Critical calpain-dependent ultrastructural alterations underlie the transformation of an axonal segment into a growth cone after axotomy of cultured Aplysia neurons.
The transformation of a stable axonal segment into a motile growth cone is a critical step in the regeneration of amputated axons. In earlier studies we found that axotomy of cultured Aplysia neurons leads to a transient and local elevation of the free intracellular Ca2+ concentration, resulting in calpain activation, localized proteolysis of submembranal spectrin, and, eventually, growth cone formation. Moreover, inhibition of calpain by calpeptin prior to axotomy inhibits growth cone formation. Here we investigated the mechanisms by which calpain activation participates in the transformation of an axonal segment into a growth cone. To that end we compared the ultrastructural alterations induced by axotomy performed under control conditions with those caused by axotomy performed in the presence of calpeptin, using cultured Aplysia neurons as a model. We identified the critical calpain-dependent cytoarchitectural alterations that underlie the formation of a growth cone after axotomy. Calpain-dependent processes lead to restructuring of the neurofilaments and microtubules to form an altered cytoskeletal region 50-150 microm proximal to the tip of the transected axon in which vesicles accumulate. The dense pool of vesicles forms in close proximity to a segment of the plasma membrane along which the spectrin membrane skeleton has been proteolyzed by calpain. We suggest that the rearrangement of the cytoskeleton forms a transient cellular compartment that traps transported vesicles and serves as a locus for microtubule polymerization. We propose that this cytoskeletal configuration facilitates the fusion of vesicles with the plasma membrane, promoting the extension of the growth cone's lamellipodium. The growth process is further supported by the radial polymerization of microtubules from the growth cone's center.
Spira
Micha E
ME
Department of Neurobiology, Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem 91904, Israel. spira@cc.huji.ac.il
Oren
Ruthi
R
Dormann
Ada
A
Gitler
Daniel
D
D000818:Animals; D001048:Aplysia; D001369:Axons / Q000378:metabolism* / Q000648:ultrastructure*; D019771:Axotomy; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D002478:Cells, Cultured; D020439:Growth Cones / Q000378:metabolism* / Q000648:ultrastructure*; D008854:Microscopy, Electron; D018715:Microscopy, Video; D009416:Nerve Regeneration; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure
D002154:Calpain; D002118:Calcium