Fig. 1

Development of intensiometric small GTPase biosensors. a Schematic of ddFP-based small GTPase sensor. b (top) Schematic depiction of KRas (G-KRas) sensor construct. (bottom) Fluorescence images showing Ras activity during sequential treatment of EGF (50 ng ml⁻¹) and EGFR inhibitor, gefitinib (400 nM). Color bar indicates range of G-KRas intensity. c Time-lapse graph represents reversible fluorescence change of G-KRas upon EGF or gefitinib treatment. n = 77 (blue), 40 (red). d Graph representing maximal fold-changes of GA-KRas intensity in HeLa cells. One way analysis of variance (ANOVA), P < 0.0001 for (EGF vs. gefitinib); P < 0.0001 for (EGF vs. pre-gefitinib + EGF); P = 0.8948 for (EGF+gefitinib vs. pre-gefitinib+EGF). n = 19, 20, 19; n.s., not significant. e Graph showing dose-dependent fold-changes of G-KRas sensor intensity upon EGF treatment. n = 20 for each concentration. f Quantification of maximal fold change of ddGFP-based Ras GTPase sensors upon EGF treatment. Each group of cells was co-transfected with plasmids as indicated. One way analysis of variance (ANOVA), P < 0.0001 for (a vs. c, d, e); P < 0.0001 for (b vs. c, d, e); P > 0.9999 for (a vs. b). n = 32, 42, 44, 27, 37; n.s., not significant. g Time-lapse graph showing dynamics of G-KRas and R-KRas sensor intensities upon EGF treatment. Images were captured at 20-s intervals. n = 17 (G-KRas), 18 (R-KRas). h Simultaneous imaging of HRas and Rac1 activities in MDA-MB-231 cell co-expressing R-HRas, G-Rac1, and Lyn-miRFP. Fluorescence ratio images showing Ras and Rac1 activity during cell migration. i (left, middle) Magnified images (area indicated by white boxes in h) showing subcellular distribution of R-HRas and G-Rac1 activities. (right) A color-coded image showing difference between normalized activities of HRas and Rac1 in subcellular regions. RFU: relative fluorescence unit; All scale bars, 20 μm; Error bars, s.e.m. Images or quantified data are representatives of multiple experiments (N > 3)