DYNAMICS OF NANOSCALE LIPID DOMAINS

描述（由申请人提供）：在细胞膜上，脂质和蛋白质形成专门的区域，“筏”，太小，无法用常规光学显微镜看到。筏状脂质构成了一种有序的液相，其物理特性与周围无序的脂质不同——更紧密地堆积和粘滞。选择性地将特定蛋白质结合到筏中对于膜运输、信号传导和特殊结构的组装（例如病毒出芽和免疫反应）是重要的。调控膜筏形成和动力特性的因素尚不清楚。细胞膜的异质性和复杂性使得研究这些结构的基本生物物理性质变得困难。由选定的脂质组分在体外形成的脂质双层膜可作为研究膜筏相关相行为的简化模型。提出的工作的长期目标是了解在生物细胞膜控制模型中决定亚微观脂质“纳米结构域”（代表筏）的大小、稳定性和动态特性的因素。第一个具体目标是测量巨型单层囊泡（GUVs）中脂质纳米结构域的大小分布和动态特性，以模拟细胞膜的物理特性。荧光波动方法，包括荧光相关光谱（FCS）和荧光强度分布分析（FIDA）将提供有关脂质纳米结构域的动态性质和大小的信息。这些测量将依赖于荧光脂质类似物，选择性地划分成不同的脂质相。第二个目标是确定选定的蛋白质如何与纳米结构域结合。FIDA测量将确定蛋白质结合程度与纳米结构域大小之间的相关性；FCS将提供有关蛋白质-纳米结构域相互作用动力学的信息。第三个目标是建立一个理论模型来解释纳米畴形成的结构基础，并改进成像技术。实验结果对理论模型进行了验证和激励。本研究建立的实验方法、测量数据和理论模型将适用于细胞膜，从而可以获得关于生物筏结构和功能的基本生物物理信息。被称为“筏”的蛋白质-脂质结构调节细胞表面的信号传导和结构形成，被认为是病毒复制和几种免疫反应的核心参与者。这项工作的目标是改进现有的成像技术，并开发计算机模拟，以提供对这种木筏如何在模型系统中形成的机制理解。

DESCRIPTION (provided by applicant): On cell membranes lipids and proteins form specialized regions, "rafts", that are too small to be seen by conventional light microscopy. The raft lipids constitute an ordered liquid phase physically distinct-more tightly packed and viscous-from the disordered surrounding lipids. The selective incorporation of specific proteins into rafts is important for membrane trafficking, signaling, and assembly of specialized structures, e.g., in virus budding and immune responses. Little is known about the factors that regulate the formation and dynamic properties of membrane rafts. The heterogeneity and complexity of cell membranes makes study of the basic biophysical properties of these structures difficult. Lipid bilayer membranes formed in vitro from selected lipid components serve as simplified models in which to study phase behavior relevant to membrane rafts. The long range goals of the proposed work are to understand the factors that determine the sizes, stabilities, and dynamic properties of submicroscopic lipid "nanodomains" (representing rafts) in controlled models of biological cell membranes. The first specific aim is to measure the distribution of sizes and dynamic properties of lipid nanodomains in Giant Unilamellar Vesicles (GUVs) that model the physical properties of cell membranes. Fluorescence fluctuation methods, including Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Intensity Distribution Analysis (FIDA) will provide information about dynamic properties and sizes of the lipid nanodomains. These measurements will rely on fluorescent lipid analogs that partition selectively into different lipid phases. The second aim is to determine how selected proteins bind to nanodomains. FIDA measurements will determine the correlation between the extent of protein binding and nanodomain size; FCS will yield information about the kinetics of protein-nanodomain interaction. The third aim is to develop a theoretical model explain the structural basis of nanodomain formation and refine imaging techniques. The experimental results both motivate and test the theoretical model. The experimental methods developed in this work and the measured data and theoretical models will be applicable to cell membranes and so could yield basic biophysical information about biological raft structure and function. PUBLIC HEALTH RELEVANCE Protein-lipid structures called "rafts" regulate signaling and structure formation on the cell surface, and are believed to be a central player in the replication of viruses and in several immune responses. The goal of this work is to refine existing imaging techniques and develop computer simulations that will provide a mechanistic understanding of how such rafts form in a model system.