Impaired binding of Rab18 to KNT1 affects FA assembly and disassembly rate. (a) Control or Rab18-depleted cells transfected with RFP-vinculin were imaged every 10 min for 40 min. Arrows show FA disassembly, and arrowheads show FA assembly. Scale bar: 10 µm. (b) Rainbow color representation of FA appearance and disappearance over time from cells shown in panel a and for U2OS cells stably expressing GFP-KNT1 K1 or Δ201–445. Each time point is shown in a different color, as indicated in the bar. Insets show magnifications of the boxed areas. Scale bars: 10 µm; insets: 2 µm. (c) Quantification of assembly and disassembly rates of FAs. The assembly and disassembly rate is expressed as percentage of focal adhesion formation or disassembly per minute. The values represent the mean ± SEM from three independent experiments, in which 15 FAs were analyzed per cell (n > 6), per condition, and per experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.