Fig. 4

a Western blot comparing the CDC42-knockout (Cdc42-KO) and control cell lines stained for β-actin and Cdc42. Blot is representative of n = 3 experiments. b The mean-squared displacement (MSD) was measured as a function of time for control and Cdc42-KO cells randomly migrating. Data are presented as mean ± s.e.m. (n_experiment = 4). A total of 10,687 control cells and 3018 Cdc42-KO cells were analyzed across the four experiments. c As a measure of directional persistence, the mean cosine of the angle between a migrating cell’s movement direction at two different time points was measured as a function of the difference in time between the two measurements. Cells migrating in a straight line would have a mean cosine angle of 1. Data are presented as mean ± s.e.m. (n_experiment = 4). A total of 4821 control cells and 1098 Cdc42-KO cells were analyzed across the four experiments. d Fraction of cells exhibiting the cytoplasmic tether phenotype. Data are presented as mean ± s.e.m. (n_experiment = 4). A total of 331 control cells and 194 Cdc42-KO cells were analyzed across the four experiments. *P = 0.0108 using two-sided t test with unequal variances. Scattered points represent relative frequency for each experimental replicate. e Time-lapse image series of control cells randomly migrating. Control cells tend to maintain one cell front at a time and migrate more persistently. Scale bar, 15 µm. Micrographs are representative of n = 331 cells. Grayscale bars indicate fluorescence intensity. f, g Time-lapse image series of Cdc42-KO cells randomly migrating. Micrographs are representative of n = 194 cells. Scale bar, 15 µm. Grayscale bars indicate fluorescence intensity. f This cell displays a range of phenotypes, including a very large, crescent-shaped leading edge in the first time point and multiple repolarization events thereafter. g This cell displays the cytoplasmic tether phenotype where the leading edge pulls away from the cell body, but remains linked by a thin cytoplasmic filament. Source data are provided as a Source Data file.