Influence of Cholesterol on Phase Behavior and Nonrandom Mixing of Membrane Components

本项目的目的是研究模拟生物膜的脂质双分子层非随机混合的热力学基础。该项目有四个主要的概念要素：(i)三组分双层脂质混合物作为哺乳动物质膜外层和内部小叶的模型。这些三组分混合物足够复杂，可以模拟真实的生物膜，但在化学上定义良好；（ii）几个独立的，互补的方法被用来彻底绘制和表征这些模型膜的组成相行为；真正的生物膜是不对称的：将研究同样不对称的模型双层，以便弥合化学简单模型和真正生物膜之间的差距；（iv）活细胞的质膜，包括天然膜筏的行为，将通过模型系统中开发的方法进行调查，并根据模型系统获得的相图进行分析。实验上，本工作涉及大量的荧光共振能量转移和苝准分子/单体测量数据集，以发现脂质相边界和荧光探针的分配行为。共聚焦荧光显微镜将用于可视化和识别共存的相。为了制备不对称脂质双分子层，将开发新的程序。胆固醇是哺乳动物质膜中含量最多的一种脂质。尽管如此，胆固醇与邻近膜分子相互作用的方式仍然不清楚。众所周知，胆固醇会与其他膜脂非随机混合，但在大多数情况下，对胆固醇的侧分布以及胆固醇影响其他膜组分侧分布的方式缺乏清晰的认识。目前生物膜中非随机混合的图景，即富含胆固醇的斑块漂浮在低胆固醇双分子层中的“筏模型”，可能会被这个研究项目所改善。此外，为了促进科学教育和培训，将对一批本科生进行脂质物理化学和荧光光谱的讲座，然后对他们进行脂质分析化学、脂质有机合成、光谱和显微镜技术的系统培训，然后将其用于指定的自主研究。

The goal of this project is to study the thermodynamic basis for nonrandom mixing in lipid bilayers that model biomembranes. The project has four main conceptual elements: (i) 3-component bilayer lipid mixtures serve as models for the outer and the inner leaflets of mammalian plasma membranes. These 3-component mixtures are sufficiently complex to model real biomembranes, yet are chemically well-defined; (ii) Several independent, complementary methods are used to thoroughly map and characterize the compositional phase behavior of these model membranes; (iii) Real biomembranes are asymmetric: model bilayers that are also asymmetric will be studied in order to bridge the gap between the chemically-simple models and real biomembranes; (iv) Behavior of the plasma membranes of living cells, including the nature membrane rafts, will be investigated by the methods developed in the model systems and analyzed in terms of the phase diagrams obtained for the model systems. Experimentally, this work involves large data sets of fluorescence resonance energy transfer and perylene excimer/monomer measurements to find the lipid phase boundaries and the partition behavior of fluorescent probes. Confocal fluorescence microscopy will be used to visualize and identify coexisting phases. New procedures will be developed in order to prepare asymmetric lipid bilayers.Cholesterol is the single most abundant lipid species in mammalian plasma membranes. Nonetheless, the way cholesterol interacts with neighboring membrane molecules has remained obscure. Cholesterol is known to mix nonrandomly with other membrane lipids, and yet in most cases a clear understanding is missing for the lateral distribution of cholesterol, and the way cholesterol influences the lateral distribution of other membrane components. A current picture of nonrandom mixing in biomembranes, the "raft model" of cholesterol-rich patches floating within a cholesterol-poor bilayer, is likely to be improved by this research project. In addition, in order to promote scientific education and training, a group of undergraduate students will receive lectures in lipid physical chemistry and fluorescence spectroscopy, followed by systematic training in lipid analytical chemistry, lipid organic synthesis, and spectroscopic and microscope techniques, which they will then use in their assigned independent research.