Development of three-colour FRET cascade for force sensing of the putative RIAM-vinculin interaction in fibroblasts.

Förster resonance energy transfer (FRET) enables the measurement of molecular interactions and conformational dynamics in biological systems. FRET-cascade, a multistep energy transfer system involving three fluorophores, enables spatial and temporal mapping of molecular interactions. Here, we leveraged FRET-cascade with time-correlated single photon counting fluorescence lifetime imaging microscopy (TCSPC-FLIM) to explore the putative interaction between Rap1-interacting Adaptor Molecule (RIAM) and vinculin in focal adhesions. We developed a novel three-fluorophore FRET-cascade system, validated using purified proteins, spectroscopic analysis, structural modelling, and negative-staining transmission electron microscopy (TEM). Putative RIAM-vinculin interactions were explored in vinculin knockout mouse embryonic fibroblasts and revealed that RIAM binds to the N-terminus of vinculin in focal adhesions. Vinculin tension‑sensing constructs report average forces of 3.0 ± 0.3 pN per focal adhesion, consistent with its role in mechano-transduction. This work establishes FRET-cascade as a powerful approach for dissecting multicomponent protein interactions and force‑sensing dynamics in live cells.