Ensemble measurements were performed as previously described (14). In summary, images of eGFP-paxillin marked cells were acquired using a Hamamatsu Orca Flash 4.0 camera and were subsequently corrected for illumination spatial inhomogeneities, background-subtracted, and intensity-normalized. The GFP image was then boxcar-averaged (“moving average v3.1” from MATLAB Central File Exchange) at 10 different rotations of the original image at 20° intervals, thresholded, and segmented using a watershed algorithm. The segmented image was then corrected to combine adjacent islands representing a single adhesion and filtered to exclude islands below a lower limit (0.5 μm2). The segmented GFP image was then used to mask the corresponding FRET signal.

FRET images were converted to FRET index values by dividing the acceptor intensity over the sum of the donor and the acceptor signal. Then, the FRET images were converted to FRET efficiency after correcting to dye labeling efficiency, bleedthrough, the measured no-load FRET efficiency, and the FRET-index measured outside the cell (14). The total integrated traction of a cell was calculated by summing the force contributions (defined as the average pixel value times number of pixels) for pixels within adhesions. For MTSlow or MTSFN9–10, forces corresponding to >7pN were set to 7.1 pN. For MTShigh measurements, calculated forces corresponding to <7 pN were set to 0 pN.

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