MOLECULAR CHARACTERIZATION OF THE ZONULA OCCLUDENS

闭合带(ZO)或紧密连接代表 血管内皮细胞旁通路的跨上皮通透性屏障 大多数脊椎动物的上皮细胞。这种细胞间的连接形成了一个 在电池的交叉处包围电池的垫圈状密封 顶端和侧方质膜，从而限制了 物质在肠腔和浆膜成分之间的扩散。 ZO是一系列细胞间最顶端的成员。 联结统称为联结复合体。一个 富含连接络合物的制剂，包括 ZO的结构元素，是由啮齿动物产生的 肝脏。以该制剂为免疫原制备抗血清 识别检测到的肝细胞连接复合区 免疫荧光染色。这些抗血清还染上了 包括人类在内的其他上皮组织的连接区 结肠癌细胞株，HT-29。一种特殊的单抗具有 已被证明与蛋白质印迹反应一个260kd的多肽 (ZO-260)。该抗体定位于超微结构水平的 ZO处的膜接触点。在此建议： 利用免疫阴性染色电子技术进一步定位该蛋白 显微镜下观察和鉴定其组织和物种分布 以及它的地形方位。我们将尝试 用免疫亲和层析法纯化ZO-260 然后在人工脂质体中重建ZO结构。这个 提纯的ZO-260还将用于产生额外的 免疫探头。这些探头将用于 以生理和机能为目标的实验 ZO的原位表征。此外，还筛选了 建立了小鼠肝脏ZO-260基因的cDNA文库。这个 原有的连接络合物富集组分将继续 用作免疫原以获得额外的单抗 定向到其他ZO-和连接络合物相关的 蛋白质。将作出特别努力以获得抗体 它们识别HT-29细胞系的连接区 由于该系统适用于结合部组件的分析 和功能。希望通过这种方式建立一个抗血清文库 可以制作，从而形成一个结构和功能目录 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.