EAGER: Calibration of novel FRET based force sensors in vitro

主要研究者：Hua，Zonglu Susan提案编号：1503109机械力是组织发育和重塑过程中组织结构的主要决定因素之一。在组织发育期间，细胞经历来自周围组织的形状变化的许多机械力。这些力不仅改变形态，而且调节遗传程序，影响新生成组织的表型和基因型，因此是用于工程组织和器官的表观遗传学的重要组成部分。到目前为止，由于缺乏测量活细胞中特定蛋白质的力的工具，我们对力传递途径的理解有限。研究小组开发了一套独特的遗传编码荧光力传感器，并证明它们可以在组织发育和重塑过程中真实的时间测量特定蛋白质中的力。校准后，这些分子力测量工具将打开重要的机会，研究力信号和转导细胞在各种条件下，EAGER赠款的总体目标是校准力在新开发的荧光共振能量转移（FRET）为基础的传感器在体外，使他们定量。该方法利用一种新的分子方法直接测量FRET探针在体外单个肌动蛋白丝中的力。将使用柔性针测量并在具有固定端的细丝上施加力，同时，将使用荧光显微镜记录FRET比率。这项研究预计将产生第一个力校准曲线的FRET探针在其自然的配置，在细胞中表达。这一结果将是一个重要的除了开发一个力传感器的“工具箱”，直接报告在细胞和组织的时间和空间的力梯度。重要的是，这项研究应该为这些探针的校准提供新的技术。关于教育的更广泛的影响，调查人员提出了一个独特的教育计划通过实验，或E2E。我们的想法是为STEM活动创造一个有形的教育工具，这些活动可以在赠款结束后很长时间内重复进行。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.