PD-L1 regulates membrane tension to localize β4 to the cell rear. Related to Fig. 4. (A) Heat-map images show Flipper-TR lifetime of WT MDA-MB-231 cells in isotonic (left) or hypo-osmotic medium (right). Scale bar: 10 μm. (B) Quantification of Flipper-TR lifetime in the same cell in isotonic or hypo-osmotic medium for 5 min. Statistical significance was determined by two-sided paired t test (n = 8 cells). ***, 0.001 > P ≥ 0.0001. (C) Pixel intensity map of time-lapse images of PD-L1^KO-PD-L1-EGFP MDA-MB-231 cells in isotonic (left) or hypo-osmotic (right) medium shown in Fig. 6 E. Top: Pixel intensity map from PD-L1 single channel only. Bottom: Pixel intensity map from integrin β4 single channel only. White dotted lines indicate rear of the cell. White arrowheads indicate location of PD-L1 (middle) or integrin β4 (bottom). Scale bar: 10 μm. (D) Time-lapse microscopy images of PC3 cells pre-labeled with an anti–PD-L1-APC antibody to show PD-L1-APC uptake during persistent cell migration. Scale bar: 10 μm. (E) Quantification of retraction fiber length and migrasome numbers/retraction fiber length in PD-L1^KO, PD-L1^KO-PD-L1-EGFP, and PD-L1^KO-PD-L1^C272A-EGFP MDA-MB-231. Data are shown as means ± SD from three independent experiments, n > 50 cells per condition. Statistical significance was determined by two-sided unpaired t test. *, 0.05 > P ≥ 0.01; ****, 0.0001 > P.