Domains in Biologically Relevant Lipid Membranes

细胞膜结构和动力学是维持细胞正常功能的关键，脂质在决定细胞膜的组织中起主导作用。自1972年“流体镶嵌”模型发表以来，人们越来越认识到膜结构的“镶嵌”方面与“流体”方面同样重要。整体膜蛋白，特别是那些连接到细胞骨架和那些可以形成信号平台的膜蛋白，当然决定了马赛克的各个方面。其他重要的镶嵌特性的出现是因为膜中的脂质自发地相互作用，可以聚集成动态簇，范围从纳米级的波动到微米级的相分离。细胞膜中被称为“筏”的短寿命小簇在体内研究是具有挑战性的。与细胞膜，特别是真核生物膜相比，脂质膜是一个容易处理的研究对象。了解已知成分的膜中脂质之间的物理和化学相互作用有助于定义生物学可以使用的比赛场地。细胞膜组织研究中一些最具影响力的概念起源于对这种“简单”系统的相行为的研究。一个非常有用的概念是富含胆固醇的液体有序相。在低相中，胆固醇抑制了脂链的波动，同时保持了脂质的旋转和横向流动，导致更紧密的包裹，因此更厚，但仍然是液晶膜。我们绘制出模型膜中磷脂、甾醇和鞘脂之间的基本相互作用。氘核磁共振（NMR）是研究膜中相共存的一种强有力的实验方法；不同的相导致清晰可区分的光谱，并且在特定相中标记的脂质的数量可以明确地测量。我们专注于测量二元脂质混合物的相图，但现在我们的工作扩展到三元混合物（如POPC， DPPC或鞘磷脂，甾醇）和三元混合物+荧光探针。进入凋亡的细胞膜被认为含有鞘磷脂转化为神经酰胺时形成的凝胶结构域。我们将测试鞘磷脂+神经酰胺是否在质膜中发现高胆固醇含量的情况下形成凝胶相。然后我们将研究含有POPC、鞘磷脂和胆固醇的“外叶”质膜模型，然后将神经酰胺添加到该系统中。有趣的是，脂质相行为对用于药物递送的脂质纳米颗粒（LNPs）的功效很重要。与Cullis和Tieleman研究小组一起，我们正在绘制用于制造LNPs的脂质的相倾向图。我们计划结合核磁共振和x射线散射来深入了解这些脂质在典型的内体膜阴离子脂质存在下形成的非双层相。

Cell membrane structure and dynamics are key to maintaining a properly functioning cell, and lipids have a dominant role in determining the organization of cell membranes. Since the "fluid mosaic" model was published in 1972, awareness has been growing that the "mosaic" aspect of membrane structure is as important as the "fluid" aspect. Integral membrane proteins, particularly those that link to the cytoskeleton and those that can form signalling platforms, certainly determine aspects of the mosaic. Other important mosaic characteristics arise because lipids in membranes spontaneously interact and can congregate in dynamic clusters ranging from nanoscale fluctuations to micron scale phase separation. Small short lived clusters known as "rafts" in cell membranes are challenging to study in vivo. In contrast to cell membranes - especially eukaryotic membranes - lipid membranes are tractable research subjects. Understanding the physical and chemical interactions among lipids in a membrane with known composition helps to define the playing field that biology can use. Some of the most influential concepts in the study of cell membrane organization originated with investigations into the phase behavior of such "simple" systems. One extremely useful concept is the cholesterol rich liquid ordered (lo) phase. In the lo phase cholesterol damps out lipid chain fluctuations while maintaining rotational and lateral lipid mobility, leading to a more tightly packed and therefore thicker, but still liquid crystalline, membrane. We map out the fundamental interactions between phospholipids, sterols and sphingolipids in model membranes. Deuterium nuclear magnetic resonance (NMR) is a powerful experimental approach to the study of phase coexistence in membranes; different phases result in clearly distinguishable spectra, and the quantity of labeled lipid in a particular phase can be measured unambiguously. We have concentrated on measuring the phase diagrams of binary lipid mixtures but are now extending our work to ternary mixtures (eg POPC, DPPC or sphingomyelin, sterol) and also ternary mixtures + fluorescent probe. Cell membranes entering apoptosis are postulated to contain gel domains formed as sphingomyelin is converted to ceramide. We will test whether gel phases form in sphingomyelin + ceramide in the presence of the high cholesterol content found in plasma membranes. We will then study an "outer leaflet" plasma membrane model containing POPC, sphingomyelin and cholesterol, then add ceramide to this system. Interestingly, lipid phase behavior is important to the efficacy of lipid nanoparticles (LNPs) used for drug delivery. With the Cullis and Tieleman research groups, we are mapping the phase propensities of lipids used to manufacture LNPs. We plan to combine NMR and X-ray scattering to gain insight into nonbilayer phases formed by these lipids in the presence of anionic lipids typical of endosomal membranes.