Gm6001

For confocal microscopy live cell imaging, we used the same microscopic setting as for FRAP. Cells were imaged for the indicated time with 20 s or 30 s intervals with 1–3% laser power depending on the fluorophores. To block MMP activities, cells were plated for 5 h with or without 50 µM GM6001 (no sc-203979, Santa Cruz) before live cell imaging. For data illustration, fluorescent images were background-subtracted and passed through low-pass filters followed by Unsharp Mask in ImageJ.
For the tracking of sliding adhesions, a time lapse of 20 min with 2 min intervals was extracted from the full Supplementary Videos. Raw images were background-subtracted. Single adhesions were identified by thresholding and tracked with the TrackMate plugin in ImageJ. Structures with sizes below 3 pixels (0.04 µm per pixel) were excluded. Adhesion sites with lifetimes above 6 min were tracked to measure their sliding velocities. Average adhesion sliding velocities were calculated on the basis of all analysed adhesions for each cell. For the determination of sliding velocities of central adhesions, vinculin-mCherry-positive adhesions were analysed within 20 µm distance from nuclei in the first frame. For the FAs in the cell protrusions, vinculin-mCherry-positive adhesions were analysed within 10 µm behind the protruding leading edge in the first frame.