Tight junctions (TJs) are composed of a claudin-based anastomosing network of TJ strands, at which plasma membranes of adjacent epithelial cells are closely attached to regulate the paracellular permeability. Although the TJ proteins occludin and tricellulin have been known to be incorporated in the TJ strand network, their molecular functions remain unknown. Here, we established tricellulin/occludin-double knockout (dKO) MDCK II cells using a genome editing technique, and evaluated the structure and barrier function of these cells. In freeze-fracture replica electron microscopy, the TJ strands of tricellulin/occludin-dKO cells had fewer branches and were less anastomosed compared with the controls. The paracellular permeability of ions and small tracers was increased in the dKO cells. A single KO of tricellulin or occludin had limited effects on the morphology and permeability of TJs. Mathematical simulation using a simplified TJ strand network model predicted that reduced cross-links in TJ strands lead to increased permeability of ions and small macromolecules. Furthermore, overexpression of occludin increased the complexity of TJ strand network and strengthened barrier function. Taken together, our data suggest that tricellulin and occludin mediate the formation and/or stabilization of TJ strand branching points, and contribute to the maintenance of epithelial barrier integrity.

紧密连接 (TJ) 由 TJ 链的基于密蛋白的吻合网络组成，相邻上皮细胞的质膜在该网络上紧密附着以调节细胞旁通透性。尽管已知 TJ 蛋白 occludin 和 tricellulin 被纳入 TJ 链网络，但它们的分子功能仍然未知。在这里，我们使用基因组编辑技术建立了三纤维素 / occludin 双敲除 (dKO) MDCK II 细胞，并评估了这些细胞的结构和屏障功能。在冷冻断裂复制电子显微镜中，与对照相比，tricellulin/occludin-dKO 细胞的 TJ 链具有更少的分支并且更少的吻合。dKO 细胞中离子和小示踪剂的细胞旁通透性增加。tricellulin 或 occludin 的单一 KO 对 TJ 的形态和渗透性的影响有限。使用简化的 TJ 链网络模型的数学模拟预测，TJ 链中减少的交联会导致离子和小分子渗透性增加。此外，occludin 的过度表达增加了 TJ 链网络的复杂性并增强了屏障功能。总之，我们的数据表明，tricellulin 和 occludin 介导了 TJ 链分支点的形成和/或稳定，并有助于维持上皮屏障的完整性。occludin 的过度表达增加了 TJ 链网络的复杂性并增强了屏障功能。总之，我们的数据表明，tricellulin 和 occludin 介导了 TJ 链分支点的形成和/或稳定，并有助于维持上皮屏障的完整性。occludin 的过度表达增加了 TJ 链网络的复杂性并增强了屏障功能。总之，我们的数据表明，tricellulin 和 occludin 介导了 TJ 链分支点的形成和/或稳定，并有助于维持上皮屏障的完整性。