Steinböck F A, Nikolic B, Coulombe P A, Fuchs E, Traub P, Wiche G
Institute of Biochemistry, University of Vienna, Vienna Biocenter, Austria.
J Cell Sci. 2000 Feb;113 ( Pt 3):483-91. doi: 10.1242/jcs.113.3.483.
Plectin, the largest and most versatile member of the cytolinker/plakin family of proteins characterized to date, has a tripartite structure comprising a central 200 nm-long (&agr;)-helical rod domain flanked by large globular domains. The C-terminal domain comprises a short tail region preceded by six highly conserved repeats (each 28-39 kDa), one of which (repeat 5) contains plectin's intermediate filament (IF)-binding site. We used recombinant and native proteins to assess the effects of plectin repeat 5-binding to IF proteins of different types. Quantitative Eu(3+)-based overlay assays showed that plectin's repeat 5 domain bound to type III IF proteins (vimentin) with preference over type I and II cytokeratins 5 and 14. The ability of both types of IF proteins to self-assemble into filaments in vitro was impaired by plectin's repeat 5 domain in a concentration-dependent manner, as revealed by negative staining and rotary shadowing electron microscopy. This effect was much more pronounced in the case of vimentin compared to cytokeratins 5/14. Preassembled filaments of both types became more and more crosslinked upon incubation with increasing concentrations of plectin repeat 5. However, at high proportions of plectin to IF proteins, disassembly of filaments occurred. Again, vimentin filaments proved considerably more sensitive towards disassembly than those composed of cytokeratins 5 and 14. In general, IFs formed from recombinant proteins were found to be slightly more responsive towards plectin influences than their native counterparts. A dose-dependent plectin-inflicted collapse and putative disruption of IFs was also observed in vivo after ectopic expression of vimentin and plectin's repeat 5 domain in cotransfected vimentin-deficient SW13 (vim(-)) cells. Our results suggest an involvement of plectin not only in crosslinking and stabilization of cytoskeletal IF networks, but also in regulation of their dynamics.
网蛋白是迄今为止已鉴定的细胞连接蛋白/片层蛋白家族中最大且功能最多样的成员，具有三方结构，由一个200纳米长的中央α螺旋杆状结构域和两侧的大球状结构域组成。C末端结构域包括一个短尾区域，其前面有六个高度保守的重复序列（每个28 - 39 kDa），其中一个（重复序列5）包含网蛋白的中间丝（IF）结合位点。我们使用重组蛋白和天然蛋白来评估网蛋白重复序列5与不同类型IF蛋白结合的影响。基于铕（III）的定量覆盖分析表明，网蛋白的重复序列5结构域优先与III型IF蛋白（波形蛋白）结合，而不是I型和II型细胞角蛋白5和14。负染色和旋转阴影电子显微镜显示，两种类型的IF蛋白在体外自组装成丝的能力都受到网蛋白重复序列5结构域的浓度依赖性损害。与细胞角蛋白5/14相比，波形蛋白的这种效应更为明显。随着网蛋白重复序列5浓度的增加，两种类型的预组装丝在孵育时交联越来越多。然而，当网蛋白与IF蛋白的比例很高时，丝会发生解聚。同样，波形蛋白丝对解聚的敏感性比由细胞角蛋白5和14组成的丝高得多。一般来说，发现由重组蛋白形成的IF对网蛋白影响的反应比天然对应物略高。在共转染的波形蛋白缺陷型SW13（vim（-））细胞中异位表达波形蛋白和网蛋白的重复序列5结构域后，在体内也观察到了网蛋白引起的剂量依赖性IF塌陷和假定的破坏。我们的结果表明，网蛋白不仅参与细胞骨架IF网络的交联和稳定，还参与其动力学的调节。
