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

Area of Science:

• Biophysics
• Cell Biology
• Molecular Mechanobiology

Background:

• Genetically encoded tension sensors (GETSs) quantify intracellular forces in mechanotransduction.
• Current GETSs use FRET pairs and elastic domains, averaging signals and masking extreme forces.
• Understanding force distribution is crucial for mechanobiology.

Purpose of the Study:

• Develop digital GETSs with tunable mechanical thresholds using coiled-coils (CCs).
• Overcome limitations of analog GETSs by resolving force distribution.
• Enable more facile and precise tension measurements in living systems.

Main Methods:

• Designed digital GETSs using coiled-coil domains.
• Validated CC probe mechanics via thermodynamic stability prediction, AlphaFold2, MD simulations, and single-molecule force spectroscopy.
• Integrated optimized CC tension sensors into vinculin in live cells.

Main Results:

• Validated the mechanical response of CC digital probes.
• Demonstrated that 13% of vinculin in focal adhesions experiences forces >9.9 pN.
• Revealed higher vinculin forces than previously reported.

Conclusions:

• Digital GETSs provide a more sensitive method for measuring cellular forces.
• CC tension sensors enable facile and precise tension measurements.
• This technology advances the study of mechanotransduction and protein dynamics.