FIGURE 6.

Expression of wild-type but not calpain-resistant FAK rescues impaired adhesion turnover in FAK-deficient cells. A, HEK 293 cells were transiently cotransfected with control (Ctlsi) or FAK (FAKsi) siRNA and mCherry (mChe), mCherry-FAK, or mCherry-FAK-V744G. Cell lysates were analyzed by immunoblotting and probed for FAK. The immunoblot shown is from one of three independent experiments. C-term, C-terminal. B, HEK 293 cells stably expressing mCherry, mCherry-FAK, or mCherry-FAK-V744G were transiently cotransfected with control or FAK siRNA and GFP-talin1. Cells were plated on fibronectin-coated glass-bottom dishes and analyzed by time-lapse fluorescence microscopy. Time-lapse montages demonstrate representative images of the dynamics of GFP-talin1 over a period of 24 min. Scale bars = 10 μm. Arrows indicate a representative adhesion. Representative movies are shown in supplemental Movies 3–6. C, duration was measured as the time elapsed between the appearance and dissolution of an observed adhesion. Rate constants for net adhesion assembly and disassembly were calculated from plots of fluorescence intensities of GFP-talin1 as described under “Experimental Procedures.” Data for each condition are the mean ± S.E. from a total of six cells over three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (by t test) compared with control siRNA.