Figure 6.
PD-L1 localizes the β4 integrin to the cell rear by generating a membrane tension gradient. (A) Left: a representative image of PD-L1-EGFP in a migrating cell. Yellow arrowhead indicates cell rear. Red arrowhead indicates cell front. Right: heat-map images show Flipper-TR lifetime. Scales of lifetime is indicated in rainbow bar. Scale bar: 10 μm. (B) Left: Quantification of PD-L1 intensity normalized to the rear of the cell. Data are shown as means ± SD (n = 46 cells, three independent experiments). Statistical significance was determined by unpaired t test. Right: quantification of Flipper-TR lifetime at the front and rear of the cell. Statistical significance was determined by two-sided, paired t test (n = 46 cells, three independent experiments). ***, 0.001 > P ≥ 0.0001; ****, 0.0001 > P. (C) A Heat-map image showing Flipper-TR lifetime in 231-PD-L1KO cells. Scale bar: 10 μm. (D) Quantification of Flipper-TR lifetime at the front and rear of the cell in 231-PD-L1KO cells. Statistical significance was determined by two-sided, paired t test (n = 40 cells, three independent experiments). (E) Time-lapse images of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells in isotonic (left) or hypo-osmotic medium (right). Top: merged images of PD-L1 (green) and integrin β4 (red). Bottom: Higher magnification images of the boxed regions in the top panels. Scale bar: 10 μm. The same images were used to generate the pixel intensity map images shown in Fig. S4 C. (F) Quantification of PD-L1 (top) and integrin β4 (bottom) fluorescent intensity correlates with the distance from cell rear in PD-L1KO-PD-L1-EGFP MDA-MB-231 cells (n = 10 cells, three independent experiments). (G) Pixel intensity map from time-lapse images of PD-L1KO-MDA-MB-231 cells in isotonic (left) or hypo-osmotic medium (right) show integrin β4 distribution. White arrowheads indicate location of the integrin β4. Scale bar: 10 μm. (H) Quantification of integrin β4 fluorescent intensity correlates with the distance from cell rear in PD-L1KO-MDA-MB-231 cells (n = 10 cells, three independent experiments). (I) Quantification of the cell rear caveolin-1 intensity in WT or PD-L1KO MDA-MB-231 cells. Data are shown as means ± SD from three independent experiments, n > 30 cells per condition. Statistical significance was determined by two-sided, unpaired t test. **, 0.01 > P ≥ 0.001. (J) Fluorescent images of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells show distribution of PD-L1 (green), integrin β4 (β4, blue), and CT-B (red). Single channel only images are shown in black-white images. Scale bar: 10 μm. (K) Fluorescent images of WT (top) or PD-L1KO (bottom) MDA-MB-231 cells show distribution of CT-B (green) and integrin β4 (β4, red). Single channel only images are shown in black-white images. White arrowhead points to accumulation of caveolae/lipid rafts at the cell rear. Scale bar: 10 μm. (L) Fluorescent images of PD-L1KO-PD-L1C272A-EGFP MDA-MB-231 cells show distribution of PD-L1C272A-EGFP (green), CT-B (red), and integrin β4 (blue). Single channel only images are shown in black-white images. Scale bar: 10 μm. (M) Left: a representative image of PD-L1C272A-EGFP in a migrating cell. Right: a heat-map image shows Flipper-TR lifetime. Scales of lifetime is indicated in rainbow bar. Scale bar: 10 μm. (N) Quantification of Flipper-TR lifetime at the front and rear of the cell in PD-L1KO-PD-L1C272A-EGFP MDA-MB-231 cells. Statistical significance was determined by two-sided, paired t test (n = 40 cells, three independent experiments).

PD-L1 localizes the β4 integrin to the cell rear by generating a membrane tension gradient. (A) Left: a representative image of PD-L1-EGFP in a migrating cell. Yellow arrowhead indicates cell rear. Red arrowhead indicates cell front. Right: heat-map images show Flipper-TR lifetime. Scales of lifetime is indicated in rainbow bar. Scale bar: 10 μm. (B) Left: Quantification of PD-L1 intensity normalized to the rear of the cell. Data are shown as means ± SD (n = 46 cells, three independent experiments). Statistical significance was determined by unpaired t test. Right: quantification of Flipper-TR lifetime at the front and rear of the cell. Statistical significance was determined by two-sided, paired t test (n = 46 cells, three independent experiments). ***, 0.001 > P ≥ 0.0001; ****, 0.0001 > P. (C) A Heat-map image showing Flipper-TR lifetime in 231-PD-L1KO cells. Scale bar: 10 μm. (D) Quantification of Flipper-TR lifetime at the front and rear of the cell in 231-PD-L1KO cells. Statistical significance was determined by two-sided, paired t test (n = 40 cells, three independent experiments). (E) Time-lapse images of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells in isotonic (left) or hypo-osmotic medium (right). Top: merged images of PD-L1 (green) and integrin β4 (red). Bottom: Higher magnification images of the boxed regions in the top panels. Scale bar: 10 μm. The same images were used to generate the pixel intensity map images shown in Fig. S4 C. (F) Quantification of PD-L1 (top) and integrin β4 (bottom) fluorescent intensity correlates with the distance from cell rear in PD-L1KO-PD-L1-EGFP MDA-MB-231 cells (n = 10 cells, three independent experiments). (G) Pixel intensity map from time-lapse images of PD-L1KO-MDA-MB-231 cells in isotonic (left) or hypo-osmotic medium (right) show integrin β4 distribution. White arrowheads indicate location of the integrin β4. Scale bar: 10 μm. (H) Quantification of integrin β4 fluorescent intensity correlates with the distance from cell rear in PD-L1KO-MDA-MB-231 cells (n = 10 cells, three independent experiments). (I) Quantification of the cell rear caveolin-1 intensity in WT or PD-L1KO MDA-MB-231 cells. Data are shown as means ± SD from three independent experiments, n > 30 cells per condition. Statistical significance was determined by two-sided, unpaired t test. **, 0.01 > P ≥ 0.001. (J) Fluorescent images of PD-L1KO-PD-L1-EGFP MDA-MB-231 cells show distribution of PD-L1 (green), integrin β4 (β4, blue), and CT-B (red). Single channel only images are shown in black-white images. Scale bar: 10 μm. (K) Fluorescent images of WT (top) or PD-L1KO (bottom) MDA-MB-231 cells show distribution of CT-B (green) and integrin β4 (β4, red). Single channel only images are shown in black-white images. White arrowhead points to accumulation of caveolae/lipid rafts at the cell rear. Scale bar: 10 μm. (L) Fluorescent images of PD-L1KO-PD-L1C272A-EGFP MDA-MB-231 cells show distribution of PD-L1C272A-EGFP (green), CT-B (red), and integrin β4 (blue). Single channel only images are shown in black-white images. Scale bar: 10 μm. (M) Left: a representative image of PD-L1C272A-EGFP in a migrating cell. Right: a heat-map image shows Flipper-TR lifetime. Scales of lifetime is indicated in rainbow bar. Scale bar: 10 μm. (N) Quantification of Flipper-TR lifetime at the front and rear of the cell in PD-L1KO-PD-L1C272A-EGFP MDA-MB-231 cells. Statistical significance was determined by two-sided, paired t test (n = 40 cells, three independent experiments).

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