Figure 4.

The Spectrin Mutants of TRIO Enhance RAC1 Signaling, Neurite Outgrowth, and Lamellipodia Formation in N1E-115 Cells, Whereas the GEFD1 Mutants Are Mostly Impaired in These Processes

(A) Immunoblot analysis of HEK293T cell lysates transfected with the indicated GFP-TRIO variants and detected with an anti-GFP antibody (lower panel). PAK1 phosphorylation amounts are detected with a phospho-Ser144 PAK1 antibody (upper panel) and compared to total PAK1 amounts detected with a PAK1 antibody (middle panel).

(B) Quantification of the ratio of phospho-PAK1 amounts over total PAK1 expression. PAK1 phosphorylation is used as a readout for the activation of the RAC1 signaling cascade. Data are presented as the mean ± SEM of at least five independent experiments.

(C) Quantification of the neurite outgrowth induced by WT or mutant TRIO. Neurite outgrowth is monitored on the basis of the number of cells harboring an extension of at least twice the length of the soma. Data are presented as n-fold change over WT TRIO, which was arbitrarily set to 1. Data are presented as the mean ± SEM of at least five independent experiments.

(D) Quantification of lamellipodia formation induced by WT or mutant TRIO. Data are presented as n-fold change over WT TRIO, which was arbitrarily set to 1. Data are presented as the mean ± SEM of at least five independent experiments.

Statistical analysis in (B), (C), and (D) were made by one-way ANOVA followed by Dunnett’s test. Asterisks indicate datasets significantly different from WT (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001).

(E) Micrographs of N1E-115 cells transfected with the indicated GFP-TRIO variants (green); rhodamine-phalloidin and Hoechst stained the actin (red) and nuclei (blue), respectively. Representative images for each variant type are presented. White arrowheads point to lamellipodia. Scale bar: 20 μm.