optogenetic activation of RhoA leads to protrusion or retraction.

(A) Scheme of the optogenetic tool. Optogenetic dimer (in gray) dimerizes upon blue light activation (blue arrow), and dissociates in the dark with an off rate of 20s (black arrow). DH-PH domain is fused to the SspB moiety (purple), which recruitment to the plasma membrane through iLID triggers RhoA activation, from GDP (light red) to GTP (dark red) state. (B) The three opto plasmids, with DH-PH domains (purple) shown in their wildtype position in the different RhoA GEFs used here. (C)Experimental timeline. Transient transfection is done at least 30 hours before local activation (blue squares). Activation is done by pulses (blue bars, top) at different frequencies, intensities and durations. Cells are observed for 30 to 60 minutes. (D) Responses distributions for each optogenetic tool. (E,F) Area over time of the cell in the activated region (E) or without activation (F), normalized by the mean initial area. t=0 is the starting point of the activation, each blue bar on top representing one light impulse. Orange: protruding cells, blue: retracting cells, gray: nonmoving cells or mixed phenotype (labeled by hand). (G,I) representative cells doing retraction (on the left) and protrusion (on the right) upon optogenetic activations on two different side of the cell. Scale bar: 10 μm. White squares: area of activation. Red color: RFPt channel (optogenetic tool). (H) Sankey diagram representing the proportion of cells doing a protrusion (orange), retraction (blue) or a mixed phenotype (gray) at one side (first activation) or the other side (second activation).

cell phenotype depends on the initial optoPRG concentration.

(A) Phenotype dependence on initial cytosolic optoPRG concentration. The normalized area in the activated region after 5 minutes is plotted against the mean fluorescence intensity for each cell, which color is labeled by hand depending on the observed phenotype. Schemes on the bottom represent high and low levels of expression (P2A plasmids implies approximately a one-to-one ratio of iLID against SspB). (B) Three representative time lapse images of transiently transfected cells, one retracting (top), one showing a mixed phenotype (middle), and one protruding (bottom). Intensities are very different, as seen by the dynamic range of the colormaps presented on the right. (C) Absolute fluorescence intensity of recruited optoPRG, before (t<0) and after (t>0) activation. Blue bars: activation pulses. (D)Phenotype depending on both optoPRG concentration and PRG DH-PH overexpression, measured both by fluorescence intensity (a.u.). Increasing recruitable and non-recruitable DH-PH domain of PRG both lead to protruding phenotypes. Phenotypes are manually labelled. (E) Cell area before activation for retracting (blue) and protruding (orange) cells. (Mann–Whitney U test. **** <0.0001)

downstream effectors show that cell phenotype is set immediately.

Distinct pathways are triggered from the first timepoint. (A,D,G) Representative timelapse images and kymographs of retracting (top) and protruding (bottom) cells labeled with Lifeact-iRFP (A), MRLC-iRFP(D), and RBD-2xTdTomato biosensor (G), activated with optoPRG starting at t=0min. White rectangles are areas of optogenetic activation. Scale bars are 10 μm. (B,E,H) Corresponding mean normalized intensities are plotted against time (mean +/-s.e.m.), blue for retracting cells and orange for protruding one. (C,F,I) Corresponding pairwise comparison for each cell of the signal inside the region of activation between the initial time and 60s (Lifeact-iRFP and RhoA biosensor) or 90s (MRCL-iRFP). Data are grouped by phenotype. *P<0.05 **P<0.01, ***P<0.001, **** P<0.0001 (Wilcoxon test to compare t=0 and t>0, independent t-test otherwise)

PH domain of PRG is triggering inhibition of RhoA at high PRG concentration and is necessary but not sufficient for protruding phenotype.

(A) Three representative cells that show very different responses to optoPRG pulsatile activation. Cell 1 has a low optoPRG expression, while Cell 2 and Cell 3 have high concentration of optoPRG. Intensities are normalized by the mean intensity before the first activation (t=0). (B) Quantification of the relative change in RhoA biosensor (RBD) after one pulse of optogenetic recruitment of the PH domain of PRG. In gray, optoPH recruitment, in red, RhoA biosensor. Light pulses are shown with blue bars. (C) Image of the corresponding cell. On the right, kymograph taken within the activation region. White dotted line shows the starting point of the activation. Scale bar: 10 μm. Region of activation is shown is the blue rectangle. (D) Scheme of the probable mechanism of PH domain dominant negative effect on RhoA. (E) Phenotype after optogenetic activation for optoPRG (bottom) and optoPRG with the PH mutated for no binding to RhoA-GTP (top). On the left, schemes of the expected behavior of the corresponding proteins. (F) Representative image of a cell transfected with the optoPRG with mutated PH, doing a retraction despite the high optoPRG concentration. (G) Quantification of protruding and retracting phenotypes in cells highly overexpressing non-recruitable PRG, comparing mutated and non-mutated optoPRG, with a scheme of the experiment on the left. See Supp Figure 6 for the selected cells. (H) Membrane displacement of optoPH cells (in blue) compared to optoPRG cells (in gray). No specific protrusion can be seen. (I) Normalized membrane area after 5 minutes in the activated for optoPRG and optoPH cells. Orange: protruding cells, gray: mixed phenotype or no movement, blue: retracting cells.

PRG activates Cdc42 and Cdc42 downstream activity is necessary for the protrusive phenotype.

(A,D,G) Representative time-lapse images and kymographs of retracting (top) and protruding (bottom) cells labeled with PBD-iRFP (A), delCMV-mCherry-WaspGBD (D), and mCherry-3xp67Phox (G) biosensors 28, activated with optoPRG starting at t=0min. White rectangles are areas of optogenetic activation. Scale bars are 10 μm. (B,E,H) Corresponding mean normalized intensities are plotted against time (mean +/-s.e.m.), blue for retracting cells and orange for protruding one. (C,F,I) Corresponding pairwise comparison for each cell of the signal inside the region of activation between the initial time and 60s (PBD biosensor) or 30s (Cdc42 and Rac1 biosensors). Data are grouped by phenotype. *P<0.05 **P<0.01, ***P<0.001, **** P<0.0001 (Wilcoxon test to compare t=0 and t>0, independent t-test otherwise). (J) Left, scheme describing the IP3 experiment. Blue bars represent optogenetic pulses (every 30s). Half an hour after the first experiment, IPA3 is added at 5 μM. Right, representative cell showing a protruding phenotype with ruffles (top), and a retracting phenotype after addition or IPA3 (bottom). (K) Quantification of phenotype switches.

a minimal model recapitulates RhoA activity dynamics and the phenotypic switch.

(A) Model for active RhoA dynamics. Interactions are represented with arrows, with the two main parameters of the model. (B) The three different RhoA dynamics are well fitted with one single free parameter, Geq/Kb. Dotted blue line: fitted curve, with gray line g(d) taken as input (optoPRG recruitment). Red line: RBD biosensor. (C) Complete model, adding Cdc42 to (A): the GEF PRG can activate both RhoA and Cdc42, but can also inhibit RhoA by directly binding to it. (D) Center, evolution of γ describing the phenotype (positive for retraction and negative for protrusion) against the free parameter Geq/Kb. Two representative dynamics are shown on the right and on the left for the same input gg(dd), for a low and high Geq/Kb. In grey, optoPRG recruitment to the membrane, in green, Cdc42 activity, in red, RhoA activity. (E) Map of the phenotype as function of the free parameter Geq/Kb and of the duration time between two pulses. (F) One example of two phenotypes controlled in the same cell. On the left, first ten minutes of the cell area in the illuminated region for different frequencies and intensities of activation (low frequency high power every 30s, high frequency low power every 15s). On the right, two representative timelapse of retraction (top) and protrusion (bottom), activation is shown with the white rectangle. Scale bar: 10μm. (G,H) Graphical conclusion on the model. (G) Balance between RhoA and Cdc42 activity is represented in function of GEF basal concentration (grey gradient), both at the basal state (top) and after optogenetic activation (bottom, with blue lightning). At low concentrations RhoA takes over. At high concentration, optoPRG binds to active RhoA and inhibits it (complex GR), which enables Cdc42 to take over. (H) Curve showing the difference between RhoA and Cdc42 activity as a function of the basal intensity of the GEF. Phenotypes are marked with the colors (blue, retraction and orange, protrusion). Optogenetic modulation happens on vertical line, with the blue range, which limits the possibility of switching from one phenotype to the other.