MOLECULAR CHARACTERIZATION OF THE TIGHT JUNCTION

提案中概述的实验将继续分子 闭合带(ZO)或紧密连接的特征。这 细胞间连接负责维持和调节 两种细胞外基质之间的跨皮细胞通透性屏障 由上皮细胞定义的隔室。紧密的结合部也 参与维持极化膜结构域特性 一种上皮细胞。这里概述的大多数研究都集中在 论肠道紧密连接的调节与组装 上皮细胞。一个目标是定义分子基础 肠道紧密连接的“松动” 中性粒细胞侵袭肠粘膜 一种炎症反应。这一过程可以通过以下方式在体外模拟 细胞因子干扰素-γ对培养的单层细胞的作用 肠道细胞系T-84。建议进行研究，以测试几个 干扰素-γ调节紧密连接通透性的假说 培养的肠道细胞单层。对紧张局势的直接影响 连接蛋白ZO-1及其生物合成谱的变化 (转录；稳定性)、异构体表达和磷酸化状态 将与其他一些“差异化”进行比较 肠细胞刷状缘(BB)膜和 细胞骨架。使用视频增强光学显微镜，我们将监测 干扰素-g在体内对连接性收缩的影响以验证这一假说 连接通透性由肌动球蛋白环调节 与粘连连接相关联。第二个目标是定义 紧密连接和组装中的早期分子事件 培养的肠道细胞中的极化膜骨骼组装。这 将通过一系列配对的延时视频和 免疫细胞化学研究，使用紧密连接的免疫探针(ZO-1， Cinglin)和BB蛋白。最后，建议进行初步研究，以 评估遗传学和分子遗传学方法用于解剖 紧密连接。一系列实验将进一步表征ZO-1 免疫基因在果蝇中枢神经系统胶质细胞致密处的表达 连接处形成了相当于血脑屏障的结构。研究到 验证ZO-1同源基因的表达，以及初始基因 提出了相应的表征方法。

The experiments outlined in the proposal will continue the molecular characterization of the zonula occludens (ZO) or tight junction. This intercellular junction is responsible for maintaining and regulating the transepithelial permeability barrier between the two extracellular compartments defined by an epithelium. The tight junction also participates in maintenance of polarized membrane domains characteristic of an epithelial cell. The majority of the studies outlined here focus on the regulation and assembly of the tight junctions in the intestinal epithelial cell. One objective is to define the molecular basis for the "loosening" of intestinal tight junctions by cells such as polymorphonuclear leukocytes as they invade the intestinal mucosa during an inflammatory response. This process can be modeled in vitro by addition of the cytokine, interferon-gamma (IFN-g) to cultured monolayers of the intestinal cell line T-84. Studies are proposed to test several hypotheses for regulation of tight junction permeability by IFN-g in cultured intestinal cell monolayers. Direct effects on the tight junction protein, ZO-1, including changes in its biosynthetic profile (transcription; stability), isoform expression, and phosphorylation state will be examined, in comparison to a number of other 'differentiation' specific proteins of the enterocyte brush border (BB) membrane and cytoskeleton. Using video-enhanced light microscopy, we will monitor IFN-g effects on junctional contractility in vivo to test the hypothesis that junctional permeability is regulated by the actomyosin ring associated with the adherens junction. A second objective is to define early molecular events in the assembly of the tight junction and polarized membrane skeletal assembly in cultured intestinal cells. This will be done by a series of paired time-lapse video and immunocytochemical studies, using immuno-probes for tight junction (ZO-1, cingulin), and BB proteins. Finally, pilot studies are proposed to assess genetic and molecular genetic approaches for the dissection of the tight junction. A series of experiments will further characterize a ZO-1 immunogen expressed in the CNS of the Drosophila where glial cell tight junctions form the equivalent of a blood-brain barrier. Studies to verify the expression of a ZO-1 homolog, together with initial genetic characterization are proposed.