Gehne Nora, Lamik Agathe, Lehmann Martin, Haseloff Reiner F, Andjelkovic Anuska V, Blasig Ingolf E
Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany.
University of Michigan, Medical School, Ann Arbor, United States of America.
PLoS One. 2017 Aug 15;12(8):e0182106. doi: 10.1371/journal.pone.0182106. eCollection 2017.
Claudins (Cldns) are transmembrane tight junction (TJ) proteins that paracellularly seal endo- and epithelial barriers by their interactions within the TJs. However, the mechanisms allowing TJ remodeling while maintaining barrier integrity are largely unknown. Cldns and occludin are heterophilically and homophilically cross-over endocytosed into neighboring cells in large, double membrane vesicles. Super-resolution microscopy confirmed the presence of Cldns in these vesicles and revealed a distinct separation of Cldns derived from opposing cells within cross-over endocytosed vesicles. Colocalization of cross-over endocytosed Cldn with the autophagosome markers as well as inhibition of autophagosome biogenesis verified involvement of the autophagosomal pathway. Accordingly, cross-over endocytosed Cldns underwent lysosomal degradation as indicated by lysosome markers. Cross-over endocytosis of Cldn5 depended on clathrin and caveolin pathways but not on dynamin. Cross-over endocytosis also depended on Cldn-Cldn-interactions. Amino acid substitutions in the second extracellular loop of Cldn5 (F147A, Q156E) caused impaired cis- and trans-interaction, as well as diminished cross-over endocytosis. Moreover, F147A exhibited an increased mobility in the membrane, while Q156E was not as mobile but enhanced the paracellular permeability. In conclusion, the endocytosis of TJ proteins depends on their ability to interact strongly with each other in cis and trans, and the mobility of Cldns in the membrane is not necessarily an indicator of barrier permeability. TJ-remodeling via cross-over endocytosis represents a general mechanism for the degradation of transmembrane proteins in cell-cell contacts and directly links junctional membrane turnover to autophagy.
紧密连接蛋白（Claudins，Cldns）是跨膜紧密连接（TJ）蛋白，通过在紧密连接内的相互作用在细胞旁密封内皮和上皮屏障。然而，在维持屏障完整性的同时允许紧密连接重塑的机制在很大程度上尚不清楚。Cldns和闭合蛋白以异嗜性和同嗜性方式通过跨细胞内吞作用进入相邻细胞，形成大的双膜囊泡。超分辨率显微镜证实了这些囊泡中存在Cldns，并揭示了跨细胞内吞囊泡中来自相对细胞的Cldns的明显分离。跨细胞内吞的Cldn与自噬体标记物的共定位以及自噬体生物发生的抑制证实了自噬体途径的参与。因此，如溶酶体标记物所示，跨细胞内吞的Cldns经历了溶酶体降解。Cldn5的跨细胞内吞作用依赖于网格蛋白和小窝蛋白途径，但不依赖于发动蛋白。跨细胞内吞作用也依赖于Cldn-Cldn相互作用。Cldn5第二个细胞外环中的氨基酸取代（F147A、Q156E）导致顺式和反式相互作用受损，以及跨细胞内吞作用减弱。此外，F147A在膜中的流动性增加，而Q156E的流动性不如F147A，但增强了细胞旁通透性。总之，紧密连接蛋白的内吞作用取决于它们在顺式和反式中相互强烈作用的能力，并且Cldns在膜中的流动性不一定是屏障通透性的指标。通过跨细胞内吞作用进行的紧密连接重塑代表了细胞间接触中跨膜蛋白降解的一般机制，并直接将连接膜更新与自噬联系起来。
