Journal of Cell Science 113, 483-491 (2000) 483 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0847 Dose-dependent linkage, assembly inhibition and disassembly of vimentin and cytokeratin 5/14 filaments through plectin’s intermediate filament-binding domain Ferdinand A. Steinböck1, Branislav Nikolic1, Pierre A. Coulombe2, Elaine Fuchs3, Peter Traub4 and Gerhard Wiche1,* 1Institute of Biochemistry and Molecular Cell Biology, University of Vienna, Vienna Biocenter, 1030 Vienna, Austria 2Departments of Biological Chemistry and Dermatology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA 3Howard Hughes Medical Institute, Departments of Molecular Genetics and Cell Biology and of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA 4Max-Planck-Institut für Zellbiologie, D-68526 Ladenburg, Germany *Author for correspondence (e-mail: [email protected]) Accepted 26 November 1999; published on WWW 19 January 2000 SUMMARY Plectin, the largest and most versatile member of the became more and more crosslinked upon incubation with cytolinker/plakin family of proteins characterized to date, increasing concentrations of plectin repeat 5. However, at has a tripartite structure comprising a central 200 nm-long high proportions of plectin to IF proteins, disassembly of α-helical rod domain flanked by large globular domains. filaments occurred. Again, vimentin filaments proved The C-terminal domain comprises a short tail region considerably more sensitive towards disassembly than preceded by six highly conserved repeats (each 28-39 kDa), those composed of cytokeratins 5 and 14. In general, IFs one of which (repeat 5) contains plectin’s intermediate formed from recombinant proteins were found to be filament (IF)-binding site. We used recombinant and native slightly more responsive towards plectin influences than proteins to assess the effects of plectin repeat 5-binding to their native counterparts. A dose-dependent plectin- IF proteins of different types. Quantitative Eu3+-based inflicted collapse and putative disruption of IFs was also overlay assays showed that plectin’s repeat 5 domain bound observed in vivo after ectopic expression of vimentin and to type III IF proteins (vimentin) with preference over type plectin’s repeat 5 domain in cotransfected vimentin- I and II cytokeratins 5 and 14. The ability of both types of deficient SW13 (vim−) cells. Our results suggest an IF proteins to self-assemble into filaments in vitro was involvement of plectin not only in crosslinking and impaired by plectin’s repeat 5 domain in a concentration- stabilization of cytoskeletal IF networks, but also in dependent manner, as revealed by negative staining and regulation of their dynamics. rotary shadowing electron microscopy. This effect was much more pronounced in the case of vimentin compared Key words: Plectin, Cytolinker protein, Intermediate filament, to cytokeratins 5/14. Preassembled filaments of both types Vimentin, Cytokeratin INTRODUCTION neurofilament proteins and lamin B), and its interaction with the actin cytoskeleton, components of the subplasma The intermediate filament-associated protein plectin (Wiche et membrane skeleton (α-spectrin/fodrin), transmembrane al., 1982) is abundantly expressed in many different tissues and integrin receptors, and high molecular mass microtubule cell types. Based on the phenotypic analyses of epidermolysis associated proteins (for recent reviews, see Fuchs and bullosa simplex (EBS)-MD patients, who carry autosomal Cleveland, 1998; Wiche, 1998; Steinböck and Wiche, 1999). recessive mutations in the plectin gene (Gache et al., 1996; Together with the neuronal and epithelial isoforms of BPAG1 McLean et al., 1996; Smith et al., 1996), as well as of plectin- (Stanley et al., 1981), dystonin/BPAG1n (Brown et al., deficient mice (Andrä et al., 1997), the protein has been 1995a,b; Yang et al., 1996) and BPAG1e (Sawamura et al., proposed to provide cells with mechanical strength by acting 1991), periplakin (Ruhrberg et al., 1997), envoplakin as a linker element of the cytoskeleton. This was supported by (Ruhrberg et al., 1996) and desmoplakin (Green et al., 1990; several previous studies showing the binding of plectin to 1992), plectin forms a family of structurally related proteins, cytoplasmic as well as nuclear intermediate filament (IF) referred to as the plakin or cytolinker protein family (Uitto et subunit proteins (vimentin, desmin, cytokeratins, GFAP, al., 1996; Wiche, 1998). 484 F. A. Steinböck and others With a molecular mass of >500 kDa, plectin is the largest urea, 50 mM Tris, pH 8.1, 2 mM DTT and 0.3 mg/ml PMSF. Further known cytolinker protein. The molecule has a dumbbell-like purification of vimentin and cytokeratins was done by ion-exchange shape with two globular domains flanking a long α-helical column chromatography using Pharmacia DEAE Sepharose CL-6B. coiled-coil rod structure (Foisner and Wiche, 1987; Wiche et Proteins were eluted by a gradient of 0-0.2 M guanidine-HCl in − al., 1991). Transient transfection of plectin cDNA expression solubilizing solution and stored at 80°C. The molecular masses of constructs into monkey (COS) and rat kangaroo (PtK2) cell the purified recombinant versions of vimentin and cytokeratins 5 and 14 were indistinguishable from their native counterparts upon analysis lines, both of which contain dense networks of vimentin as well by SDS-PAGE (data not shown). The purity of protein preparations as cytokeratin filaments, indicated that only the carboxy- was as documented previously (Coulombe and Fuchs, 1990; Nikolic terminal globular domain, and not the amino-terminal globular et al., 1996). or the central rod domain of the molecule, was indispensable for IF association (Wiche et al., 1993). In subsequent studies Purification of IF proteins from cells and tissues the IF-binding site of plectin could be mapped to a stretch of Vimentin was purified from rat glioma C6 cells following the protocol approximately 50 amino acid residues residing within repeat 5, of Foisner et al. (1988). Samples of vimentin were stored frozen in 6 the penultimate of six homologous repeats constituting M urea at −80˚C. Cytokeratins (K5, K6, K14, K17 and small amounts plectin’s carboxy-terminal globular domain (Nikolic et al., of K16) were isolated from human skin and purified by Mono Q 1996). This stretch is located just downstream of a highly column according to Wawersik et al. (1997). conserved central region approximately 200 amino acids long In vitro assembly of IFs (consisting of tandem repeats of a 19-amino-acid motif), which Prior to assembly, samples of recombinant or native vimentin were is common to all six carboxy-terminal repeats (Wiche et al., dialyzed against 5 mM Tris, pH 8.5, 1 mM EDTA and 1 mM DTT 1991; Liu et al., 1996). (overnight at 4°C), and centrifuged at 200000 g for 10 minutes at 4°C, To investigate the binding and networking capacities of using a Beckman benchtop ultracentrifuge. After addition of 0.1 plectin for different types of IFs we have carried out volumes of 200 mM Tris, pH 7.0, and 1.6 M NaCl, assembly mixtures biochemical and ultrastructural studies using native and/or were incubated for 1 hour at 37°C. Cytokeratins 5 and 14 were bacterially expressed recombinant forms of vimentin (type III coassembled by dialysis first against 5 M urea in 50 mM Tris, pH 8.1, IF protein) and cytokeratins 5 and 14 (type I and II IF proteins). 2 mM DTT, 0.3 mg/ml PMSF, for 2 hours at room temperature, then We report here that plectin’s repeat 5 domain was able to bind against 2.5 M urea (in the same solution) for 2 hours at room temperature, and finally against 2 mM Tris, pH 9.0, overnight at 4°C. to and crosslink all types of IF proteins, but showed higher Filament assembly was induced by addition of 0.1 volumes of 100 affinity for type III IF proteins. Interestingly, we found that this mM Tris, pH 7.0, followed by incubation for 2 hours at room domain could also inhibit filament assembly in vitro in a temperature. concentration-dependent manner, with distinct efficiencies depending on the type of IF subunit protein, and that it could Electron microscopy cause the disruption of assembled IFs. For negative staining, 6 µl of in vitro assembly mixtures pipetted onto Formvar/carbon-coated and glow-discharged 400 mesh copper grids, were stained with 10 drops of 1% uranyl acetate for approximately 90 MATERIALS AND METHODS seconds. For rotary shadowing, samples (100 µl, mixed 1:1 with glycerol) were sprayed onto freshly cleaved mica and prepared for Expression plasmids electron microscopy essentially as described by Tyler and Branton For bacterial expression, full-length mouse vimentin cDNA (GenBank (1980). Shadowing with platinum (0.74 nm) occurred at an angle of accession number M26251), including the stop codon at position bp 7°, with carbon (10 nm) at 90°. Specimens were visualized in a JEOL 1879-1881, was excised from mammalian expression vector pMC- 1210 electron microscope operated at 80 kV. V21 (Ming Chen, 1992) and subcloned in several steps into a modified version of plasmid pET23a (Novagen), yielding plasmid pFS129. Tag- Europium overlay binding assay less vimentin encoded by pFS129 was used in all experiments. Urea-solubilized IF proteins were dialyzed against 50 mM NaHCO3, pET15b-derived plasmids pBN135 and pBN132 (encoding pH 8.5, and labeled with Eu3+ overnight at room temperature, using plectin’s repeat domains 4 and 5, respectively) have been described in 10 µl Eu3+-labeling reagent per 100 µl protein (0.5-1.5 mg/ml) Nikolic et al. (1996), and plasmids pET-K5 and pET-K14 (encoding according to the protocol of the manufacturer (Wallac, Turku, human cytokeratins K5 and K14, respectively) in Coulombe and Finland). Fuchs (1990). For binding assays, 96-well microtiter plates were coated For transfection of cultured cells, we used plasmids pBN36 and (overnight at 4°C) with 100 µl of recombinant plectin proteins (100 pBN47, both derived from mammalian expression vector pAD29 nM) or BSA type H1 (Gerbu, Gaiberg, Germany), all in 25 mM (Nikolic et al., 1996), encoding amino acid residues leu4069-leu4367 Na2B4O7, pH 9.3.