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Fig. 6. Adhesion-site dynamics and PIP₂ signals. In B16-F1 cells expressing GFP-vinculin variants, individual adhesions were analysed using confocal laser-scanning microscopy. (A) Representative images of adhesions before and after bleaching (red arrows) and kymographs from line scans parallel to the length axis of bleached adhesion. The half-time of fluorescence recovery was calculated based on the kymographs. Notice that vinculin variants wt and LD have comparable incorporation rates. Scale bar, 5 µm. (B) Movement of adhesions was followed over 20 minutes. Representative areas of cells expressing vinculin-wt or vinculin-LD are given, showing start position of adhesions (t=0 minutes) and tracks of adhesions tips marked in red (t=0-20 minutes). Tracks were used to calculate the average speed of retrograde movement. Scale bar, 5 µm. (C) B16-F1 cells were seeded onto fibronectin, transfected with murine PIP 5-kinase , wild type (wt) or kinase deficient (KD), and fixed 12 hours after transfection. Localization of GFP-vinculin variants (wt or LD) is shown individually (greyscale, left) and in merged images (green) together with PIP 5-kinase (blue) and F-actin (red). Scale bar, 10 µm. Notice that only cells expressing wild-type constructs of both PIP 5-kinase and vinculin show a localization of vinculin that is entirely perinuclear and start to detach.

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