Figure 3. Pak inhibition affects the organization and dynamics of paxillin in FAs. (A) Paxillin immunofluorescence imaged by TIRF microscopy in migrating PtK1 cells expressing PID WT or an inactive mutant of the PID (PID L107F, noted control). Insets show GFP expression of the GFP–PID WT or GFP–PID L107F constructs. White boxes in the paxillin column indicate the positions of insets for higher magnifications shown in the right column. (B–D) Quantification of paxillin fluorescence intensity in the protrusion (B), the average adhesion area in the protrusion (C), and the percentage of ventral cell area containing paxillin (D) for each condition ± SEM. *, P < 1.10−6 compared with control (Ct) cells; Student’s t test. a.u., arbitrary units. (E) Frequency histogram of paxillin foci length for each condition ± SEM. Numbers at the top right are the average lengths of the adhesion sites ± SEM. For quantifications in B–E, the experiment was repeated at least three times with n ≥ 30 cells for each condition, and all detectable FAs were quantified in each cell (corresponding to a minimum of 30 FAs per cell). (F and G) Example of GFP-paxillin fluorescence time-lapse images imaged by TIRF microscopy in PtK1 cells expressing GFP-paxillin alone (noted as a control; F) or in combination with PID (G). White boxes in the whole area images (left) indicate the localization of the magnified regions shown in the right panels. Elapsed time in minutes is shown. Arrowheads and arrows indicate assembling and disassembling FAs, respectively. Note that FAs in Pak-inhibited cells (G) were sparsely stained and assembled/disassembled slower than those in control cells (F). (H) Average rate constants of FA assembly and disassembly measured from 8–15 FAs per cell ± SEM, and n ≥ 7 cells per condition. *, P < 1.10−5 compared with control cells; Student’s t test. (I) Average FA lifetime measured from the initiation of a new GFP-paxillin cluster to complete disappearance (±SEM). *, P < 1.10−6 compared with PID-expressing cells. FAs in PID-expressing cells never disassembled during the 30 min of imaging.
Figure 3.

Pak inhibition affects the organization and dynamics of paxillin in FAs. (A) Paxillin immunofluorescence imaged by TIRF microscopy in migrating PtK1 cells expressing PID WT or an inactive mutant of the PID (PID L107F, noted control). Insets show GFP expression of the GFP–PID WT or GFP–PID L107F constructs. White boxes in the paxillin column indicate the positions of insets for higher magnifications shown in the right column. (B–D) Quantification of paxillin fluorescence intensity in the protrusion (B), the average adhesion area in the protrusion (C), and the percentage of ventral cell area containing paxillin (D) for each condition ± SEM. *, P < 1.10−6 compared with control (Ct) cells; Student’s t test. a.u., arbitrary units. (E) Frequency histogram of paxillin foci length for each condition ± SEM. Numbers at the top right are the average lengths of the adhesion sites ± SEM. For quantifications in B–E, the experiment was repeated at least three times with n ≥ 30 cells for each condition, and all detectable FAs were quantified in each cell (corresponding to a minimum of 30 FAs per cell). (F and G) Example of GFP-paxillin fluorescence time-lapse images imaged by TIRF microscopy in PtK1 cells expressing GFP-paxillin alone (noted as a control; F) or in combination with PID (G). White boxes in the whole area images (left) indicate the localization of the magnified regions shown in the right panels. Elapsed time in minutes is shown. Arrowheads and arrows indicate assembling and disassembling FAs, respectively. Note that FAs in Pak-inhibited cells (G) were sparsely stained and assembled/disassembled slower than those in control cells (F). (H) Average rate constants of FA assembly and disassembly measured from 8–15 FAs per cell ± SEM, and n ≥ 7 cells per condition. *, P < 1.10−5 compared with control cells; Student’s t test. (I) Average FA lifetime measured from the initiation of a new GFP-paxillin cluster to complete disappearance (±SEM). *, P < 1.10−6 compared with PID-expressing cells. FAs in PID-expressing cells never disassembled during the 30 min of imaging.

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