Dynamics of Raft Formation and Growth

描述（由申请人提供）：对细胞信号传导和其他生物过程至关重要的膜蛋白存在于筏中，但筏很难研究，因为它们具有亚微观尺寸，动态结构和没有固定化学计量的成分。筏的组成——蛋白质和脂质——必须确定，以便了解哪些蛋白质在细胞级联中相互作用，因为它们在筏中居住而彼此接近。现在，一种新的实验程序允许在370℃的生理温度下分离细胞筏，而不是以前必须的40℃，这使得筏研究中的基本问题得以解决。哺乳动物体温（370℃）下细胞膜中存在的许多结构域的组成可能与保存在冰上（40℃）的细胞膜的组成不同，因此许多结构域无法在低温下可靠地确定。现在可以确定生物温度下筏的组成，并且可以将筏中偏爱的蛋白质的膜锚点与40℃时获得的数据进行比较。蛋白质类型（gpi锚定、跨膜结构域、戊酰化）与筏中胆固醇含量之间的关系将被分类。比较不同类型木筏中的胆固醇水平将揭示导致胆固醇在木筏之间移动的机制。将评估筏内一种蛋白质被另一种蛋白质所取代，以及由此产生的对细胞过程的影响（如果有的话）。控制胆固醇和鞘磷脂含量之间关系的物理化学将在模型筏系统中进行研究。该模型系统还允许以具体的方式接近蛋白质如何促进筏形成的实验难以捉摸的问题。对于细胞研究，将采用一种新方法，利用破裂囊泡所需的压力直接取决于其膜的脂质组成这一事实。将这种方法与传统的膜密度分离方法相结合，将允许收集大范围的域。这将首次提供蛋白质和脂质筏含量的分析，而不受低温、洗涤剂和/或碱性pH值的影响，而这些都是之前所有筏分离程序所要求的。分离含有肽的模型双层结构域和测量组成将确定创建这些结构域的物理机制。这将产生可实验验证的生物结构域形成机制的假设。公共卫生相关性：胆固醇是细胞膜中最丰富的分子，其在筏和其他结构域内的分布对膜内蛋白质的位置至关重要。高胆固醇水平与动脉粥样硬化和中风等疾病有关。筏和细胞膜的其他区域内蛋白质的不适当分布改变了细胞生长，与癌症有关。因此，确定膜筏内蛋白质和脂质（包括胆固醇）之间的关系直接关系到健康和疾病中的细胞功能。

DESCRIPTION (provided by applicant): Membrane proteins that are critical for cell signaling and other biological processes reside in rafts, but rafts have been difficult to study because they have submicroscopic sizes, dynamic structures, and components that do not have fixed stoichiometries. The compositions of rafts - proteins and lipids - must be determined in order to understand which proteins interacting in cellular cascades come into proximity with each other as a result of their residence in a raft. A new experimental procedure now allows cellular rafts to be isolated at the physiological temperature of 370C, rather than the previously necessary temperature of 40C, enabling basic question in raft studies to be addressed. The compositions of many domains that exist within a cell membrane at mammalian body temperature (370C) are likely to be different from those of cell membranes that are kept on ice (40C), so many domains cannot be reliably determined at low temperature. The composition of rafts at biological temperature can now be determined, and membrane anchors of proteins favored in rafts can be compared to data derived at 40C. The relationship between types of proteins (GPI-anchored, transmembrane domain, prenylated) and amounts of cholesterol in a raft will be classified. Comparison of cholesterol levels in different types of rafts will uncover mechanisms that cause cholesterol to move between rafts. The displacement of one protein by another inside a raft, and the resulting effect on cellular processes, if any, will be assessed. The physical chemistry that controls the relationship between cholesterol and sphingomyelin content will be studied in a model raft system. The model system also allows the experimentally elusive question of how proteins contribute to raft formation to be approached in a concrete manner. For cellular studies, a new method will be employed that exploits the fact that the pressure needed to rupture a vesicle depends directly on the lipid composition of its membrane. Combining this method with the traditional approach of separation by membrane density will allow collection of a large range of domains. This will provide, for the first time, analysis of protein and lipid raft content without alteration caused by low temperature, detergents, and/or alkaline pH required by all previous raft isolation procedures. Isolating model bilayer domains containing a peptide and measuring compositions will determine the physical mechanisms that create these domains. This will yield experimentally testable hypotheses of mechanisms of biological domain formation. PUBLIC HEALTH RELEVANCE: Cholesterol is the most abundant molecule in cell plasma membranes, and its distribution within rafts and other domains is critical to the location of proteins within membranes. High cholesterol levels are implicated in diseases, including atherosclerosis and strokes. Improper distribution of proteins within rafts and other domains of cell membranes alters cell growth, related to cancers. Thus, determining the relationship between proteins and lipids, including cholesterol, within membrane rafts bears directly on cell function in health and disease.