Speckles and tracks were identified using quantitative fluorescent speckle microscopy software made available by the Danuser laboratory (53). The localizations, which are given with pixel precision, are fit to subpixel positions by Gaussian fitting.

Every frame was used to track speckles, but velocities were calculated from speckle displacements over five frames (10 s), giving velocities on the time scale of our FRET measurements. Adhesions were masked by an Otsu threshold of the eGFP-paxillin image after background subtraction to remove the diffuse cytoplasmic signal. F-actin stress fibers were masked as the brightest 3% of pixels of a time-series projection of the F-actin tracks. We measure the actin velocities of tracks that originate both over stress fibers and adhesions.

From our mean velocity measurement, <d>, we calculate a corrected velocity, s, using the following relation (50)s=d24σ2where σ is the localization error (SD) of single-molecule localizations (estimated to be 47 nm here).