DEFINING PRINCIPLES AND FUNCTIONS OF MEMBRANE ORGANIZATION USING ASYMMETRIC VESICLES

描述(申请人提供)：了解膜脂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。由膜脂组成的人工囊泡已被广泛应用于膜的研究，但普遍缺乏脂质的不对称性，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，可以制备各种高度可控的脂质不对称的脂泡。不对称囊泡将被用来定义与生物医学相关的膜组织和功能的原理。利用显微镜和荧光光谱学，可以确定脂质组成和不对称性对膜脂和蛋白质组织成具有不同分子组成的区域的影响。1994年，我们和Deborah Brown(Stony Brook)博士一起提出了被广泛使用的真核细胞结构域的工作模型：它们是富含鞘脂和类固醇的液体有序结构域，与富含不饱和脂的无序结构域共存。由于鞘磷脂只在外层小叶中含量丰富，因此内层小叶中的结构域形成可能涉及到小叶之间的相互作用，即内部和外部小叶的物理性质之间的耦合。初步研究证实，这种小叶间的结合是存在的。首先， 假设在模拟质膜的不对称膜中，外叶中的脂域诱导内叶脂域的自发形成将得到检验。然后，将定义脂类结构对结构域形成和小叶间耦合的影响。脂肪酰链的长度、不饱和度、甾醇浓度和甾醇结构都会有所不同。这应该定义什么细胞膜具有能够自发地在外部和/或内部小叶中形成结构域的脂类。这些研究将具有生物医学意义。验证一个假设，即具有一个很长的酰基链的脂类，允许从一个小叶渗透到另一个小叶(即交错)，增强物理性质的偶联将产生影响 对于肾上腺脑白质腺病，这是一种导致脂质大量过度积聚的疾病。此外，这些研究还将验证一种假设，即膜组织在涉及胆固醇生物合成缺陷的疾病中受到影响，这些疾病导致前体类固醇的大量过度积聚，例如Smith-Lemli-Opitz综合征和结缔组织退行性变。下一步的研究将研究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，我们在上述研究中发现的调节结构域形成并可以交换到细胞中的脂类将被用作工具来探索结构域形成和细胞内小叶间耦合的功能后果。内吞作用和细胞聚集性假说 参与信号转导的内叶脂类需要与外叶相连的小叶间偶联 将测试小叶域的形成。

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.