Rap1 dynamics in ruffles. (A) The ruffles at upper left (box) are cut by the planes shown at different times. Note the changing distribution of active Rap1 and Rap1 activity distributed from the ventral surface all the way to the top of the ruffles (upper right plane). Color scale indicates corrected FRET/CFP, excluding the highest and lowest 2% of ratio values to eliminate spurious pixels. Scale bars, 5 µm. (B) A concave ruffle is cut by a series of planes parallel to the coverslip, at increasing height above the coverslip. Highest Rap1 activity is seen on the surface of the ruffle facing the direction of motion (Video 4). The shape of the ruffle is evident in the pattern of Rap1 activity on the bottom of the Cos7 cell (lowest plane). Scale bar, 5 µm. (C) A dorsal ruffle is cut by a single plane showing Rap1 activity throughout the structure and in connections to a dense region of high activity on the bottom of the Cos7 cell. Scale bar, 5 µm.