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，或紧密 交界处。 该细胞间连接负责形成 大多数细胞旁细胞途径中的半透性屏障 脊椎动物上皮细胞。 连接形成血液组织的基础 大脑、胸腺和睾丸的屏障，构成正常的 密封允许上皮细胞边界分离生理 隔间。 ZO-1，第一个被描述为一部分的蛋白质 闭合带的结构已被确定 特点。 基于这些研究，本提案将 专注于两个具体目标。 首先，ZO-1 将用作 特异性探针来识别与相关的其他分子 紧密连接，长期目标是提供完整的 这种细胞间连接的分子描述。 特别的 重点将放在鉴定整合膜蛋白上 其与 ZO-1 特别相关。 两种策略 计划鉴定这些蛋白质。 分离、纯化的 ZO-1， 已被证明能够重新粘合到紧密的 形态正确位点处的连接点将被琼脂糖凝胶- 偶联用于亲和层析。 ZO-1耗尽的紧密连接 膜，用已知不会干扰的洗涤剂溶解 Z0-1/质膜结合，会与这些ZO-1-发生反应 耦合珠子，并测定膜蛋白的特异性结合。 第二种策略是直接溶解 ZO-1/膜 在不从其中提取 ZO-1 的条件下进行蛋白质组装 天然结合位点，以及梯度分离和免疫 沉淀这些组装体并研究它们的蛋白质组成。 在该提案的第二个具体目标中，细胞生物学 ZO-1 将在载珠 MDCK 细胞中进行研究，其中细胞 单层培养物可在细胞内负载抗 ZO-I 抗体，以及这些抗体对生物发生的影响 以及跨上皮通透性和上皮细胞的稳定性 细胞极性直接显示。

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.