RhoA activity is associated with both extension and retraction of the membrane at the leading edge. (a–g) Images and kymographs (of regions marked with red/blue arrows) from time-lapse videos. (f and g) RhoA knockdown cell images show ruffling (starting at arrow; 5 s) perpendicular to long extension. (f) Arrowheads, phase dark spherical accumulations (SA). Boxed region in f is magnified in g. (h) Overlays of consecutive time-lapse images of RhoA Raichu probe lifetime (color scale) and probe localization on plasma membrane (white). Cell 1 shows RhoA activity before membrane retraction, and cell 2 shows before both retraction and extension. Solid arrow, extension; dashed arrows, retraction. Cell outlines show 0- (dark blue) and 45-s (light blue) frames. (i) mDIA1 and F-actin localization in CEM cells on EC imaged by STED microscopy. (j) p-MLC and F-actin localization in CEM cells after 30 min on EC. Arrows, p-MLC at leading edge; dashed line, transmigrated region. LE, leading edge; U, uropod; CB, cell body; SA, phase dark spherical accumulations along protrusions. Bars, 10 µm.