Bader B L, Magin T M, Freudenmann M, Stumpp S, Franke W W
Institute of Cell and Tumor Biology, German Cancer Research Center, Heidelberg.
J Cell Biol. 1991 Dec;115(5):1293-307. doi: 10.1083/jcb.115.5.1293.
The roles of the different molecular domains of intermediate filament (IF) proteins in the assembly and higher order organization of IF structures have recently been studied by various groups but with partially controversial results. To examine the requirement of the aminoterminal (head) and the carboxyterminal (tail) domain of cytokeratins (CKs) for de novo IF formation in the living cell, we have constructed cDNAs coding for intact as well as head- and/or tail-less human CKs 8 and 18 and the naturally tail-less human CK 19, all under the control of the human beta-actin promoter. After transient and stable transfections of mouse 3T3-L1 cells, which are devoid of any CKs, we have studied, with such constructs, the resulting gene products by gel electrophoresis and immunolocalization techniques. By light and electron microscopy we show that extended cytoplasmic IF meshworks are formed from pairs of the type II CK 8 with the type I CKs 18 or 19 as well as from pairs of tail-less CK 8 with tail-less CKs 18 or 19 in the transfected cells, proving that the absence of the tail domain in both types of CKs does not prevent the de novo formation of regular IFs. Most surprisingly, however, we have observed spectacular alterations in the nucleocytoplasmic distribution of the IFs formed from tail-less CKs. In many of the transfected cells, a large part, or all, of the detectable CKs was found to occur in extensive IF bundles in the nucleoplasm. Intranuclear accumulations of CK deposits, however mostly nonfibrillar, were also observed when the cells had been transfected with cDNAs encoding tail-less CKs also lacking their head domains, whereas CKs deleted only in the head domain were found exclusively in the cytoplasm. The specific domain requirements for the assembly of cytoplasmic IF bundles are discussed and possible mechanisms of intranuclear accumulation of IFs are proposed.
近期，多个研究小组对中间丝（IF）蛋白不同分子结构域在IF结构组装和高级组织中的作用进行了研究，但结果存在部分争议。为了检测细胞角蛋白（CKs）的氨基末端（头部）和羧基末端（尾部）结构域在活细胞中从头形成IF的必要性，我们构建了在人β-肌动蛋白启动子控制下，编码完整以及无头和/或无尾的人CK 8、18和天然无尾的人CK 19的cDNA。在用这些构建体瞬时和稳定转染缺乏任何CKs的小鼠3T3-L1细胞后，我们通过凝胶电泳和免疫定位技术研究了所得的基因产物。通过光学和电子显微镜，我们发现转染细胞中由II型CK 8与I型CKs 18或19形成的对以及由无尾CK 8与无尾CKs 18或19形成的对形成了延伸的细胞质IF网络，证明两种类型的CKs中尾部结构域的缺失并不妨碍规则IF的从头形成。然而，最令人惊讶的是，我们观察到由无尾CKs形成的IF在核质分布上发生了显著变化。在许多转染细胞中，大部分或所有可检测到的CKs都出现在核质中的广泛IF束中。当用编码也缺乏头部结构域的无尾CKs的cDNA转染细胞时，也观察到了CK沉积物的核内积累，然而大多是非纤维状的，而仅在头部结构域缺失的CKs仅在细胞质中发现。讨论了细胞质IF束组装的特定结构域要求，并提出了IF核内积累的可能机制。
