Dynamics of Raft Formation and Growth

总结 对于细胞信号和其他生物过程至关重要的膜蛋白存在于筏中， 筏很难研究，因为它们具有亚微观的尺寸，动态结构， 不具有固定化学计量的组分。筏的组成-蛋白质和脂质- 为了了解哪些蛋白质在细胞级联中相互作用， 因为他们住在木筏上而彼此接近。一个新的实验程序 允许细胞筏在37 ℃的生理温度下分离，而不是以前的 必要的温度4 º C，使筏研究中的基本问题得到解决。的 在哺乳动物体温下存在于细胞膜内的许多结构域的组合物 (37这些细胞膜可能与保存在冰上的细胞膜（4 ℃）不同，因此许多 在低温下不能可靠地确定畴。生物学中筏的组成 温度现在可以确定，膜锚蛋白有利于筏可以， 与在4 ℃下获得的数据相比。蛋白质类型之间的关系（GPI锚定， 跨膜结构域，异戊二烯化）和筏中胆固醇的量进行分类。 比较不同类型的筏中的胆固醇水平将揭示导致 胆固醇在木筏之间移动。一种蛋白质被另一种蛋白质取代， 将评估对细胞过程产生的影响（如果有的话）。物理化学控制着 将在模型筏中研究胆固醇和鞘磷脂含量之间关系 系统这个模型系统也允许蛋白质如何贡献的实验性难题 以具体方式接近筏形。对于细胞研究，一种新的方法将是 采用的是利用这样一个事实，即破裂囊泡所需的压力直接取决于 其膜的脂质组成。将该方法与传统的分离方法相结合， 通过膜密度将允许收集大范围的域。这将为第一个 时间、蛋白质和脂筏含量分析无低温引起的变化， 洗涤剂和/或所有先前的筏隔离程序所需的碱性pH。隔离模型 含有肽的双层结构域和测量组合物将确定物理性质， 创造这些领域的机制。这将产生实验可检验的假设， 生物领域形成的机制。

Summary 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 37¿C, rather than the previously necessary temperature of 4¿C, enabling basic question in raft studies to be addressed. The compositions of many domains that exist within a cell membrane at mammalian body temperature (37¿C) are likely to be different from those of cell membranes that are kept on ice (4¿C), 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 4¿C. 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.