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

This is the authors' abstract. We don't add key points for this paper.

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.