Extended Data Fig. 7: RasGAP recruitment cannot overcome blebbistatin inhibition but partially overcomes a sub-optimal dose.
From: Ras suppression potentiates rear actomyosin contractility-driven cell polarization and migration
(a) Time-lapse images of Dictyostelium expressing mRFPmars-SspB R73Q-C2GAPB (red; top panel), before or after global recruitment. 50 µM blebbistatin was added during imaging. Pink arrows denote cell of interest (DIC). Time in min:sec format. Scale bars: 5 µm. (b) Time-lapse images of neutrophil expressing LifeAct-miRFP703 (cyan; lower panel), before or after low-dose blebbistatin (20 µM) treatment. Time in min:sec format. Scale bars: 5 µm. (c) Strategy for testing low-dose blebbistatin on RASAL3-directed myosin II contraction and migration. (d) Time-lapse images of 20 µM blebbistatin-treated neutrophil expressing CRY2PHR-mCherry-RASAL3 (red; upper panel) and LifeAct-miRFP703 (cyan; lower panel), before or after recruitment. Pink arrow denotes cell of interest. Time in min:sec format. Scale bars: 5 µm. (e) Colour-coded (1-min intervals) outlines of cell in (d). (f) 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 16 mins. Cartoon summarizes recruitment, actin polymerization or cell shape corresponding to kymograph. Box-and-whisker plots of (g) average speed, (j) basal area, and (k) aspect ratio, before (black) and after (red) recruitment in blebbistatin-treated cells. n = 11 cells examined over 3 independent experiments; asterisks indicate significant difference, ***P = 0.001 (Two-sided Wilcoxon signed-rank test). 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 blebbistatin-treated cell, before or after RASAL3 recruitment (GraphPad Prism 8). Centroid tracks of 20 µM blebbistatin-treated neutrophils (n = 10 cells from 3 independent experiments) showing motility before (h) or after (i) RASAL3 recruitment. Each track lasts 5 mins and was reset to same origin. Source numerical data are provided.
