Microscope for ultrasensitive measurement of single-molecule interaction and conformation

用于超灵敏测量单分子相互作用和构象的显微镜

• 批准号: 9219009
• 负责人: Sanjeevi Sivasankar
• 金额: $ 27.05万
• 依托单位: IOWA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9219009
• 关键词: Adhesions; Adhesives; Adopted; Area; Back; Benchmarking; Binding; Biological; Biophysical Process; Biophysics; Cell-Cell Adhesion; Cells; Data; Development; E-Cadherin; Electronics; Feedback; Fluorescence Resonance Energy Transfer; Goals; Individual; Lasers; Legal patent; Light; Maintenance; Maps; Measurement; Measures; Mechanics; Mediating; Methods; Microscope; Molecular; Molecular Conformation; Molecular Structure; Monitor; Neoplasm Metastasis; Pathologic Processes; Physiological; Physiological Processes; Play; Positioning Attribute; Proteins; Publications; Research; Resolution; Role; Sampling; Scanning Probe Microscopes; Signal Transduction; Stimulus; Structure; Techniques; Technology; Temperature; Tertiary Protein Structure; Testing; Time; Tissues; base; conformational conversion; experimental study; feeding; high energy physics; insight; instrument; mechanical force; nanometer; operation; particle physics; prototype; response; single molecule; single-molecule FRET; soft tissue; stem cell differentiation

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.

抽象的 机械信号在调节生理和病理过程（例如组织形成）中起关键作用 以及维持，干细胞分化和癌症转移。但是，分子机制 机械力诱导生物反应在很大程度上未知。这主要是由于缺乏 自动化，高吞吐量，高分辨率，探索机械力之间关系的技术， 分子结构和生理功能。该提议的第一个目标是开发一个超稳的目标 自动化显微镜可以测量单分子之间的相互作用力，同时又 监视他们的构象。该仪器称为单个单个超敏化测量的显微镜 分子相互作用和构象（音乐）将整合超稳的原子力显微镜（AFM） 荧光共振能量转移（FRET）。如我们的初步数据中所述，我们已经 开发了用于超稳定AFM操作的原型技术以及整合单分子fret和 AFM方法。我们建议的第二个目的是使用音乐来确定e-的生物物理基础 钙粘蛋白是一种介导所有软组织完整性的必不可少的细胞粘附蛋白 机械力。基于广泛的初步数据，我们假设e-钙蛋白在多个中结合 通过在这些结构之间切换构象和调节粘附。但是，该机制通过 哪些不同的电子钙粘蛋白结构的形成是未知的，并且直接证据证明其相互转换是 缺乏。音乐将用于绘制电子钙蛋白采用的不同粘合剂构象，测量 它们的力引起的互转换并将机械作用分配给E-钙粘蛋白的单个蛋白质结构域 粘附。这项研究的结果将提供对细胞相互作用，附着， 分离并转移。