New strategy to measure forces at the molecular scale in vivo

项目总结/摘要 机械力是许多细胞过程的中心，例如细胞分裂、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 将生化能转化为机械能的机器。尽管细胞中的力无处不在， 关于体内力产生和力传递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在分子水平上施加的力 在活细胞中。本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。为了制造这些新的力传感器， 合理设计在约1至约15皮牛顿的特定力下解压缩的线圈（CC）（目标1）。 我们将使用光镊校准选定的CC（目标2）。为了测量感兴趣的蛋白质（POI）上的力， 在体内，我们将创建一个菌株库，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开CC的力，蛋白质将失去功能性。 通过确定每个嵌合构建体的功能性，我们将能够确定施加在 POI的二分法。作为原理证明，我们将使用此策略来测量两个 在裂殖酵母中参与网格蛋白介导的内吞作用的细胞骨架蛋白（aim 3）。如果成功，我们 战略有可能极大地影响和改变对力量产生机制、力量 几乎所有生命形式和所有细胞和发育过程中的传感和机械转导。

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.