FIBER CELL JUNCTIONS IN NORMAL AND CATARACTOUS LENSES

这种相互竞争的延续是一项关于超微结构和 晶状体纤维细胞连接的功能研究。这两种主要类型的 连接是介导细胞间连接的缝隙连接(GJS) 离子和小分子的输运和方阵列结(SAJ) 它们是由起伏的膜对紧密结合而成 在舌头和沟槽的交错中。SAJ的一个建议角色 就是限制低折射率的细胞外空间。 在第一期支助期间，SAJ牢牢地确立了 在正常的老化过程中表现出明显的变化 牛晶状体和人类老年性核性白内障。结果表明， SAJ在核不透明的形成中发挥了关键作用。这个 SAJ的数量也在#年的早期显著增加 糖尿病兔晶状体白内障的形成。我们计划测试一下 假设纤维细胞膜是损伤的初始位置 多种白内障形成的超微结构观察 纤维细胞膜中的连接特化。这项提议是为了 牛和人晶状体的研究主要采用薄片和 冷冻-断裂-刻蚀电子显微镜进一步表征 老化过程和散射中心的形成。薄片 图像将从整个晶状体的厚可控震源组织切片中获得 使用在初始赠款期间改进的方法。这些切片 允许在整个镜头中进行良好的保存，便于 透明和不透明区域的比较。冷冻-断裂-蚀刻 技术将给出膜蛋白的分布，这是 对于识别交界处及其随年龄的变化很有价值。新的 将启动对人类糖尿病晶状体的研究，特别是那些患有糖尿病的晶状体 后囊下白内障，因为此白内障包含广泛的 SAJ的集合。还提出了新的实验来研究 用激光扫描技术研究荧光染料的胞间运动 共焦显微镜。共焦显微镜是聚焦在 在厚可控震颤器切片中选择完整的纤维细胞 来自切割表面的其他单元的干扰。染料的运输 通过GJS对于理解为什么GJS在 镜片在外围或内部具有与GJS不同的通道特性 其他纸巾。我们还将评估SAJ提供一个 用于通过MIP26的通道的细胞间传输的替代路线， 形成正方形阵列的主要膜蛋白。水和离子 可以通过一个正方形阵列中的MIP26通道进入 然后通过其他MIP26进入相邻的纤维细胞 频道。离体牛SAJ的结构继续研究 建议使用高分辨率的钽复制品来制作薄膜 确定单个MIP26亚基的大小并表征 大型晶体阵列中的通道。膜的分离 将作为离子强度的函数来研究SAJ中的 二价阳离子的浓度，以确定其机理 粘附力。这些研究将提供重要的新信息 关于细胞间连接在衰老中的作用以及关于 人类白内障形成的一般机制。

This competing continuation is a proposal for ultrastructural and functional studies of lens fiber cell junctions. The two major types of junctions are gap junctions (GJs) which mediate the intercellular transport of ions and small molecules and square array junctions (SAJs) which are formed by the close association of undulating membrane pairs in the tongue-and-groove interdigitations. One suggested role for SAJs is to limit the low refractive index extracellular space compartment. During the first period of support, it was firmly established that SAJs show distinctive and pronounced modifications during aging in normal bovine lenses and nuclear human senile cataracts. The results suggest that SAJs play a key role in the formation of nuclear opacities. The amount of SAJ also increased significantly in the early stages of cataract formation in diabetic rabbit lenses. We plan to test the hypothesis that the fiber cell membrane is the initial site of damage in the formation of many cataracts by examining the ultrastructure of junctional specializations in fiber cell membranes. This proposal is for the study of bovine and human lenses primarily employing thin-section and freeze-fracture-etch electron microscopy to further characterize the aging process and the formation of scattering centers. The thin-section images will be derived from thick Vibratome tissue slices of whole lenses using methods refined during the initial grant period. These slices permit excellent preservation throughout the lens and facilitate comparisons of transparent and opaque regions. The freeze-fracture-etch technique will give the distribution of membrane proteins which is valuable for identifying junctions and their alterations with age. New studies will be initiated on human diabetic lenses, especially those with posterior subcapsular cataract because this cataract contains extensive aggregations of SAJs. New experiments are also proposed to investigate the intercellular movement of fluorescent dyes using the laser scanning confocal microscope. The confocal microscope is ideal for focusing on selected intact fiber cells within thick Vibratome sections without interference from other cells of the cut surfaces. The transport of dye through GJs is important for understanding why GJs in the interior of the lens have different channel properties than GJs in the periphery or in other tissues. We will also evaluate the hypothesis that SAJs offer an alternate route for intercellular transport through channels of MIP26, the major membrane protein that forms the square arrays. Water and ions could pass through MIP26 channels in one square array into the extracellular space then into adjacent fiber cells through other MIP26 channels. continued structural studies of SAJs in isolated bovine membranes are proposed employing high resolution tantalum replicas to determine the size of single MIP26 subunits and to characterize the channels in large crystalline arrays. The separation of the membranes in SAJs will be investigated as a function of ionic strength and the concentration of divalent cations in order to determine the mechanism of adhesion. These studies will provide important new information concerning the role of intercellular junctions in aging and about the general mechanisms underlying cataract formation in humans.