Integrin trafficking suppresses Rac activity and activates RhoA. (A and B) A2780 cells expressing Raichu-Rac were seeded onto CDMs and stimulated with cRGDfV as indicated. Fluorescence lifetime images were captured at 1-min intervals, and representative lifetime maps are shown. (C) FRET efficiency was calculated for ROIs at the cell periphery at the front, middle (mean of the two sides), or back, from lifetime maps generated as in A and B (single images or means of all frames from time-lapse videos, n > 30/condition). (D) FRET efficiency at the cell front was calculated as in C (for each frame of time-lapse videos, n > 9/condition). (E and F) A2780 cells expressing Raichu-RhoA were analyzed as in A and B. (G) FRET efficiency was calculated as in C (n > 35/condition). (H) FRET efficiency at the cell front was calculated as in D (n > 15/condition). (I) H1299 cells stably expressing mutant p53 (273H) or control vector (VEC) were transfected with Raichu-Rac or Raichu-RhoA. FLIM was performed as in A and B, and FRET efficiency at the cell front was calculated as in C (n > 8/condition). (J) A2780 cells were transfected with control or RCP-specific siRNA and allowed to recover for 24 h. Cells were then transfected with Raichu-Rac or Raichu-RhoA. FLIM was performed as in A and B, and FRET efficiency at the cell front was calculated as in C (n > 13/condition). (K) A2780 cells expressing Raichu-RhoA were seeded onto CDMs and treated with vehicle or the Rac inhibitor NSC-23766 for 2 h. FLIM was performed as in A and B, and FRET efficiency at the cell front was calculated as in C (control, n = 8; NSC-23766, n = 10). Yellow lines represent the baseline activity as determined by an inactive mutant of the probe. Data represent means ± SEM from at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Zoomed images from videos are shown in the time sequence and correspond to areas indicated by dotted ROIs. Bars, 10 µm.