Dynamics of Raft Formation and Growth

筏形成和生长的动力学

• 批准号: 6838820
• 负责人: FREDRIC S COHEN
• 金额: $ 26.12万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6838820
• 关键词: bacterial toxins; binding proteins; cholesterol; cytolysins; fluorescence microscopy; fluorescent dye /probe; glycosylphosphatidylinositols; green fluorescent proteins; intermolecular interaction; lipid bilayer membrane; membrane biogenesis; membrane model; membrane proteins; method development; molecular dynamics; phospholipids; sphingolipids; sphingomyelins

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.

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胆固醇和鞘脂的相分离结构域组成。许多细胞过程——例如信号转导和细胞内运输——被认为是由筏行为控制的。筏的广泛重要性也将它们与许多疾病联系起来，一些病毒甚至似乎在筏部位融合和/或萌芽。但筏形成、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对于发现控制筏这些方面的物理化学原理具有许多优势；脂相分离、蛋白质划分为胆固醇/鞘脂结构域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。通过在双层中包含胆固醇、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，其选择性和灵敏度是使用细胞不可能实现的。荧光和非荧光（淬灭剂）探针将被构建为划分为选定的域并以高浓度放置在形成双层的筏中。这些技术将允许检测小的脂质微域筏，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，并将研究与相对单层中的液体有序域的任何耦合。将确定相对单层中的脂质酰基链之间的接触控制偶联的程度。在被认为划分成筏的蛋白质中，GPI 锚定蛋白是最突出的。 GPI-GFP 将用作模型蛋白来评估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筏成核中心的假设将得到检验。实验目标的结果将用于适应为其他系统中的相创建和生长而开发的理论，以便可以基于基本物理原理对筏的综合理解。