Dose-dependent linkage, assembly inhibition and disassembly of vimentin and cytokeratin 5/14 filaments through plectin's intermediate filament-binding domain

Plectin, the largest and most versatile member of the cytolinker/plakin fam ily of proteins characterized to date, has a tripartite structure comprisin g a central 200 nm-long alpha-helical rod domain flanked by large globular domains. The C-terminal domain comprises a short tail region preceded by si x highly conserved repeats (each 28-39 kDa), one of which (repeat 5) contai ns plectin's intermediate filament (IF)-binding site. We used recombinant a nd native proteins to assess the effects of plectin repeat 5-binding to IF proteins of different types. Quantitative Eu3+-based overlay assays showed that plectin's repeat 5 domain bound to type III IF proteins (vimentin) wit h 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 reveal ed by negative staining and rotary shadowing electron microscopy. This effe ct was much more pronounced in the case of vimentin compared to cytokeratin s 5/14. Preassembled filaments of both types became more and more crosslink ed upon incubation with increasing concentrations of plectin repeat 5, Howe ver, at high proportions of plectin to IF proteins, disassembly of filament s occurred. Again, vimentin filaments proved considerably more sensitive to wards disassembly than those composed of cytokeratins 5 and 14. In general, IFs formed from recombinant proteins were found to be slightly more respon sive towards plectin influences than their native counterparts. A dose-depe ndent plectin-inflicted collapse and putative disruption of Ifs was also ob served in vivo after ectopic expression of vimentin and repeat 5 domain in cotransfected deficient SW13 (vim(-)) cells. Our results suggest an involve ment of plectin not only in crosslinking and plectin's deficient involvemen t stabilization of cytoskeletal regulation of their dynamics.