In Vivo Fluorescence Fluctuation Spectroscopy

体内荧光波动光谱

• 批准号: 8616072
• 负责人: JOACHIM D MUELLER
• 金额: $ 26.93万
• 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 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8616072
• 关键词: Accounting; Address; Area; Basic Science; Bifidobacterium longum BIF protein; Binding; Binding Proteins; Biological; Biological Models; Cell membrane; Cell physiology; Cells; Cellular biology; Color; Complex; Computer software; Cytoplasm; Data; Development; Diffusion; Disease; Drug Design; Elements; Environment; Epidermal Growth Factor Receptor; Event; Exhibits; Fluorescence; Fluorescence Resonance Energy Transfer; Gagging; Geometry; Goals; Homo; In Vitro; Kinetics; Knowledge; Label; Lateral; Lead; Life; Measurement; Measures; Membrane; Membrane Proteins; Methodology; Methods; Modeling; Molecular; Optics; Performance; Pharmacologic Substance; Photobleaching; Preclinical Drug Evaluation; Principal Investigator; Process; Property; Protein Binding; Proteins; Regulation; Research; Resolution; Sampling; Scanning; Signal Transduction; Sorting - Cell Movement; Spectrum Analysis; Structure; System; Techniques; Thick; Transport Vesicles; Tsg101 protein; Vesicle; Viral; Work; Writing; base; color detection; drug development; fighting; fluorescence imaging; in vivo; novel; novel strategies; particle; programs; protein complex; protein function; research study; single molecule; stoichiometry; submicron; theories; tool

DESCRIPTION (provided by applicant): 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. A unique feature of FFS is the ability to measure the stoichiometry and binding curve of fluorescently labeled protein complexes through brightness analysis. Brightness is the average fluorescence intensity of a single protein complex and is directly proportional to the number of labeled protein molecules. Soluble homo- and hetero- protein complexes have been successfully characterized by brightness analysis of cellular FFS data. While we have made enormous progress in the characterization of soluble protein complexes by FFS, our ability to investigate membrane-bound proteins by FFS remains woefully inadequate. This deficit is especially egregious because more than half of all proteins interact with the membrane. Data from structural studies show that membrane proteins function in complexes, but our ability to detect and quantify the interactions is very limited. This proposal seeks to extend FFS capabilities to the characterization of membrane-bound proteins by capitalizing on recent advances in FFS methodology. We first focus on proteins at the plasma membrane. The technical approach is based on z-scan FFS where the optical observation volume is scanned axially through the sample. Z-scan FFS takes the geometry of the sample into account and separates between cytoplasmic and membrane signal. We develop and characterize the performance of z-scan FFS and extend the technique to include correlation functions, lateral imaging, and fluorescence lifetime. Both single- and dual-color z-scan FFS are developed in order to characterize both homo- and hetero-protein interactions. In addition we will explore the potential of FFS to characterize proteins at vesicles inside the living cell. Vesicles transport, sort, digest and stor proteins. The regulation of these diverse processes is not well understood but involves specific proteins that associate with vesicles. FFS experiments of such vesicles carrying fluorescently-labeled proteins lead to bright, but infrequent peaks on top of background. Characterization of such data is a daunting challenge, but recent advances in brightness analysis offer a quantitative approach to separate the background from the bright peaks. We will investigate this approach with the goal of determining the copy number of proteins and the coexistence of two proteins on the same vesicles. This development of new FFS methods fills a critical need, because we still lack methods that quantify proteins at membranes and at vesicles. The impact of the new methodology will be felt in many biological areas with applications ranging from basic research in cell biology to pharmaceutical drug screening. In vivo FFS could help fighting diseases by providing detailed information about protein interactions and may lead to the identification of targets for drug development.

描述（由申请人提供）：荧光波动光谱（FFS）是一种用于细胞应用的有吸引力的技术。它确定具有亚微米分辨率和单分子敏感性的蛋白质的动力学和分子特性。 FFS的一个独特特征是能够通过亮度分析测量荧光标记的蛋白质复合物的化学计量和结合曲线。亮度是单个蛋白质复合物的平均荧光强度，并且与标记的蛋白质分子的数量成正比。可溶性同型和杂种蛋白复合物已成功地通过细胞FFS数据的亮度分析来表征。尽管我们在FFS对可溶性蛋白复合物的表征方面取得了巨大进展，但我们研究FFS膜结合的蛋白的能力仍然不足。这种赤字尤其严重，因为所有蛋白质中有超过一半与膜相互作用。来自结构研究的数据表明，膜蛋白在复合物中起作用，但是我们检测和量化相互作用的能力非常有限。该建议旨在通过利用FFS方法的最新进展来扩展FFS能力，以扩展膜结合蛋白的表征。我们首先关注质膜的蛋白质。技术方法基于Z扫描FFS，其中光学观察量通过样品轴向扫描。 Z-SCAN FFS考虑了样品的几何形状，并在细胞质和膜信号之间分离。我们开发和表征Z扫描FF的性能，并将技术扩展到包括相关功能，横向成像和荧光寿命。为了表征同型和异蛋白相互作用，单色和双色Z-SCAN FFS均被开发。此外，我们将探索FFS表征活细胞内囊泡的蛋白质的潜力。囊泡运输，排序，摘要和Stor蛋白。这些不同过程的调节尚不清楚，但涉及与囊泡相关的特定蛋白质。携带荧光标记蛋白的此类囊泡的FFS实验导致背景顶部明亮但很少峰。这种数据的表征是一个艰巨的挑战，但是亮度分析的最新进展提供了一种定量方法，可以将背景与亮峰区分开。我们将研究这种方法，目的是确定蛋白质的拷贝数以及在同一囊泡上的两种蛋白质的共存。新FFS方法的这种开发填补了一个关键的需求，因为我们仍然缺乏量化膜和囊泡上蛋白质的方法。新方法的影响将在许多生物学领域都可以感受到，从细胞生物学的基础研究到药物筛查的应用。体内FFS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疾病，并可能导致鉴定药物开发目标。