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

 描述（由申请人提供）：本竞争性续批申请旨在支持进一步分析人眼透镜纤维细胞膜中与年龄相关的变化，以阐明透明晶状体和白内障晶状体中发生的主要差异。该提案旨在将膜研究的方法改进到单透镜水平，这将使我们能够考虑眼库提供的供体健康史信息。长期目标是了解眼睛透镜膜在维持透镜透明度中的作用。该透镜膜具有独特的脂质组成和结构，被认为在透镜内部保持低氧浓度，从而防止白内障形成。我们的研究将 为开发替代策略以预防透镜混浊的发生或减缓其进展提供了基础。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透镜内环境稳定中的作用。上一个资助期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使晶状体-脂质膜的整体物理性质保持一致，并且独立于磷脂组成的变化。因此，CBD有助于维持晶状体-膜稳态，而膜磷脂组成随年龄显著变化。我们将（i）继续调整，完善和改进我们最近开发的方法，用于量化来自单个透镜的完整纤维细胞膜中的脂质结构域，以及（ii）基于单个透镜测量，检查随着年龄和白内障形成而发生的纤维细胞膜的变化。将特别注意确定白内障患者和年龄匹配的透明晶状体的透镜膜中脂质组织的主要差异，以及将透明晶状体保留到80岁和90岁的人的透镜膜的结构。最后（3），我们将测试 透镜中氧分压的增加（白内障形成的原因之一）引发脂质过氧化并急剧改变纤维中脂质的组织的假说 细胞膜，包括CBD和胆固醇晶体的形成。我们的研究将基于使用最先进的EPR技术和方法，并在威斯康星州医学院的国家生物医学EPR中心开发。EPR自旋标记方法允许识别膜结构域，给出关于结构和分子动力学的信息作为共存结构域中膜深度的函数，并允许量化这些结构域中的脂质。它们还能够测量膜域内和跨膜域的氧转运。

 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.