In Vivo Fluorescence Fluctuation Spectroscopy

描述(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决定蛋白质的动力学和分子性质。FFS的一个独特功能是能够通过亮度分析测量荧光标记蛋白质络合物的化学计量比和结合曲线。亮度是单个蛋白质复合体的平均荧光强度，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，成功地表征了可溶的同种和异种蛋白质复合体。虽然我们已经在FFS表征可溶蛋白质复合体方面取得了巨大的进展，但我们用FFS研究膜结合蛋白的能力仍然严重不足。这一缺陷尤其严重，因为超过一半的蛋白质与膜相互作用。来自结构研究的数据表明，膜蛋白在复合体中发挥作用，但我们检测和量化相互作用的能力非常有限。这项提议试图通过利用FFS方法的最新进展，将FFS的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。该技术方法基于z-扫描FFS，其中光学观察体通过样品轴向扫描。Z-Scan FFS考虑了样品的几何形状，并将细胞质信号和膜信号分开。我们开发和表征了z-扫描FFS的性能，并将该技术扩展到包括关联函数、横向成像和荧光寿命。单色和双色z-扫描荧光光谱都是为了表征同种和异种蛋白质的相互作用而开发的。此外，我们还将探索FFS在描述活细胞内小泡上的蛋白质方面的潜力。囊泡运输、分类、消化和储存蛋白质。对这些不同过程的调控还不是很清楚，但涉及到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罕见的峰。这类数据的表征是一项艰巨的挑战，但亮度分析的最新进展提供了一种将背景与亮峰分开的定量方法。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相同小泡上的共存。这种新的FFS方法的发展填补了一个关键的需求，因为我们仍然缺乏对膜和囊泡上的蛋白质进行量化的方法。新方法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。在体内，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，并可能导致识别药物开发的目标。

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.