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

Förster resonance energy transfer (FRET) is a powerful technique for probing molecular interactions and conformational changes in biological systems. Cascade-FRET, a multistep energy transfer system involving three fluorophores, enables spatial and temporal mapping of molecular interactions. Here, we leveraged Cascade-FRET with time-correlated single photon counting fluorescence lifetime imaging microscopy (TCSPC-FLIM) to explore the putative interaction between Rap1-interacting Adapter Molecule (RIAM) and vinculin in focal adhesions. We developed a novel three-fluorophore Cascade-FRET system connected by flexible peptide linkers comprising mTurquoise2, mVenus, and mScarlet-I, validated using purified proteins, spectroscopic analysis, structural modeling, 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. This complex requires an intact microtubule cytoskeleton. Vinculin tension-sensing constructs quantified intracellular forces, with an average force of 3.0 ± 0.3 pN per focal adhesion. These findings corroborate the mechanosensitive role of vinculin and its interaction with RIAM in a force-independent manner. This study demonstrates the utility of Cascade-FRET and TCSPC-FLIM in investigating multicomponent molecular complexes. Our findings provide novel insights into RIAM-vinculin interactions and their regulation by intracellular tension, paving the way for advanced applications of Cascade-FRET in dynamic cellular systems.