Fig. 2: Differential effects of N- and C-terminal switch II variants on cellular RAC1 activity.
From: Distinct sub-clusters of developmental disorder-associated variants in the switch II region of RAC1
A Representative images of NIH 3T3 fibroblasts transfected with the indicated RAC1 variants illustrate the most common morphology for each variant. Cells stained with anti-myc, labelling transfected cells (green) and Alexa405-Phalloidin to label F- actin (magenta). B Images of NIH 3T3 fibroblasts transfected with indicated RAC1 variants and the GEF Tiam1. Cells stained with anti-myc to label cells transfected with RAC1 variants, anti-flag to label cells transfected with the GEF Tiam1 and Alexa405-Phalloidin to label F- actin. All scale bars indicate 10 μm. C Graph shows cell circularity values of individual transfected cells, with lines indicating group means. Each point represents one cell. n > 140 cells from 9 coverslips. Statistical analysis was performed using the Kruskal-Wallis test (H = 466.5, df = 5, P < 0.0001), followed by Dunn’s multiple comparisons test (α = 0.05). Comparisons to WT, the P-values: T17N (P = 0.0019), Q61L (P < 0.0001), R66G (P < 0.0001), L70V (P > 0.9999) and S71F (P = 0.8749). Controls are shown in grey, activating variants in blue and dominant-negative variants in orange. D Graphs show the cell circularity values of individual transfected cells and the line indicates the mean value. Statistical analysis was performed using the Kruskal-Wallis test (H = 200.5, df = 7, P < 0.0001), followed by Dunn’s multiple comparisons test (α = 0.05). Comparisons to GEF alone, P-values: WT + GEF, R66G + GEF and R68G + GEF (P > 0.9999), T17N + GEF (P < 0.0001), L70V + GEF (P = 0.0037), S71F + GEF (P < 0.0001) and P73L + GEF (P < 0.0001),. n > 90 cells from 9 coverslips. Controls shown in grey, activating variants in blue and dominant negative variants in orange. E Graph showing emission ratio of YFP to CFP for populations of HEK293 cells expressing RAC1 Raichu biosensor modified to include the indicated variant. n = 30 cell populations. Statistical analyses were performed using one-way ANOVA (F(7232) = 114.8, P < 0.0001). Comparisons to WT, the P-values: T17N (P < 0.0001), Q61L (P < 0.0001), R68G (P < 0.0001), L70V (P = 0.0376) and S71F (P = 0.0018), both R66G and P73L (P > 0.9999). Asterisks indicate the significant differences in the data from WT-RAC1 Raichu. F Representative GST-PAK1 pulldown assay blot showing total (TL) and pulled-down (PD) myc-RAC1. αTubulin is a loading control. G Quantitation of pulled-down RAC1/Total RAC1 from three independent experients and normalised to RAC1-WT. Statistically analysed using one-way ANOVA. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.
