Inai T, Kobayashi J, Shibata Y
Department of Developmental Molecular Anatomy, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
Eur J Cell Biol. 1999 Dec;78(12):849-55. doi: 10.1016/S0171-9335(99)80086-7.
Tight junctions (TJs) create a paracellular permeability barrier and also act as a fence preventing intermixing of proteins and lipids between the apical and basolateral plasma membranes. Recently, claudin-1 has been identified as an integral membrane protein localizing at TJs, and introduced claudin-1 can form TJ-like networks in fibroblasts. To investigate the function of claudin-1, MDCK cells were transfected with a mammalian expression vector containing myc-tagged mouse claudin-1, and four stable clones were obtained. The myc-tagged claudin-1 precisely colocalized with both occludin and ZO-1 at cell-cell contact sites, indicating that exogenous claudin-1 was properly targeted to the TJs. Immunoblot analysis revealed that overexpression of claudin-1 increased expression of ZO-1 but not of occludin or ZO-2. The barrier functions of these cells were evaluated by transepithelial electrical resistance (TER) and paracellular flux. Claudin-1-expressing cells exhibited about four times higher TER than wild-type MDCK cells. Consistent with the increase of TER, the cells overexpressing claudin-1 showed reduced paracellular flux, estimated at 4 and 40 kD FITC-dextrans. These results suggest that claudin-1 is involved in the barrier function at TJs.
紧密连接（TJs）形成细胞旁通透性屏障，还起到栅栏的作用，防止蛋白质和脂质在顶端和基底外侧质膜之间相互混合。最近，claudin-1已被鉴定为一种定位于紧密连接的整合膜蛋白，导入的claudin-1可在成纤维细胞中形成类似紧密连接的网络。为了研究claudin-1的功能，用含有myc标签的小鼠claudin-1的哺乳动物表达载体转染MDCK细胞，并获得了四个稳定克隆。带有myc标签的claudin-1与闭合蛋白和ZO-1在细胞间接触部位精确共定位，表明外源性claudin-1正确靶向紧密连接。免疫印迹分析显示，claudin-1的过表达增加了ZO-1的表达，但没有增加闭合蛋白或ZO-2的表达。通过跨上皮电阻（TER）和细胞旁通量评估这些细胞的屏障功能。表达claudin-1的细胞的TER比野生型MDCK细胞高约四倍。与TER的增加一致，过表达claudin-1的细胞显示细胞旁通量降低，以4kD和40kD异硫氰酸荧光素标记的葡聚糖进行评估。这些结果表明claudin-1参与紧密连接的屏障功能。
