Formation, Elasticity, and Permeability of Raft Bilayers

描述(由申请人提供)：膜微域(“筏”)在许多重要的生物功能和病理过程中起着关键作用。这项建议的目的是调查一些基本的尚未解决的问题，关于筏的性质和筏的形成机制。我们的中心假设是，双层结构和弹性性质，由碳氢链组成和胆固醇含量控制，在RAFT膜和非RAFT膜之间的脂类和蛋白质的分类以及调节RAFT通道的功能方面都是至关重要的。为了研究相关脂质和脂质-蛋白质系统的物理化学、结构和通透性，本申请建议使用一系列生物物理和生化技术，包括共聚焦显微镜、域间分配系数测量、通过吸管吸气直接测量浮筏机械性能的新应用、冷冻断裂电子显微镜、X射线衍射和电压钳实验。这种方法应该产生关于特定RAFT分子之间相互作用的直接和详细的信息。 具体目标包括确定信号转导中涉及的RAFT和非RAFT磷脂双层、不同跨膜长度的多肽和膜通道蛋白之间的分选机制。为了测试在双层微区之间分选的分子机制模型，将测量脂类和多肽在双层微区中的分布，并将确定包含：(1)RAFT和非RAFT区域，(2)仅RAFT双层，和(3)仅非RAFT双层的囊泡的双层结构和弹性。此外，作为对筏子的直接功能测试，这三个双层系统的通透性和结构将在存在和不存在两个与筏子相关的膜通道，缝隙连接的连接子和水通道Aquaporin-0的情况下确定。这些数据将提供关于脂-脂、脂-蛋白质和蛋白质-蛋白质相互作用在这些通道的分类、聚集和通透性中的作用的见解。

DESCRIPTION (provided by applicant): Membrane microdomains ("rafts") play critical roles in a number of important biological functions and pathological processes. The goal of this proposal is to investigate some of the fundamental open questions concerning raft properties and mechanisms of raft formation. Our central hypothesis is that bilayer structural and elastic properties, as controlled by hydrocarbon chain composition and cholesterol content, are critical in the sorting of both lipids and proteins between raft and non-raft membranes, as well as in regulating the function of raft channels. To study the physical-chemical, structural, and permeability properties of relevant lipid and lipid-protein systems, this application proposes to use a battery of biophysical and biochemical techniques, including confocal microscopy, interdomain partition coefficient measurements, direct measurements of raft mechanical properties by a novel application of pipette aspiration, freeze-fracture electron microscopy, X-ray diffraction, and voltage clamp experiments. This approach should yield direct and detailed information on the roles of interactions between specific raft molecules. Specific aims include determining the mechanisms involved in the sorting between raft and non-raft bilayers of phospholipids involved in signal transduction, peptides of different transbilayer lengths, and membrane channel proteins. To test models of the molecular mechanisms involved in sorting between bilayer microdomains, the distribution of lipids and peptides in bilayer microdomains will be measured, and bilayer structure and elasticity will be determined for vesicles containing: (1) both raft and non-raft domains, (2) only raft bilayers, and (3) only non-raft bilayers. In addition, as a direct functional test of rafts, the permeability and structure of these three bilayer systems will determined in the presence and absence of two raft-associated membrane channels, the connexon of gap junctions and the water channel aquaporin-0. These data will provide insights concerning the roles of lipid-lipid, lipid-protein, and protein-protein interactions in the sorting, aggregation, and permeability of these channels.