Fig. 6
From: Optogenetic dissection of Rac1 and Cdc42 gradient shaping
Cdc42 and Rac1 drive different cellular responses. a, b Trajectories of cells stimulated as represented in Fig. 5 were analyzed quantitatively. a Cell speed defined as the instantaneous velocity of the cell displacement averaged over five consecutive time frames (top scheme). Box plots show instantaneous velocity of cells expressing CIBN-GFP-CAAX together with ITSN-CRY2-mCherry (blue) or TIAM-CRY2-mCherry (red) stimulated with various gradients of light (ITSN: n = 25 (ctrl), n = 16 (1×), n = 20 (2×), n = 19 (3×), n = 16 (4×), TIAM: n = 19 (ctrl), n = 18 (1×), n = 18 (2×), n = 17 (3×), n = 18 (4×)). Box plots represent the median, interquartile (box), 1.5 IQR (whiskers). b Directionality defined as the angular precision of cell displacement: the angle of displacement was measured using the initial position averaged over the first three frames and the final position averaged over the last three frames (top scheme). Angles were then bootstrapped and angular precision was calculated with the formula \(r = \sqrt {( {1}/{n}^{\ast} \sum _{i = 1}^{n} \sin\theta _{i} )^{2} + ( {1}/{n}^{\ast} \sum _{i = 1}^{n} \cos\theta _{i} )^{2}} .\) Box plots represent the median, interquartile (box), 1.5 IQR (whiskers). Statistical significance between consecutive conditions was evaluated using Wilcoxon’s rank sum test. *p ≤ 0.05. n.s., nonsignificant (p > 0.05). c Speed and d directionality measurements on HeLa cells expressing CIBN-GFP-CAAX and ITSN-CRY2-mCherry after Rac1 inhibition with 100 µM NSC 23766 and stimulated with various Cdc42 gradients. n = 8 (1×), n = 16 (2×) or n = 17 (4×). e Effects of slope, amplitude, and spatial extent of Cdc42 gradients on cell velocity and angular precision. HeLa cells expressing CIBN-GFP-CAAX and ITSN-CRY2-mCherry were stimulated with varying gradients of light. Gradients in blue (a, b) and red (c, d) have two distinct spatial extents. Gradients in light (a, c) and dark (b, d) color have two distinct amplitudes. Two gradients (b, c) have the same slope. n = 13 (a), n = 16 (b), n = 22 (c), n = 16 (d). *p ≤ 0.05, **p ≤ 0.01, n.s., nonsignificant (p > 0.05) (Wilcoxon rank sum test)
