Fig. 5. Phosphorylation of WAVE2 is required to maintain the directional persistence of cell migration. (A) NIH 3T3 cells stably expressing EGFP, EGFP-WAVE2-wild-type (WT) or EGFP-WAVE2-AAA (AAA) were treated with control siRNA (C) and grown to confluence in a chamber slide. Monolayers were scratch-wounded and cell migration was filmed using time-lapse microscopy. Frames were taken from a representative movie (see supplementary material Movies 1-3 for originals) showing migration at the indicated time points. (B) Cells were tracked every 30 minutes from 8-16 hours post-wounding and their paths overlayed. Paths are oriented such that the start point is normalised to the origin and the wound face runs parallel to the x-axis. All tracks from several fields in this representative experiment are shown. (C,D) Experiment performed as for A and B except that cells were treated with WAVE2 siRNA 48 hours before wounding (see supplementary material Movies 4-6 for originals). (E) Cells from the experiment described in panels A-D were plated in parallel and lysates immunoblotted as indicated.