IN VIVO FLUORESCENCE FLUCTUATION SPECTROSCOPY

描述(由申请人提供)：由于其独特的特性，荧光波动光谱(FFS)对于细胞应用是一种有吸引力的技术。它以亚微米分辨率和单分子灵敏度决定蛋白质的动力学和分子性质。特别是，将FFS应用于用荧光蛋白标记的细胞蛋白质，有可能提供关于它们在活细胞中相互作用的定量信息。我们引入了双色多激发荧光光谱来定量两个标记了不同荧光颜色的蛋白质的同质和异质相互作用。双色多激发荧光光谱通过利用荧光蛋白的激发和发射性质的差异来实现必要的灵敏度。我们将开发用于活体研究的双色多激发荧光光谱，实施全局分析方法，并彻底表征该技术。到目前为止，大多数FFS亮度实验都局限于细胞核。我们将通过开发一种考虑细胞形状的技术，将FFS亮度分析的范围扩展到细胞质和质膜。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。双色多激发荧光光谱技术将用于研究核受体RXR与其辅调节因子SRC-1在体内的相互作用。受体-辅调节分子复合体齐聚状态的定量表征将是本研究的重点。此外，我们还研究了动力蛋白的齐聚作用以及它与胞浆和质膜上的亲内素的相互作用。核受体和动力素与许多疾病有关，如癌症、糖尿病和神经退行性疾病。在体内，FFS研究可以通过提供有关蛋白质相互作用的详细信息来帮助抗击这些疾病，并可能导致确定药物开发的靶点。该项目的目标是开发一种光谱工具，具有直接量化活细胞内蛋白质相互作用的独特能力。蛋白质相互作用的知识有助于确定疾病的分子原因或机制。它还提供了可能有助于开发治疗方法的信息。

DESCRIPTION (provided by applicant): Because of its unique features, fluorescence fluctuation spectroscopy (FFS) is an attractive technique for cellular applications. It determines kinetic and molecular properties of proteins with submicron resolution and single molecule sensitivity. Especially, the application of FFS to cellular proteins tagged with a fluorescent protein has the potential to provide quantitative information about their interactions in a living cell. We introduce dual-color multi-excitation FFS to quantify the homo- and hetero-interactions of two proteins labeled with distinct fluorescent colors. Dual-color multi-excitation FFS achieves the necessary sensitivity by exploiting the differences in the excitation and emission properties of the fluorescent proteins. We will develop dual-color multi excitation FFS for in vivo studies, implement global analysis methods and thoroughly characterize the technique. So far most FFS brightness experiments have been limited to the cell nucleus. We will extend the reach of FFS brightness analysis to the cytoplasm and to the plasma membrane by developing a technique that takes the cell shape into account. 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 multi-excitation FFS will be applied to study the interactions between the nuclear receptor RXR and its coregulators SRC-1 in vivo. The quantitative characterization of the oligomerization state of the receptor-coregulator complex will be at the focus of this study. In addition, we characterize the oligomerization of dynamin and it interaction with endophilin both in the cytoplasm and on the plasma membrane. Nuclear receptors and dynamin are implicated in a number of diseases, such as cancer, diabetes, and neurodegenerative diseases. In vivo FFS studies could help in fighting these diseases by providing detailed information about the protein interactions and may lead to the identification of targets for drug development. The goal of the project is the development of a spectroscopic tool with the unique ability to quantify protein interactions directly inside a living cell. Knowledge of protein interactions helps to identify the molecular cause or mechanism underlying a disease. It also provides information that may aid in the development of therapies.