Fig. 2: Migrating cells display an intrinsic membrane tension gradient.
a Representative Flipper-TR intensity images of a WT-Hs578t cell under isotonic (Iso) condition. White dashed boxes indicate front and rear regions of interest (ROIs). Color bar: Flipper-TR intensity (photons per pixel). Scale bar, 20 µm. b Fluorescence decay curves from front (green) and rear (red) ROIs of a representative cell. Experimental data (dots) were fitted with a two-exponential decay function (blue line) to calculate Flipper-TR lifetime (ns) and adjusted R² values. Insets show ROI locations. c Flipper-TR lifetimes at the front (green) and rear (red) of individual cells. Gray lines connect paired measurements from the same cell. b, c Data are presented as mean values +/- SEM of n = 44 cells from three independent experiments. Statistical analysis: One-tailed, Mann–Whitney test; ****P < 0.0001, **P < 0.001. d Per-cell differences in lifetime (Δ = Front - Rear); each blue circle represents one cell. e Representative Flipper-TR intensity images of a WT-Hs578t cell under isotonic (Iso, left) and hypotonic (Hypo, right) conditions. ROIs at the cell front and rear indicated by dashed boxes. Color bar as in panel a. Scale bar, 10 µm. f Flipper-TR lifetimes at the front and rear under Iso and Hypo conditions. (g) Per-cell differences in lifetime (Δ = Front - Rear) under Iso (dark blue circles) and Hypo (light blue circles). Gray lines link paired values; circles show per-cell differences. f, g Data are presented as mean values +/- SEM., n = 19 cells from three independent experiments. One-tailed, Mann–Whitney test; ****P < 0.0001. h Representative time-lapse images of WT-Hs578t cells co-expressing opto-PI3K (iSH2), SiR-actin, and Cav1-RFP. Insets show optogenetically activated ROIs. Timepoints span from -2 min (pre-activation) to +16 min (post-activation). i Quantification of normalized signal intensity and cell area in the activated ROI over time for actin (orange), Cav1 (gray), and cell area (blue). Data are presented as mean values +/- SEM of n = 17 cells from three independent experiments.
