Fig. 4. WAVE2 is essential for lamellipodium and ruffle formation in mouse embryonic fibroblasts (MEFs). (A) Sub-confluent serum-starved wild-type (WT) and WAVE2 knockout (KO) MEFs were left unstimulated or stimulated with 20 ng/ml PDGF for 10 min. The leading edge and other areas of lamellipodia and ruffles are more obvious by cortactin staining (green panels) and detection of F-actin cytoskeleton by TRITC-phalloidin (red panels). KO cells show more distinct cytoskeletal defects. Note the absence of leading edge and aberrant lamellipodia formation (yellow arrows point to leading edge of lamellipodia) in unstimulated KO cells compared with WT cells. PDGF induces lamellipodia (yellow arrows), and circular ruffles (blue arrows) in KO MEFs are structurally malformed. Scale bar, 10 µm. (B) WAVE2 retroviral expression rescues the defect in lamellipodium formation. WAVE2 KO MEFs transduced with WAVE2 retrovirus (RVWAVE2), left unstimulated or stimulated with 20 ng/ml PDGF for 10 min. Cortactin staining is shown in red and F-actin in blue. Rescued KO MEFs exhibit a normal morphology with well-defined lamellipodium areas. (C) Membrane ruffle formation of WT, KO and Rescue (KO MEFs rescued with the WAVE2 retrovirus). One hundred cells of each condition were counted. Membrane ruffles are broken down into two distinct categories: lamellipodia and circular ruffles. Numbers represent an average of more than eight independent experiments performed for WT and KO and more than three experiments for the Rescue conditions. The average of these conditions with standard error bars is shown. (D) The chemotactic response of WT and KO primary MEFs to PDGF was examined under conditions containing (+PDGF) or lacking (-PDGF) PDGF at 10 ng/ml. The graph depicts the average of three experiments (42 fields of cells per condition) counted at two magnifications.