Lipid Domains in Lens Membranes of a Single Eye: EPR Spin-Labeling Studies

 描述(由申请人提供)：这项竞争性续期拨款是为了支持进一步分析与年龄相关的人眼晶状体纤维细胞膜的变化，以阐明透明晶状体和白内障晶状体中存在的主要差异。这项建议旨在将膜研究的方法改进到单晶状体水平，这将使我们能够考虑眼库提供的捐赠者健康史信息。长期目标是了解眼镜膜在维持晶状体透明度方面的作用。晶状体膜具有独特的脂质成分和结构，被认为可以保持晶状体内部的低氧浓度，从而防止白内障的形成。我们的研究将 为制定预防或减缓晶状体混浊进展的替代策略提供依据。重点将放在纤维细胞膜的脂质双层部分在维持纤维细胞和晶状体内稳态方面的作用。前一批赠款期间的重要进展包括确定胆固醇的作用，特别是胆固醇双层结构域(CBD)的关键作用。CBD的存在确保了周围的磷脂双层被胆固醇饱和。纤维细胞膜中饱和的胆固醇含量保持了晶状体类脂膜的整体物理性质的一致性，并与磷脂组成的变化无关。因此，CBD有助于维持晶状体-膜的动态平衡，而膜磷脂成分随着年龄的增长而发生显著变化。我们将(I)继续调整、改进和改进我们最近开发的用于对完整的纤维细胞膜中的脂类结构域进行量化的方法，以适应来自单个晶状体的膜，以及(Ii)基于单个晶状体的测量，研究纤维细胞膜随年龄和白内障形成的变化。将特别注意确定白内障患者和年龄匹配的透明晶状体的晶状体膜中脂质组织的主要差异，以及80岁和90岁仍保留透明晶状体的人的晶状体膜结构。最后(Iii)，我们将测试 假设晶状体中氧分压升高(白内障形成的原因之一)会引发脂质过氧化，并极大地改变纤维中脂质的组织。 细胞膜，包括CBD和胆固醇晶体的形成。我们的研究将基于威斯康星医学院国家生物医学EPR中心可用和开发的最先进的EPR技术和方法。EPR自旋标记方法可以识别膜结构域，提供作为共存结构域中膜深度的函数的结构和分子动力学信息，并允许对这些结构域中的脂类进行定量。它们还能够测量膜区域内和跨膜区域的氧传输。

 DESCRIPTION (provided by applicant): This competitive renewal grant is a request to support further analysis of age-related changes in membranes of human eye lens fiber-cells in order to elucidate major differences occurring in transparent and cataractous lenses. This proposal seeks to improve the methodology of membrane studies to the single lens level, which will allow us to consider donor health history information provided by the Eye Bank. The long-range goal is to understand the role of eye lens membranes in maintaining lens transparency. The lens membranes have unique lipid compositions and structures thought to maintain low oxygen concentration in the lens interior, and thus, protect against cataract formation. Our research will provide a basis to develop alternative strategies to prevent the onset or slow the progression of lens opacification. The emphasis will be on the role of the lipid bilayer portion of fiber-cell membranes in maintaining fiber-cell and lens homeostasis. Important progress in the previous grant period includes the identification of the role of cholesterol and the crucial role of cholesterol bilayer domains (CBDs) in particular. The presence of the CBD ensures that the surrounding phospholipid bilayer is saturated with cholesterol. The saturating cholesterol content in fiber-cell membranes keeps the bulk physical properties of lens-lipid membranes consistent and independent of changes in phospholipid composition. Thus, the CBD helps to maintain lens-membrane homeostasis while the membrane phospholipid composition changes significantly with age. We will (i) continue to adapt, refine, and improve our recently developed methods for the quantification of lipid domains in intact fiber-cell membranes to membranes derived from a single lens, and (ii) based on single lens measurements, examine changes in fiber-cell membranes occurring with age and cataract formation. Special attention will be paid to determine major differences in the organization of lipids in lens membranes of people with cataracts and age-matched clear lenses, as well as to the structure of lens membranes of people who retain clear lenses into their eighth and ninth decades. Finally (iii), we will test the hypothesis that an increase in oxygen tension in the lens (one of the causes of cataract formation) initiates lipid peroxidation and drastically changes the organization of lipids in fiber cell membranes, including the formation of CBDs and cholesterol crystals. Our studies will be based on the use of state-of-the-art EPR techniques and methods available and developed at the National Biomedical EPR Center at the Medical College of Wisconsin. EPR spin-labeling methods permit identification of membrane domains, give information about structure and molecular dynamics as a function of the membrane depth in coexisting domains, and allow quantification of lipids in these domains. They also are capable of measuring oxygen transport within and across membrane domains.