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 和维持、干细胞分化和癌症转移。然而， 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，技术来探索机械力之间的关系， 分子结构和生理功能。这项提案的第一个目标是开发一种超稳定的， 自动化，显微镜，可以测量单个分子之间的相互作用力，同时 监测它们的形态。这种仪器，称为显微镜超灵敏测量单， 分子相互作用和构象（MUSIC），将整合超稳定原子力显微镜（AFM） 荧光共振能量转移（FRET）。正如我们的初步数据所描述的，我们已经 开发了超稳定AFM操作和整合单分子FRET的原型技术， AFM方法。我们建议的第二个目的是使用MUSIC来确定E- 钙粘蛋白是一种调节所有软组织完整性的重要细胞间粘附蛋白， 机械力基于广泛的初步数据，我们假设E-钙粘蛋白与多种 构象并通过在这些结构之间切换来调节粘附。然而， 不同的E-钙粘蛋白结构形成的是未知的，它们相互转化的直接证据是 缺乏MUSIC将用于绘制出E-钙粘蛋白所采用的不同粘附构象，测量 它们的力诱导的相互转换，并分配一个机械作用的个别蛋白质结构域在E-钙粘蛋白 粘连这项研究的结果将提供一个生物物理的理解细胞如何相互作用，附着， 分离并转移

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.