Figure 5

Polycystin-1 regulates focal adhesion dynamics. (A-B) Quantification of focal adhesion disassembly in MDCK^Zeo (clones F2 and F6) and MDCK^PKD1Zeo (clones 36 and 68) cells (A), and in Pkd1 ^+/+ and Pkd1 ^−/− fibroblasts (B). Each graph is representative of three independent experiments; in each experiment 3 groups of at least 100 cells were counted. Averages and SD are shown. Statistical analysis: ANOVA; NS (non-significant) p > 0.05, ** p < 0.01,***p < 0.001, ****p < 0.0001, referred to the relative control bars. (C-G) MDCK^Zeo (clone F6) and MDCK^PKD1Zeo (clone 36), as well as Pkd1 ^+/+ and Pkd1 ^−/− fibroblasts, were transfected with GFP-paxillin. GFP-paxillin-positive adhesions were subjected to FRAP analysis. The panel (C) show images of whole cells (bar: 10 μm) and zoom-in of the individual focal adhesions (bar: 2 μm) at crucial time points: pre-bleaching time (−10s), at bleaching completed time (0 s), post-bleaching corresponding at about t1/2 recovery times of the different cell lines (8 s, 13 s), and at long time point (200 s). Bleached focal adhesions are indicated with arrows in the first image and asterisks in the higher magnification images. The results showing the half time (t1/2) of fluorescence recovery are reported in the box plots (D, F); average values are indicated above each box. Statistical analysis: t-test, ****p < 0.0001. F6, n = 22; 36, n = 30, Pkd1 ^+/+, n = 16; Pkd1 ^−/−, n = 28 (n, number of focal adhesion analyzed). In the box plot: the line indicates the median value, the box indicates the 1st and 3rd quartile values, the points indicate the minimum and maximum values. Sample fluorescence recovery curves of FRAP are shown in (E) and (G). The fluorescence intensity in the recovery curves corresponds to the fluorescence at each time point after photobleaching, background subtracted, and normalized to the pre-bleaching intensity.