DEFINING PRINCIPLES AND FUNCTIONS OF MEMBRANE ORGANIZATION USING ASYMMETRIC VESICLES

使用不对称囊泡定义膜组织的原理和功能

• 批准号: 8990997
• 负责人: Erwin London
• 金额: $ 30.74万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8990997
• 关键词: Adrenoleukodystrophy; Affect; Affinity; Amino Acid Sequence; Amino Acids; Bacteria; Behavior; Cell membrane; Cell physiology; Cells; Characteristics; Cholesterol; Collaborations; Coupling; Cyclodextrins; Defect; Dependence; Disease; Endocytosis; Eukaryotic Cell; Event; Fatty Alcohols; Fluorescence Resonance Energy Transfer; Fluorescence Spectroscopy; Health; Integral Membrane Protein; Knowledge; Laboratory Finding; Length; Life; Link; Lipids; Liquid substance; Mammalian Cell; Membrane; Membrane Biology; Membrane Lipids; Membrane Proteins; Membrane Structure and Function; Methods; Microscopy; Modeling; Molecular; Names; Nature; Pathway interactions; Penetration; Peptide Sequence Determination; Peptides; Phosphatidylinositol 4,5-Diphosphate; Play; Preparation; Property; Proteins; Role; Signal Pathway; Signal Transduction; Smith-Lemli-Opitz Syndrome; Sphingolipids; Sterols; Structure; Testing; To specify; Unsaturated Fats; Very Long Chain Fatty Acid; Vesicle; Virus; Work; artificial vesicle; base; cholesterol biosynthesis; design; insight; lipid biosynthesis; lipid metabolism; lipid structure; membrane model; monolayer; novel; physical property; tool; uptake

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.

描述（由申请人提供）：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膜小叶和外膜小叶（单层）中脂质成分的差异。我们的实验室开发了一种突破性方法，可以制备多种具有高度可控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，将定义脂质组成和不对称性对膜脂质和蛋白质组织成具有不同分子组成的域的影响。 1994 年，我们与 Deborah Brown 博士（Stony Brook）一起提出了广泛使用的真核细胞结构域性质工作模型：它们是富含鞘脂和甾醇的液体有序结构域，与富含无序结构域共存。存在于不饱和脂质中。由于鞘脂仅在外部小叶中丰富，因此内部小叶中的域形成可能涉及小叶之间的相互作用，即内部和外部小叶物理性质之间的耦合。初步研究证实存在这种小叶间耦合。首先， 将测试在模拟质膜的不对称膜中，外叶中的脂质结构域诱导内叶脂质结构域的自发形成的假设。然后将定义脂质结构对结构域形成和叶间偶联的影响。脂酰基链长度、不饱和度、甾醇浓度和甾醇结构将会变化。这应该定义哪些细胞膜具有能够在外叶和/或内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。测试以下假设：具有很长酰基链的脂质可以从一个小叶渗透到另一小叶（即叉指），从而增强物理特性的耦合，这将产生影响 用于肾上腺脑白质腺病，这是一种导致脂质大量过度积累叉指酰基链的疾病。此外，这些研究还将检验这样的假设：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，例如胆固醇。 Smith-Lemli-Opitz 综合征和去睾酮症。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调节结构域形成并且可以交换到细胞中的脂质将被用作探测细胞中结构域形成和叶间偶联的功能后果的工具。内吞作用和聚类的假设 参与信号转导的内叶脂质需要与外叶相连的叶间偶联 将测试小叶结构域的形成。