B-50 GAP-43-INDUCED FORMATION OF FILOPODIA DEPENDS ON RHO-GTPASE/

Ln the present study we show that expression of the neural PKC-substra te B-50 (growth-associated protein [GAP-43]) in Rat-1 fibroblasts indu ced the formation of filopodial extensions during spreading. This morp hological change was accompanied by an enhanced formation of periphera l actin filaments and by accumulation of vinculin immunoreactivity in filopodial focal adhesions, colocalizing with B-50. In time lapse expe riments, the B50-induced filopodial extensions were shown to stay in c lose contact with the substratum and appeared remarkably stable, resul ting in a delayed lamellar spreading of the fibroblasts. The morphogen etic effects of the B-50 protein were entirely dependent on the integr ity of the two N-terminal cysteines involved in membrane association ( C3C4), but were not significantly affected by mutations of the PKC-pho sphorylation site (S41) or deletion of the C terminus (177-226). Cotra nsfection of B-50 with dominant negative Cdc42 or Rac did not prevent B-50-induced formation of filopodial cells, whereas this process could be completely blocked by cotransfection with dominant negative Rho or Clostridium botulinum C3-transferase. Conversely, constitutively acti ve Rho induced a similar filopodial phenotype as B-50. We therefore pr opose that the induction of surface extensions by B-50 in spreading Ra t-1 fibroblasts depends on Rho-guanosine triphosphatase function.