MOLECULAR CHARACTERIZATION OF THE ZONULA OCCLUDENS

闭锁小带（ZO）或紧密连接代表了 细胞旁途径的跨上皮通透性屏障 大多数脊椎动物的上皮细胞 这种细胞间连接形成一个 密封圈状密封，在 顶端和侧面的质膜，从而限制 物质在内腔和血清成分之间扩散。 ZO是一系列细胞间的最顶端的成员。 这些连接统称为连接复合体。 一 富含连接复合物的制剂，包括 ZO的结构元件，已经从啮齿动物中产生 肝脏 用该制剂作为免疫原制备的抗血清 识别肝细胞的连接复合体区域， 免疫荧光染色。 这些抗血清还可以染色 包括人在内的其他上皮组织的连接区 结肠细胞系HT-29。 一种特殊的单克隆抗体 在Western印迹上显示与260 kd多肽反应 （ZO-260）。 这种抗体定位于超微结构水平， ZO处的膜接触点。 这里提出 使用免疫阴性染色电子显微镜进一步定位该蛋白 显微镜和表征其组织和物种分布 以及其地形定向。 将试图 使用免疫亲和色谱法在ZO-260中纯化， 然后在人工脂质体中重建ZO结构。 的 纯化的ZO-260也将用于产生额外的 免疫探针 这些探头将用于 实验的目的是一个生理和功能 Z 0的原位表征。 此外，筛选 建立了ZO-260基因的小鼠肝脏cDNA文库。 的 最初的连接复合物富集级分将继续被 用作免疫原以获得额外的单克隆抗体 针对其他ZO和连接复合物相关的 proteins. 将特别努力获得抗体 其识别HT-29细胞系的连接区 因为该系统适合于分析连接组件 和功能 希望通过这种方式， 可以产生，从而产生结构和功能目录 连接复合体的分子组成。

The zonula occludens (ZO) or tight junction represents the transepithelial permeability barrier of the paracellular pathway in most vertebrate epithelia. This intercellular junction forms a gasket-like seal that encircles the cell at the intersection of the apical and lateral plasma membranes and thereby limits the diffusion of substances between lumenal and serosal components. The ZO is the apical most member of a series of intercellular junctions collectively known as the junctional complex. A preparation enriched for junctional complexes, including the structural elements of the ZO, has been generated from rodent liver. Antisera produced using this preparation as immunogen recognize the junctional complex region of hepatocytes as assayed by immunofluorescent staining. These antisera also stain the junctional region of other epithelial tissues including the human colonic cell line, HT-29. One particular monoclonal antibody has been shown to react on Western blots with a 260kd polypeptide (ZO-260). This antibody localizes at the ultrastructural level at the points of membrane contact at the ZO. It is proposed here to further localize this protein using immunonegative stain electron microscopy and to characterize its tissue and species distribution as well as its topographical orientation. Attempts will be made to purify in ZO-260 using immuno-affinity chromatography and to then reconstitute the ZO structure in artificial liposomes. The purified ZO-260 will also be used to generate additional immunological probes. These probes will be utilized in experiments aimed at a physiological and functional characterization of the ZO in situ. In addition, screening of mouse liver cDNA libraries for the ZO-260 gene is proposed. The original junctional complex enriched fraction will continue to be used as immunogen to obtain additional monoclonal antibodies directed toward other ZO- and junctional complex-associated proteins. Particular effort will be made to procure antibodies which recognize the junctional region of the HT-29 cell line because this system is amenable to analysis of junction assembly and function . It is hoped that in this manner a library of antisera can be produced, resulting in a structural and functional catalog of the molecular constituents of the junctional complex.