IN VIVO FLUORESCENCE FLUCTUATION SPECTROSCOPY

体内荧光涨落光谱

• 批准号: 7937181
• 负责人: JOACHIM D MUELLER
• 金额: $ 23.09万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937181
• 关键词: Accounting; Area; Basic Science; Binding; Biological; Biological Models; Cell Nucleus; Cell Shape; Cell membrane; Cells; Cellular biology; Color; Complex; Computer software; Cyan Fluorescent Protein; Cytoplasm; Development; Diabetes Mellitus; Diffusion; Disease; Dynamin; Endocytosis; Eukaryotic Cell; Fluorescence; Fluorescence Spectroscopy; Gene Expression; Goals; Guanosine Triphosphate Phosphohydrolases; Homo; Interphase Cell; Investigation; Kinetics; Knowledge; Label; Lead; Life; Malignant Neoplasms; Measurement; Membrane; Methods; Modeling; Molecular; Neurodegenerative Disorders; Nuclear Receptors; Performance; Pharmacologic Substance; Preclinical Drug Evaluation; Process; Property; Proteins; RXR; Research; Resolution; Scanning; Scientist; Signal Transduction; Spectrum Analysis; Techniques; Testing; Thick; Work; Writing; biological systems; drug development; fighting; in vivo; instrument; interest; new technology; nuclear receptor coactivator 1; protein complex; protein protein interaction; receptor; red fluorescent protein; research study; single molecule; stoichiometry; submicron; theories; therapy development; tool; two-photon

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.

描述（由申请人提供）：由于其独特的功能，荧光波动光谱法（FFS）是一种有吸引力的细胞应用技术。它以亚微米分辨率和单分子灵敏度测定蛋白质的动力学和分子特性。特别是，FFS的应用程序与荧光蛋白标记的细胞蛋白质有可能提供定量信息，它们在活细胞中的相互作用。我们引入双色多激发FFS来量化两种不同荧光颜色标记的蛋白质的同源和异源相互作用。双色多激发FFS通过利用荧光蛋白的激发和发射特性的差异来实现必要的灵敏度。我们将开发用于体内研究的双色多激发FFS，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亮度实验仅限于细胞核。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波动技术，使其在体内研究的全部潜力得以实现。这项新技术的影响将在许多生物领域感受到，其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FFS将用于研究核受体RXR与其辅调节因子SRC-1之间的相互作用。受体辅调节复合物的寡聚化状态的定量表征将是本研究的重点。此外，我们还研究了发动蛋白的寡聚化及其与细胞质和细胞膜上的内亲蛋白的相互作用。核受体和动力蛋白与许多疾病有关，如癌症、糖尿病和神经退行性疾病。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对抗这些疾病，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，该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。