Abstract
The cytoarchitecture, synaptic connectivity, and physiological properties of neurons are determined during their development by the interactions between the intrinsic properties of the neurons and signals provided by the microenvironment through which they grow. Many of these interactions are mediated and translated to specific growth patterns and connectivity by specialized compartments at the tips of the extending neurites: the growth cones (GCs). The mechanisms underlying GC formation at a specific time and location during development, regeneration, and some forms of learning processes, are therefore the subject of intense investigation. Using cultured Aplysia neurons we studied the cellular mechanisms that lead to the transformation of a differentiated axonal segment into a motile GC. We found that localized and transient elevation of the free intracellular calcium concentration ([Ca2+] i ) to 200–300 μM induces GC formation in the form of a large lamellipodium that branches up into growing neurites. By using simultaneous on-line imaging of [Ca2+] i and of intraaxonal proteolyticactivity, we found that the elevated [Ca2+] i activate proteases in the region in which a GC is formed. Inhibition of the calcium-activated proteases prior to the local elevation of the [Ca2+] i blocks the formation of GCs. Using retrospective immunofluorescent methods we imaged the proteolysis of the submembrane spectrin network, and the restructuring of the cytoskeleton at the site of GC formation. The restructuring of the actin and microtubule network leads to local accumulation of transported vesicles, which then fuse with the plasma membrane in support of the GC expansion.
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REFERENCES
Ambron, R. T.,Dulin, M. F.,Zhang, X. P.,Schmied, R., andWalters, E. T. (1995). Axoplasm enriched in a protein mobilized by nerve injury induces memory-like alterations in Aplysia neurons. J. Neurosci. 15: 3440-3446.
Ambron, R. T.,Zhang, X. P.,Gunstream, J. D.,Povelones, M., andWalters, E. T. (1996). Intrinsic injury signals enhance growth, survival, and excitability of Aplysia neurons. J. Neurosci. 16: 7469-7477.
Ashery, U.,Penner, R., andSpira, M. E. (1996). Acceleration of membrane recycling by axotomy of cultured aplysia neurons. Neuron 16: 641-651.
Aunis, D., andBader, M. F. (1988). The cytoskeleton as a barrier to exocytosis in secretory cells. J. Exp. Biol. 139: 253-266.
Bailey, C. H., andKandel, E. R. (1993). Structural changes accompanying memory storage. Annu. Rev. Physiol. 55: 397-426.
Benbassat, D., andSpira, M. E. (1993). Survival of isolated axonal segments in culture: Morphological, ultrastructural, and physiological analysis. Exp. Neurol. 122: 295-310.
Borgens, R. B.,Jaffe, L. F., andCohen, M. J. (1980). Large and persitent electrical currents enter the transected lamprey spinal cord. Proc. Natl. Acad. Sci. U.S.A. 77: 1209-1213.
Dash, P. K., Tian, L. M., and Moore, A. N. Sequestration of cAMP response element-binding protein by transcription factor decoys causes collateral elaboration of regenerating Aplysia motor neuron axons. Proc. Natl. Acad. Sci. U.S.A. 395: 8339-8344.
Eberhard, D. A., andHolz, R.W. (1988). Intracellular CaCC activates phospholipase C. Trends Neurosci. 11: 517-520.
Eddlemann, C. S.,Bittner, G. D., andFishman, H. M. (2000). Barrier permeability at cut axonal ends progressively decreases until an ionic seal is formed. Biophysical J. 79: 1883-1890.
Gabso, M.,Neher, E., andSpira, M. E. (1997). Low mobility of the Ca2C buffers in axons of cultured Aplysia neurons. Neuron 18: 473-481.
Gitler, D., andSpira, M. E. (1998). Real time imaging of calcium-induced localized proteolytic activity after axotomy and its relation to growth cone formation. Neuron 20: 1123-1135.
Glanzman, D. L.,Kandel, E. R., andSchacher, S. (1990). Target-dependent structural changes accompanying long-term synaptic facilitation in Aplysia neurons. Science 249: 799-802.
Gordon-Weeks, P. R. (2000). Neuronal Growth Cones. In Bard, J. B. L.,Barlow, P. W., andKirk, D. L. (eds.), Developmental and Cell Biology Series, Vol. 37, Cambridge University Press. Cambridge.
Kandel, E. R.,Schwartz, J. H., andJessell,T. M. (1991). Principles of Neuronal Science. Elsevier, NewYork.
Kosaka, T.,Kosaka, K.,Nakayama, T.,Hunziker, W., andHeizmann, C. W. (1993). Axons and axon terminals of cerebellar Purkinje cells and basket cells have higher levels of parvalbumin immunoreactivity than somata dendrites: Quantitative analysis by immunogold labeling. Exp. Brain Res. 93: 483-491.
Lankford, K. L.,Waxman, S. G., andKocsis, J. D. (1998). Mechanisms of enhancement of neurite regeneration in vitro following a conditioning sciatic nerve lesion. J. Comp. Neurol. 391: 11-29.
Leibovitch, D. (2001). Exogenous protease intracellular microinjections induce ectopic growth cone formation and neuritogenesis. MSc Thesis. The Hebrew University of Jerusalem, Jerusalem, Israel.
Leytus, S. P.,Melhado, L. L., andMangel, W. F. (1983a). Rhodamine-based compounds as fluorogenic substrates for serine proteinases. Biochem. J. 209: 299-307.
Leytus, S. P.,Patterson,W. L., andMangel,W. F. (1983b). New class of sensitive and selective fluorogenic substrates for serine proteinases. Amino acid and dipeptide derivatives of rhodamine. Biochem. J. 215: 253-260.
Lichstein, J.W.,Ballinger, M. L.,Blanchette, A. R.,Fishman, H. M., andBittner, G. D. (2000). Structural changes at cut ends of earthworm giant axons in the interval between dye barrier formation and neuritic growth. J. Compar. Neurobiol. 416: 143-157.
Neher, E. (1995). The use of fura-2 for estimating Ca buffers and Ca fluxes. Neuropharmacology 34: 1423-1442.
Perrin, D.,Moller, K.,Hanke, K., andSoling, H. D. (1992). cAMP and Ca(2C)-mediated secretion in parotid acinar cells is associated with reversible changes in the organization of the cytoskeleton. J. Cell Biol. 116: 127-134.
Roberts,W. M. (1993). Spatial calcium buffering in saccular hair cells. Nature 363: 74-76.
Saido, T. C.,Sorimachi, H., andSuzuki, K. (1994). Calpain: New perspectives in molecular diversity and physiological-pathological involvement. FASEB J. 8: 814-822.
Spira, M. E.,Benbassat, D., andDormann, A. (1993). Resealing of the proximal and distal cut ends of transected axons: Electrophysiological and ultrastructural analysis. J. Neurobiol. 24: 300-316.
Spira, M. E.,Dormann, A.,Ashery, U.,Gabso, M.,Gitler, D.,Benbassat, D.,Oren, R., andZiv, N. E. (1996). Use of Aplysia neurons for the study of cellular alterations and the resealing of transected axons in vitro. J. Neurosci. Methods 69: 91-102.
Spira, M. E.,Ziv, N. E.,Oren, R.,Dormann, A., andGitler, D. (2000). High calcium concentration, calpain activation and cytoskeleton remodeling in neuronal regeneration after axotomy. In Pochet, R.,Donato, R.,Haiech, J.,Heizmann, C., andGerke, V. (eds.), Calcium: The Molecular Basis of Calcium Action in Biology and Medicine, Kluwer, Dordrecht, pp. 589-605.
Strautman, A. F.,Cork, R. J.,Robinson, K. R. (1990). The distribution of free calcium in transected spinal axons and its modulation by applied electrical fields. J. Neurosci. 10: 3564-3575.
Walters, E. T.,Alizadeh, H., andCastro, G. A. (1991). Similar neuronal alterations induced by axonal injury and learning in Aplysia. Science 253: 797-799.
Walters, E. T., andAmbron, R. T. (1995). Long-term alterations induced by injury and by 5-HT in Aplysia sensory neurons: Convergent pathways and common signals? Trends Neurosci. 18: 137-142.
Welch, M. D.,Mallavarapu, A.,Rosenblatt, J., andMitchison, T. J. (1997). Actin dynamics in vivo. Curr. Opin. Cell Biol. 9: 54-61.
Yawo, H., andKuno, M. (1983). How a nerve fiber repairs its cut end: Involvement of phospholipase A2. Science 222: 1351-1353.
Yawo, H., andKuno, M. (1985). Calcium dependence of membrane sealing at the cut end of the cockroach giant axon. J. Neurosci. 5: 1626-1632.
Ziv,N. E., andSpira, M. E. (1993). Spatiotemporal distribution of Ca2C following axotomy and throughout the recovery process of cultured Aplysia neurons. Eur. J. Neurosci. 5: 657-668.
Ziv, N. E., andSpira, M. E. (1995). Axotomy induces a transient and localized elevation of the free intracellular calcium concentration to the millimolar range. J. Neurophysiol. 74: 2625-2637.
Ziv, N. E., andSpira, M. E. (1997). Localized and transient elevations of intracellular Ca2C induce the dedifferentiation of axonal segments into growth cones. J. Neurosci. 17: 3568-3579.
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Spira, M.E., Oren, R., Dormann, A. et al. Calcium, Protease Activation, and Cytoskeleton Remodeling Underlie Growth Cone Formation and Neuronal Regeneration. Cell Mol Neurobiol 21, 591–604 (2001). https://doi.org/10.1023/A:1015135617557
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DOI: https://doi.org/10.1023/A:1015135617557