Cholesterol Crystalline Domain Function in Eye Lens: EPR Spin-Labeling Studies

描述（由申请人提供）：人眼晶状体的纤维细胞膜被胆固醇饱和（显示胆固醇与磷脂的摩尔比从皮质的1到细胞核的高达4），这导致这些膜内形成不混溶的胆固醇结晶结构域（CCDs）。在胆固醇含量最高的情况下，高达50%的细胞膜表面可被ccd占据。这些情况存在于晶状体核中，那里的胆固醇含量可以显著超过膜的溶解度阈值。ccd的出现通常是一种病理征兆；然而，只有在晶状体中，ccd才能发挥积极的生理功能，维持晶状体的透明度，并可能防止白内障的形成。这项建议的长期目标是对胆固醇在纤维细胞膜中的功能有更深入的了解。在短期内，我们将(i)研究高胆固醇含量如何影响磷脂晶状体膜的横向组织，特别关注CCDs的形成，以及（ii）检验CCDs的存在决定周围散装磷脂-胆固醇膜性质的假设。此外，实验将揭示晶状体膜的磷脂组成是否会影响ccd的形成、大小和稳定性。（iv）最后，在模型膜系统中开发和测试的方法将用于检测和表征成熟、衰老和白内障形成过程中晶状体纤维细胞膜中共存的胆固醇结晶和散装磷脂-胆固醇域。对于从晶状体分离的膜中共存结构域的研究，以及在纤维细胞膜模型中，将使用氧运输（DOT）方法进行区分。DOT方法基于电子顺磁共振（EPR）自旋标记技术，可以区分不同的膜结构域，并在不需要分离的情况下，将共存结构域的结构和分子动力学信息作为膜深度的函数。它还允许获得膜域内和膜域间氧运输的信息。

DESCRIPTION (provided by applicant): Fiber-cell membranes of the human eye lens are saturated with cholesterol (showing cholesterol-to- phospholipid mole ratios from 1 in the cortex to as high as 4 in the nucleus), which leads to the formation of immiscible cholesterol crystalline domains (CCDs) within these membranes. At the highest cholesterol content, up to 50% of the cell-membrane surface can be occupied by CCDs. These conditions exist in the lens nucleus, where the cholesterol content can significantly exceed the solubility threshold of the membrane. The appearance of CCDs is usually a sign of pathology; however, only in the eye lens can CCDs play a positive physiological function, maintaining lens transparency and possibly protecting against cataract formation. The long-term objective of this proposal is to achieve a greater understanding of cholesterol's function in fiber-cell membranes. In the short-term, we will (i) examine how high cholesterol content affects the lateral organization of phospholipid lens membranes, with special attention paid to the formation of CCDs, and (ii) test the hypothesis that the presence of CCDs determines the properties of the surrounding bulk phospholipid- cholesterol membranes. (iii) Additionally, experiments will reveal if the phospholipid composition of the lens membrane affects the formation, size, and stability of CCDs. (iv) Finally, the methodology developed and tested in model-membrane systems will be applied to detect and characterize coexisting cholesterol crystalline and bulk phospholipid-cholesterol domains in lens fiber-cell membranes during maturation, aging, and cataract formation. For studies of coexisting domains in membranes isolated from the eye lens, as well as in models of fiber-cell membranes, the discrimination by oxygen transport (DOT) method will be used. The DOT method, which is based on electron paramagnetic resonance (EPR) spin-labeling techniques, permits discrimination of different membrane domains and gives information about structure and molecular dynamics as a function of the membrane depth in coexisting domains without the need for their separation. It also allows information about oxygen transport within and across membrane domains to be obtained. PUBLIC HEALTH RELEVANCE: Age-related cataracts are a major cause of blindness in developing countries. The reason for the onset of cataracts is unknown, but a great deal of evidence suggests that the presence of the cholesterol crystalline domain in the eye lens helps to maintain transparency and prevent cataract formation. The proposed studies will generate important fundamental information about topographical and age-related differences in cholesterol- membrane interactions in the eye lens that will increase our understanding of the role cholesterol plays and, in turn, help contribute to the prevention of age-related nuclear cataracts.