Extended Data Fig. 1: C2GAPB or RasG S17N regulates Ras and protrusive activities.
From: Ras suppression potentiates rear actomyosin contractility-driven cell polarization and migration
(a) Representative images of vegetative Dictyostelium before (top panel, ‘-DOX’) and after (bottom panel, ‘+DOX’) doxycycline-induced GFP-C2GAPB (green) expression. Cells (DIC) were treated with TRITC-dextran (red) before imaging. Scale bars: 10 µm. (b) Macropinocytosis uptake measurements, before (black) and after (red) C2GAPB expression. n = 103 cells for ‘-DOX’ and n = 103 cells for ‘+DOX’, over 3 independent experiments; asterisks indicate significant difference, ****P ≤ 0.0001 (Two-sided Mann-Whitney test). Boxes extend from 25th to 75th percentiles, median is at the centre, and whiskers and outliers are graphed according to Tukey’s convention (GraphPad Prism 8). (c) Time-lapse images of vegetative Dictyostelium expressing mRFPmars-RasG S17N (red, bottom panel, ‘+DOX’) and RBD-GFP (green) after doxycycline treatment. Pink arrows denote RBD patches without RasG S17N. Time in ‘min’ format. Scale bars represent 5 µm. (d) Time-lapse images of C2GAPB-null Dictyostelium expressing RBD-GFP. Time in ‘min’ format. Scale bars: 5 µm. Time-lapse images of Dictyostelium or neutrophil expressing mRFPmars-SspB R73Q-RasG S17N ΔCAAX (e) or CRY2PHR-mCherry-KRAS4B S17N ΔCAAX (red; upper panel) and LifeAct-miRFP703 (cyan; lower panel) (g). RasG or KRAS4B S17N ΔCAAX was recruited to cell front by applying laser near it, as shown by dashed white box. White arrows denote existing protrusions whereas pink arrows highlight newer protrusions. Time in min:s format. Scale bars: 5 µm. (h) Polar histogram demonstrates higher probability of fresh protrusion formation away from recruitment area; n = 32 protrusions pooled from 14 cells, examined over 3 independent experiments. (f) Time-lapse images of latrunculin A-treated Dictyostelium expressing mRFPmars-SspB R73Q-C2GAPB (red; bottom panel) and RBD-YFP (yellow; top panel) before or after global recruitment. Pink arrows indicate RBD patches whereas white arrows denote recruitment. Time in min:s format. Scale bars: 5 µm. (i, j) Colour-coded (2-mins intervals) outlines of cells in (c). Source numerical data are provided.
