MOLECULAR CHARACTERIZATION OF THE TIGHT JUNCTION

提案中概述的实验将继续进行分子 闭合小带（ZO）或紧密连接的表征。 这 细胞间连接负责维持和调节 两种细胞外基质之间的跨上皮通透性屏障 由上皮界定的隔室。 紧密连接还 参与维持极化的膜结构域特性 一个上皮细胞。 这里列出的大多数研究都集中在 对肠道紧密连接的调节和组装 上皮细胞 一个目标是确定的分子基础， 肠紧密连接被细胞“松动”， 多形核白细胞，因为它们侵入肠粘膜， 炎症反应 这个过程可以在体外模拟， 向培养的单层细胞中加入细胞因子干扰素-γ（IFN-g） 肠细胞系T-84 研究建议测试几个 IFN-γ调节细胞紧密连接通透性假说 培养的肠细胞单层。 直接影响紧 连接蛋白ZO-1，包括其生物合成谱的变化 （转录;稳定性）、同种型表达和磷酸化状态 将检查，在比较其他一些'分化' 肠上皮细胞刷状缘（BB）膜的特异性蛋白质， 细胞骨架 使用视频增强光学显微镜，我们将监测 IFN-g对体内连接收缩性的影响以检验假设 连接通透性是由肌动球蛋白环调节的 与粘附连接相关。 第二个目标是定义 紧密连接组装中的早期分子事件， 极化膜骨架组装在培养的肠细胞。 这 将通过一系列成对的延时视频来完成， 免疫细胞化学研究，使用紧密连接的免疫探针（ZO-1， 扣带蛋白）和BB蛋白。 最后，建议开展试点研究， 评估遗传和分子遗传学方法， 紧密连接 一系列的实验将进一步表征ZO-1 免疫原在果蝇的中枢神经系统中表达， 连接形成了相当于血脑屏障的结构。 研究来 验证ZO-1同源物的表达，以及初始遗传修饰， 特征提出。

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.