Dynamics of Raft Formation and Growth

描述（由申请人提供）：筏是膜的专门区域，由富含特定蛋白质的胆固醇和鞘脂的相分离结构域组成。大量的细胞过程-如信号转导和细胞内运输-被认为是由筏行为控制。木筏的广泛重要性也将它们与许多疾病联系在一起，有些病毒甚至出现在木筏上融合和/或发芽。但是，筏的形成、生长和组成的具体结构和动力学还不清楚。平面脂质双层模型系统具有许多优势，发现的物理化学原理，管理这些方面的筏;脂相分离，蛋白质的分区成胆固醇/鞘脂域，并控制这些域的形成蛋白质都可以在双层膜进行调查。通过包括胆固醇，鞘磷脂和荧光探针的双层，动力学方面的相分离的胆固醇/鞘磷脂域将研究荧光显微镜的选择性和灵敏度不可能使用细胞。将构建荧光和非荧光（猝灭剂）探针以分配到选定的结构域中，并以高浓度置于筏形成双层中。这些技术将允许检测小的脂质微区筏，量化它们的生长和溶解，并表征它们的稳定性。将研究单个单层小叶内的筏，并研究与相对单层中的液体有序域的任何耦合。将确定相对单层中脂质的酰基链之间的接触控制偶联的程度。在被认为是分割成筏的蛋白质中，GPI锚定蛋白是突出的; GPI-GFP将被用作模型蛋白，以评估在不同条件下蛋白质和筏之间的关系。胆固醇结合蛋白可以作为一个中心的筏成核的假设将被测试。实验目标的结果将被用来适应理论发展阶段的创建和其他系统的增长，使一个完整的理解筏可以基于基本的物理原理。

DESCRIPTION (provided by applicant): Rafts are specialized regions of membranes that consist of phase-separated domains of cholesterol and sphingolipids enriched in particular proteins. A large number of cellular processes - such as signal transduction and intracellular trafficking - are thought to be controlled by raft behavior. The wide-ranging importance of rafts has also linked them to many diseases, and some viruses even appear to fuse and/or bud at raft sites. But specific structures and dynamics of raft formation, growth, and composition are as yet unknown. The planar lipid bilayer model system has many advantages for discovering the physical chemical principles that govern these aspects of rafts; lipid phase separation, partitioning of proteins into cholesterol/sphingolipid domains, and control of formation of these domains by proteins can all be investigated in bilayer membranes. By including cholesterol, sphingomyelin, and fluorescent probes in bilayers, kinetic aspects of phase-separated cholesterol/sphingomyelin domains will be studied by fluorescence microscopy with selectivity and sensitivity not possible using cells. Fluorescent and non-fluorescent (quencher) probes will be constructed to partition into selected domains and placed in raft forming bilayers at high concentrations. These techniques will allow small lipid-microdomain rafts to be detected, their growth and dissolution to be quantified, and their stability to be characterized. Rafts within a single monolayer leaflet will be studied and any coupling to a liquid-ordered domain in the opposite monolayer will be investigated. The extent to which contact between acyl chains of lipids in opposite monolayers controls coupling will be determined. Among the proteins thought to partition into rafts, GPI anchored proteins are prominent; GPI-GFP will be used as a model protein to assess relationships between proteins and rafts under varying conditions. The hypothesis that cholesterol-binding protein can serve as a center for nucleation of rafts will be tested. The results of experimental aims will be used to adapt theory developed for phase creation and growth in other systems, so that an integrated understanding of rafts can be based on fundamental physical principles.