IN VIVO FLUORESCENCE FLUCTUATION SPECTROSCOPY

体内荧光涨落光谱

• 批准号: 6870930
• 负责人: JOACHIM D MUELLER
• 金额: $ 17.19万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6870930
• 关键词: HeLa cells; bioengineering /biomedical engineering; bioimaging /biomedical imaging; biomedical equipment development; biophysics; cell biology; charge coupled device camera; chemical kinetics; fluorescence microscopy; fluorescence spectrometry; green fluorescent proteins; intermolecular interaction; intravital microscopy; method development; model design /development; molecular assembly /self assembly; nuclear receptors; optics; physical model; protein structure function; statistics /biometry; structural biology; time resolved data; transcription factor

DESCRIPTION (provided by applicant): The unique features of fluorescence fluctuation spectroscopy (FFS) make this technique attractive for cellular applications. FFS determines kinetic and molecular properties of proteins with submicron resolution and single molecule sensitivity. Especially, the application of FFS to proteins tagged with a variant of green fluorescent protein has the potential to revolutionize in vivo studies. We will focus here on dual-color photon counting histogram (PCH) analysis. PCH is a relatively new FFS technique with the potential to provide quantitative information about protein association in living cells. Dual-color PCH exploits two fluorescent protein labels with distinctively different emission spectra. Separating the two colors into different detection channels strongly increases the sensitivity of PCH in detecting protein association. We will adept dual-color PCH for in vivo studies, implement global analysis methods and thoroughly characterize the technique. The long-term objective of the proposed research lies in the concurrent development and application of fluorescence fluctuation techniques, so that their full potential for in vivo studies is realized. The impact of this new technology will be felt in many biological areas with applications ranging from basic research in cell biology to pharmaceutical drug screening. Dual-color PCH will be applied to study the interactions between nuclear receptors and their coregulators in vivo. Nuclear receptors are transcription factors that turn gene expression on and off. They inhibit or enhance transcription by recruiting an array of coactivat6r or corepressor proteins. The quantitative characterization of the oligomerization state of the receptorcoregulator complex by dual-color PCH will be at the focus of this study. Nuclear receptors affect processes as diverse as reproduction, development, and general metabolism, and have been implicated in cancer, diabetes, and many other diseases. Dual-color PCH could help in fighting these diseases by providing detailed information about the protein interactions that govern gene expression in cells.

描述（由申请人提供）： 荧光波动光谱（FFS）的独特特征使该技术对细胞应用有吸引力。 FFS确定蛋白质的动力学和分子特性，具有亚微米分辨率和单分子敏感性。特别是，将FFS应用于用绿色荧光蛋白变体标记的蛋白质具有革新体内研究的潜力。我们将在此关注双色光子计数直方图（PCH）分析。 PCH是一种相对较新的FFS技术，具有有关活细胞中蛋白质关联的定量信息的潜力。双色PCH利用具有截然不同的发射光谱的两个荧光蛋白标记。将两种颜色分离为不同的检测通道可强烈提高PCH在检测蛋白质关联中的灵敏度。我们将熟练的双色PCH用于体内研究，实施全球分析方法并彻底表征该技术。拟议的研究的长期目标在于同时开发和应用荧光波动技术，以便实现它们在体内研究的全部潜力。在许多生物学领域，将感受到这项新技术的影响，其应用从细胞生物学的基础研究到药物筛查。双色PCH将用于研究核受体与其核心调节剂体内之间的相互作用。核受体是转录基因表达开关和关闭基因的转录因子。它们通过募集一系列共抗菌6R或Corepressor蛋白来抑制或增强转录。本研究的重点，双色PCH将通过双色PCH对受体调节剂复合物的寡聚状态的定量表征。核受体会影响与繁殖，发育和一般代谢一样多样化的过程，并与癌症，糖尿病和许多其他疾病有关。双色PCH可以通过提供有关控制细胞基因表达的蛋白质相互作用的详细信息来帮助对抗这些疾病。