Figure 1. Focal adhesion size is a unique predictor of cell migration speed. (A–C) Effect of changes in substrate compliance—rigid glass (black), stiff (gray), and soft (white) polyacrylamide gels coated with collagen I, and depletion of focal adhesion proteins (FAK, paxillin, talin, and zyxin) on focal adhesion size (A), cell size (B), and cell migration speed (C). At least 30 cells per condition were analyzed to assess focal-adhesion and cell morphology and >50 cells per condition were tracked to assess cell motility. Error bars represent SEM. Multiple comparison to the control (i.e., WT cells on stiff substrates) was performed by 1-way analysis of variance (ANOVA) using the Dunnett post test. Significant statistical difference are shown as follows, ***P < 0.001, **P < 0.005, *P < 0.01. (D–F) Assessment of regression among focal adhesion size, cell size, and cell speed. Mean size of focal adhesion is biphasically and linearly correlated with cell speed (D) and cell size (E), respectively, while cell size is weakly correlated with cell speed either biphasically (r² = 0.51) or linearly (r² = 0.32). Gaussian (nonlinear) and linear models were tested to the data set ranged between 0 and 1 after normalization as (x-x_min)/(x_max-x_min). Error bars represent SEM. Note that cell size is not statistically related to cell speed. (G) Schematic of prediction of cell speed by focal adhesion size. Cell speed is predicted by the mean size of focal adhesion not through regulation of cell size. Panels (A, C, and D) were reprinted from ref. 8.