Hanada Shinichiro, Harada Masaru, Kumemura Hiroto, Omary M Bishr, Kawaguchi Takumi, Taniguchi Eitaro, Koga Hironori, Yoshida Takafumi, Maeyama Michiko, Baba Shinji, Ueno Takato, Sata Michio
Second Department of Medicine, Kurume University School of Medicine, Liver Cancer Division, Research Center for Innovative Cancer Therapy, Kurume University, 67 Asahi-machi, Kurume 830-0011, Japan.
Exp Cell Res. 2005 Apr 1;304(2):471-82. doi: 10.1016/j.yexcr.2004.12.009. Epub 2005 Jan 21.
Many neurodegenerative diseases are characterized by the presence of protein aggregates bundled with intermediate filaments (IFs) and similar structures, known as Mallory bodies (MBs), are observed in various liver diseases. IFs are anchored at desmosomes and hemidesmosomes, however, interactions with other intercellular junctions have not been determined. We investigated the effect of IF inclusions on junction-associated and cytosolic proteins in various cultured cells. We performed gene transfection of the green fluorescent protein (GFP)-tagged cytokeratin (CK) 18 mutant arg89cys (GFP-CK18 R89C) in cultured cells and observed CK aggregations as well as loss of IF networks. Among various junction-associated proteins, zonula occludens-1 and beta-catenin were colocalized with CK aggregates on immunofluorescent analyses. Similar results were obtained on immunostaining for cytosolic proteins, 14-3-3 zeta protein, glucose-6-phosphate dehydrogenase and DsRed. E-cadherin, a basolateral membrane protein in polarized epithelia, was present on both the apical and basolateral domains in GFP-CK18 R89C-transfected cells. Furthermore, cells containing CK aggregates were significantly larger than GFP-tagged wild type CK18 (GFP-WT CK18)-transfected or non-transfected cells (P < 0.01) and sometimes their morphology was significantly altered. Our data indicate that CK aggregates affect not only cell morphology but also the localization of various cytosolic components, which may affect the cellular function.
许多神经退行性疾病的特征是存在与中间丝（IFs）捆绑在一起的蛋白质聚集体，并且在各种肝脏疾病中也观察到了类似结构，即马洛里小体（MBs）。IFs锚定在桥粒和半桥粒上，然而，其与其他细胞间连接的相互作用尚未确定。我们研究了IF内含物对各种培养细胞中与连接相关和胞质蛋白的影响。我们在培养细胞中进行了绿色荧光蛋白（GFP）标记的细胞角蛋白（CK）18突变体arg89cys（GFP-CK18 R89C）的基因转染，并观察到CK聚集以及IF网络的丧失。在各种与连接相关的蛋白中，紧密连接蛋白-1和β-连环蛋白在免疫荧光分析中与CK聚集物共定位。对胞质蛋白、14-3-3ζ蛋白、葡萄糖-6-磷酸脱氢酶和DsRed进行免疫染色也得到了类似结果。E-钙黏蛋白是极化上皮细胞中的一种基底外侧膜蛋白，在GFP-CK18 R89C转染的细胞的顶端和基底外侧结构域均有表达。此外，含有CK聚集物的细胞明显大于GFP标记的野生型CK18（GFP-WT CK18）转染或未转染的细胞（P < 0.01），有时其形态也会发生显著改变。我们的数据表明，CK聚集不仅影响细胞形态，还影响各种胞质成分的定位，这可能会影响细胞功能。
