N-WASP-induced filopodial formation requires and synergizes with
Toca-1. A, N1E115 cells were transfected with N-WASP alone,
N-WASP with Toca-1 mutants, and N-WASP with Toca-1 (a). All
transfections were carried out with GFP-actin. Panels show GFP and DIC images
with a merge. The bar chart shows a statistical analysis of
the experiments (b). Neurites and filopodial formation were scored as
described under “Materials and Methods.” Toca-1 F-BAR domain and
Cdc42 binding mutants were as described in the legend to
Fig. 2. Three independent
experiments were performed (with n = 10-15). Data shown are mean
± S.D. Bar = 10 μm. B, Toca-1 was KD
using RNAi and the affect of N-WASP on filopodial formation examined.
a, Toca-1 protein levels with control RNAi and Toca-1 RNAi. b,
left panel, N-WASP transfection with control RNAi. Right panel,
N-WASP transfection with Toca-1 RNAi. Lower panel shows tracing of
filopodia in red. c, statistical analysis of experiments similar to
those presented in b of single cells. Data shown are mean ±
S.D. (n = 20).