EAGER: Calibration of novel FRET based force sensors in vitro

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PI: Hua, Zonglu Susan Proposal Number: 1503109Mechanical force is one of the major determinants of tissue organization during its development and remodeling. During tissue development, cells experience numerous mechanical forces from shape change of the surrounding tissues. These forces not only modify the morphology, but also regulate genetic programs, affecting phenotype and genotype of newly generated tissue, and are thus a significant component of epigenetics for engineering tissues and organs. To date, our understanding of force transmission pathways has been limited due to lack of tools to measure the forces in specific proteins in live cells. The investigators' group has developed a unique set of genetically coded fluorescence force sensors and demonstrated that they can measure forces in specific proteins in real time during tissue development and remodeling. When calibrated, these molecular force measurement tools will open significant opportunities to study force signaling and transduction in cells under a variety of conditions.The overall objective of this EAGER grant is to calibrate forces in newly developed fluorescence resonance energy transfer (FRET)-based sensors in vitro to make them quantitative. The approach utilizes a novel molecular method to directly measure the force in FRET probes in individual actin filaments in vitro. A flexible needle will be used to measure and exert forces on the filament with fixed ends, and simultaneously, FRET ratio will be recorded using fluorescence microscopy. This research is expected to generate the first force calibration curves for FRET probes in their natural configuration as expressed in cells. This result will be an important addition to the development of a force sensor 'toolbox' to directly report temporal and spatial force gradients in cells and tissues. Importantly, this research should provide new techniques for calibration of these probes. With regard to educational broader impacts, the investigators propose a unique program on Education through Experimentation, or E2E. The idea is to create a tangible educational tool for STEM activities that can be repeated long after the grant is over. This one-year EAGER grant will serve as a pilot run on this novel E2E idea.