcb.127.5.1461

Ca2+ influx and neurite growth in response to purified N-cadherin and laminin

PMCID: PMC2120265  PMID: 7962102

Abstract

The signaling mechanisms underlying neurite growth induced by cadherins and integrins are incompletely understood. In our experiments, we have examined these mechanisms using purified N-cadherin and laminin (LN). We find that unlike the neurite growth induced by fibroblastic cells expressing transfected N-cadherin (Doherty, P., and F.S. Walsh. 1992. Curr. Opin. Neurobiol. 2:595-601), growth induced by purified N- cadherin in chick ciliary ganglion (CG), sensory, or forebrain neurons is not sensitive to inhibition by pertussis toxin. Using fura-2 imaging of single cells, we show that soluble N-cadherin induces Ca2+ increases in CG neuron cell bodies, and, importantly, in growth cones. In contrast, N-cadherin can induce Ca2+ decreases in glial cells. N- cadherin-induced neuronal Ca2+ responses are sensitive to Ni2+, but are relatively insensitive to diltiazem and omega-conotoxin. Similarly, neurite growth induced by purified N-cadherin is inhibited by Ni2+, but is unaffected by diltiazem and conotoxin. Soluble LN also induced small Ca2+ responses in CG neurons. LN-induced neurite growth, like that induced by N-cadherin, is insensitive to diltiazem and conotoxin, but is highly sensitive to Ni2+ inhibition. K+ depolarization experiments suggest that voltage-dependent Ca2+ influx pathways in CG neurons (cell bodies and growth cones) are largely blocked by the combination of diltiazem and Ni2+. Our results demonstrate that cadherin signaling involves cell type-specific Ca2+ changes in responding cells, and in particular, that N-cadherin can cause Ca2+ increases in neuronal growth cones. Our findings are consistent with the current idea that distinct neuronal transduction pathways exist for cell adhesion molecules compared with integrins, but suggest that the involvement of Ca2+ signals in both of these pathways is more complex than previously appreciated.

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Selected References

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  1. Appel F., Holm J., Conscience J. F., Schachner M. Several extracellular domains of the neural cell adhesion molecule L1 are involved in neurite outgrowth and cell body adhesion. J Neurosci. 1993 Nov;13(11):4764–4775. doi: 10.1523/JNEUROSCI.13-11-04764.1993. [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., Jhabvala P. Extracellular matrix molecules and cell adhesion molecules induce neurites through different mechanisms. J Cell Biol. 1990 Dec;111(6 Pt 1):2725–2732. doi: 10.1083/jcb.111.6.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Bixby J. L. Protein kinase C is involved in laminin stimulation of neurite outgrowth. Neuron. 1989 Sep;3(3):287–297. doi: 10.1016/0896-6273(89)90253-5. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Borasio G. D., John J., Wittinghofer A., Barde Y. A., Sendtner M., Heumann R. ras p21 protein promotes survival and fiber outgrowth of cultured embryonic neurons. Neuron. 1989 Jan;2(1):1087–1096. doi: 10.1016/0896-6273(89)90233-x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Connor J. A., Kater S. B., Cohan C., Fink L. Ca2+ dynamics in neuronal growth cones: regulation and changing patterns of Ca2+ entry. Cell Calcium. 1990 Feb-Mar;11(2-3):233–239. doi: 10.1016/0143-4160(90)90074-5. [DOI] [PubMed] [Google Scholar]
  12. 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]
  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., 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]
  15. 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]
  16. Doherty P., Walsh F. S. Cell adhesion molecules, second messengers and axonal growth. Curr Opin Neurobiol. 1992 Oct;2(5):595–601. doi: 10.1016/0959-4388(92)90024-f. [DOI] [PubMed] [Google Scholar]
  17. Dryer S. E., Dourado M. M., Wisgirda M. E. Properties of Ca2+ currents in acutely dissociated neurons of the chick ciliary ganglion: inhibition by somatostatin-14 and somatostatin-28. Neuroscience. 1991;44(3):663–672. doi: 10.1016/0306-4522(91)90086-4. [DOI] [PubMed] [Google Scholar]
  18. Frei T., von Bohlen und Halbach F., Wille W., Schachner M. Different extracellular domains of the neural cell adhesion molecule (N-CAM) are involved in different functions. J Cell Biol. 1992 Jul;118(1):177–194. doi: 10.1083/jcb.118.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gower H. J., Barton C. H., Elsom V. L., Thompson J., Moore S. E., Dickson G., Walsh F. S. Alternative splicing generates a secreted form of N-CAM in muscle and brain. Cell. 1988 Dec 23;55(6):955–964. doi: 10.1016/0092-8674(88)90241-3. [DOI] [PubMed] [Google Scholar]
  20. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  21. Gundersen R. W. Response of sensory neurites and growth cones to patterned substrata of laminin and fibronectin in vitro. Dev Biol. 1987 Jun;121(2):423–431. doi: 10.1016/0012-1606(87)90179-5. [DOI] [PubMed] [Google Scholar]
  22. Hall D. E., Neugebauer K. M., Reichardt L. F. Embryonic neural retinal cell response to extracellular matrix proteins: developmental changes and effects of the cell substratum attachment antibody (CSAT). J Cell Biol. 1987 Mar;104(3):623–634. doi: 10.1083/jcb.104.3.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hammarback J. A., McCarthy J. B., Palm S. L., Furcht L. T., Letourneau P. C. Growth cone guidance by substrate-bound laminin pathways is correlated with neuron-to-pathway adhesivity. Dev Biol. 1988 Mar;126(1):29–39. doi: 10.1016/0012-1606(88)90235-7. [DOI] [PubMed] [Google Scholar]
  24. Hatta K., Takagi S., Fujisawa H., Takeichi M. Spatial and temporal expression pattern of N-cadherin cell adhesion molecules correlated with morphogenetic processes of chicken embryos. Dev Biol. 1987 Mar;120(1):215–227. doi: 10.1016/0012-1606(87)90119-9. [DOI] [PubMed] [Google Scholar]
  25. Hatten M. E., Lynch M., Rydel R. E., Sanchez J., Joseph-Silverstein J., Moscatelli D., Rifkin D. B. In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor. Dev Biol. 1988 Feb;125(2):280–289. doi: 10.1016/0012-1606(88)90211-4. [DOI] [PubMed] [Google Scholar]
  26. Holz G. G., 4th, Kream R. M., Spiegel A., Dunlap K. G proteins couple alpha-adrenergic and GABAb receptors to inhibition of peptide secretion from peripheral sensory neurons. J Neurosci. 1989 Feb;9(2):657–666. doi: 10.1523/JNEUROSCI.09-02-00657.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Igarashi M., Strittmatter S. M., Vartanian T., Fishman M. C. Mediation by G proteins of signals that cause collapse of growth cones. Science. 1993 Jan 1;259(5091):77–79. doi: 10.1126/science.8418498. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Kocsis J. D., Rand M. N., Lankford K. L., Waxman S. G. Intracellular calcium mobilization and neurite outgrowth in mammalian neurons. J Neurobiol. 1994 Mar;25(3):252–264. doi: 10.1002/neu.480250306. [DOI] [PubMed] [Google Scholar]
  30. Lagenaur C., Lemmon V. An L1-like molecule, the 8D9 antigen, is a potent substrate for neurite extension. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7753–7757. doi: 10.1073/pnas.84.21.7753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lampidis T. J., Kolonias D., Savaraj N., Rubin R. W. Cardiostimulatory and antiarrhythmic activity of tubulin-binding agents. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1256–1260. doi: 10.1073/pnas.89.4.1256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Leavesley D. I., Schwartz M. A., Rosenfeld M., Cheresh D. A. Integrin beta 1- and beta 3-mediated endothelial cell migration is triggered through distinct signaling mechanisms. J Cell Biol. 1993 Apr;121(1):163–170. doi: 10.1083/jcb.121.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lemmon V., Burden S. M., Payne H. R., Elmslie G. J., Hlavin M. L. Neurite growth on different substrates: permissive versus instructive influences and the role of adhesive strength. J Neurosci. 1992 Mar;12(3):818–826. doi: 10.1523/JNEUROSCI.12-03-00818.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lemmon V., McLoon S. C. The appearance of an L1-like molecule in the chick primary visual pathway. J Neurosci. 1986 Oct;6(10):2987–2994. doi: 10.1523/JNEUROSCI.06-10-02987.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Letourneau P. C. Cell-to-substratum adhesion and guidance of axonal elongation. Dev Biol. 1975 May;44(1):92–101. doi: 10.1016/0012-1606(75)90379-6. [DOI] [PubMed] [Google Scholar]
  36. Martini R., Schachner M. Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and MAG) and their shared carbohydrate epitope and myelin basic protein in developing sciatic nerve. J Cell Biol. 1986 Dec;103(6 Pt 1):2439–2448. doi: 10.1083/jcb.103.6.2439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Meriney S. D., Gray D. B., Pilar G. R. Somatostatin-induced inhibition of neuronal Ca2+ current modulated by cGMP-dependent protein kinase. Nature. 1994 May 26;369(6478):336–339. doi: 10.1038/369336a0. [DOI] [PubMed] [Google Scholar]
  39. Narahashi T., Herman M. D. Overview of toxins and drugs as tools to study excitable membrane ion channels: I. Voltage-activated channels. Methods Enzymol. 1992;207:620–643. doi: 10.1016/0076-6879(92)07045-p. [DOI] [PubMed] [Google Scholar]
  40. Neugebauer K. M., Emmett C. J., Venstrom K. A., Reichardt L. F. Vitronectin and thrombospondin promote retinal neurite outgrowth: developmental regulation and role of integrins. Neuron. 1991 Mar;6(3):345–358. doi: 10.1016/0896-6273(91)90244-t. [DOI] [PubMed] [Google Scholar]
  41. Ng-Sikorski J., Andersson R., Patarroyo M., Andersson T. Calcium signaling capacity of the CD11b/CD18 integrin on human neutrophils. Exp Cell Res. 1991 Aug;195(2):504–508. doi: 10.1016/0014-4827(91)90402-g. [DOI] [PubMed] [Google Scholar]
  42. Paradies N. E., Grunwald G. B. Purification and characterization of NCAD90, a soluble endogenous form of N-cadherin, which is generated by proteolysis during retinal development and retains adhesive and neurite-promoting function. J Neurosci Res. 1993 Sep 1;36(1):33–45. doi: 10.1002/jnr.490360105. [DOI] [PubMed] [Google Scholar]
  43. Reichardt L. F., Bossy B., Carbonetto S., de Curtis I., Emmett C., Hall D. E., Ignatius M. J., Lefcort F., Napolitano E., Large T. Neuronal receptors that regulate axon growth. Cold Spring Harb Symp Quant Biol. 1990;55:341–350. doi: 10.1101/sqb.1990.055.01.035. [DOI] [PubMed] [Google Scholar]
  44. Roark E. F., Paradies N. E., Lagunowich L. A., Grunwald G. B. Evidence for endogenous proteases, mRNA level and insulin as multiple mechanisms of N-cadherin down-regulation during retinal development. Development. 1992 Apr;114(4):973–984. doi: 10.1242/dev.114.4.973. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Schuch U., Lohse M. J., Schachner M. Neural cell adhesion molecules influence second messenger systems. Neuron. 1989 Jul;3(1):13–20. doi: 10.1016/0896-6273(89)90111-6. [DOI] [PubMed] [Google Scholar]
  47. Schwartz M. A. Spreading of human endothelial cells on fibronectin or vitronectin triggers elevation of intracellular free calcium. J Cell Biol. 1993 Feb;120(4):1003–1010. doi: 10.1083/jcb.120.4.1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shankar G., Davison I., Helfrich M. H., Mason W. T., Horton M. A. Integrin receptor-mediated mobilisation of intranuclear calcium in rat osteoclasts. J Cell Sci. 1993 May;105(Pt 1):61–68. doi: 10.1242/jcs.105.1.61. [DOI] [PubMed] [Google Scholar]
  49. Shirayoshi Y., Hatta K., Hosoda M., Tsunasawa S., Sakiyama F., Takeichi M. Cadherin cell adhesion molecules with distinct binding specificities share a common structure. EMBO J. 1986 Oct;5(10):2485–2488. doi: 10.1002/j.1460-2075.1986.tb04525.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sorimachi M. Caffeine- and muscarinic receptor agonist-sensitive Ca2+ stores in chick ciliary ganglion cells. Brain Res. 1993 Nov 5;627(1):34–40. doi: 10.1016/0006-8993(93)90745-9. [DOI] [PubMed] [Google Scholar]
  51. Tomaselli K. J., Neugebauer K. M., Bixby J. L., Lilien J., Reichardt L. F. N-cadherin and integrins: two receptor systems that mediate neuronal process outgrowth on astrocyte surfaces. Neuron. 1988 Mar;1(1):33–43. doi: 10.1016/0896-6273(88)90207-3. [DOI] [PubMed] [Google Scholar]
  52. Tomaselli K. J., Reichardt L. F., Bixby J. L. Distinct molecular interactions mediate neuronal process outgrowth on non-neuronal cell surfaces and extracellular matrices. J Cell Biol. 1986 Dec;103(6 Pt 2):2659–2672. doi: 10.1083/jcb.103.6.2659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Von Bohlen Und Halbach Friedrich, Taylor Joanne, Schachner Melitta. Cell Type-specific Effects of the Neural Adhesion Molecules L1 and N-CAM on Diverse Second Messenger Systems. Eur J Neurosci. 1992;4(10):896–909. doi: 10.1111/j.1460-9568.1992.tb00116.x. [DOI] [PubMed] [Google Scholar]
  54. Williams E. J., Doherty P., Turner G., Reid R. A., Hemperly J. J., Walsh F. S. Calcium influx into neurons can solely account for cell contact-dependent neurite outgrowth stimulated by transfected L1. J Cell Biol. 1992 Nov;119(4):883–892. doi: 10.1083/jcb.119.4.883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Williams E. J., Walsh F. S., Doherty P. The production of arachidonic acid can account for calcium channel activation in the second messenger pathway underlying neurite outgrowth stimulated by NCAM, N-cadherin, and L1. J Neurochem. 1994 Mar;62(3):1231–1234. doi: 10.1046/j.1471-4159.1994.62031231.x. [DOI] [PubMed] [Google Scholar]
  56. Williams E. J., Walsh F. S., Doherty P. Tyrosine kinase inhibitors can differentially inhibit integrin-dependent and CAM-stimulated neurite outgrowth. J Cell Biol. 1994 Mar;124(6):1029–1037. doi: 10.1083/jcb.124.6.1029. [DOI] [PMC free article] [PubMed] [Google Scholar]

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