Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan.
J Biol Chem. 2012 Mar 16;287(12):9525-33. doi: 10.1074/jbc.M111.327064. Epub 2012 Jan 31.
The role of tight junctions (TJs) in the establishment and maintenance of lipid polarity in epithelial cells has long been a subject of controversy. We have addressed this issue using lysenin, a toxin derived from earthworms, and an influenza virus labeled with a fluorescent lipid, octadecylrhodamine B (R18). When epithelial cells are stained with lysenin, lysenin selectively binds to their apical membranes. Using an artificial liposome, we demonstrated that lysenin recognizes the membrane domains where sphingomyelins are clustered. Interestingly, lysenin selectively stained the apical membranes of epithelial cells depleted of zonula occludens proteins (ZO-deficient cells), which completely lack TJs. Furthermore, the fluorescent lipid inserted into the apical membrane by fusion with the influenza virus did not diffuse to the lateral membrane in ZO-deficient epithelial cells. This study revealed that sphingomyelin-cluster formation occurs only in the apical membrane and that lipid polarity is maintained even in the absence of TJs.
紧密连接（TJs）在维持上皮细胞脂类极性中的作用一直存在争议。我们使用从蚯蚓中提取的毒素 lysenin 和用荧光脂质标记的流感病毒来解决这个问题。当用 lysenin 染色上皮细胞时，lysenin 选择性地结合到它们的顶膜上。利用人工脂质体，我们证明了 lysenin 识别鞘磷脂聚集的膜域。有趣的是，lysenin 选择性地染色了去封闭蛋白（ZO-缺陷细胞）的上皮细胞的顶膜，这些细胞完全缺乏 TJs。此外，与流感病毒融合插入顶膜的荧光脂质在 ZO-缺陷的上皮细胞中不会扩散到侧膜。这项研究表明，鞘磷脂簇的形成仅发生在上皮细胞的顶膜上，并且即使在没有 TJs 的情况下，脂类极性也能得到维持。
