DEFINING PRINCIPLES AND FUNCTIONS OF MEMBRANE ORGANIZATION USING ASYMMETRIC VESICLES

描述（由申请人提供）：了解膜脂质如何影响膜组织和功能仍然是膜生物学中最重要的挑战之一。由膜脂质组成的人工囊泡已被广泛用于研究膜，但通常缺乏脂质不对称性，即大多数生物膜的内外膜小叶（单层）中脂质组成的差异。我们的实验室开发了一种突破性的方法，可以制备各种具有高度可控的脂质不对称性的脂质囊泡。不对称囊泡将用于定义具有生物医学相关性的膜组织和功能的原理。使用显微镜和荧光光谱的脂质组合物和不对称性后，组织的膜脂质和蛋白质的结构域具有不同的分子组成的影响将被定义。1994年，我们与Deborah Brown博士（斯托尼布鲁克）一起提出了一种广泛使用的真核细胞结构域性质的工作模型：它们是富含鞘脂和甾醇的液体有序结构域，与富含不饱和脂质的无序结构域共存。由于鞘脂仅在外瓣叶中丰富，因此内瓣叶中的结构域形成可能涉及瓣叶之间的相互作用，即内外瓣叶物理性质之间的耦合。初步研究证实发生了这种瓣叶间偶联。一是 将测试在模拟质膜的不对称膜中，外小叶中的脂质结构域诱导内小叶脂质结构域的自发形成的假设。然后定义脂质结构对结构域形成和小叶间偶联的影响。脂酰基链长度、不饱和度、甾醇浓度和甾醇结构将变化。这应该定义什么样的细胞膜具有能够在外部和/或内部小叶中自发形成结构域的脂质。这些研究将具有生物医学意义。检验具有一个非常长的酰基链的脂质（允许从一个小叶渗透到另一个小叶（即交错））增强物理性质的耦合的假设将具有影响 肾上腺脑白质病，一种导致脂质大量过度积累的交叉酰基链的疾病。此外，这些研究将检验以下假设：膜组织在涉及胆固醇生物合成缺陷的疾病中受到影响，导致前体甾醇大量过度积累，例如Smith-Lemli-Opitz综合征和桥粒甾醇病。接下来的研究将检查膜蛋白序列如何控制结构域的形成以及膜蛋白与特定结构域的关联。最后，我们发现在上述研究中调节结构域形成的脂质，可以交换到细胞中，将被用作探测结构域形成和细胞中小叶间偶联的功能后果的工具。内吞作用和聚集的假说 参与信号转导的内部小叶脂质需要与外部小叶连接的小叶间偶联。 将测试小叶结构域形成。

DESCRIPTION (provided by applicant): Understanding how membrane lipids influence membrane organization and function remains one of the most important challenges in membrane biology. Artificial vesicles composed of membrane lipids have been widely used to study membranes, but generally lack lipid asymmetry, the difference in lipid composition in the inner and outer membrane leaflets (monolayers) characteristic of most biomembranes. Our lab developed a breakthrough method allowing preparation of a wide variety of lipid vesicles with highly controllable lipid asymmetry. Asymmetric vesicles will be used to define principles of membrane organization and function that are of biomedical relevance. Using microscopy and fluorescence spectroscopy the effect of lipid composition and asymmetry upon the organization of membrane lipids and proteins into domains with different molecular compositions will be defined. In 1994 we proposed, together with Dr. Deborah Brown (Stony Brook), the widely used working model for the nature of eukaryotic cell domains: that they are sphingolipid- and sterol-rich liquid ordered-like domains co-existing with disordered domains rich in unsaturated lipids. Since sphingolipids are only abundant in outer leaflets, domain formation in inner leaflets is likely to involve interactions between the leaflets, i.e. coupling between inner and outer leaflet physical properties. Preliminary studies confirm that this interleaflet coupling occurs. First, the hypothesis that in asymmetric membranes that mimic plasma membranes lipid domains in the outer leaflet induce spontaneous formation of inner leaflet lipid domains will be tested. Then the effect of lipid structure upon the domain formation and interleaflet coupling will be defined. Lipi acyl chain length, unsaturation, sterol concentration, and sterol structure will be varied. This should define what cell membranes have lipids with the capacity to spontaneously form domains in the outer and/or inner leaflets. These studies will have biomedical implications. Testing the hypothesis that lipids with one very long acyl chain, which allows penetration from one leaflet into another (i.e. interdigitation), enhance coupling of physical properties will have implications for adrenoleukoadenopathy, a disease which causes lipids to massively overaccumulate interdigitating acyl chains. In addition, the studies will test the hypothesis that membrane organization is impacted in diseases involving cholesterol biosynthesis defects resulting in massive overaccumulation of precursor sterols, e.g. Smith-Lemli-Opitz syndrome and desmosterolosis. The next studies will examine how membrane protein sequence controls domain formation and the association of membrane proteins with specific domains. Finally, lipids which we find modulate domain formation in the studies above and which can be exchanged into cells will be used as tools to probe the functional consequences of domain formation and interleaflet coupling in cells. The hypothesis that endocytosis and the clustering of inner leaflet lipids involved in signal transduction requires interleaflet coupling linked to outer leaflet domain formation will be tested.