Regulation of Microdomain Structure in Living Cells

描述（由申请人提供）：细胞膜含有特定脂质和蛋白质组成的局部区域，称为膜微区。最近，一类被称为脂筏的膜微区的性质已成为极大的兴趣，由于他们提出的作用，膜运输，细胞信号，和病原体的进出细胞。脂筏通常被定义为胆固醇和鞘脂富集结构域，被认为通过将一些蛋白质浓缩成筏，同时将其他蛋白质分离在膜的非筏区域来调节蛋白质功能。然而，脂筏的许多基本性质仍然未知，包括特定蛋白质靶向脂筏的机制。一个常见的引用模型与脂筏蛋白质的协会绘制类比的液体有序和液体无序的脂质相的混合物中的脂质探针的分区。我们假设这种分配模型不足以解释细胞中筏蛋白的行为。相反，我们假设筏蛋白是有针对性的，以胆固醇依赖的方式积极维护的结构域，膜锚定的模式是一个主要的决定因素的机制筏协会为任何给定的蛋白质。为了验证这一假设，我们建议调查的机制，管理筏协会的三个常用的研究筏蛋白：霍乱毒素B亚基（糖脂结合蛋白），血凝素（跨膜蛋白），和GFP-GPI（糖基磷脂酰肌醇锚定蛋白）。要做到这一点，我们将进行生物物理测量筏和非筏蛋白在活细胞结合筏形成的计算机模拟。这些研究的具体目的是（1）确定三种代表性筏蛋白的亚微米分布和扩散流动性是否与筏形成的主动维持模型一致;（2）确定不同类型的筏蛋白是否共定位或竞争在相同筏中的驻留;以及（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靶向干扰或增强脂筏功能以改善人类健康的疗法至关重要。这些研究也将有助于我们理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.