Eger A, Stockinger A, Wiche G, Foisner R
Institute of Biochemistry and Molecular Cell Biology, University of Vienna, Biocenter, Austria.
J Cell Sci. 1997 Jun;110 ( Pt 11):1307-16. doi: 10.1242/jcs.110.11.1307.
The intermediate filament-binding protein plectin and cytokeratin were localised at the cellular periphery of fully polarised Madin-Darby canine kidney (MDCK) cells, whereas vimentin was primarily found in a perinuclear network. Confocal and immunoelectron microscopy revealed that plectin was restricted to areas underlying the lateral plasma membrane. It colocalised with fodrin, a component of the submembrane skeleton, and was closely associated with desmosomal plaque structures. Biochemically, plectin was shown to interact directly with immunoprecipitated desmoplakin in vitro. Upon loss of cell polarity in low calcium medium, plectin redistributed to a cytoplasmic vimentin- and cytokeratin-related network, clearly distinct from diffusely distributed fodrin and internalised desmoplakin structures. The structural reorganisation of plectin was also reflected by an increased solubility of the protein in Triton X-100/high salt, and a decrease in its half-life from approximately 20 to approximately 5 hours. Furthermore, unlike cytokeratins and vimentin, desmoplakin and fodrin did not associate with plectin attached to magnetic beads in cell lysates of unpolarised cells, while all proteins formed a stable complex in polarised cells. Altogether, these data indicate that plectin is involved in the anchorage of intermediate filaments to desmosomes and to the submembrane skeleton in polarised MDCK cells.
中间丝结合蛋白网蛋白和细胞角蛋白定位于完全极化的犬肾（MDCK）细胞的细胞周边，而波形蛋白主要存在于核周网络中。共聚焦显微镜和免疫电子显微镜显示，网蛋白局限于外侧质膜下方的区域。它与膜下骨架的一个成分血影蛋白共定位，并与桥粒斑结构紧密相关。生化分析表明，网蛋白在体外可直接与免疫沉淀的桥粒芯蛋白相互作用。在低钙培养基中细胞极性丧失后，网蛋白重新分布到与波形蛋白和细胞角蛋白相关的细胞质网络中，明显不同于弥散分布的血影蛋白和内化的桥粒芯蛋白结构。网蛋白的结构重组还表现为该蛋白在Triton X-100/高盐中的溶解度增加，其半衰期从约20小时缩短至约5小时。此外，与细胞角蛋白和波形蛋白不同，在未极化细胞的细胞裂解物中，桥粒芯蛋白和血影蛋白不与附着在磁珠上的网蛋白结合，而在极化细胞中所有蛋白质形成稳定的复合物。总之，这些数据表明网蛋白参与了极化MDCK细胞中中间丝与桥粒以及膜下骨架的锚定。
