Activated Cdc42 colocalizes with linear invadosomes, DDR1, and collagen I fibrils. (A and B) MDA-MB-231 cells transiently transfected with GFP-V12Cdc42 or GFP-N17Cdc42 were cultured for 4 h on collagen I. Tks5 (green) colocalizes with GFP-V12Cdc42 (red) at linear invadosomes whereas GFP-N17Cdc42 (red) does not. Panels on the right show enlarged views of the boxed regions. Correlation coefficient of colocalization: Tks5/V12Cdc42 r = 0.21; Tks5/N17Cdc42 r = −0.11 (n = 25). (C) Quantification of the percentage of GFP-WTCdc42–, GFP-V12Cdc42–, and GFP-N17Cdc42–transfected cells able to form linear invadosomes. Data are mean ± SEM (n > 500, three independent experiments; ***, P < 0.001). (D) Active Cdc42 level was monitored in MDA-MB-231 cells treated as indicated. Data are mean ± SEM, three independent experiments. **, P < 0.005; ***, P < 0.001. (E) MDA-MB-231 stably expressing DDR1-GFP transiently transfected with myc-V12Cdc42 were seeded for 4 h on collagen I (DDR1-GFP, green; myc-V12Cdc42, red). Panels on the right show an enlarged view of the boxed region. Correlation coefficient of colocalization: DDR1/mycV12Cdc42 r = 0.15 (n = 10). (F) MDA-MB-231 cells were transfected with Raichu-Cdc42 and seeded on labeled collagen I. Shown are representative images: on the left, the pseudocolored ratio image generated from YFP/CFP ratio images represents the FRET ratio, which correlates with the Cdc42 activity (in yellow); and on the right, a collagen I image. Arrows represent colocalization of active probe and collagen I fibrils. Bars: (A and B, left) 5 µm; (A and B, right) 2.5 µm; (E, left) 7 µm; (E, enlarged panels on the right) 2 µm; (F) 3 µm.