Domains in Biologically Relevant Lipid Membranes

生物学相关脂质膜中的结构域

• 批准号: RGPIN-2019-07229
• 负责人: Thewalt, Jenifer
• 金额: $ 2.62万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=762595
• 关键词: Domains; Biologically; Relevant; Lipid; Membranes

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.

细胞膜的结构和动力学是维持细胞正常运作的关键，而脂质在决定细胞膜的组织结构中起着主导作用。自从1972年“流体马赛克”模型发表以来，人们越来越意识到膜结构的“马赛克”方面和“流体”方面一样重要。完整的膜蛋白，特别是那些与细胞骨架相连的蛋白和那些可以形成信号平台的蛋白，当然决定了嵌合体的各个方面。其他重要的马赛克特征的产生是因为膜中的脂类自发地相互作用，并可以聚集在从纳米级波动到微米级相分离的动态簇中。细胞膜上被称为“木筏”的短命小簇在活体研究中是具有挑战性的。与细胞膜--尤其是真核膜--形成鲜明对比的是，脂膜是易于处理的研究对象。了解已知组成的膜中脂类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竞争环境。细胞膜组织研究中一些最有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(LO)相。在LO阶段，胆固醇抑制了脂链的波动，同时保持了脂类的旋转和侧向流动性，导致了更紧密的堆积，因此更厚，但仍然是液晶的。我们绘制了模型膜中磷脂、甾醇和鞘磷脂之间的基本相互作用。氘核磁共振是研究膜中相共存的一种强有力的实验方法；不同的相可以得到清晰的光谱，并且可以毫不含糊地测量特定相中标记的脂类的数量。我们一直专注于测量二元脂质混合物的相图，但现在正在将我们的工作扩展到三元混合物(如POPC、DPPC或鞘磷脂、甾醇)以及三元混合物+荧光探针。推测进入细胞凋亡的细胞膜中含有神经鞘磷脂转化为神经酰胺时形成的凝胶域。我们将测试在质膜中发现的高胆固醇含量存在的情况下，鞘磷脂+神经酰胺中是否形成凝胶相。然后，我们将研究一个含有POPC、鞘磷脂和胆固醇的“外叶”质膜模型，然后在这个系统中添加神经酰胺。有趣的是，脂相行为对脂类纳米粒(LNPs)用于药物输送的有效性是重要的。与库利斯和蒂勒曼的研究小组一起，我们正在绘制用于制造LNPs的脂类的相倾向。我们计划将核磁共振和X射线散射结合起来，以深入了解这些脂类在阴离子脂类存在的情况下形成的非双层相，这些脂类是典型的内膜。