REGULATED TYROSINE PHOSPHORYLATION AT THE TIPS OF GROWTH CONE FILOPODIA

Several types of evidence suggest that protein-tyrosine phosphorylatio n is important during the growth of neuronal processes, but few specif ic roles, or subcellular localizations suggestive of such roles, have been defined. We report here a localization of tyrosine-phosphorylated protein at the tips of growth cone filopodia. Immunocytochemistry usi ng a mAb to phosphorylated tyrosine residues revealed intense staining of the tips of most filopodia of Aplysia axons growing slowly on a po lylysine substrate, but of few filopodia of axons growing rapidly on a substrate coated with Aplysia hemolymph, which has growth-promoting m aterial. Cytochalasin D, which causes F-actin to withdraw rapidly from the growth cone, caused the tyrosine-phosphorylated protein to withdr aw rapidly from filopodia, suggesting that the protein associates or i nteracts with actin filaments. Phosphotyrosine has previously been fou nd concentrated at adherens junctions, where bundles of actin filament s terminate, but video-enhanced contrast-differential interference con trast and confocal interference reflection microscopy demonstrated tha t the filopodial tips were not adherent to the substrate. Acute applic ation of either hemolymph or inhibitors of protein-tyrosine kinases to neurons on polylysine resulted in a rapid loss of intense staining at filopodial tips concomitant with a lengthening of the filopodia (and their core bundles of actin filaments). These results demonstrate that tyrosine-phosphorylated protein can be concentrated at the barbed end s of actin filaments in a context other than an adherens junction, ind icate an association between changes in phosphorylation and filament d ynamics, and provide evidence for tyrosine phosphorylation as a signal ing mechanism in the filopodium that can respond to environmental cues controlling growth cone dynamics.