Physical and Chemical Properties of Lipid Rafts

描述（由申请人提供）：脂筏是生物膜中一个重要的新概念，但仍然存在争议，因为在这个节点上，细胞生物学家对筏的定义——抗洗涤剂膜和胆固醇提取诱导的筏功能的废除——没有明确的细胞相关性，通过显微镜方法确定。在Specific Aim I中，我们将比较应用于活细胞的复杂且高度合适的光显微镜方法的结果与细胞膜中脂质微域的三种极端模型的预测。方法包括荧光相关光谱法、单粒子跟踪法、单染料示踪法和单畴成像法。模型和实验之间的比较结果应该导致我们对细胞表面脂质微域的看法有相当大的改进。具体目标II旨在探索可以提供功能的木筏的物理和化学特性。跨层通信以及为什么某些分子成分驻留在筏中是当今该领域未解决的关键问题。使用不对称平面支撑的双层，我们将确定外部单层上的液体有序结构域如何与内部单层通信，以促进筏介导的信号转导过程。还将定义决定各种筏状偏好和交联膜组分划分为液体有序结构域的因素，以及筏状驻留组分之间对这些结构域中位置的竞争。由于筏状洗涤剂阻力的概念对该概念至关重要，我们将使用荧光显微镜成像各种洗涤剂对平面支撑膜筏状结构域的作用。

DESCRIPTION (provided by applicant): Lipid rafts are an important new concept in biomembranes but one that remains controversial because at this juncture the cell biologist's definitions of rafts--detergent resistant membranes and cholesterol extraction-induced abrogation of raft function--have no unambiguous cellular correlate as determined by microscopic methods. In Specific Aim I, we will compare the results of sophisticated and highly appropriate light microscopic methods applied to living cells to the predictions of three extreme models for lipid microdomains in cell membranes. The methods include fluorescence correlation spectroscopy, single particle tracking, single dye tracing, and single domain imaging. The results of the comparison between models and experiments should lead to a considerable refinement in our views of lipid microdomains on cell surfaces. Specific Aim II is directed towards exploring the physical and chemical properties of rafts that could provide functionality. Transbilayer communication and why certain molecular components reside in rafts are key unanswered questions in the field today. Using asymmetric planar supported bilayers, we will determine how liquid-ordered domains on the outer monolayer communicate with the inner monolayer to facilitate raft-mediated signal transduction processes. The factors that determine partitioning of various raft-preferring and cross-linked membrane components into liquid-ordered domains and the competition between raft-resident components for sites in these domains will also be defined. Because the concept of raft detergent resistance is so crucial to the concept, we will image the action of various detergents on raft domains existing in planar supported membranes using fluorescence microscopy.