WIP is an essential component of the Nck–N-WASp–Arp2/3 pathway. (A–D) Confocal images of either WIP WT and KO or N-WASp WT and KO MEFs transfected with a combination of mCherry-actin, membrane-bound Nck SH3 fusion, and GFP-WIP or GFP–N-WASp demonstrate the necessity of WIP for Nck-induced, N-WASp–dependent actin polymerization. Higher magnifications of clusters are shown in the insets. Bars, 10 µm. (A) Antibody-induced aggregation of Nck SH3 domains (green) induces the formation of actin comet tails (red) in WIP WT MEFs (top) but does not induce actin polymerization in WIP KO MEFs (bottom). (B) Aggregation of Nck SH3 domains (cyan) in WIP KO MEFs rescued with GFP-WIP (green) induces actin comet tails (red) similar to those seen in WIP WT MEFs. (C) Nck SH3 aggregates (cyan) neither recruit GFP–N-WASp (green) nor induce actin polymerization (red) in WIP KO MEFs (top), whereas Nck SH3 aggregates in WIP WT MEFs both recruit GFP–N-WASp and induce actin polymerization (bottom). (D) Nck SH3 aggregates (cyan) recruit GFP-WIP (green) in both N-WASp KO MEFs (top) and N-WASp WT MEFs (bottom) but only induce actin polymerization (red) in N-WASp WT MEFs.