Mechanism of the paracellular molecular transport

为了了解紧密连接(TJ)作为细胞旁空间分子传递和生理屏障的工具，我们分析了组成TJ的Claudins的功能以及细菌毒力因子影响TJ1细胞旁屏障功能的作用方式。Claudins的功能分析。(1)我们分别建立了claudin-1缺陷和claudin-5缺陷小鼠，发现claudin-1构成TJ，首先在表皮中被证实，而claudin-5主要形成TJ在血脑屏障。(2)我们通过使用表达GFP标记的Claudin的Eph4上皮细胞来分析TJ链的动态行为。结果发现，TJ链在重塑过程中保持连续性，伴随着细胞的运动，当TJ链缩短时，多余的Claudin被内吞到相邻的一个细胞中。这种动态行为被认为是TJ2本构势垒函数的基础。细菌毒力因子影响TJ屏障功能的机制分析(1)我们研究了致病性大肠杆菌(EPEC)如何在上皮细胞模型中降低细胞旁屏障功能，发现EPEC毒力因子之一MAP对此起作用。此外，EPEC与上皮细胞之间的密切联系被发现触发(A)毒力因子(S)的分泌，而MAP不是EPEC屏障破坏活动所必需的。(2)我们分析了Claudins和Clostridium perfiivens肠毒素(CPE)之间的结合性质，这种结合被认为是特异性识别Claudins作为受体的。结果表明，CPE除与claudin 3和4结合外，还与claudin 6、7、8和14结合，并识别claudins的第二个胞外环。体外和体内实验都表明，CPE的Claudin结合片段打开了上皮细胞的细胞旁通路。

To understand the tight junction (TJ) as the apparatus for molecular transfer and physiological barrier at the paracellular space, we analyzed function of claudins composing TJ and the mode of action by which bacterial virulence factors affect the paracellular barrier function of TJ.1. Functional analyses of claudins.(1) We generated claudin-1-deficient and claudin-5-deficient mice, respectively, and found that claudin-1 constitutes TJ that was first demonstrated in the epidermis, and claudin-5 essentially forms TJ at the blood-brain barrier.(2) We analyzed dynamic behavior of the TJ strand by using Eph4 epithelial cells expressing GFP-tagged claudin. It was found that the TJ strands retain their continuities during their remodeling accompanied by the cell movement When the TJ strands shortened, the superfluous claudins were endocytosed into one of the adjacent cells. This dynamic behavior is considered to underlie the constitutive barrier function of TJ.2. Analyses for mechanisms by which bacterial virulence factors affect the barrier function of TJ(1) We examined how enteropathogenic Escherichia coli (EPEC) reduces the paracellular barrier function in the epithelial cell model, and found that Map, one of EPEC virulence factors, is responsible for it Moreover, the intimate association between EPEC and epithelial cells was found to trigger the secretion of (a) virulence factor(s) other than Map, which are essential for the barrier-disrupting activity of EPEC.(2) We analyzed the binding nature between claudins and Clostridium perfiivens enterotoxin (CPE), which is known to specifically recognize claudins as receptors. The results demonstrated that CPE binds to claudin 6,7,8, and 14 besides claudin 3 and 4, which had been reported so far, and that CPE recognizes the second extracellular loop of the claudins. Both in vitro and in vivo experiments revealed that a claudin-binding fragment of CPE opened the paracellular pathways of epithelial cells.