Engineering a single-fluorophore tension sensor to reveal molecule-specific force heterogeneity in vivo

Introduction Mechanical forces are fundamental to cardiovascular development, homeostasis, and pathogenesis. However, visualizing real-time cellular tension in living tissues remains challenging. While existing FRET-based tension sensors have advanced mechanobiology, cumbersome image analyses, uncertain probe abundance, and intermolecular artifacts limit their in vivo application. To overcome these barriers, we developed a novel, FRET-free single-fluorophore tension sensor designed for straightforward, high-resolution force imaging in living organs. Methods We engineered the single-fluorophore tension sensor by inserting an elastic linker into a circularly permuted EGFP (cpEGFP). This tension sensor module was inserted in the flexible region of either α-Actinin or α-Catenin and C-terminally fused to mCherry, allowing simplified, simultaneous readout of tension (green) and probe abundance (red). These probes were validated in mammalian cells. We also generated α-Actinin or α-Catenin tension indicator knock-in mice for live-tissue imaging. Force–fluorescence relationships were characterized using optical tweezers. Results Blebbistatin-mediated relaxation increased the tension sensor’s green fluorescence in a dose-dependent manner while the mCherry fluorescence remained constant, confirming that the sensor responds to reduced intracellular tension. Purified tension sensor molecules stretched with optical tweezers showed a progressive fluorescence loss over 0–4 pN. In cardiomyocytes from α-Actinin tension indicator mice, the indicators showed a mosaic pattern of higher and lower tension along Z-discs, and blebbistatin treatment markedly increased green fluorescence. In living mouse tissues, the α-Actinin and α-Catenin indicators revealed distinct spatial tension patterns. Notably, while the α-Catenin tension indicator reported uniformly high tension at most adherens junctions (e.g., in the liver), the α-Actinin tension indicator showed a mosaic tension pattern even within the same adherens junctions—uncovering molecule-specific heterogeneity previously unseen in vivo. Conclusion These findings indicate that tissue tension is regulated in a more complex, molecule-specific manner than previously considered. Our engineered single-fluorophore tension indicators enable the visualization of subtle changes in tension with high sensitivity and simplified analysis for in vivo force imaging, enabling quantitative comparison across molecular contexts. This tool provides a powerful, highly accessible platform for exploring dynamic mechanical regulation in cardiovascular biology in vivo.