MOLECULAR BIOLOGY OF ZONULAE OCCLUDENTES

本提案中概述的实验旨在研究 闭锁小带的细胞和分子生物学 交界处。 这种细胞间连接负责形成 在大多数情况下， 脊椎动物上皮 这种连接形成了血液组织的基础 脑、胸腺和睾丸中的屏障，并构成正常的 密封允许上皮细胞边界分离的生理 隔间 ZO-1，第一个被描述为 咬合小带的结构已经被确定， 表征了 根据这些研究，本提案将 专注于两个具体目标。 首先，ZO-1将被用作 特异性探针，以鉴定与所述抗体相关的其他分子。 紧密连接，长期目标是提供一个完整的 这种细胞间连接的分子描述。 特别 重点将放在确定完整的膜蛋白 与ZO-1特异性结合 两种策略， 鉴定这些蛋白质的计划。 分离纯化的ZO-1， 已被证明能够重新绑定到紧 在形态学上正确的位点的连接处，将是琼脂糖凝胶- 偶联用于亲和层析。 ZO-1缺失紧密连接 膜，用已知不干扰 ZO-1/质膜结合，将与这些ZO-1- 偶联的珠，并测定膜蛋白的特异性结合。 第二种策略将是直接溶解ZO-1/膜 蛋白质组装体在不提取ZO-1的条件下， 天然结合位点，并梯度分离和免疫 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， 将在珠加载的MDCK细胞中研究ZO-1，其中细胞为 单层培养物可以在细胞内负载抗ZO-I 抗体，以及这些抗体对生物合成的影响 和稳定跨上皮渗透性和上皮细胞 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.