IFs limit the generation of traction forces at FAs. (A) Immunofluorescence confocal images in two distinct focus planes of astrocytes stained for vinculin. The white dotted line indicates the position of the wound. (B) Graph showing the percentage of junctions showing positive vinculin staining. (C) Fluorescence images of migrating astrocytes expressing VinTS (Green Fire Blue LUT). Representative frequency distribution of frequencies of FRET index (central graph) and quantification of the mean FRET index (inversely related to vinculin tension) of vinculin in FAs (right graph). Each dot represents a FA. The white dotted line indicates the position of the wound. (D) Immunofluorescence and intensity projection (fire LUT) of paxillin (magenta) and actin (green) on micropatterned cells. Inset shows the crossbow-shaped micropattern. Projections are calculated on one experiment representative of three. (E) Left: Tractions fields of astrocytes plated on collagen-coated crossbows. Arrows indicate the orientation, color, and length of the local magnitude of the force in Pa. Right: Quantification of traction forces in single micropatterned cells. Data are from n = 3 independent experiments. Histograms show mean ± SEM. The sample size for each repeat: si ctl 137, 247, 150 and si triple IF 222, 200, 151 for B; si ctl 2,537, 3,312, 1,473 adhesions and si triple IF 1,447, 2,548, 2,081 adhesions for C; si ctl 32, 21, 25 and si triple IF 30, 46, 38 for E. ***, P < 0.001. Bar, 10 µm.