Development of three-color FRET measurement of force-dependent sensing of RIAM-Vinculin interactions in focal adhesions

Forster 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 2.95 {+/-} 0.97 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. Significance StatementFocal adhesions are used by all adherent cells to attach to their surroundings and transmit mechanical signals. This paper uses a biosensor to measure force changes within the focal adhesion-associated protein called vinculin. The fluorescence lifetime of the biosensor changes when force is applied across vinculin, allowing us to report on changes in force within developing focal adhesions. We show that another protein, RIAM, interacts with vinculin in a force-independent manner. This was achieved by developing a three-color cascade FRET model, showing how the three proteins--talin, vinculin, and RIAM--interact over time. This research has furthered our understanding of the order and mechanism in which these components assemble in cell adhesions.