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The formation of a growth cone at the tip of a transected axon is a crucial step in the subsequent regeneration of the amputated axon. During this process, the transected axon is transformed from a static segment into a motile growth cone. Despite the importance of this process for regeneration of the severed axon, little is known about the mechanisms underlying this transformation. Recent studies have suggested that Ca2+-activated proteinases underlay the morphological remodeling of neurons after injury. However, this hypothesis was never tested directly. Here we tested the ability of transient and localized increases in intracellular proteolytic activity to induce growth cone formation and neuritogenesis. Minute amounts of the proteinase trypsin were microinjected into intact axonal segments or somata of cultured Aplysia neurons, transiently elevating the intracellular protease concentration to 13-130 nM in the vicinity of the injection site. Such microinjections were followed by the formation of ectopic growth cones and irreversible neuritogenesis. Growth cones were not formed after external application of trypsin, microinjection of the carrier solution, or inactivated trypsin. Growth cone formation was not preceded by increases in free intracellular Ca2+ or changes in passive membrane properties, and was blocked by inhibitors of actin and tubulin polymerization. Trypsin-induced neuritogenesis was associated with ultrastructural alterations similar to those observed by us after axotomy. We conclude that local and transient elevations of cytoplasmic proteolytic activity can induce growth cone formation and neuritogenesis, and suggest that localized proteolytic activity plays a role in growth cone formation after axotomy.

Induction of Growth Cone Formation by Transient and Localized Increases of Intracellular Proteolytic Activity