TRANSFORMATIVE LIPID EXCHANGE APPROACHES TO STUDY MEMBRANE ORGANIZATION

该项目旨在借助最近实现的一项实验室来了解生物膜的结构和功能 环糊精催化控制膜磷脂和鞘磷脂组成的突破 脂肪交换。这使得可以制备在两者方面都接近自然膜的脂泡 脂肪组成和，第一次，脂质不对称，在内部和 许多天然膜所特有的外层脂层。该方法也正在扩展到控制 活细胞中的脂类成分。这些方法正被应用于解决长期存在的膜问题 区域形成。我们和合作者黛博拉·布朗博士在1994年提出了仍然存在的工作模式 在野外：由于神经鞘脂脂-胆固醇富集液(LO)的有序分离而形成细胞膜域 结构域来自不饱和富脂液体无序结构域。尽管这些域很容易在 人工脂质囊泡，以及仔细的研究证明它们存在于活细胞中，领域仍然存在争议 而且特征不佳。该项目将利用脂质交换来克服研究进展的障碍。 膜结构域。首先，研究使用了更多模仿细胞膜的不对称脂泡 比过去使用的对称泡更紧密地定义了管理区域属性的规则，并且 由脂类和蛋白质形成。这包括检验长期存在的假设，即脂质诱导的信号 跨膜转导可由体内和膜内脂类的物理性质之间的耦合而产生 膜的外层脂层。从这些研究中获得的知识将揭示如何操纵血脂和 蛋白质在细胞中控制结构域的形成和蛋白质与结构域的结合，从而如何探索 它们的功能。其次，将用更易处理的生命系统和方法来研究领域。我们发现 莱姆病的病原体伯氏疏螺旋体含胆固醇细菌具有结构域 尺寸足以通过电子显微镜可视化和使用FRET进行简单的荧光域检测， 此外，还可以改变甾醇的化学结构，以允许对结构域形成进行可控的调制。这 使得在体外和体内明确地识别细菌的LO结构域成为可能。研究范围将扩大到 其他可能含有LO结构域的致病菌：幽门螺杆菌、溃疡的原因和一些 癌症和金黄色葡萄球菌，耐药菌株(耐甲氧西林金黄色葡萄球菌)是主要的公共卫生威胁。研究将会 定义细菌域的特性、形成原理和潜在的生物医学 这意味着什么。然后研究将扩展到哺乳动物细胞，利用我们发现的一种 环糊精，可与外源磷脂和外源磷脂完全交换质膜外叶脂肪 神经鞘脂脂，不干扰细胞类固醇。利用这一点，改变脂质是如何调节结构域形成的， 属性，并将定义函数。该方法也将用于质膜脂质的研究。 不对称性及其作用。该方法的进一步发展将对生物膜研究产生广泛影响。

This project aims to understand biomembrane structure and function with the aid of a recent lab-achieved breakthrough in control of membrane phospholipid and sphingolipid composition via cyclodextrin-catalyzed lipid exchange. This permits preparation of lipid vesicles mimicking natural membranes closely in terms of both lipid composition and, for the first time, lipid asymmetry, the difference in the lipid composition in the inner and outer lipid layers characteristic of many natural membranes. The method is also being extended to control of lipid composition in living cells. These methods are being applied to solve long-standing issues of membrane domain formation. We and collaborator Dr. Deborah Brown proposed in 1994 what remains the working model in the field: cell membrane domains form due to segregation of sphingolipid-cholesterol rich liquid ordered (Lo) domains from unsaturated lipid rich liquid disordered domains. Although such domains are readily observed in artificial lipid vesicles, and careful studies document their presence in living cells, domains remain controversial and poorly characterized. This project will use lipid exchange to overcome roadblocks to progress in studies of membrane domains. First, studies using asymmetric lipid vesicles that mimic cell membranes much more closely than the symmetric vesicles employed in the past will define the rules governing domain properties and formation by lipids and proteins. This includes testing the long-standing hypothesis that lipid-induced signal transduction across membranes can result from coupling between physical properties of lipids in the inner and outer lipid layers of a membrane. Knowledge gained from these studies will reveal how to manipulate lipids and proteins in cells to control domain formation and protein association with domains, and thus how to explore their function. Second, domains will be studied with more tractable living systems and methods. We found that the cholesterol-containing bacterium Borrelia burgdorferi, the causative agent of Lyme disease, has domain size sufficient for visualization by electron microscopy and facile fluorescence domain detection using FRET, plus accessibility to altering sterol chemical structure to allow controlled modulation of domain formation. This made it possible to unequivocally identify bacterial Lo domains in vitro and in vivo. Studies will be extended to other pathogenic bacteria likely to contain Lo domains: Helicobacter pylori, the cause of ulcers and some cancers, and S. aureus, drug-resistant strains of which (MRSA) are a major public health threat. Studies will define the properties of bacterial domains, the principles behind their formation, and potential biomedical implications. Studies will then be extended to mammalian cells taking advantage of our discovery of a cyclodextrin that can fully exchange plasma membranes outer leaflet lipids with exogenous phospholipid and sphingolipid without disturbing cell sterols. Using this, how altering lipids modulate domain formation, properties, and function will be defined. This method will also be used to investigate plasma membrane lipid asymmetry and its function. Further development of the method will broadly impact biomembrane studies.