Microscope for ultrasensitive measurement of single-molecule interaction and conformation

摘要 机械信号在调节组织形成等生理和病理过程中起着关键作用 以及维持、干细胞分化和癌症转移。然而，其分子机制 机械力引起的生物反应在很大程度上是未知的。这主要是由于缺乏 自动化，高通量，高分辨率，探索机械力之间的关系的技术， 分子结构和生理功能。这项提议的第一个目标是开发一种超稳定的， 自动化显微镜，可以同时测量单个分子之间的相互作用力 监测他们的构象。这种仪器，称为显微镜，用于超灵敏地测量单个… 分子相互作用和构象(MUSIC)，将集成一个超稳定原子力显微镜(AFM) 与荧光共振能量转移(FRET)。正如我们的初步数据所描述的那样，我们已经 开发了超稳定AFM操作的原型技术，并将单分子FRET和 原子力显微镜方法。我们提议的第二个目标是使用音乐来确定E- 钙粘附素是一种重要的细胞-细胞黏附蛋白，它调节所有软组织的完整性，对 机械力。基于大量的初步数据，我们假设E-钙粘附素结合在多个 构象并通过在这些结构之间切换来调节粘附性。然而，这些机制通过 目前尚不清楚形成了哪些不同的E-钙粘附素结构，它们相互转化的直接证据是 缺乏。MUSIC将用来绘制E-钙粘蛋白所采用的不同黏附构象，MEASure 它们的力诱导相互转换以及在E-钙粘附素中对单个蛋白结构域的机械作用 粘附力。这项研究的结果将提供对细胞如何相互作用、附着、 分离和转移。

ABSTRACT Mechanical signals play a critical role in regulating physiological and pathological processes like tissue formation and maintenance, stem cell differentiation and cancer metastasis. However, the molecular mechanisms by which mechanical forces induce biological responses are largely unknown. This is primarily due to the lack of automated, high throughput, high resolution, techniques to explore the relationship between mechanical force, molecular structure and physiological function. The first goal of this proposal is to develop an ultra-stable, automated, microscope that can measure interaction forces between single molecules while simultaneously monitoring their conformation. This instrument, called the Microscope for Ultrasensitive-measurement of Single- molecule Interaction and Conformation (MUSIC), will integrate an ultra-stable atomic force microscope (AFM) with fluorescence resonance energy transfer (FRET). As described in our preliminary data, we have already developed prototype technologies for ultra-stable AFM operation and for integrating single molecule FRET and AFM methods. The second aim of our proposal is to use MUSIC to determine the biophysical basis by which E- cadherin, an essential cell-cell adhesion protein that mediates the integrity of all soft tissue, responds to mechanical force. Based on extensive preliminary data, we hypothesize that E-cadherins bind in multiple conformations and modulate adhesion by switching between these structures. However, the mechanisms by which different E-cadherin structures are formed is unknown and direct evidence for their interconversion is lacking. MUSIC will be used to map out the different adhesive conformations adopted by E-cadherin, measure their force-induced interconversion and to assign a mechanistic role to individual protein domains in E-cadherin adhesion. The results of this research will provide a biophysical understanding of how cells interact, attach, detach and metastasize.