Volume 137, Issue 8 e202407359
Forschungsartikel

Digital and Tunable Genetically Encoded Tension Sensors Based on Engineered Coiled-Coils

Shuhong Liu

Shuhong Liu

Department of Chemistry, Emory University, Atlanta, Georgia, 30322 United States

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Jinchan Liu

Jinchan Liu

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, 06520 United States

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Alexander Foote

Alexander Foote

Department of Chemistry, Emory University, Atlanta, Georgia, 30322 United States

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Hiroaki Ogasawara

Hiroaki Ogasawara

Department of Chemistry, Emory University, Atlanta, Georgia, 30322 United States

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Sarah Al Abdullatif

Sarah Al Abdullatif

Department of Chemistry, Emory University, Atlanta, Georgia, 30322 United States

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Prof. Victor S. Batista

Prof. Victor S. Batista

Department of Chemistry, Yale University, New Haven, Connecticut, 06520 United States

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Prof. Khalid Salaita

Corresponding Author

Prof. Khalid Salaita

Department of Chemistry, Emory University, Atlanta, Georgia, 30322 United States

Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, 30322 United States

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First published: 13 January 2025

Abstract

Genetically encoded tension sensors (GETSs) allow for quantifying forces experienced by intracellular proteins involved in mechanotransduction. The vast majority of GETSs are comprised of a FRET pair flanking an elastic “spring-like” domain that gradually extends in response to force. Because of ensemble averaging, the FRET signal generated by such analog sensors conceals forces that deviate from the average, and hence it is unknown if a subset of proteins experience greater magnitudes of force. We address this problem by developing digital GETSs comprised of coiled-coils (CCs) with tunable mechanical thresholds. We validate the mechanical response of CC digital probes using thermodynamic stability prediction, AlphaFold2 modeling, steered molecular dynamics simulations, and single-molecule force spectroscopy. Live cell measurements using optimized CC tension sensors that are inserted into vinculin demonstrate that 13 % of this mechanosensor experiences forces >9.9 pN within focal adhesions. This reveals greater magnitudes of vinculin force than had previously been reported and demonstrates that CC tension sensors enable more facile and precise tension measurements in living systems.

Conflict of Interests

The authors declare no conflict of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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