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<p>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.</p><p></p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月26日

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  • Conor A. Treacy,
  • Tommy L. Pallett,
  • Tam T. T. Bui,
  • Simon P. Poland,
  • Mark A. Pfuhl,
  • Maddy Parsons,
  • Simon M. Ameer‑Beg

摘要

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.