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

## TL;DR

The paper introduces a new three-fluorophore FRET system to study how RIAM and vinculin interact in cells, revealing vinculin's role in sensing mechanical forces.

## Contribution

A novel three-fluorophore FRET-cascade system was developed and validated to study protein interactions and force sensing in live cells.

## Key findings

- RIAM binds to the N-terminus of vinculin in focal adhesions.
- Vinculin tension-sensing constructs report an average force of 3.0 ± 0.3 pN per focal adhesion.
- FRET-cascade is effective for studying multicomponent protein interactions and force-sensing dynamics in live cells.

## Abstract

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.

Förster resonance energy transfer (FRET) is essential for studying molecular interactions, yet challenges remain in mapping complex interactions in cellular environments. Here, the authors employ a three-fluorophore FRET-cascade system with time-correlated single photon counting fluorescence lifetime imaging microscopy to elucidate Rap1-interacting adaptor molecule (RIAM)-vinculin interactions in focal adhesions, revealing mechanosensitive roles of vinculin and its interaction with RIAM in a force-independent manner.