Fig. 3: Three-colour FRET model applied to the vinculin tension-sensing biosensor with mScarlet-RIAM in fixed MEF^Vinc-/- cells.

A Structural representations of the VincTS biosensor in complex with mScarlet–RIAM (1–27 aa). The left panel shows a schematic model created in BioRender®, and the right panel shows a molecular model generated in PyMOL using PDB structures (7PNN, mVenus; 4Q9W, mTurquoise2; 5LK4, mScarlet-I) and AlphaFold3 predictions of vinculin, RIAM, and the spider-silk linker (GPGGA)₈. B Fluorescence lifetime data for VincTS with and without mScarlet–RIAM. Representative FLIM and epifluorescence images are shown on the right. Scale bar = [insert µm if known]. C Average corrected force per cell for MEF^Vinc−/− cells transfected with VincTS ± mScarlet–RIAM. Each data point represents the mean force per cell; bars indicate the pooled population mean. Right panels show corrected lifetime and force maps of a representative VincTS + mScarlet–RIAM cell, with corresponding lifetime and force distributions below. Red = Gaussian fit; blue = first component of bimodal fit; black = second component. Statistical analysis was performed by one-way ANOVA with Tukey’s correction for multiple comparisons: P ≥ 0.123 ns (ns), P ≤ 0.0332 (*), P ≤ 0.0021 (**), P ≤ 0.0002 (***), P ≤ 0.0001 (****).