Occludin, tricellulin, and marvelD3 belong to the tight junction (TJ)-associated MARVEL protein family. Occludin and tricellulin jointly contribute to TJ strand branching point formation and epithelial barrier maintenance. However, whether marvelD3 has the same function remains unclear. Furthermore, the roles of the carboxy-terminal cytoplasmic tail, which is conserved in occludin and tricellulin, on the regulation of TJ strand morphology have not yet been explored in epithelial cells. We established tricellulin/occludin/marveld3 triple-gene knockout (tKO) MDCK II cells and evaluated the roles of marvelD3 in the TJ strand structure and barrier function using MDCK II cells and a mathematical model. The complexity of TJ strand networks and paracellular barrier did not change in tKO cells compared to that in tricellulin/occludin double-gene knockout (dKO) cells. Exogenous marvelD3 expression in dKO cells did not increase the complexity of TJ strand networks and epithelial barrier tightness. The expression of the carboxy-terminal truncation mutant of tricellulin restored the barrier function in the dKO cells, whereas occludin lacking the carboxy-terminal cytoplasmic tail was not expressed on the plasma membrane. These data suggest that marvelD3 does not affect the morphology of TJ strands and barrier function in MDCK II cells and that the carboxy-terminal cytoplasmic tail of tricellulin is dispensable for barrier improvement.

中文翻译：

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TAMP家族对上皮细胞紧密连接链网络和屏障功能的影响

Occludin、tricellulin 和 marvelD3 属于紧密连接 (TJ) 相关的 MARVEL 蛋白家族。Occludin 和三纤维素共同促进 TJ 链分支点的形成和上皮屏障的维持。然而，marvelD3是否具有相同的功能尚不清楚。此外，尚未在上皮细胞中探索在 occludin 和三纤维素中保守的羧基末端细胞质尾部对 TJ 链形态调节的作用。我们建立了tricellulin / occludin / marveld3三基因敲除 (tKO) MDCK II 细胞，并使用 MDCK II 细胞和数学模型评估了 marvelD3 在 TJ 链结构和屏障功能中的作用。与三纤维素/ occludin相比， tKO细胞的 TJ 链网络和细胞旁屏障的复杂性没有变化双基因敲除 (dKO) 细胞。dKO 细胞中的外源性 marvelD3 表达不会增加 TJ 链网络的复杂性和上皮屏障的紧密度。三纤维素的羧基末端截断突变体的表达恢复了 dKO 细胞的屏障功能，而缺少羧基末端细胞质尾部的 occludin 则不在质膜上表达。这些数据表明 marvelD3 不影响 MDCK II 细胞中 TJ 链的形态和屏障功能，并且三纤维素的羧基末端细胞质尾部对于屏障改善是可有可无的。