Extended Data Fig. 3: Global recruitment of dominant negative Ras isoforms improves polarity and migration in Dictyostelium and neutrophils.
From: Ras suppression potentiates rear actomyosin contractility-driven cell polarization and migration
(a) Time-lapse confocal images of vegetative Dictyostelium expressing mRFPmars-SspB R73Q-RasG S17N ΔCAAX before or after laser was switched on globally. Pink arrows denote recruitment causing increased movement. Time in min:sec format. Scale bars: 5 µm. Box-and-whisker plots of (b, d) average cell speed, and (c, e) cell area, before (black) or after (red) opto-Ctrl (control) or opto-RasG S17N ΔCAAX recruitment. n = 10 (Ctrl) or n = 7 cells (RasG S17N ΔCAAX) examined over 3 independent experiments; asterisks indicate significant difference, *P = 0.0313 (d), *P = 0.0156 (e); ns denotes non-significant difference, P = 0.2730 (b), P = 0.7394 (c) (Two-sided Wilcoxon signed-rank test). (f) Time-lapse images of neutrophil expressing CRY2PHR-mCherry-KRAS4B S17N ΔCAAX (red; upper panel) and LifeAct-miRFP703 (cyan; lower panel), before or after recruitment. Pink arrows denote recruited cell; white arrows indicate control cell where there was no recruitment. Time in min:sec format. Scale bars: 5 µm. (g) Colour-coded (1-min intervals) outlines of recruited cell in (f). (h) Representative kymograph of cortical LifeAct intensity before or after recruitment. Linear colour map shows that blue is the lowest LifeAct intensity whereas yellow is the highest. Duration of kymograph is 22 mins. Cartoon depicts recruitment, F-actin polymerization or cell shape corresponding to kymograph. Box-and-whisker plots of (i) cell area, (l) average speed, and (m) aspect ratio, before (black) or after (red) recruitment. n = 14 cells examined over 3 independent experiments; asterisks indicate significant difference, ****P ≤ 0.001 (Two-sided Wilcoxon signed-rank test). Centroid tracks (n = 13 cells from 3 independent experiments) showing motility before (j) or after (k) recruitment. Each track lasts 5 mins and was reset to same origin. (b-e,i,l,m) Boxes extend from 25th to 75th percentiles, median is at the centre, and whiskers and outliers are graphed according to Tukey’s convention. Connecting lines are provided between paired data points obtained from the same cell, before or after KRAS4B S17N ΔCAAX recruitment (GraphPad Prism 8). Source numerical data are provided.
