Milatz Susanne, Piontek Jörg, Hempel Caroline, Meoli Luca, Grohe Christoph, Fromm Anja, Lee In-Fah M, El-Athman Rukeia, Günzel Dorothee
Institute of Clinical Physiology, Charité - Universitätsmedizin Berlin, Berlin, Germany.
Institute of Physiology, Christian-Albrechts-University Kiel, Kiel, Germany.
Ann N Y Acad Sci. 2017 Oct;1405(1):102-115. doi: 10.1111/nyas.13393. Epub 2017 Jun 20.
Claudins are integral components of tight junctions (TJs) in epithelia and endothelia. When expressed in cell lines devoid of TJs, claudins are able to form TJ-like strands at contacts between adjacent cells. According to a current model of TJ strand formation, claudin protomers assemble in an antiparallel double row within the plasma membrane of each cell (cis-interaction) while binding to corresponding double rows from the neighboring cells (trans-interaction). Cis-interaction was proposed to involve two interfaces of the protomers' first extracellular segment (extracellular loop (ECL)1). In the current study, three naturally occurring claudin-10 isoforms and two claudin-10 chimeras were used to investigate strand formation. All constructs were able to interact in cis (Förster/fluorescence resonance energy transfer (FRET)), to integrate into TJs of MDCK-C7 cells (confocal laser scanning microscopy), and to form TJ-like strands in HEK293 cells (freeze-fracture electron microscopy). Strand formation occurred despite the fact that isoform claudin-10a_i1 lacks both structural ECL1 elements reported to be crucial for cis-interaction. Furthermore, results from FRET experiments on claudin-10 chimeras indicated that identity of the first transmembrane region rather than ECL1 is decisive for claudin-10 cis-interaction. Therefore, in addition to the interaction interfaces suggested in the current model for TJ strand assembly, alternative interfaces must exist.
闭合蛋白是上皮细胞和内皮细胞中紧密连接（TJ）的组成成分。当在缺乏紧密连接的细胞系中表达时，闭合蛋白能够在相邻细胞间的接触部位形成类似紧密连接的链。根据目前关于紧密连接链形成的模型，闭合蛋白原聚体在每个细胞的质膜内以反平行双排形式组装（顺式相互作用），同时与相邻细胞的相应双排结合（反式相互作用）。顺式相互作用被认为涉及原聚体第一个细胞外区段（细胞外环（ECL）1）的两个界面。在本研究中，使用了三种天然存在的闭合蛋白-10亚型和两种闭合蛋白-10嵌合体来研究链的形成。所有构建体都能够进行顺式相互作用（荧光共振能量转移（FRET）），整合到MDCK-C7细胞的紧密连接中（共聚焦激光扫描显微镜），并在HEK293细胞中形成类似紧密连接的链（冷冻蚀刻电子显微镜）。尽管闭合蛋白-10a_i1亚型缺乏据报道对顺式相互作用至关重要的两个ECL1结构元件，但仍发生了链的形成。此外，对闭合蛋白-10嵌合体进行的FRET实验结果表明，第一个跨膜区域的一致性而非ECL1对闭合蛋白-10的顺式相互作用起决定性作用。因此，除了当前模型中提出的紧密连接链组装的相互作用界面外，必定还存在其他界面。
