Fig. 3.

PA-Rac1 ruffles are lamellar structures that are formed and maintained by actin polymerization. a Representative montage of ruffle formation and elongation in response to optogenetic stimulation in RPE1 cells. Red arrows indicate highly curved membrane ruffles stimulated by PMA; blue arrows indicate large ruffles formed in response to optogenetic stimulation. Scale Bar = 10 μm. b Representative montage of PA-Rac1 stimulated ruffle dynamics. Red arrows show the growing end of the ruffle, blue arrows show branching points. Scale bar = 3 μm. c Kymograph of PA-Rac1 signal at a lamellipodial edge during Rac1 stimulation. The red arrows represent lift-offs of lamellipodia (LOL) and blue arrow points at a merger of two ruffles. X scale bar = 5 μm, Y scale bar = 5 min. d Representative screen capture showing measurements of membrane ruffle contours using Syglass VR. e Representative diagram showing squared end-to-end length (R), and contour length (L) overlaid on a top view of a ruffle. f Squared end-to-end length vs. Contour length of ruffles stimulated by PMA (grey) or PA-Rac1 (black). Curve of fit (red) corresponds to the persistence length derived from the 2D worm-like chain model. g Representative images showing the measurement of ruffle thickness (blue arrows). Image panels show slices through a macropinocytic cup as portrayed in the top left panel. Scale bar = 3 μm. h Boxplot of membrane ruffle thickness (μm). Outer range shows total range, box height shows the standard deviation, and inner bar shows mean. i Representative images showing extraction of ruffle protrusion height. Measured height shown by blue bars top right, overlay top left. Bottom shows ruffle height (μm) vs length (μm). Measurements were taken for 9 representative ruffles