INHIBITION OF FORMATION OF FILOPODIA AFTER AXOTOMY BY INHIBITORS OF PROTEIN-TYROSINE KINASES

The activity of motile protrusions of the growth cone-filopodia, veils , and lamellipodia-is essential for directed growth of a neuronal proc ess. The regulation of the formation of these protrusions is not well understood. Numerous filopodia and veils or lamellipodia form within m inutes of transection of an Aplysia axon in culture, as the initial co mponents of growth cones of regenerating neurites. Axotomy, therefore, provides a robust and reliable protocol for analyzing the formation o f these protrusions. We evaluated the involvement of protein phosphory lation in the regulation of protrusive activity. Of the inhibitors of protein kinases assayed, only the inhibitors of protein tyrosine kinas es-genistein, lavendustin A, herbimycin A, and erbstatin analogue-supp ressed the formation of protrusions, as assessed by high magnification video microscopy. These drugs did not work by preventing resealing of the axon, as evident from visual inspection and by the unimpaired eff ectiveness of genistein or lavendustin in preventing formation of filo podia when applied after resealing. Inhibition of protein tyrosine kin ases not only prevented the formation of actin-based protrusions, but also caused deterioration of the actin network underlying the protrusi ve area of preexisting growth cones. Consistent with an involvement of protein tyrosine phosphorylation in the generation of protrusive stru ctures, immunocytochemistry revealed that aggregates of phosphotyrosin e appeared at the margins of the axon, from which protrusions emerge s hortly after axotomy. These results suggest a role for protein tyrosin e phosphorylation in the formation and maintenance of actin-based prot rusive structures. (C) 1995 John Wiley and Sons, Inc.