INDUCTION OF GROWTH CONE FORMATION BY TRANSIENT AND LOCALIZED INCREASES OF INTRACELLULAR PROTEOLYTIC ACTIVITY

The formation of a growth cone at the tip of a transected axon is a cr ucial step in the subsequent regeneration of the amputated axon, Durin g this process, the transected axon is transformed from a static segme nt into a motile growth cone. Despite the importance of this process f or regeneration of the severed axon, little is known about the mechani sms underlying this transformation. Recent studies have suggested that Ca2+-activated proteinases underlay the morphological remodeling of n eurons after injury, However, this hypothesis was never tested directl y. 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 microin jected into intact axonal segments or somata of cultured Aplysia neuro ns, 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 irreversibl e neuritogenesis. Growth cones were not formed after external applicat ion 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-ind uced neuritogenesis was associated with ultrastructural alterations si milar to those observed by us after axotomy. We conclude that local an d transient elevations of cytoplasmic proteolytic activity can induce growth cone formation and neuritogenesis, and suggest that localized p roteolytic activity plays a role in growth cone formation after axotom y.