Dynamics of Raft Formation and Growth

描述(由申请人提供)：木筏是膜的特殊区域，由富含特定蛋白质的胆固醇和鞘脂组成的相分离区域。大量的细胞过程--如信号转导和细胞内运输--被认为是由木筏行为控制的。木筏的广泛重要性也将它们与许多疾病联系在一起，一些病毒甚至似乎在木筏部位融合和/或发芽。但木筏的形成、生长和组成的具体结构和动力学尚不清楚。平面脂质双层模型体系具有许多优点，可以用来发现支配筏子这些方面的物理化学原理；在双层膜中，可以研究脂相分离、蛋白质到胆固醇/鞘磷脂结构域的分配以及蛋白质对这些结构域形成的控制。通过在双层中加入胆固醇、鞘磷脂和荧光探针，相分离的胆固醇/鞘磷脂结构域的动力学方面将通过荧光显微镜进行研究，而选择性和灵敏度是使用细胞无法实现的。将构建荧光和非荧光(猝灭剂)探针，将其划分为选定的区域，并放置在形成高浓度双层的筏子中。这些技术将能够检测到小的微脂微域木筏，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中的浮筏，并将研究与相反单层中的液体有序结构域的任何耦合。将确定相对单层中脂类的酰基链之间的接触控制偶联的程度。在被认为可以划分为RAFT的蛋白质中，GPI锚定蛋白是突出的；GPI-GFP将被用作模型蛋白，以评估不同条件下蛋白质与RAFT之间的关系。胆固醇结合蛋白可以作为木筏成核中心的假设将得到检验。实验目标的结果将被用来改编为在其他系统中产生和生长相而开发的理论，以便能够基于基本的物理原理对木筏进行完整的理解。

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.