REGULATION OF CELL SHAPE AND DIFFERENTIATION OF THE RPE

这项提议的长期目标是阐明分子 以及指导、调节和维护结构性和系统性的机制 视网膜色素上皮(RPE)的功能分化通过 筛选一组针对RPE细胞产生的单抗，两个 抗体被确定为这些疾病的分化标志物 研究，3C4和F22。3C4抗原是一种跨膜蛋白，它是 仅在RPE细胞中表达。在发育过程中，3C4变得极化为 成熟雏鸡RPE的基侧表面，在那里它增加了 与膜下细胞骨架结合。这种联系似乎是 直接通过肌动蛋白进行调节。为了进一步确定结构的特征 和这种独特的蛋白质的功能，3C4抗原将从一个 构建RPE表达文库并测序。克隆将被用来寻找 原位杂交法检测3C4在哺乳动物RPE细胞中的表达 真核表达技术将用于功能分析 克隆的3C4基因。F22单抗识别细胞外基质 沉积在光感受器间基质(IPM)中的分子，在 玻璃体-视网膜交界处和巩膜软骨板内。这个 抗原的分布与它在神经节中的作用是一致的。 眼发育过程中的细胞分化和轴突引导。这个 F22在IPM和玻璃体-视网膜交界处的定位提示 它对维持视网膜与RPE的连接可能很重要 玻璃体。F22抗原的结构特征将是 通过从RPE cDNA文库中克隆和测序获得。这个 F22抗原的功能作用将通过抗体封闭来确定 在眼睛发育的关键阶段的实验。 β-1整合素受体在引导和维持RPE细胞中的作用 我们还将考察它们之间的区别。整合素是一类 提供细胞外基质之间联系的跨膜蛋白 和细胞骨架。整合素传递的信号来自 细胞外基质对细胞骨架的影响细胞附着、迁移 和差异化。免疫细胞化学技术将用于 鉴定和定位特定整合素异源二聚体的表达 发展。直接参与连接RPE的整合素 Bruch膜上的细胞将使用二价交联进行鉴定 试剂和差示萃取法。以确定是否可逆 黏附斑块蛋白的磷酸化调节整合素信号转导 在发育过程中，磷酸酪氨酸蛋白与 整合素将使用一组产生的抗体进行检测 磷酸酪氨酰黏附斑块蛋白。特定的表达方式 层粘连蛋白亚型的研究将在发育中的鸡眼使用 抗体和cdna探针的细胞化学方法。他们在这方面的作用 极化RPE表型的发展将被确定。

The long-term objectives of this proposal are to elucidate the molecules and the mechanisms which direct, regulate and maintain structural and functional differentiation of the retinal pigment epithelium (RPE). By screening a panel of monoclonal antibodies produced against RPE cells, two antibodies were identified to use as differentiation markers in these studies, 3C4 and F22. The 3C4 antigen is a transmembrane protein which is expressed only in RPE cells. During development, 3C4 becomes polarized to the basal-lateral surface of mature chick RPE where it increases its association with the submembranous cytoskeleton. This linkage appears to be mediated directly through actin. To further characterize the structure and function of this unique protein, the 3C4 antigen will be cloned from an RPE expression library and sequenced. The clones will be used to look for expression of the 3C4 in mammalian RPE cells by in situ hybridization. Eukaryotic expression technology will be used for functional analysis of the cloned 3C4 gene. The F22 mAb recognizes an extracellular matrix molecule which is deposited in the interphotoreceptor matrix ( IPM), at the vitreoretinal junction and in the cartilaginous plate in the sclera. The distribution of the antigen is consistent with it having a role in ganglion cell differentiation and axonal guidance during ocular development. The localization of F22 in the IPM and the vitreoretinal juncture suggests that it could be important for maintaining retinal attachment to the RPE and vitreous. The structural characterization of the F22 antigen will be obtained by cloning and sequencing the gene from a RPE cDNA library. The functional roles of the F22 antigen will be determined by antibody blocking experiments at critical stages in ocular development. The role of beta-1 integrin receptors in directing and maintaining RPE cell differentiation will also be examined. Integrins are a family of transmembrane proteins that provide a link between the extracellular matrix and the cytoskeleton. Signals transmitted by integrins from the extracellular matrix to the cytoskeleton effect cell attachment, migration and differentiation. Immunocytochemical techniques will be used to identify and map the expression of specific integrin heterodimers during development. Integrins which are directly involved in linking the RPE cells to Bruch's membrane will be identified using bivalent crossinglinking reagents and differential extraction methods. To determine if reversible phosphorylation of adhesion plaque proteins modulates integrin signalling during development, the association of phosphotyrosine proteins with integrins will be examined using a panel of antibodies produce against phosphotyrosyl adhesion plaque proteins. The expression of specific laminin isoforms will be investigated in the developing chick eye using cytochemical approaches with antibodies and cDNA probes. Their role in the development of a polarized RPE phenotype will be determined.