Dynamics of Raft Formation and Growth

描述(申请人提供)：对细胞信号和其他生物过程至关重要的膜蛋白存在于木筏中，但木筏一直很难研究，因为它们具有亚微观大小、动态结构和没有固定化学计量的成分。必须确定木筏的组成--蛋白质和脂类--以便了解在细胞级联中相互作用的哪些蛋白质由于它们在木筏上的停留而彼此接近。一项新的实验程序现在允许在370摄氏度的生理温度下分离出细胞筏，而不是以前所需的40摄氏度，这使得筏子研究中的基本问题得以解决。在哺乳动物体温(370摄氏度)下，细胞膜内存在的许多结构域的组成可能与冰冻(40℃)的细胞膜的组成不同，因此许多结构域在低温下无法可靠地确定。现在可以确定木筏在生物温度下的组成，可以将木筏中有利于蛋白质的膜锚定与40摄氏度下的数据进行比较。将对RAFT中蛋白质类型(GPI锚定、跨膜结构域、异丙基化)与胆固醇含量之间的关系进行分类。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。我们将评估在木筏内一种蛋白质被另一种蛋白质置换，以及由此对细胞过程产生的影响。控制胆固醇和鞘磷脂含量之间关系的物理化学将在模型RAFT系统中进行研究。该模型系统还允许以一种具体的方式来探讨蛋白质如何对RAFT形成起作用这一实验上难以捉摸的问题。对于细胞研究，将采用一种新的方法，该方法利用这样一个事实，即破裂小泡所需的压力直接取决于其膜的脂质成分。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结构域。这将首次提供蛋白质和脂筏含量的分析，而不会因所有先前的RAFT分离程序所需的低温、洗涤剂和/或碱性pH而引起改变。分离含有多肽的模型双层结构域并测量组合物将确定创建这些结构域的物理机制。这将产生生物域形成机制的可实验检验的假说。与公共健康相关：胆固醇是细胞膜中含量最丰富的分子，它在筏子和其他区域的分布对蛋白质在膜内的位置至关重要。高胆固醇水平与包括动脉粥样硬化和中风在内的疾病有关。蛋白质在筏和细胞膜的其他区域内的不适当分布会改变细胞的生长，这与癌症有关。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细胞在健康和疾病中的功能。

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.