(4aw) Development of a FRET-Based Tension Sensor for Measuring Forces Across Proteins in Living Cells

Force and other mechanical variables are important regulators in many biological processes. For instance, forces from blood flow are key factors in the development of disease states, like atherosclerosis, and the mechanical environment cells experience can determine the differentiation state of stem cells and the metastatic potential of cancer cells. But how cells detect and interpret these forces, a process called mechanotransduction, is not well understood.

Most research on mechanotransduction has focused on cataloging cellular responses and identifying proteins that change activity or localization in response to applied force. This is mainly due to a lack of tools to study force at a molecular level in a living cell. We therefore developed a calibrated, genetically encoded Forster Resonance Energy Transfer (FRET) based tension sensor with pico-Newton sensitivity consisting of a spring-like peptide sequence between two fluorescent proteins. When tension is applied to the sensor the spring extends and the energy transfer between the fluorescent proteins decreases. The sensor has been put into the protein vinculin, as it is a major component of the mechanical link between integrins, which mediate cell adhesion to the extra-cellular matrix, and the actin cytoskeleton, the primary force generation system of the cell. The sensor detects changes in response to inhibition of cellular contractility, in different areas of a migrating cell, and as focal adhesions assemble or disassemble.

With this new tool, we can visualize the spatio-temporal dynamics protein deformation in live cells to study the relationships between forces and cell signaling in ways that were previously impossible. Initial efforts will focus on kinases that have been strongly implicated in mechanotransduction. Additional efforts include development of a polymer physics based model of the sensor to further optimize this sensor and aid in the development of new sensors for other proteins. Future projects include developing a microscopy-based screening system to detect proteins that localize to areas of high force in focal adhesions and are therefore likely key players in mechanotransduction.