IN VIVO FLUORESCENCE FLUCTUATION SPECTROSCOPY

体内荧光涨落光谱

• 批准号: 7010752
• 负责人: JOACHIM D MUELLER
• 金额: $ 19.84万
• 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/7010752
• 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)： 荧光涨落光谱(FFS)的独特特性使这项技术在细胞应用中具有吸引力。FFS以亚微米分辨率和单分子灵敏度确定蛋白质的动力学和分子性质。特别是，将荧光荧光技术应用于标记了绿色荧光蛋白变体的蛋白质，可能会给体内研究带来革命性的变化。这里我们将重点介绍双色光子计数直方图(PCH)分析。PCH是一种相对较新的FFS技术，有可能提供关于活细胞中蛋白质结合的定量信息。双色PCH使用两个发射光谱截然不同的荧光蛋白质标记。将这两种颜色分离到不同的检测通道中，大大提高了PCH在检测蛋白质结合时的灵敏度。我们将熟练地将双色五氯环己烷用于体内研究，实施全球分析方法，并彻底表征这项技术。拟议研究的长期目标在于同时开发和应用荧光涨落技术，以实现其在体内研究的全部潜力。这项新技术的影响将在许多生物学领域感受到，应用范围从细胞生物学的基础研究到药物筛选。双色PCH将在体内用于研究核受体及其辅助调节因子之间的相互作用。核受体是启动和关闭基因表达的转录因子。它们通过招募一系列共激活的6R或共抑制蛋白来抑制或增强转录。通过双色PCH对受体辅调节复合体齐聚状态的定量表征将是本研究的重点。核受体影响生殖、发育和一般新陈代谢等多种过程，并与癌症、糖尿病和许多其他疾病有关。双色PCH通过提供控制细胞中基因表达的蛋白质相互作用的详细信息，可以帮助抗击这些疾病。