Report |
Address correspondence to Pierre A. Coulombe, Dept. of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205. Tel.: (410) 614-0510. Fax: (410) 614-0510. E-mail: coulombe{at}jhmi.edu
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Abstract |
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Key Words: keratin; intermediate filament; tail domain; rheology; cross-linker
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Introduction |
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In vitro, keratin filaments can be induced to form large bundles after minor changes to the buffer conditions, in the absence of exogenous proteins. As expected, this significantly enhances the mechanical resilience of keratin gels (Ma et al., 2001). Along with other data (Coulombe et al., 2000), this suggests that keratin IFs can modulate their organization and mechanical properties though self-interaction. Here we provide evidence that the nonhelical tail domain of K14, the predominant type I keratin in basal cells of skin epithelia, binds keratin filaments in vitro and ex vivo, and contributes to the remarkable properties of keratin filaments.
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Results and discussion |
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There is evidence suggesting that the COOH-terminal tail domain of cytoplasmic IF proteins may influence filamentfilament interactions (Herrmann and Aebi, 1998). Moreover, the tail domain of K14 is not required for efficient copolymerization with K5 (Coulombe et al., 1990; Wilson et al., 1992). We compared the properties of wt K5K14 and K5K14T assemblies at pH 7.0 to assess the role of K14's tail domain toward bundling. Whereas wt K5K14 displays the texture of a gel under this condition, the K5K14
T polymer (Fig. 1 A) flows like a liquid. In a high-speed pelleting assay designed to measure polymerization efficiency, >95% of both the wt K5K14 and K5K14
T protein pools are retrieved in the pellet (Fig. 1 B). In a low-speed pelletting assay devised to assess the formation of cross-linked networks (Pollard and Cooper, 1982), nearly all the wt K5K14 proteins end up in the pellet, compared with only
20% for K5K14
T. DIC light microscopy confirms the presence of extensive bundling in the wt K5K14 sample, whereas few bundles can be seen for K5K14
T (unpublished data).
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The K14T protein we used has a 12-amino-acid-long epitope tag at its COOH terminus (Albers and Fuchs, 1987). To rule out that its rheological properties stem in part from this short tag, we repeated these studies with wt K5K19 polymer (Fradette et al., 1998). K19 features a short tail domain (13 amino acids) and is coexpressed with K5 in a subset of progenitor basal cells in the skin (Stasiak et al., 1989). Rheological studies show that K5K19 behaves like a weak viscoelastic solid when subjected to small deformations at pH 7.0. However, as is the case for K5K14
T (Fig. 1 B), the K5K19 polymer rapidly softens when subjected to progressively larger deformations. At 200% strain amplitude, for instance, the wt K5K14 and K5K19 assembled at 0.2 mg/ml (4 µm) exhibit elastic moduli G' of 38 ± 7 and 4.2 ± 0.2 dynes/cm2, respectively. Interestingly, immunoelectron microscopy studies involving human skin epithelia showed that K5K19-rich basal cells exhibit a looser keratin network than K5K14-rich basal cells (Dr. Lucie Germain, personal communication). The data we report here suggest that although the nonhelical tail domain of K14 is dispensable for 10-nm filament assembly, it contributes to the intrinsic potential of K5K14 for interaction with self, which enhances its ability to withstand large deformations.
The purified K14 tail domain binds to keratin filaments in vitro
To assess whether the short nonhelical tail domain of K14 (50 residues) can interact directly with keratin filaments, we added an NH2-terminal histidine (His) tag to facilitate its purification. Purified His-T14 migrates with a mass of
7 kD on SDS-PAGE, reacts with antibodies directed against the His tag and the K14 COOH terminus (Fig. 2 A), and behaves as a monomer with an extended shape in solution (unpublished data). His-T14 cosediments with wt K5K14 filaments produced under standard buffer conditions at pH 7.4 (Fig. 2, B and B', kD
2 µM). In contrast, His-NPT (control for His tag) and insulin (size control) do not cosediment (Fig. 2 B), establishing specificity. His-T14 also cosediments with K8K18 filaments and K5K14
T filaments (Fig. 2 C), but not with vimentin filaments (Fig. 2 C) or F-actin (unpublished data). Therefore, the tail domain of K14 binds directly to determinant(s) shared between K5K14 and K8K18 filaments.
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The K14 tail domain associates with keratin filaments in epithelial cells in culture
We performed transient transfection assays with constructs in which the K14 tail domain is fused to a myc epitope tag. In PtK2 cells, an epithelial cell line that displays keratin and vimentin IF networks, Myc-T14 is detected in the cytoplasm, where it occurs as a filamentous pattern, as well as in the nucleus, where it does not exhibit any pattern (Fig. 3, A and B). Double-immunofluorescence staining shows that in the cytoplasm, the signal for myc-T14 colocalizes with K8K18 filaments to a significant extent (Fig. 3, A', A'', B', and B''), but not with vimentin IFs (unpublished data). The best instances of colocalization involved bundles of keratin filaments whose shape and organization are reminiscent of stress fibers. Dual immunostaining of untransfected PtK2 cells for F-actin and K8K18 shows that the two types of filaments are closely apposed in a significant fraction of cells, indicating that this phenomenon is not a function of myc-T14 expression (unpublished data). Myc-T14 may thus show a greater affinity for a specific subset of keratin filaments, or weakly promote the formation of straight K8K18 bundles. Alternatively, this observation may simply stem from the sensitivity of the assay. Transfected myc-PTE1, used as a control, exhibits a punctate cytoplasmic pattern that is independent from keratin filaments (Fig. 3, CC''). Similar results were obtained in mouse 308 cells, a K5K14-expressing skin keratinocyte line (unpublished data).
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The K14 tail domain interacts with type I keratin sequences in a yeast two-hybrid screen
In an effort to isolate tail domain binding proteins, the COOH termini of both human K14 and K16 were used as baits in a yeast two-hybrid screen involving a human cDNA library made from a squamous cell carcinoma of the leg. Among 2,000,000 cfus screened with the K14 tail domain, 121 scored positive for both growth on His-minus medium and lacZ expression. Among the 40 clones sequenced, 32 (80%) corresponded to type I keratin sequences, and an additional 2 clones corresponded to a keratin-related sequence. The 5' boundary of all the interacting type I keratin clones is given in Fig. 4 A. These findings can be interpreted in two ways. First, the K14 tail domain binds multiple sites along type I keratin sequences (Fig. 4 B). Some of these sites would be shared among them (i.e., linker L12), and others potentially unique (e.g., the head domain of K14). Second, the tail domain binds a restricted number of sites, perhaps a single one, located within the COOH-terminal half of the rod domain of many type I sequences (Fig. 4, A and B). In this case, the heterogeneity observed at the 5' end would reflect the manner with which the reverse transcriptase operated while making the library. Of note, all type I sequences contained at least coil 2 from the rod, and no type II keratin clones were pulled out. Among 3,500,000 cfus screened with the K16 tail domain, 581 scored positive. Among 143 clones that were sequenced, 132 encoded elongation factor 1 , and none encoded a keratin. The complete results of this screen will be published elsewhere. Meanwhile, these studies provide genetic evidence that K14's tail domain, but not K16's, can bind sequence determinant(s) shared by several type I keratins. These findings are interesting given the functional differences observed between K14 and K16 in the context of a protein replacement study in transgenic mouse skin (Paladini and Coulombe, 1999; Wawersik and Coulombe, 2000). These differences are, in part, due to the COOH-terminal 105 amino acids in these two homologous type I keratins. We have rheological evidence that suspensions of K5K16 filaments are mechanically weaker than K5K14 ones when prepared and tested under bundling-promoting conditions (unpublished data).
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The binding site(s) for the K14 tail domain on the keratin polymer is displayed by K5K14, K5K14T, and K8K18 filaments, and may be shared by several type I keratins (as suggested by the yeast two-hybrid screen). Electrostatic forces may well play a role in these interactions, as filamentfilament contacts can be modulated by changes in pH and in salt conditions. Filaggrin, a known keratin cross-linker, binds keratin filaments via electrostatic interactions (Mack et al., 1993). Likewise, the 50-residue-long IF binding motif contained within the tail domain of plectin is rich in basic residues (Nikolic et al., 1996). Given the estimate of
700 keratin monomers per µm of filament length (Herrmann and Aebi, 1998), only a subset of polymer-bound tail domains need be exposed and involved in these interactions to account for a filament cross-bridging effect (Fig. 4 C). Additional studies are needed to define the binding sites and mechanism(s) involved, determine whether this applies to other keratin polymers, and assess the importance of this phenomenon for the mechanical scaffolding function of keratin polymers in vivo.
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Materials and methods |
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Preparation of Myc fusion proteins.
For expression in mammalian cells, the K14 tail sequence was transferred to vector pcDNA3-Nmyc (Jones et al., 1999), which has a CMV promoter and a polyadenylation signal. The NH2 terminus of the resulting fusion proteins is MAEQKLISEEDLLGSGSTM427S, where the tag is underlined and 427S correspond to the first residue of K14's tail domain. Myc-PTE1, which codes for a myc fusion to acyl-CoA thioesterase (Jones et al., 1999), was used as a control.
Production, expression, and purification of recombinant proteins
Plasmids pET-K5, pET-K14 (Coulombe and Fuchs, 1990), pET-K14T (Coulombe et al., 1990), and pET-K19 (Fradette et al., 1998) were used to generate recombinant human proteins. For His-tagged proteins, the plasmids pET-His-T14 and pET-His-NPT were transformed into E. coli strain BL21 (DE3), and protein purification was done using a nickel column (Novagen). Routine electrophoretic assays were used to assess protein size, purity, and identity.
Filament assembly and analysis
Keratin IFs (0.5 or 1 mg/ml) were reconstituted by serial dialysis starting from type Itype II heterotypic complexes (Ma et al., 2001) using the following three buffers at room temperature: (a) 9 M urea, 25 mM Tris-HCl, pH 7.4, 25 mM ß-ME for 4 h; (b) 2 M urea, 5 mM Tris-HCl, pH 7.4, 5 mM ß-ME, for 1 h; and (c) 5 mM Tris-HCl, 5 mM ß-ME, for >12 h (overnight). The pH of the last buffer was adjusted at values of 7.4 or 7.0, depending on the experiment. The physical state of the polymer and polymerization efficiency were assessed as described (Ma et al., 2001). Filament morphology was examined by negative staining (1% uranyl acetate) and electron microscopy (Philips CM120, Germany). Sampling was restricted to regions of the sample where individual filaments could be seen. Larger polymer structures (i.e., bundles) were examined using DIC light microscopy (Eclipse; Nikon). Bundling was assessed by subjecting the assemblies to low-speed centrifugation (8,200 x g for 20 min) (Pollard and Cooper, 1982) followed by SDS-PAGE. Human recombinant vimentin was assembled as described (Herrmann and Aebi, 1998). Purified actin and protocols were provided by Enrique de la Cruz (Yale University, New Haven, CT). For copelletting assays, 50 µl of assembled keratin (10 µM), vimentin (10 µM), and actin filaments (5 µM) were mixed with either His-T14, His-NPT (Geisbrecht et al., 1999), insulin (Sigma-Aldrich), or vinculin's tail domain, a gift from Dr. Susan Craig (Johns Hopkins University, Baltimore, MD). After 2 h, the mixtures were centrifuged at 150,000 x g for 30 min, and the pellet and supernatant fractions were analyzed by SDS-PAGE (Ma et al., 2001).
Rheological studies
All measurements (Coulombe et al., 2000) were obtained using a strain-controlled 50-mm cone-and-plate ARES 100 rheometer (Rheometrics, Inc.) as described (Ma et al., 2001). The linear equilibrium values of the elastic modulus G' () and loss (viscous) modulus G''(
) of the suspensions were measured by setting oscillatory strain amplitude at
= 1% and sweeping from low to high frequency
. Strain-dependent viscoelastic moduli were measured by subjecting polymers to three cycles of deformation of increasing amplitude at 1 rad/sec-1; G' and G'' were computed from the maximum magnitude of the measured stress. To assess strain hardening, we conducted assays in which the stress was continuously monitored as eight cycles of oscillatory shear deformation were applied (Ma et al., 2001). These studies were repeated three times using independent protein preparations.
Yeast two-hybrid screen
PCR products corresponding to the human K14 tail domain, starting at 425His (Marchuk et al., 1984), and the K16 tail domain, starting at 426Ser (Paladini et al., 1995), were fused in frame to the GAL4 DNA binding domain in the yeast expression vector pAS21 (ClONTECH Laboratories, Inc.). For library screening, the Saccaromyces cerevisiae strain Y190 was transformed with AS-T14 or AS-T16. Single colonies were selected after growth in synthetic minimal carbon source (2% sucrose) medium lacking Trp, grown, and retransformed with a human skin cDNA library fused in GAL4 activator domain in pACT2 by a PEG-based method. This library was constructed from poly(A) mRNAs extracted from a squamous cell carcinoma including adjacent normal tissue. mRNA priming for reverse transcription was done using random hexamers, and products of 0.52.0-kb size were used to make the library. Transformed yeast cells were plated on SC medium lacking Trp, Leu, and His in the presence of 25 mM 3-amino-1, 2, 4-triazole, incubated at 30°C for 7 d. Growing colonies (His+) were subjected to ß-galactosidase assay. Plasmids harboring cDNA were recovered from positive colonies and sequenced. As negative controls, AS-T14 and AS-T16 were tested for their interaction with K5 or K6 protein.
Cell lines, transfections, and immunological reagents
Cell lines were maintained as recommended by their sources: PtK2 (kidney epithelium of the rat kangaroo; American Type Culture Collection); 308 (mouse epidermal keratinocytes), a gift of Dr. Stuart Yuspa (National Cancer Institute, Bethesda, MD); and BHK-21 (baby hamster kidney fibroblasts; American Type Culture Collection). Transient transfections were performed using polyethylenimine (Sigma-Aldrich) as described (Boussif et al., 1995). At 24 or 48 h posttransfection, cells were fixed with 3.0% paraformaldehyde for 15 min, extracted with 0.01% Triton-X in PBS buffer for 5 min, and processed for indirect immunofluorescence. We used rabbit polyclonal antisera against K18 and K14, obtained from E. Fuchs (University of Chicago, Chicago, IL); mouse monoclonals against vimentin (V9; Sigma-Aldrich); the myc epitope (American Type Culture Collection) K14, a gift from Dr. Irene Leigh (Imperial Cancer Research Fund, London, UK), and K8K18, a gift from Dr. Bishr Omary (Stanford university, Stanford, CA); and chicken polyclonal against K14 (Wawersik and Coulombe, 2000). For immunofluorescence microscopy, rhodamine- or FITC-conjugated goat antimouse or antirabbit secondary antibodies were used (Kirkegaard and Perry Labs). For Western blotting, we used enhanced chemiluminescence (Amersham Pharmacia Biotech) or alkaline phosphatase (Bio-Rad Laboratories) detection.
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Footnotes |
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* Abbreviations used in this paper: His, histidine; IF, intermediate filament.
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Acknowledgments |
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These studies were supported by National Institutes of Health grants AR44232 and AR42047 to P.A. Coulombe, and National Science Foundation Grants CTS9812624 and DB19729358 to D. Wirtz.
Submitted: 16 April 2001
Revised: 8 October 2001
Accepted: 9 October 2001
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References |
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