Development of a BRET-Based Molecular Tension Sensor to Study Altered Tensions in Disease Pathogenesis

Abstract

Alteration of mechanical forces is an emerging factor in diseases like cancer. Changes in macroscopic stiffness in disease are accompanied by a wealth of changes in a cell's tensional homeostasis at a molecular level where mechanotransduction signaling pathways are aberrantly activated. However, the lack of tools to measure tensions at a molecular level in the cellular environment has crippled the identification of mechanosensing proteins and characterization of magnitudes of physiologic force in normal and disease contexts. We have developed a new bioluminescence resonance energy transfer (BRET)-based molecular tension sensor that responds predictably to changes in forces and exhibits an enhanced dynamic range compared to other FRET-based molecular tension sensors. We show that our tension sensor exhibits a distance-dependent change in BRETthrough the use of rigid alpha-helical linkers of varying length. As a proof of concept, we inserted our sensor into the canonical mechanosensing focal adhesion protein vinculin, and observed expected tension changes. We also encoded it into the mechanosensing cell-surface receptor Notch. Upon presentation of Notch's ligand on a neighboring cell, we detected a decrease in BRET signal corresponding to a stretching of the sensor due to the force induced by endocytosis of the ligand. This signal can be measured in bulk by a luminometer and in individual cells via imaging. We anticipate our BRET-based molecular tension sensor will be well suited to identify novel mechanosensing proteins and characterize the magnitude of change in tension sensed during disease pathogenesis.