Fig. 2: Analysis of Rho GTPase crosstalk in living cells.

a Schematic of rapid activity perturbation and combined activity measurement strategy. b–e Analysis of perturbation-response relationships of Rho GTPase activity in Neuro-2a cells. b, c Representative TIRF images before dimerizer addition (b, top) and Rac1 perturbation and uncorrected, raw Rho sensor and raw control sensor signal kinetics (c, bottom) corresponding to orange boxes (see also Supplementary Movie 2). All constructs are predominantly cytosolic and homogenously distributed in the cell bodies and neurite-like protrusions. d Average perturbation and control-corrected activity sensor signal kinetics for selected crosstalk combinations (see Supplementary Fig. 2 for all combinations). e Quantification of average sensor signal changes during Rho GTPase activity perturbation. Responses at time points before (pre), and 5 min after dimerizer addition (on) are shown. f Influence diagram that summarizes significant activity response measurements at 5 min after dimerizer addition. All observations and measurements are based on at least 3 independent repetitions with a total of at least 28 cells per condition (exact numbers of cells are indicated in individual panels). Scale bars: 5 µm; 0.26 μm/pixel; ****P < 0.0001; Student’s t-Test. Error bars represent standard error of the mean. YFP yellow fluorescent protein, GBD GTPase-binding domain, All statistical tests were two-sided. Source data are provided as a Source Data file.