Vol. 9, Issue 6, 1279-1292, June 1998

B-50/GAP-43-induced Formation of Filopodia Depends on Rho-GTPase

Lambertus H. J. Aarts,^* Loes H. Schrama,^* Willem J. Hage,^¶ Johannes L. Bos,^* Willem Hendrik Gispen,^§ and Peter Schotman^*

 ^*Department of Physiological Chemistry,  Rudolf Magnus Institute for Neurosciences, Utrecht University, 3584 CG, Utrecht, The Netherlands;  ^§Department of Medical Pharmacology, Rudolf Magnus Institute for Neurosciences, Utrecht University, 3584 CG, Utrecht, The Netherlands; and  ^¶Hubrecht Laboratory, Netherlands Institute for Developmental Biology, 3584 CG, Utrecht, The Netherlands

In the present study we show that expression of the neural PKC-substrate B-50 (growth-associated protein [GAP-43]) in Rat-1 fibroblasts induced the formation of filopodial extensions during spreading. This morphological change was accompanied by an enhanced formation of peripheral actin filaments and by accumulation of vinculin immunoreactivity in filopodial focal adhesions, colocalizing with B-50. In time lapse experiments, the B-50-induced filopodial extensions were shown to stay in close contact with the substratum and appeared remarkably stable, resulting in a delayed lamellar spreading of the fibroblasts. The morphogenetic effects of the B-50 protein were entirely dependent on the integrity of the two N-terminal cysteines involved in membrane association (C3C4), but were not significantly affected by mutations of the PKC-phosphorylation site (S41) or deletion of the C terminus (177-226). Cotransfection 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 active Rho induced a similar filopodial phenotype as B-50. We therefore propose that the induction of surface extensions by B-50 in spreading Rat-1 fibroblasts depends on Rho-guanosine triphosphatase function.