New strategy to measure forces at the molecular scale in vivo

测量体内分子尺度力的新策略

• 批准号: 9926921
• 负责人: Julien Berro
• 金额: $ 25.13万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9926921
• 关键词: Actins; Biochemical; Biochemical Pathway; Biological; Biological Assay; Cell division; Cell physiology; Cells; Chimera organism; Clathrin; Collaborations; Complement; Complex; Cytoskeletal Proteins; Developmental Process; Disease; Dynein ATPase; Endocytosis; Engineering; Eukaryota; Fimbrin; Fission Yeast; Fluorescence; Fluorescence Resonance Energy Transfer; Focal Adhesions; Gene Expression Regulation; Health; Homologous Gene; Individual; Letters; Libraries; Life; Measurement; Measures; Mechanics; Mediating; Membrane; Methodology; Methods; Molecular; Monitor; Morphogenesis; Mutate; Names; Neoplasm Metastasis; Organelles; Organism; Pathway interactions; Phenotype; Play; Production; Prokaryotic Cells; Proteins; Spectrum Analysis; System; Tertiary Protein Structure; Testing; Tissues; Ursidae Family; Vinculin; alpha helix; base; cell motility; design; flexibility; force sensor; in vivo; interest; laser tweezer; mechanical force; mechanotransduction; molecular scale; novel therapeutic intervention; organ growth; protein crosslink; protein function; quantum; stem cell differentiation; tool; trafficking; transmission process; virtual

PROJECT SUMMARY/ABSTRACT Mechanical forces are central to a large number of cellular processes, such as cell division, cell motility, organelle morphogenesis and stem cell differentiation, to name a few. Forces are produced by molecular machineries that convert biochemical energy into mechanical energy. Despite the ubiquity of forces in cells, little is known about the molecular mechanisms of force production and force transmission in vivo, essentially, because there is a lack of universal tools and methods to directly measure forces applied at the molecular level in live cells. This project aims to develop new universally applicable molecular force sensors to measure the magnitude of forces produced on individual proteins in vivo. To create these new force sensors we will rationally design coiled-coils (CCs) that unzip under specific forces between ~1 to ~15 piconewtons (aim 1). We will calibrate select CCs using optical tweezers (aim 2). To measure forces on a protein of interest (POI) in vivo, we will create a library of strains where individual CCs are inserted between key domains of the POI. If the force applied on the POI chimera is larger than the force to unzip the CC, the protein will lose functionality. By determining the functionality of each chimeric construct, we will be able to determine the forces applied on the POI by dichotomy. As a proof of principle, we will use this strategy to measure the forces on two cytoskeletal proteins involved in clathrin-mediated endocytosis in fission yeast (aim 3). If successful, our strategy has the potential to highly impact and transform the study of mechanisms of force production, force sensing and mechanotransduction in virtually all life forms, and all cellular and developmental processes.

项目摘要/摘要 机械力是许多细胞过程的中心，如细胞分裂、细胞运动、 细胞器形态发生和干细胞分化，仅举几例。力是由分子产生的 将生化能转化为机械能的机器。尽管细胞中的力量无处不在， 关于在体内产生力量和传递力量的分子机制知之甚少，基本上， 因为缺乏通用的工具和方法来直接测量分子水平上的作用力 在活细胞中。该项目旨在开发新的普遍适用的分子力传感器来测量 在体内对单个蛋白质产生的力的大小。为了创造这些新的力传感器，我们将 合理设计可在~1至~15皮牛顿(目标1)的比力作用下解压的盘绕线圈(CCS)。 我们将使用光学镊子校准选定的CCS(目标2)。测量感兴趣蛋白质(POI)上的力 Vivo，我们将创建一个菌株文库，在其中单个CC插入POI的关键结构域之间。如果 施加在POI嵌合体上的力大于解压CC的力，蛋白质将失去功能。 通过确定每个嵌合结构的功能，我们将能够确定施加在其上的力 用二分法计算POI。作为原则的证明，我们将使用这一策略来测量两个 分裂酵母中参与网状蛋白介导的内吞作用的细胞骨架蛋白(目标3)。如果成功，我们的 战略有可能极大地影响和改变力量生产机制、力量机制的研究 感知和机械转导几乎在所有的生命形式以及所有的细胞和发育过程中。