Linking molecular tension and cellular tractions: a multiscale approach to focal adhesion mechanics

Focal adhesions (FAs) are mechanosensitive structures that mediate force transmission between cells and the extracellular matrix. While Traction Force Microscopy (TFM) quantifies cellular tractions exerted on deformable substrates, Förster Resonance Energy Transfer (FRET)-based tension probes, such as vinculin tension sensors, measure molecular-scale forces within FA proteins. Despite their potential synergy, these methods have rarely been combined to explore the interplay between molecular tension and cellular tractions. Here, we introduce a framework integrating TFM and FRET-based vinculin tension sensors to investigate FA mechanics across scales. At cell level, tractions and vinculin tension increased with substrate stiffness. At FA level, vinculin tension correlated solely with vinculin density, while tractions scaled with FA area, orientation, total vinculin content and vinculin density. Direct comparison of tractions to vinculin tension revealed a complex, heterogenous relationship between these forces, possibly linked to diverse cell and FA maturation states. Sub-FA analysis revealed conserved spatial patterns, with both tension and traction increasing towards the cell periphery. This multiscale approach provides an integrated workflow for studying focal adhesion forces, helping to bridge the gap between vinculin tension and cellular tractions.