Extended Data Fig. 6: Ras suppression at the back improves polarity and contraction-driven migration.
From: Ras suppression potentiates rear actomyosin contractility-driven cell polarization and migration
Representative images of Dictyostelium expressing (a) mRFPmars-SspB R73Q-C2GAPB or (c) mRFPmars-SspB R73Q-RasG S17N ΔCAAX recruited to the back by applying laser near it (dashed white box). Time in min:sec format. Scale bars: 5 µm. (b) Cartoon demonstrates phenomenon in (a) and (c). (d) Time-lapse images of macrophage expressing CRY2PHR-mCherry-RASAL3 (red; upper panel) and LifeAct-miRFP703 (cyan; lower panel), before or after global recruitment. Time in min:sec format. Scale bars represent 5 µm. (e) Time-lapse images of neutrophil expressing CRY2PHR-mCherry-RASAL3 (red; upper panel) and LifeAct-miRFP703 (cyan; lower panel), placed in fMLP gradient, post-global recruitment. Chemoattractant was located on right-hand side of the field. Time in min:sec format. Scale bars: 5 µm. (f) Centroid tracks of chemotaxing opto-RASAL3-recruited neutrophils (n = 10 cells pooled from 3 independent experiments). Tracks last 2 mins and was reset to same origin. (g) Time-lapse images of CK666-treated neutrophil expressing CRY2PHR-mCherry-RASAL3 (red; upper panel) and LifeAct-miRFP703 (cyan; lower panel), before or after recruitment. Pink arrows denote long blebs which appear after recruited RASAL3 localized to back. Time in min:sec format. Scale bars: 5 µm. (h) Quantification of number of blebs per cell within a minute, before (black) or after (red) recruitment. n = 37 CK666-treated cells examined over 3 independent experiments; asterisks indicate significant difference, ****P ≤ 0.0001 (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 CK666-treated cell, before or after RASAL3 recruitment (GraphPad Prism 8). Time-lapse images of Dictyostelium expressing mRFPmars-C2GAPB (red; top panel) and myosin II-GFP (green; middle panel) before (j) or after (i) doxycycline treatment. CK666 was added during imaging. Since there is no C2GAPB expression without doxycycline, red panel is not shown in (j). Pink arrows denote long blebs (DIC) in C2GAPB-expressing cells after CK666 was added. Time in min:sec format. Scale bars: 5 µm. Source numerical data are provided.
