EAGER: Calibration of novel FRET based force sensors in vitro

在组织发育和重塑过程中，机械力是组织组织的主要决定因素之一。在组织发育过程中，细胞受到来自周围组织形状变化的众多机械力。这些力量不仅改变形态，而且调节遗传程序，影响新生成组织的表型和基因型，因此是工程组织和器官表观遗传学的重要组成部分。迄今为止，由于缺乏测量活细胞中特定蛋白质中的力的工具，我们对力传递途径的理解受到限制。研究小组开发了一套独特的基因编码荧光力传感器，并证明它们可以在组织发育和重塑过程中实时测量特定蛋白质的力。经过校准后，这些分子力测量工具将为研究各种条件下细胞中的力信号传导和转导提供重要机会。这项EAGER拨款的总体目标是在体外校准新开发的基于荧光共振能量转移（FRET）的传感器的力，使其定量。该方法利用一种新颖的分子方法直接测量FRET探针在体外单个肌动蛋白细丝中的力。将使用一根柔性针来测量并对具有固定末端的灯丝施加力，同时使用荧光显微镜记录FRET比率。这项研究有望生成FRET探针在细胞中表达的自然构型的第一个力校准曲线。这一结果将是力传感器“工具箱”发展的重要补充，以直接报告细胞和组织中的时间和空间力梯度。重要的是，本研究将为这些探针的校准提供新的技术。关于教育的广泛影响，研究人员提出了一个独特的计划，通过实验的教育，或E2E。这个想法是为STEM活动创建一个有形的教育工具，可以在资助结束后很长一段时间内重复使用。这项为期一年的EAGER赠款将作为这一新颖的E2E想法的试点。

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.