Dynamics of Raft Formation and Growth

描述（由申请人提供）：筏是膜的特殊区域，由富含特定蛋白质的相分离的胆固醇和鞘脂结构域组成。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的。木筏的广泛重要性也将它们与许多疾病联系起来，有些病毒甚至似乎在木筏上融合和/或发芽。但是筏子形成、生长和组成的具体结构和动力学尚不清楚。平面脂质双层模型系统在发现筏的这些方面的物理化学原理方面有许多优点；脂相分离，蛋白质进入胆固醇/鞘脂结构域的分配，以及蛋白质对这些结构域形成的控制，都可以在双层膜中进行研究。通过在双层中加入胆固醇、鞘磷脂和荧光探针，相分离胆固醇/鞘磷脂结构域的动力学方面将通过荧光显微镜进行研究，其选择性和灵敏度是使用细胞无法实现的。荧光和非荧光（淬灭剂）探针将被构建到选定的区域，并在高浓度下放置在筏形形成的双层结构层中。这些技术将允许小的脂质微结构筏被检测，它们的生长和溶解被量化，它们的稳定性被表征。将研究单层传单内的筏，并研究与相反单层中液体有序域的任何耦合。将确定相对单层中脂质酰基链之间的接触控制耦合的程度。在被认为分裂成筏的蛋白质中，GPI锚定蛋白是突出的；GPI-GFP将被用作模型蛋白来评估不同条件下蛋白质和筏之间的关系。关于胆固醇结合蛋白可以作为筏形核中心的假设将被验证。实验目标的结果将用于适应其他系统中相产生和生长的理论，以便基于基本物理原理对筏的综合理解。

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.