Depletion of ADF or cofilin changes the lamellipodia history of migratory MTLn3 cells. Representative kymographs of control (A), ADF KD (B) and cofilin KD (C). The lamellipodium history (protrusion, pausing, and retraction) of polar migratory MTLn3 was analyzed using kymograph images. The kymograph image was created from a line that crosses the cell centroid, and the reference point from which the lamellipodium is measured, is the position of the lamellipodium at time point zero. Arrow shows protrusion, dashed arrow shows retraction. D and E. The lamellipodia of ADF KD and cofilin KD cells spent the majority of their time (60.7% and 64.4%, respectively) protruding as compared to control cells (44.3%). This was rescued to a significant degree by re-expressing the untagged human ADF but not cofilin in ADF KD cells and human cofilin but not ADF in cofilin KD cells. F and G. The lamellipodia of ADF KD cells protruded significantly more frequently than in control and cofilin KD cells. Re-expressing of untagged human ADF in ADF KD cells restored the control phenotype. H and I. The protrusion of lamellipodia in cofilin KD cells was significantly more persistent than in control and ADF KD cells. Re-expressing huADF.RedTrack or huCofilin.mRFP or huCofilin.RedTrack restored the control phenotype in cofilin KD cells. n ≥ 10 cells in each treatment, three independent experiments, an average of 110 measurements. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control.