Engineering PresTen: a novel membrane tension biosensor for probing cellular mechanotransduction - PROJECT SUMMARY Mechanical forces are crucial regulators of diverse biological processes, such as sensory perception (e.g., hearing, balance, touch), blood pressure regulation, and essential cellular behaviors like migration, division, and differentiation. As a pivotal biophysical parameter, membrane tension critically governs cell behavior by dynamically modulating ion channel activity, cytoskeletal organization, and mechanotransduction pathways. Current tools for measuring dynamic membrane tension changes in living cells, especially in rapid sensory systems like the cochlea, are limited in speed, sensitivity, and specificity. This project addresses this critical gap by developing PresTen, a novel, cutting-edge, genetically encoded fluorescent biosensor. PresTen is engineered from prestin (SLC26A5), a unique motor protein in cochlear outer hair cells responsible for rapid electromotility. Prestin’s inherent mechanosensitive properties and conformational changes make it exceptionally suited for detecting dynamic membrane tension changes without perturbing ion gradients. Our recent finding that prestin responds to membrane tension and thinning provides an intrinsic advantage, offering superior sensitivity and speed over conventional reporters. This exploratory tool-development project aims to establish PresTen’s feasibility and utility. We will engineer and optimize PresTen for enhanced performance by uncoupling prestin’s voltage/chloride sensitivities and integrating circularly permuted fluorescent reporters into mechanosensitive domains. Subsequently, we will calibrate PresTen’s fluorescence response against defined membrane tension using reconstituted systems and live-cell measurements. Finally, we will validate PresTen in key mechanotransduction systems, applying it to cochlear outer hair cells to capture high-frequency force dynamics and in cardiomyocytes to demonstrate broader applicability. Upon its successful development, PresTen will revolutionize our ability to precisely measure dynamic membrane tension in live cells and tissues, offering an unprecedented high-speed, non-invasive platform. This novel bioengineered tool will transform our understanding of mechanotransduction, opening new avenues for discovery in diverse areas from sound amplification and cardiac disease to immune cell activation and cancer cell metastasis. Crucially, PresTen also creates novel technological opportunities, serving as a precise mechanical force indicator for nanodrug delivery systems and for high-throughput drug screening, where direct membrane tension monitoring is currently a major challenge. This R21 project represents a critical, innovative step towards unraveling the dynamic roles of mechanical forces in health and disease.
