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.

摘要