Shono Akemi, Tsukaguchi Hiroyasu, Yaoita Eishin, Nameta Masaaki, Kurihara Hidetake, Qin Xiao-Song, Yamamoto Tadashi, Doi Toshio
Department of Clinical Biology and Medicine, The University of Tokushima Graduate School of Medical Sciences, 3-18-15 Kuramoto, Tokushima, 770-0042, Japan.
J Am Soc Nephrol. 2007 Sep;18(9):2525-33. doi: 10.1681/ASN.2006101084. Epub 2007 Aug 5.
The predominant type of cellular junction between normal podocyte foot processes is the slit diaphragm. Under nephrotic conditions,however, foot process effacement leads to the loss of slit diaphragms and the new formationof tight junctions composed of the proteins coxsackievirus and adenovirus receptor (CAR) and zonula occludens 1 (ZO-1). Podocin, a protein that plays a key role in maintaining the integrity of the slit diaphragm, has also been localized to these tight junctions, but its function at this site is unknown. In this study, we confirmed that podocin colocalizes with CAR and ZO-1 at the tight junction between foot processes in nephrotic rats. Using primary cultures of rat podocytes, as well as cell lines that co-expressed podocin and CAR, we observed that podocin was recruited to sites of cell-cell contact and that it co-localized with CAR and ZO-1. Immunoprecipitation suggested that these three junctional proteins from a multi-protein complex. Consistent with this, we found that podociin facilitated the coalescence of preassembled lipid rafts containing CAR and restricted their lateral mobility, the latter likely a result of dynamic actin reorganization and subsequent tethering of CAR-podocin complexes to the cytoskeleton. In conclusion, in addition to serving as a structural protein of the slit diaphragm of normal podocytes, our data suggest that podocin may also serve as a scaffold that links tight junction proteins to the actin cytoskeleton in nephrotic foot processes.
正常足细胞足突之间主要的细胞连接类型是裂孔隔膜。然而，在肾病状态下，足突消失导致裂孔隔膜丧失，取而代之的是由柯萨奇病毒和腺病毒受体（CAR）及紧密连接蛋白1（ZO-1）组成的紧密连接的新形成。足蛋白在维持裂孔隔膜完整性中起关键作用，也定位于这些紧密连接，但它在该位点的功能尚不清楚。在本研究中，我们证实足蛋白在肾病大鼠的足突间紧密连接处与CAR和ZO-1共定位。利用大鼠足细胞原代培养物以及共表达足蛋白和CAR的细胞系，我们观察到足蛋白被募集到细胞-细胞接触位点，且与CAR和ZO-1共定位。免疫沉淀提示这三种连接蛋白形成一个多蛋白复合物。与此一致，我们发现足蛋白促进了含有CAR的预组装脂筏的聚集，并限制其侧向移动，后者可能是动态肌动蛋白重组以及随后CAR-足蛋白复合物与细胞骨架连接的结果。总之，除了作为正常足细胞裂孔隔膜的结构蛋白外，我们的数据表明足蛋白在肾病足突中可能还作为一种支架，将紧密连接蛋白与肌动蛋白细胞骨架相连。
