Regulation of Microdomain Structure in Living Cells

描述（由申请人提供）：细胞膜包含特定的脂质和蛋白质组成的局部区域，称为膜微域。最近，一类被称为脂筏的膜微结构域的性质由于其在膜运输，细胞信号传导和病原体从细胞进入和退出中的作用而引起了极大的兴趣。脂筏通常被定义为胆固醇和鞘脂富集域，被认为通过将一些蛋白质集中到筏中而将其他蛋白质分离到膜的非筏区来调节蛋白质功能。然而，脂筏的许多基本特性仍然未知，包括特定蛋白质靶向脂筏的机制。一个通常被引用的蛋白质与脂筏的关联模型是通过类比液体有序和液体无序脂相混合物中脂质探针的分配而得出的。我们假设这种分配模型不足以解释筏蛋白在细胞中的行为。相反，我们假设筏蛋白以胆固醇依赖的方式被靶向积极维持结构域，并且膜锚定模式是任何给定蛋白质筏关联机制的主要决定因素。为了验证这一假设，我们建议研究三种常见的筏状蛋白：霍乱毒素b亚基（一种糖脂结合蛋白）、血凝素（一种跨膜蛋白）和GFP-GPI（一种糖基磷脂酰肌醇锚定蛋白）的筏状结合机制。为此，我们将结合计算机模拟木筏形成过程，对活细胞中的木筏蛋白和非木筏蛋白进行生物物理测量。这些研究的具体目的是：(1)确定三种代表性筏体蛋白的亚微米分布和扩散迁移率是否与筏体形成的主动维持模型一致；(2)确定不同类型的筏蛋白是否在同一筏中共同定位或竞争居住；(3)生成脂筏组装机制的计算机模拟。这些研究的完成将为蛋白质靶向细胞筏的机制提供新的信息。这些信息对于最终设计针对干扰或增强脂质筏功能以改善人类健康的治疗方法至关重要。这些研究也将有助于我们了解膜结构如何调节细胞功能的长期目标。

DESCRIPTION (provided by applicant): Cell membranes contain localized regions of specialized lipid and protein composition known as membrane microdomains. Recently, the properties of a class of membrane microdomains termed lipid rafts have become of great interest due to their proposed role in membrane trafficking, cell signaling, and the entry and exit of pathogens from cells. Commonly defined as cholesterol and sphingolipid-enriched domains, lipid rafts are thought to regulate protein function by concentrating some proteins into rafts, while segregating others in non-raft regions of the membrane. However, many of the fundamental properties of lipid rafts remain unknown, including the mechanism(s) by which specific proteins are targeted to lipid rafts. A commonly cited model for the association of proteins with lipid rafts is drawn by analogy to the partitioning of lipid probes in mixtures of liquid-ordered and liquid-disordered lipid phases. We hypothesize that this partitioning model is insufficient to explain the behavior of raft proteins in cells. Instead, we postulate that raft proteins are targeted to actively maintained domains in a cholesterol-dependent manner, and that the mode of membrane anchorage is a major determinant of the mechanism of raft association for any given protein. To test this hypothesis, we propose to investigate the mechanisms that govern the raft association of three commonly studied raft proteins: cholera toxin B-subunit (a glycolipid-binding protein), hemagglutinin (a transmembrane protein), and GFP-GPI (a glycosylphosphatidylinositol-anchored protein). To do so, we will perform biophysical measurements of raft- and non-raft proteins in living cells in combination with computer simulations of raft formation. The specific aims of these studies are (1) to determine if the sub-micron distribution and diffusional mobility of three representative raft proteins are consistent with an actively maintained model for raft formation; (2) to determine if different types of raft proteins co-localize or compete for residence in the same rafts; and (3) to generate in silico simulations of lipid raft assembly mechanisms. Completion of these studies will provide new information regarding the mechanisms that underlie targeting of proteins to rafts in cells. Such information is critical to the eventual design of therapies targeted at interfering with or enhancing lipid raft function to improve human health. These studies will also contribute to our long- term goal of understanding how membrane structure regulates cellular functions.