Fig. 4: Sequential Rac and Rho activation is tightly coupled to cell protrusion-retraction cycles in space and time.

a Representative A431 cell expressing a cytosolic cell volume marker (mCitrine) and an improved Rac activity sensor (mCherry-3xp67^phoxGBD; top; see also Supplementary Movie 3; n = 23 cells from 3 independent experiments). The white arrow marks the direction of a local cell protrusion. b Automated tracing of the cell border using a modified version of the ADAPT plugin⁴³. c Maps generated by the ADAPT plugin that represent the spatio-temporal dynamics of the Rac sensor signal between the green lines in (b) (left) and of the cell edge velocity (right). The yellow arrows in (c) point to the local protrusion that occurs at the position of the cell area marked by the yellow arrow in (b). Red areas in the velocity map correspond to local cell protrusions, blue areas to local cell retractions. d Plot of Rac sensor signals and cell edge velocity corresponding to the yellow dotted line in (c). e Representative TIRF images (left) of A431 cells that generate spontaneous protrusion-retraction cycles and express the Rac or Rho GTPase activity sensors and the cell volume marker (see also Supplementary Movies 4 and 5). White arrows represent the protrusion direction. Kymographs (right) correspond to white arrows in TIRF images. f Crosscorrelation between Rac sensor signal and cell edge velocity plotted against the time shift between these measurements. g Enrichment of Rac and Rho sensor signals in protrusions (>0.075 μm/min) and retractions (<−0.075 μm/min). Values are normalized to average control sensor enrichment measurements. n = 3 independent experiments with >21 cells per condition. Error bars represent standard error of the mean. Measurements corresponding to individual cells are shown in Supplementary Fig. 5c, d. Scale bars: 10 µm; 0.26 μm/pixel. PH Pleckstrin homology domain, DH Dbl homology domain, Source data are provided as a Source Data file.