N-WASP is required for cup formation. (a–i) Inhibition of N-WASP activity attenuates protrusion induction and cup maturation. Green and white arrowheads highlight POS adhesion sites. (j–n) Inhibition of actin polymerization using the inhibitor Latrunculin A or siRNA of N-WASP prevents F-actin–rich protrusion overgrowth due to MRCKβ depletion. Green and white arrowheads highlight POS adhesion sites. (m and n) Heat maps signify F-actin intensity. (o) Schematic figure illustrates a model in which Dbl3 activates Cdc42 at POS-membrane adhesions to drive N-WASP–dependent cup formation that is regulated by MRCKβ to control the size and shape and wrapping of POS as nascent protrusions mature into cups. Protein staining was quantified as mean intensity. Scale bars represent 10 μm, unless highlighted otherwise. All quantifications are based on n = 3 independent experiments. Shown are the data points and means ±1SD for b, c, f, g, k, l, and n, scatter plots for d and h, where the median and upper and lower quartiles are highlighted represents the total number of cells analyzed for each type of sample across all experiments, and P values derived from t tests.