Fig. 2

Cdc42 and Rac1 have different responses to GEF activation. a We imposed GEF activity gradients of different slopes using optogenetics. Patterned illumination with grayscale levels (left) was shone onto the samples, imposing linear light gradients of the same amplitude but different spatial extents on cells attached on round micropatterns of 35 µm in diameter (top right). The reference gradient called 1× spans over the whole diameter of the cell. The gradient 2× spans over a half cell diameter, and therefore has a slope twice as sharp as for the gradient 1× (bottom right). b Membrane recruitment of the optogenetic partner CRY2-mCherry to the basal side of cells on round micro-patterns following 30 min of illumination with 4× (top left) and 2× (bottom left) gradients. mCherry fluorescence (solid lines) was measured along the cell diameter following illumination with 1× (light gray), 2× (medium gray) and 4× (black) gradients (dashed lines). Error bars indicate the s.d. of n = 15 (1×), n = 24 (2×) and n = 15 (4×) cells. c We activated GEFs of Cdc42 (ITSN) or Rac1 (TIAM) with light gradients and measured the fluorescence pattern of PAK1-iRFP. Schemes at the top represent the fusion proteins used. d PAK1-iRFP recruitment following 4x (top) or 2× (bottom) activation gradients of ITSN. Fluorescence was recorded using TIRFM in HeLa cells on round micro-patterns, and initial fluorescence was subtracted for normalization. Micrographs represent the averaged fluorescence of n = 15 (4×, top) or n = 12 (2×, bottom) cells. Insets show the illumination patterns (not to scale). e Normalized fluorescence of ITSN-CRY2-mCherry (blue) and PAK1-iRFP (purple) was measured along the cell diameter and averaged (solid lines). Error bars: s.d. Gray lines at the top show positions at which the curves are statistically different (p < 0.05, Wilcoxon’s rank sum test). f PAK1-iRFP recruitment following 4× (top, n = 15) or 2× (bottom, n = 19) activation gradients of TIAM. g Normalized fluorescence of TIAM-CRY2-mCherry (red) and PAK1-iRFP (purple) along the cell diameter of n = 15 (4×, top) or n = 19 (2×, bottom) cells, Error bars: s.d. Gray: Wilcoxon’s rank sum test (p < 0.05). Single cell data for e and g are presented in Supplementary Figure 3. Scale bars: 20 µm