MOLECULAR BIOLOGY OF ZONULAE OCCLUDENTES

本提案中概述的实验旨在研究 闭锁带的细胞和分子生物学 交叉口。这种细胞间连接负责形成 多发性骨髓瘤细胞旁通路中的半透屏障 脊椎动物的上皮细胞。交界处构成了血液组织的基础 脑、胸腺和睾丸的屏障，并构成正常 允许上皮细胞与生理上皮细胞分离的密封 车厢。ZO-1，第一个被描述为 已确定闭合带的结构，并 特色化的。根据这些研究，本提案将 把重点放在两个具体目标上。在第一次发射中，ZO-1将被用作 特定的探针，用于识别与该基因相关的其他分子 紧密结合，长期目标是提供完整的 这种细胞间连接的分子描述。特例 重点将放在鉴定完整的膜蛋白上 它与ZO-1特别相关。以下两个策略 鉴定这些蛋白质是有计划的。分离纯化的ZO-1， 已被证明有能力重新绑定到TECH 在形态正确的位置上的连接，将是琼脂糖凝胶- 偶联后用于亲和层析。ZO-1-耗尽的紧密连接 膜，用已知不会干扰的洗涤剂增溶 Z0-1/质膜结合，将与这些ZO-1- 偶联微球，并检测膜蛋白的特异性结合。 第二种策略是直接增溶ZO-1/膜 在不提取ZO-1的条件下的蛋白质组装 天然结合部位，并进行梯度分离和免疫 沉淀这些集合体并研究它们的蛋白质组成。 在这项提议的第二个具体目标中，细胞生物学 ZO-1将在载珠的MDCK细胞中进行研究，其中细胞 单层培养细胞内可负载抗ZO-I抗体 抗体，以及这些抗体对生物发生的影响 和稳定跨上皮通透性和上皮细胞 细胞的极性直接显示出来。

The experiments outlined in this proposal are designed to study the cell and molecular biology of the zonula occludens, or tight junction. This intercellular junction is responsible for forming a semi-permeable barrier in the paracellular pathway in most vertebrate epithelia. The junction forms the basis of blood-tissue barriers in brain, thymus and testis, and constitutes the normal seal permitting epithelial cells to boundary separate physiological compartments. ZO-1, the first protein to be described as part of the structure of the zonula occludens has been identified and characterized. Based on these studies, the present proposal will focus on two specific aims. In the first, ZO-1 will be used as a specific probe to identify additional molecules associated with the tight junction, with the long-term aim of providing a complete molecular description of this intercellular junction. Particular emphasis will be placed on identifying integral membrane proteins which specifically associate with ZO-1. Two strategies for identifying these proteins are planned. Isolated, purified ZO-1, which has been demonstrated to be competent to rebind to tight junctions at the morphologically correct sites, will be sepharose- coupled for affinity chromatography. ZO-1-depleted tight junction membranes, solubilized with detergents known not to interfere with Z0-1/plasma membrane binding, will be reacted with these ZO-1- coupled beads, and specific binding of membrane proteins assayed. The second strategy will be to directly solubilize ZO-1/membrane protein assemblies under conditions which do not extract ZO-1 from native binding sites, and to gradient isolate and immune precipitate these assemblies and study their protein composition. In the second specific aim of this proposal, the cell biology of ZO-1 will be studied in bead-loaded MDCK cells, wherein cells in monolayer culture may be intracellularly loaded with anti-ZO-I antibodies, and the effects of these antibodies on the biogenesis and stabilization of transepithelial permeability and epithelial cell polarity directly demonstrated.