EAGER: Calibration of novel FRET based force sensors in vitro

EAGER：基于 FRET 的新型力传感器的体外校准

• 批准号: 1503109
• 负责人: Zonglu Susan Hua
• 金额: $ 9.99万
• 依托单位: SUNY at Buffalo
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2017-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1503109&HistoricalAwards=false
• 关键词: EAGER; Calibration; novel; FRET; based

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.

PI：Hua, Zonglu Susan 提案编号：1503109 机械力是组织组织发育和重塑过程中的主要决定因素之一。在组织发育过程中，细胞因周围组织的形状变化而受到大量机械力。这些力不仅改变形态，而且调节遗传程序，影响新产生的组织的表型和基因型，因此是工程组织和器官的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的力的工具，我们对力传递途径的理解受到限制。研究人员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 资助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) 的传感器中的力，使其定量。该方法利用一种新颖的分子方法直接测量体外单个肌动蛋白丝中 FRET 探针的力。将使用柔性针测量并向具有固定端的灯丝施加力，同时使用荧光显微镜记录 FRET 比率。这项研究预计将为 FRET 探针以其在细胞中表达的自然配置生成第一条力校准曲线。这一结果将是对力传感器“工具箱”开发的重要补充，以直接报告细胞和组织中的时间和空间力梯度。重要的是，这项研究应该为这些探针的校准提供新技术。关于更广泛的教育影响，研究人员提出了一项独特的实验教育计划（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时间内重复使用。这笔为期一年的 EAGER 资助将作为这一新颖的 E2E 想法的试点。