Abstract
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.
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Data availability
Source Data files including the data points for the generation of figures and graphs, and the corresponding statistical analysis, are accessible at Zenodo (https://doi.org/10.5281/zenodo.14692589)76. Raw microscopy files are available from the corresponding authors upon request.
Code availability
The TFM code to analyze 2D traction microscopy data, as well as the custom-written FRET code to calculate FRET efficiencies from FLIM-Phasor data, and the custom-written TFM-FRET code for the simultaneous analysis of molecular tension and cell traction is open source and can be accessed through Zenodo (https://doi.org/10.5281/zenodo.14692589)76.
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Acknowledgements
The authors thank Professor Jelle Hendrix (Hasselt University, Belgium) for constructive discussions on pixel-based FRET efficiency analysis, and Rik Nuyts for assistance with microscopy measurements and imaging optimization. They are grateful to the following funding sources: S.R. and S.A. were supported by KU Leuven internal funding: IDN/20/021, KA/20/026 and C14/22/085. S.D. was supported by KU Leuven internal funding: ZB/22/028. S.R. and H.V.O. received funding from the Research Foundation Flanders (FWO) through a project with grant number G0C2422N. H.V.O. and L.K. were supported by the iBOF project 21/083C. H.V.O. received the FWO infrastructure grant I009718N. In addition, S.A. and J.B.F. are recipients of FWO fellowships with grant numbers 1S95125N and 1259223 N, respectively.
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Experiments and data analysis were performed by S.A. and L.K. S.A. and S.V. generated the DNA constructs used in the study. The rheological characterization of the hydrogels was performed by D.L. and S.D., under the supervision of R.C. and H.V.O. The customized Matlab algorithms were developed by L.K., Q.C. and J.B.F. Q.C., M.C., H.V.O., and S.R. developed the initial concept. The work was supervised by H.V.O. and S.R. The original draft was written by S.A., under the supervision of S.R. All authors contributed to the writing of the final version.
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Communications Biology thanks Brenton Hoffman, Andrea Ravasio, Catalina Soto-Montandon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Ophelia Bu and Christina Karlsson Rosenthal. A peer review file is available.
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Aytekin, S., Kimps, L., Coucke, Q. et al. Linking molecular tension and cellular tractions: a multiscale approach to focal adhesion mechanics. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09514-0
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DOI: https://doi.org/10.1038/s42003-026-09514-0
