Nck1 is required for cell edge protrusion. (A) Graphical depiction of the Nck1 adapter protein. Nck1 consists of three SH3 domains followed by a single SH2 domain. (B and C) Representative frames from the 10-min time-lapse videos of cells plated on 10 µg/ml fibronectin-coated coverslips. The three rightmost panels are representative kymographs constructed as in Fig. 2. Nck1 KD cells (n = 29 cells; B) and Nck1 KD cells expressing Nck-GFP (n = 19 cells; C) are shown. (D and E) Quantification of the number of protrusions (D) and retractions (E) per 10-min video. Mean ± SEM. ANOVA between all cell types: protrusions, P < 0.0001; retractions, P < 0.0001. Fisher’s PLSD for all cells versus WT: *, P ≤ 0.001. (F) Localization of Nck1-GFP and cortactin in Nck1 KD cells. Nck1 KD cells were infected with Nck1-GFP (a and f) and endogenous cortactin was visualized by immunostaining (b and g). Cells were plated on fibronectin-coated coverslips and fixed 45 min after plating. F-actin was visualized with Alexa Fluor 350 phalloidin (c and h). The merged images of Nck1-GFP and cortactin images (d and i) are shown. The merged images of Nck1, cortactin, and actin (e and j) are also shown. f–j are enlargements of the areas indicated in a. (G) Immunoblot of WT and Nck1 KD cell lysates for Nck1. The HSP-70 panel represents a loading control. Bars, 10 µm.