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性质。FFS的一个独特特点是能够通过亮度分析测量荧光标记蛋白复合物的化学计量学和结合曲线。亮度是单个蛋白质复合物的平均荧光强度，与标记的蛋白质分子的数量成正比。通过细胞FFS数据的亮度分析，已经成功地表征了可溶性的同源蛋白和异质蛋白复合物。虽然我们在利用FFS表征可溶性蛋白复合物方面取得了巨大进展，但我们利用FFS研究膜结合蛋白的能力仍然严重不足。这种缺陷尤其严重，因为超过一半的蛋白质与细胞膜相互作用。结构研究的数据表明，膜蛋白在复合物中起作用，但我们检测和量化相互作用的能力非常有限。本提案旨在通过利用FFS方法的最新进展，将FFS能力扩展到膜结合蛋白的表征。我们首先关注质膜上的蛋白质。技术方法是基于z-扫描FFS，其中光学观测体通过样品轴向扫描。z扫描FFS考虑了样品的几何形状，并将细胞质和膜信号分开。我们开发并表征了z扫描FFS的性能，并将该技术扩展到包括相关函数、横向成像和荧光寿命。开发了单色和双色z扫描FFS，以表征homo-和hetero-protein相互作用。此外，我们将探索FFS在活细胞内囊泡中表征蛋白质的潜力。小泡运输、分类、消化和储存蛋白质。这些不同过程的调控尚不清楚，但涉及与囊泡相关的特定蛋白质。这种携带荧光标记蛋白质的囊泡的FFS实验导致背景顶部出现明亮但不常见的峰。对这些数据进行表征是一项艰巨的挑战，但最近在亮度分析方面的进展提供了一种定量方法，可以将背景与明亮的峰值分开。我们将研究这种方法，目的是确定蛋白质的拷贝数和两种蛋白质在同一囊泡上的共存。这种新的FFS方法的发展填补了一个关键的需求，因为我们仍然缺乏定量膜和囊泡蛋白的方法。从细胞生物学的基础研究到药物筛选，新方法的影响将在许多生物学领域得到应用。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对抗疾病，并可能导致确定药物开发的靶点。