CELL STRUCTURE AND FUNCTION 39: 31–43 (2014) © 2014 by Japan Society for Cell Biology

In vitro Assembly Properties of Human Type I and II Hair

Yuko Honda1, Kenzo Koike2, Yuki Kubo3, Sadahiko Masuko1, Yuki Arakawa4, and Shoji Ando4* 1Department of Anatomy and Physiology, Faculty of Medicine, Saga University, Nabeshima 5-1-1, Saga 849-8501, Japan, 2Beauty Research Center, KAO Corporation, Bunka 2-1-3, Sumida-ku, Tokyo 131-8501, Japan, 3Department of Biomolecular Sciences, Faculty of Medicine, Saga University, Nabeshima 5-1-1, Saga 849-8501, Japan, 4Faculty of Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Nishi-ku, Kumamoto 860-0082, Japan

ABSTRACT. Multiple type I and II hair keratins are expressed in hair-forming cells but the role of each protein in hair fiber formation remains obscure. In this study, recombinant proteins of human type I hair keratins (K35, K36 and K38) and type II hair keratins (K81 and K85) were prepared using bacterial expression systems. The heterotypic subunit interactions between the type I and II hair keratins were characterized using two- dimensional gel electrophoresis and surface plasmon resonance (SPR). Gel electrophoresis showed that the heterotypic complex-forming urea concentrations differ depending on the combination of keratins. K35-K85 and K36-K81 formed relatively stable heterotypic complexes. SPR revealed that soluble K35 bound to immobilized K85 with a higher affinity than to immobilized K81. The in vitro (IF) assembly of the hair keratins was explored by negative-staining electron microscopy. While K35-K81, K36-K81 and K35-K36-K81 formed IFs, K35-K85 afforded tight bundles of short IFs and large paracrystalline assemblies, and K36-K85 formed IF tangles. K85 promotes lateral association rather than elongation of short IFs. The in vitro assembly properties of hair keratins depended on the combination of type I and II hair keratins. Our data suggest the functional significance of K35-K85 and K36-K81 with distinct assembly properties in the formation of macrofibrils.

Key words: , hair keratins, intermediate filaments, protein-protein interaction, self-assembly

Introduction Rogers et al., 2004, 2005; Schweizer et al., 2007). Like other IF proteins, keratins have a characteristic tripartite Keratins are the most complex subgroup of intermediate structure that includes a central α-helical rod domain filament (IF) proteins (Coulombe and Omary, 2002; flanked by non-α-helical N-terminal head and C-terminal Langbein et al., 1999, 2001; Langbein and Schweizer, tail domains (Fuchs and Weber, 1994). The rod domain, 2005; Langbein et al., 2007; Rogers et al., 2004, 2005; consisting of approximately 310 amino acids, has relatively Schweizer et al., 2007). In the human genome, IF proteins conserved sequences at the N- and C-terminal ends, and include at least 37 (epithelial ) genes displays long heptad repeat patterns (abcdefg) of hydro- expressed in the different types of epithelia and at least 17 phobic residues (a and d). In contrast, the head and tail genes expressed in hair- or nail-forming cells domains of IF proteins have variable length, sequences and (Coulombe and Omary, 2002; Langbein et al., 1999, 2001; chemical characteristics. From the extent of sequence Langbein and Schweizer, 2005; Langbein et al., 2007; homology of the rod domain, acidic keratins are classified into as type I, whereas neutral-basic keratins are type II. *To whom correspondence should be addressed: Shoji Ando, Faculty of Both type I and II keratins are required to form IFs in vivo Biotechnology and Life Science, Sojo University, Ikeda 4-22-1, Nishi-ku, and in vitro (Coulombe and Omary, 2002; Fuchs and Kumamoto 860-0082, Japan. Weber, 1994; Herrmann and Aebi, 2004). Tel/Fax: +81–96–326–3927 E-mail: [email protected] According to a recently introduced nomenclature for Abbreviations: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio] mammalian keratins, type I hair keratins are numbered propanesulfonate; IEF, isoelectric focusing; IF, intermediate filament; KAPs, from 31 to 40 (33a and 33b are isoforms), while type II hair keratin-associated proteins; MF, macrofibril; PMSF, phenylmethylsulfonyl keratins are from 81 to 86 (Schweizer et al., 2006). Here fluoride; proto-MF, proto-macrofibril; RU, resonance unit; SPR, surface plasmon resonance; TCEP, tris(2-carboxyethyl)phosphine; TEM, transmission we describe these proteins as, for example, keratin 81 as electron microscopy; ULF, unit-length filament. K81. The complex expression patterns of hair keratins as

31 Y. Honda et al. well as in human hair follicles have been between particular type I and II hair keratins has not been determined (Langbein et al., 1999, 2001; Langbein and characterized. Furthermore, the fundamental significance of Schweizer, 2005; Langbein et al., 2007; Rogers et al., the multiplicity of hair keratins in hair-forming compart- 2004, 2005; Schweizer et al., 2007). The tissue units out- ments remains unknown. side the hair-forming compartments are the outer root The formation of macrofibrils (MFs) is not well under- sheath, companion layer and inner root sheath, each of stood. MFs are the hair keratin IF bundles that are observed which expresses specific sets of cytokeratins. By contrast, in the cortex of hair fibers and wool by transmission elec- the hair-forming compartments such as the matrix (just tron microscopy (TEM), and have maximum lengths of above the germinative cells), cortex (differentiated from the approximately 10 μm (Bryson et al., 2009; Chapman and matrix and constituting the bulk of hair fibers) and cuticle Gemmell, 1971; Jones and Pope, 1985; Marshall et al., (surrounding the cortex) show complex expression patterns 1991; Morioka, 2009; Orwin, 1979; Orwin and Woods, of hair keratins depending on hair differentiation. In the 1982; Plowman et al., 2007). In MFs, hair keratin IFs are early stage of differentiation, the matrix/pre-cortex ex- embedded in a matrix material composed of hair keratin- presses K35 and K85, while the early cuticle expresses associated proteins (KAPs) that link each other and to IFs K32, K35 and K85. During the middle stage of differentia- (Rogers et al., 2006). Three kinds of MFs have been tion, the lower cortex begins to express K31, and the cuticle observed and their distributions across hair fibers and wool begins to express K82. The lower cortex also weakly may affect hair curvature or crimp (Bryson et al., 2009; expresses K38 at this stage, and the K38-positive cells scat- Marshall et al., 1991; Plowman et al., 2007). ter heterogeneously in the cortex. At an advanced stage of In this study, we prepared recombinant human type I hair differentiation, the mid- to upper cortex expresses almost keratins K35, K36, K38, and type II hair keratins K81 and simultaneously K33a, K33b, K36, K81, K83, K86, and K85, and examined their protein-protein interactions with finally K34 and K39. The cuticle layer expresses K39 and an emphasis on the specificity of interactions between par- K40. The complex mechanism of hair keratin expression ticular type I and II proteins. The proteins were also used to makes it difficult to clearly determine the specific type I explore the characteristics of in vitro IF formation by and II hair keratin pairs used to form IFs, in contrast to the changing the subunit combinations of the type I and II hair specific heterotypic pair expression of cytokeratins in vari- keratins. K35 and K85 are the first pairs expressed in the ous types of epithelia (Fuchs and Weber, 1994). It is also human hair matrix/pre-cortex in the early stages of differ- unknown why gene mutations have been reported only in entiation, while K36 and K81 are expressed in the mid- to type II hair keratins such as K81, K83, K85 and K86. These upper cortex at an advanced stage of differentiation mutations are the underlying cause of inherited diseases (Langbein et al., 1999, 2001; Langbein and Schweizer, including monilethrix and ectodermal dysplasia of hair and 2005; Schweizer et al., 2007). K38 is expressed in cells nail types (Schweizer et al., 2007; Shimomura et al., 2010). scattered randomly throughout the entire cortex (Langbein For cytokeratins, many point mutations have been discov- et al., 1999; Langbein and Schweizer, 2005; Yu et al., ered in both the type I and II cytokeratin genes in patients 2009), and is suggested to be related to the curvature of a suffering from epithelial disorders such as epidermolysis hair fiber (Thibaut et al., 2007). We observed that the affin- bullosa simplex and epidermolytic hyperkeratosis (Fuchs ities in the heterotypic subunit interactions depend on the and Weber, 1994). combination of type I and II hair keratins. In IF formation IF formation in vivo and in vitro have been well docu- experiments, while K81 afforded IFs by copolymerization mented for cytokeratins (Coulombe and Omary, 2002; with type I hair keratins, the combination of K85 and K35 Fuchs and Weber, 1994; Herrmann et al., 2002; Herrmann gave tight bundles of short IFs and their large paracrystal- and Aebi, 2004). Both type I and II cytokeratins are needed line assemblies. The importance of the different assembly to form heterotypic coiled-coil dimers that then polymerize properties of K81 and K85 in the formation of MFs in the into full-width short filaments, i.e., unit-length filaments cortex is discussed. (ULFs), with a diameter of approximately 15 nm and length of 50–70 nm (Herrmann et al., 2002). The ULFs anneal longitudinally and compact radially to form mature IFs Materials and Methods with a diameter of approximately 10 nm and length of sev- eral micrometers (Herrmann et al., 2002). Few studies on Preparation of recombinant human hair keratins and IF assembly of hair keratins have been undertaken. Wang et cytokeratins al. (Wang et al., 2000) reported efficient in vitro IF assem- bly of a mouse hair keratin pair. Hofmann et al. (Hofmann The DNAs coding for complete sequences of human type I hair et al., 2002) reported that in vitro IF formation of human keratins (K35: NM_002280, 1.4 kb; K36: NM_003771, 1.4 kb; hair keratins K31, K83 and K86 differs from that of human K38: NM_006771, 1.4 kb) and type II hair keratins (K81: cytokeratins because of the requirement of a relatively high NM_002281, 1.5 kb; K85: NM_002283, 1.5 kb), flanked by 5' ionic strength. However, the specificity of the interactions NdeI and 3' BamHI restriction sites, were chemically synthesized.

32 Assembly of Hair Keratins

The codon usage was adapted to the codon usage bias of E. coli concentration of each protein was determined spectrophotometri- genes. The DNAs sequences were cloned into appropriate plas- cally using a Quant-iT Protein Assay kit (Life Technologies, mids (pBluescript, pMK or pMA) and then confirmed by sequenc- Carlsbad, CA, USA) and Quant-iT Protein standard. ing. The constructs were digested with NdeI and BamHI, and the resulting DNA fragments were isolated and then subcloned into Surface plasmon resonance measurements pET3a plasmid (Merck, Darmstadt, Germany) that had been pre- viously digested with NdeI and BamHI. The full-length human Homo- and heterotypic subunit interactions of the type I and II cytokeratin 14 cDNA (1.4 kb) and cytokeratin 5 cDNA (1.8 kb), hair keratins were determined using surface plasmon resonance flanked by 5' NdeI and 3' BamHI restriction sites, were construc- (SPR) technology on a Biacore 1000 (GE Healthcare). Activation ted by PCR amplification using DNAs from the human mammary of the carboxyl groups of a CM5 sensor chip was carried out gland cDNA library (Takara, Otsu, Japan) as a template. The according to manufacturer’s instructions. Type II hair keratin was resulting DNA samples were cloned into pT7blue (Merck), con- immobilized on the sensor chip as a ligand through the amino firmed by sequencing, and then subcloned into pET24c plasmid group as follows. K81 or K85 was injected at a concentration of (Merck). 165 or 172 μg/ml, respectively, in 10 mM sodium acetate, pH 4–5, The identity of each construct was confirmed by restriction 5 mM DTT and 6 M urea, at a flow rate of 5 μl/min. The amount enzyme analysis and by expression in E. coli strain Rosetta(DE3) of protein immobilized on the sensor chip was adjusted by con- pLysS (Merck) using isopropyl-β-D-thiogalactopyranoside (1 mM) trolling the contact time of the sample with the activated surface induction. Recombinant protein was enriched in the inclusion of the sensor chip. As a result, 7279 resonance units (RU) of K81 body, which was isolated by the method of Nagai and Thøgersen or 7260 RU of K85 was immobilized on the sensor chip (1000 (Nagai and Thøgersen, 1987), as follows. After expression for 3–5 RU=1 ng/mm2). Unreacted carboxyl groups of the sensor chip h at 37°C, approximately 4 g of bacteria were harvested and were blocked using 1 M ethanolamine (pH 8.5). Soluble type I or resuspended in 10 ml of lysis buffer (50 mM Tris-HCl, pH 8.0, II hair keratin to be used as an analyte was dialyzed against a buf- 25% sucrose and 1 mM EDTA). The cells were lysed by addition fer solution (2 mM Tris-HCl, pH 9.0, 5 mM DTT and protease of 25 mg of lysozyme (Sigma-Aldrich, St. Louis, MO, USA) dis- inhibiter cocktail), and then diluted to a protein concentration of solved in 2.5 ml of the lysis buffer and then aging the mixture for 125–500 nM in 10 mM HEPES-NaOH, pH 7.4–8.5, containing

30 min on ice. MgCl2, MnCl2 and DNase I (Sigma-Aldrich) were 50–200 mM NaCl, 3.4 mM EDTA, 5 mM DTT and 0.005% then added to give final concentrations of 10 mM, 1 mM and 10 Tween 20. The analyte solution was injected over the sensor chip μg/ml, respectively. After 30 min, 25 ml of detergent buffer (20 at a flow rate of 5 μl/min at 25°C. The surface of the sensor chip mM Tris-HCl, pH 7.5, 0.2 M NaCl, 1% deoxycholic acid, 1% was regenerated by washing with a basic solution (10 mM Nonidet P-40, and 2 mM EDTA) was added to the lysate, which HEPES-NaOH, pH 8.0, 50 mM NaCl, 5 mM DTT and 7 M urea). was then centrifuged at 5000×g for 10 min at 4°C. The resulting A control experiment was performed using a surface that did not pellet was resuspended in 30 ml of washing buffer (20 mM Tris- contain immobilized protein. The blank sensorgram was subtrac- HCl, pH 7.5, 0.5% Triton X-100, and 1 mM EDTA) and then cen- ted from the assay curve using BIAevaluation 3.0 software (GE trifuged at 5000×g for 10 min at 4°C. The supernatant was Healthcare). discarded. This washing step was repeated until a tight pellet of inclusion body was obtained. Recombinant protein was extracted Two-dimensional gel electrophoresis from the inclusion body using 30 ml of buffer 1 (10 mM Tris-HCl, pH 7.5, 8 M urea, 2 mM EDTA, 5 mM DTT, 1 mM phenylmethyl- Equimolar amounts of the purified type I and II hair keratins were sulfonyl fluoride (PMSF), and an adequate amount of protease mixed to give a total protein concentration of approximately 0.1 inhibiter cocktail) at 10°C. After centrifugation at 100,000×g at mg/ml in 400 μl of 10 mM Tris-HCl, pH 8.0, 9.5 M urea, 2 mM 10°C for 1 h, the supernatant was loaded on a Source 15Q (GE EDTA, 10 mM DTT, and then dialyzed against lower concentrations Healthcare, Buckinghamshire, UK) column (1×9 cm) equilibrated (4–7 M) of urea in 10 mM Tris-HCl, pH 7.5, 1% Triton X-100, with buffer 2 (10 mM Tris-HCl, pH 8.0, 6 M urea, 1 mM EDTA, 5 1% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate mM DTT, 0.5 mM PMSF, and protease inhibitor cocktail). Pro- (CHAPS), 64 mM DTT and protease inhibitor cocktail. After teins were eluted from the column by application of a linear con- addition of Pharmalyte (GE Healthcare) and Orange G (Sigma- centration gradient of NaCl (0–0.3 M over 70 min) in buffer 2. Aldrich) to give final concentrations of 1% and 0.0025%, respec- The fractions containing desired proteins were collected, dialyzed tively, the sample solution was used to swell an Immobiline against buffer 3 (10 mM Tris-HCl, pH 8.0, 8.5 M urea, 2 mM DryStrip (pH 4–7, 7 cm long, GE Healthcare) at 20°C for 16 h. EDTA, 10 mM DTT, and protease inhibitor cocktail), cleared of Isoelectric focusing (IEF) in the first dimension was performed on aggregates by centrifugation at 100,000×g at 10°C for 1 h, and a CoolPhorestar Type-P (Anatech, Tokyo, Japan) using the manu- stored at –80°C. The authenticity of the protein was verified by facturer’s program. After IEF, the gel strip was treated in 25 mM SDS-PAGE, amino terminal sequence analysis using a PPSQ sys- Tris-HCl, pH 6.8, 6 M urea, 30 mM DTT, 2% SDS, 25% glycerol tem (Shimadzu, Kyoto, Japan), and peptide mass fingerprinting and 0.0025% bromophenol blue for 30 min, and then treated with using an Autoflex II matrix-assisted laser desorption/ionization 10% SDS-PAGE in the second dimension. The separated proteins mass spectrometer (Bruker Daltonics, Billerica, MA, USA). The were visualized by staining with Coomassie Brilliant Blue.

33 Y. Honda et al.

Bovine serum albumin (BSA, Sigma-Aldrich) was used as a marker protein.

In vitro IF assembly

Equimolar amounts of purified type I and II hair keratins were mixed at protein concentrations of 0.05–0.1 mg/ml, in 10 mM Tris-HCl, pH 8.0, 9.5 M urea, 2.5 mM EDTA, 10 mM DTT, and an adequate amount of protease inhibitor cocktail at 25°C. The urea concentration was lowered by stepwise dialysis against 4 M and 2 M urea in the same buffer, and then against 25 mM Tris- HCl, pH 7.5, 10 mM DTT or 5 mM tris(2-carboxyethyl)phosphine Fig. 1. SDS-PAGE of recombinant human hair keratins and cytokeratins (TCEP), 2.5 mM EDTA, and protease inhibitor cocktail at 25°C. prepared in this study. The purified polypeptides were resolved on 10% Final dialysis was performed against “filament formation buffer” polyacrylamide gel, and stained with Coomassie brilliant blue: lane 1, hair keratin 35 (K35); lane 2, hair keratin 36 (K36); lane 3, hair keratin 38 consisiting of 25 mM Tris-HCl, pH 7.2, 1 mM MgCl2, 180 mM NaCl, 10 mM DTT or 5 mM TCEP, and protease inhibitor cock- (K38); lane 4, hair keratin 81 (K81); lane 5, hair keratin 85 (K85), lane 6, cytokeratin 14 (K14); lane 7, cytokeratin 5 (K5). tail, at 37°C. In these procedures, all buffers were degassed and then stored under N2 atmosphere before use, and the dialysis was carried out in anaerobic bags filled with N2 gas. For cytokeratins 14 and 5, IF assembly was carried out as follows. Equimolar ion-exchange column chromatography as described under amounts of both proteins were mixed at protein concentrations of “Materials and Methods”. The authenticities of the proteins 0.1–0.2 mg/ml, in 25 mM Tris-HCl, pH 7.5, 9.0 M urea, and 5 were verified by SDS-PAGE (Fig. 1), amino terminal mM DTT. The urea concentration was lowered by dialysis against sequence analysis, and mass spectrometry. Yields of the 2 M urea in the same buffer. Final dialysis was performed against purified proteins were 6–56 mg/l of bacterial growth 5 mM Tris-HCl, pH 7.5, 20 mM NaCl, and 5 mM DTT. medium.

Electron microscopy Binding measurements of human hair keratins using SPR For negative staining, an aliquot (20 μl) of each sample was adsorbed for 3 min to a collodion film on a copper grid. Then the We used surface plasmon resonance (SPR) technique to grid was exposed to 2% uranyl acetate for 30 s. After removal of examine the molecular interactions between the human hair excess liquid with filter paper, the sample was allowed to air dry. keratins (Fig. 2). First, K81 or K85 was immobilized on the Samples were examined using a transmission electron microscope dextran surface of a sensor chip as described in the “Mate- (TEM, JEM-1210, JEOL, Tokyo, Japan) that was operated at an rials and Methods” section. The amount of K81 or K85 accelerating voltage of 80 kV. immobilized on a sensor chip was adjusted to approxi- mately 7300 RU (Fig. 2b–d), by controlling the contact time of the sample with the activated surface of the sensor Results chip. When soluble K35 was injected as an analyte over the K81-immobilized sensor chip at a concentration in the Preparation of recombinant human hair keratins and range of 125–500 nM, sensorgrams were obtained that cytokeratins showed the concentration-dependent binding of K35 to the immobilized K81 (Fig. 2a). Conversely, when soluble K81 We expressed, in E. coli, the DNA sequences encoding in the same concentration range was injected over the same human type I hair keratins 35 (K35; total 455 amino acid sensor chip, concentration-dependent binding was observed residues containing 23 cysteine residues, calculated pI with very weak resonance signals (Fig. 2a). Similarly, solu- 4.77), 36 (K36; total 467 residues containing 20 cysteine ble K35 bound to the immobilized K85 with much more residues, pI 4.75) and 38 (K38; total 456 residues contain- intense resonance signals compared with that for the bind- ing 28 cysteine residues, pI 4.64), type II hair keratins 81 ing of soluble K85 to the immobilized K85 (data not (K81; total 505 residues containing 34 cysteine residues, pI shown). Thus, the heterotypic interaction between the type 5.37) and 85 (K85; total 507 residues containing 27 cys- I and II hair keratins was apparently superior to the homo- teine residues, pI 6.42), human type I cytokeratin 14 (K14; typic interaction between the type II hair keratins, as expec- total 472 residues containing 5 cysteine residues, pI 4.94), ted from previous SPR measurements reported for and type II cytokeratin 5 (K5; total 590 residues containing cytokeratins (Hofmann and Franke, 1997). Unfortunately, 4 cysteine residues, pI 7.77). The proteins were extracted because K36 and K38 readily aggregated in the running from inclusion bodies using 8 M urea, and then purified by buffer used here, their binding to the immobilized K81 or

34 Assembly of Hair Keratins

Fig. 2. SPR of heterotypic and homotypic subunit interactions of human hair keratins. In (a), K81 was immobilized (7.8 ng/mm2) on a sensor chip as described in the “Materials and Methods” section. In (b–d), K81 (b) or K85 (c, d) was immobilized (7.3 ng/mm2), by controlling the contact time of each protein with the activated surface of the sensor chip. Specific binding of soluble samples to the immobilized K81 or K85 was obtained by subtracting the blank sensorgram from the assay curve as explained in the “Materials and Methods” section. (a) Soluble K35 at a concentration of 125 nM (black dotted line), 250 nM (black solid line) or 500 nM (black dashed line), and soluble K81 at a concentration of 125 nM (gray dotted line), 250 nM (gray solid line) or 500 nM (gray dashed line) were injected over a K81-immobilized sensor chip under the conditions of pH 8.0 and 50 mM NaCl. (b) Soluble K35 at a concentration of 250 nM was injected over a K81-immobilized sensor chip, under the conditions of pH 7.4 (dotted line), 8.0 (solid line) or 8.5 (dashed line) and 50 mM NaCl. (c) Soluble K35 at a concentration of 250 nM was injected over a K85-immobilized sensor chip under the conditions of pH 7.4 (dotted line), 8.0 (solid line) or 8.5 (dashed line) and 50 mM NaCl. (d) Soluble K35 at a concentration of 250 nM was injected over a K85-immobilized sensor chip under the conditions of pH 8.0 and 50 mM (black dotted line), 100 mM (black solid line), 150 mM (black dashed line) or 200 mM (gray dashed line) of NaCl.

K85 was unable to be determined. both types of hair keratins from 9.5 M urea down to various The effects of pH and ionic strength on the interaction lower urea concentrations as described in the “Materials between soluble K35 and immobilized K81 or K85 were and Methods” section. By lowering the urea concentration examined (Fig. 2b–d). Under the conditions used here, from 9.5 M, the type I and II hair keratins associated to maximum binding of K35 to the immobilized K81 or K85 form a heterotypic complex at a certain urea concentration. was obtained at pH 8.0 in the presence of 50 mM NaCl. Complex formation and dissociation occur reversibly at the Interestingly, the resonance signal obtained for the binding same urea concentration (Franke et al., 1983). Because the of K35 to the immobilized K85 at pH 8.0 was almost twice type I and II proteins have different isoelectric points, for- that observed for the binding of K35 to the immobilized mation of a heterotypic complex is observed as a shift in K81 at the same pH (Fig. 2b and c). Because the amounts isoelectric point in IEF measurements at the first dimen- of K81 and K85 immobilized on the sensor chips were sion. SDS-PAGE at the second dimension dissociates the almost equal (Fig. 2b and c), these results suggest that K35 complex and hence allows identification of the proteins prefers K85 rather than K81 as a heterotypic partner. involved. This method has been used to characterize the interactions between type I and II cytokeratins in urea, and Heterotypic complex formation between human type I has revealed that cytokeratins obtained from various tissues and II hair keratins and cultured cells form heterotypic complexes at 6.0–9.0 M urea depending on the combination of type I and II subunit To characterize the formation of heterotypic complexes proteins (Eichner et al., 1986; Franke et al., 1983). In this between the type I and II hair keratins, two-dimensional gel study, we observed that heterotypic complex formation electrophoresis was performed after dialysis of mixtures of occurred at different urea concentrations depending on the

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Fig. 3. Two-dimensional gel electrophoretic analysis of heterotypic subunit interactions of human hair keratins. IEF in the first dimension (horizontal direction) was performed at different urea concentrations, as indicated in the lower right corner. After IEF, 10% SDS-PAGE in the second dimension (vertical direction) was carried out. The separated proteins were visualized by staining with Coomassie brilliant blue. Square brackets with solid lines and an underlined number indicate the position of the hair keratin that is forming a heterotypic complex. Square brackets with dashed lines and a number without an underline indicate the position of the hair keratin that is not forming a heterotypic complex. The combinations of K35-K81 (a–c) and K36-K81 (d–f) formed in 5.0 and 5.5 M urea, respectively. The combination of K35-K85 (j–l) occurred partly in 6.0 M urea, and fully in 5.5 M urea. The complex K36-K85 (m–o) formed partly in 5.5 M urea, and fully in 5.0 M urea. The combination of K38-K81 (g–i) formed in 4 M urea, while that of K38-K85 (p–r) formed a complex partly in 5.0 M urea and fully in 4.0 M urea. BSA (bovine serum albumin, pI 5.5) was co-electrophoresed as a reference protein.

combination of type I and II hair keratins (Fig. 3). K81 expressed in the matrix or cortex at the same stage of dif- formed a complex with K35 at 5.0 M urea (Fig. 3a–c), with ferentiation have a higher affinity than other combinations. K36 at 5.5 M urea (Fig. 3d–f), and with K38 at 4.0 M urea In contrast, K38 showed a relatively low affinity for K81 or (Fig. 3g–i). K85 formed a complex with K35 partly at 6.0 K85. M urea and fully at 5.5 M urea (Fig. 3j–l), with K36 partly at 5.5 M urea and fully at 5.0 M urea (Fig. 3m–o), and with In vitro IF assembly conditions of human hair K38 partly at 5.0 M and fully at 4.0 M (Fig. 3p–r). In an keratins equivalent experiment, human cytokeratins K14 and K5 formed a complex at 7.0 M urea (data not shown). Thus, In general, in vitro IF formation is influenced primarily by the human hair keratins formed heterotypic complexes at pH and ionic strength (Coulombe and Omary, 2002; Fuchs urea concentrations lower than 6.0 M, suggesting lower and Weber, 1994; Herrmann and Aebi, 2004). As a refer- compatibility between the type I and II hair keratins than ence, cytokeratin K14 and K5 formed IFs with widths of between cytokeratin pairs. Among the combinations 9±2 (mean±standard deviation) nm and lengths of several examined here, those of K35-K85 and K36-K81 formed micrometers at neutral pH and low ionic strength such as 5 relatively stable complexes compared with the other combi- mM Tris-HCl, pH 7.5 and 20 mM NaCl (Fig. 4e), as repor- nations. This suggests that the combinations that are ted previously (Herrmann et al., 2002; Herrmann and Aebi,

36 Assembly of Hair Keratins

Fig. 4. Electron microscopy of the assembly products formed by the combination of (a) K35-K81, (b) K36-K81, (c) K35-K36-K81, (d) K38-K81 and (e) cytokeratin K14-K5. The assembly experiments were carried out as described in the “Materials and Methods” section. The samples were contrasted by negative staining. (a) The combination K35-K81 formed IFs with lengths of 400–800 nm. ULFs were observed at low ionic strength (inset). (b) The combination K36-K81 produced IFs with lengths of 200–500 nm. (c) The combination K35-K36-K81 formed IFs with lengths of up to 1 μm. (d) The combination K38-K81 formed only aggregates. (e) Cytokeratin K14 and K5 formed wavy IFs. Scale bars, 200 nm (a–e) and 50 nm (a, inset).

2004; Yano et al., 1991). In the current study, various con- by K14 and K5 (Fig. 4e). The combination of K36-K81 ditions for IF assembly of the human hair keratins prepared gave IFs with widths of 7±1 nm and lengths of 200–500 nm in this study were examined. It was observed that IFs form (Fig. 4b). During IF assembly experiments, it was observed at neutral pH and relatively high ionic strengths (25 mM that at a low ionic strength (i.e., 25 mM Tris-HCl, pH 7.5) Tris-HCl, pH 7.2 and 180 mM NaCl) compared with the after removal of urea by dialysis, the combinations of type I conditions used for cytokeratins (e.g., 5 mM Tris-HCl, pH and II hair keratins formed unit-length filaments (ULFs) 7.5 and 0–20 mM NaCl) (Herrmann et al., 2002; Herrmann with a diameter of 11±2 nm and lengths of 61±12 nm, as and Aebi, 2004; Yano et al., 1991) or type III and IV IF shown in the inset of Fig. 4a for the combination of K35- proteins (e.g., 20 mM Tris-HCl, pH 7.5 and 100–150 mM K81 and in Fig. 5a and b for the combination of K35-K85. NaCl) (Ando et al., 2004; Gohara et al., 2008; Herrmann et To connect the ULFs longitudinally to form IFs, the fila- al., 1992; Hisanaga and Hirokawa, 1990). To prevent ment formation buffer needed to contain 180 mM NaCl. unfavorable oxidation of cysteine residues contained in Changing the NaCl concentration in the filament formation both types of human hair keratins, either 10 mM DTT or 5 buffer from 180 to 135 or 200 mM resulted in formation of mM TCEP was used as a reducing reagent. No obvious dif- shorter IFs or an increased amount of aggregates, respec- ference in the effects of DTT and TCEP on IF formation tively. When K35, K36 and K81 were mixed with a molar has been observed (Burns et al., 1991; Hofmann et al., 2002). ratio of 0.5:0.5:1.0 in 9.5 M urea, this combination gave IFs with widths of 7±1 nm and lengths of up to 1 μm (Fig. 4c). In vitro assembly characteristics of combinations Because all three proteins were observed in the pellet frac- using K81 tion after centrifugation (20,000×g) of the reaction mixture followed by SDS-PAGE (data not shown), both K35 and Under the conditions used, the combination of K35-K81 K36 appeared to copolymerize with K81 to form IFs. The formed IFs with widths of 7±1 nm and lengths of 400–800 combination of three proteins always afforded longer IFs nm (Fig. 4a), slightly narrower and shorter than IFs formed compared with those formed by the combination of K35-

37 Y. Honda et al.

Fig. 5. Electron microscopy of the assembly products formed by the combination of (a–h) K35-K85, (i) K36-K85 and (j) K38-K85. (a, b) The combination of K35-K85 formed ULFs at low ionic strength. (c) The combination of K35-K85 formed short IFs with lengths of 200–400 nm in 180 mM NaCl. (d) The short IFs tended to associate laterally and tightly, and concatenated with each other to form IF bundles with lengths of 300–400 nm. The dashed lines indicate two short IF bundles that are concatenating with each other. (e, h) The IF bundles appeared to further associate longitudinally and laterally to form large paracrystalline assemblies. The dashed lines indicate two IF bundle units that are connected to each other. (f, g) In the paracrystalline assemblies, periodic patterns of approximately 20 nm (square brackets) were sometimes observed. (i) The combination of K36-K85 formed loosely associated IF tangles. (j) The combination of K38-K85 gave only aggregates. Scale bars, 50 nm (a and b), 100 nm (f and g), 200 nm (c, d, i and j), 500 nm (e), and 5 μm (h).

K81 or K36-K81, so the type I proteins K35 and K36 seem compaction-type reorganization might have occurred in to cooperate to elongate IFs, although the reason for this is each IF during the bundle formation. The IF bundles unknown. Overall, IFs formed by the combination of K35- appeared to further associate tightly to form large paracrys- K81, K36-K81 or K35-K36-K81 seemed to be more linear talline assemblies (Fig. 5e–h), in which many IF bundle (Fig. 4a–c) than the IFs containing K14 and K5 (Fig. 4e). units with widths of 40–100 nm and lengths of 300–500 nm The combination of K38-K81 failed to form IFs under the appeared to be connected to each other (Fig. 5e). A single conditions examined here; only aggregates were observed IF bundle unit might be formed from a short IF bundle like (Fig. 4d). that shown in Fig. 5d, and its length corresponded to 5–8 times that of an ULF. Interestingly, patterns with a period In vitro assembly characteristics of combinations of approximately 20 nm were observed in some places using K85 along the paracrystalline assemblies (Fig. 5f and g). These patterns might reflect a structural feature of hair keratin IFs, The combination of K35-K85 formed relatively short IFs because some kinds of IFs have a repeated segment struc- with widths of 7±1 nm and lengths of 200–400 nm (Fig. ture of 22 nm along the filament axis (Ando et al., 2004; 5c). Again, these shorter IFs appear to be more linear than Herrmann and Aebi, 2004; Hisanaga and Hirokawa, 1990). those formed between K14 and K5 (Fig. 4e). The shorter The combination of K36-K85 afforded IFs with widths of IFs tended to associate laterally and tightly, and concaten- 6±1 nm, loosely associated IF bundles, and large tangles ated longitudinally with each other to form IF bundles with that appeared to be linked to each other by many IFs (Fig. widths of 79±37 nm and lengths of 300–400 nm (Fig. 5d). 5i). In contrast, the combination of K38-K85 gave only The shorter IFs in the bundle were further narrower (the aggregates (Fig. 5j), as observed for the combination of widths are 6±1 nm) and straight, suggesting that radial K38-K81 (Fig. 4d).

38 Assembly of Hair Keratins

Fig. 6. Electron microscopy of the assembly products formed by the combination of (a, b) K35-K36-K81-K85 and (c, d) preformed ULFs composed of K35-K85 and K36-K81. (a and b) The combination K35-K36-K81-K85 afforded many IF tangles. (c and d) Two kinds of ULFs composed of K35-K85 and K36-K81 were preformed, mixed and dialyzed against filament formation buffer containing 180 mM NaCl. Note the tightly associated IF bundles. Scale bars, 200 nm.

In vitro assembly characteristics of combinations In vitro assembly characteristics of K38 using both K81 and K85 In contrast to the other type I human keratins, K38 When equimolar amounts K35, K36, K81 and K85 were appeared to lack an ability to form IFs de novo through pol- mixed in 9.5 M urea for IF formation, mainly large tangles ymerization with type II hair keratins (Fig. 4d and 5j). This composed of many IFs with widths of 6±1 nm were might be partly caused by the low affinity of K38 for K81 observed, as shown in Fig. 6a and b. Compared with the IFs or K85, as shown in the two-dimensional gel electrophore- obtained by mixing K35, K36 and K81 (Fig. 4c), this result sis investigations (Fig. 3g–i and p–r). In the presence of indicates that the addition of K85 promoted lateral associa- K35 and K36, however, K38 appeared to be involved in IF tion of IFs, although detailed knowledge of how each sub- formation. For example, when a mixture of K35, K36, K38, unit protein is assembled into dimers, tetramers, ULFs and K81 and K85 in 9.5 M urea was prepared for IF polymeri- finally IFs is not known. We also preformed two kinds of zation, IF tangles similar to those shown in Fig. 6a and b ULFs composed of the combinations of K35-K85 and K36- were observed (data not shown). All of the proteins were K81 independently by dialysis, mixed both ULF samples, recovered in the pellet fraction after centrifugation of the and then undertook the final dialysis against the filament reaction mixture at 20,000×g followed by SDS-PAGE anal- formation buffer containing 180 mM NaCl. This method ysis, suggesting that K38 was incorporated into the IFs and afforded primarily IF bundles with widths of 52±25 nm IF tangles. (Fig. 6c and d). Although both types of ULFs probably associate longitudinally with each other to form an IF, the ULFs composed of K35-K85 seemed to determine the Discussion assembly behavior of the reaction mixture. In this study, recombinant human type I hair keratins (K35, K36 and K38), type II hair keratins (K81 and K85), human type I cytokeratin K14 and type II cytokeratin K5 were pre-

39 Y. Honda et al. pared using the bacterial expression systems. K35 and K85 5 M urea in two-dimensional gel electrophoresis (Hofmann are the first pair expressed in the matrix/pre-cortex during et al., 2002). Similarly, hair keratins extracted from wool the early stages of differentiation in human hair follicles, formed heterotypic complexes at urea concentrations of less while K38 is produced in the lower cortex following the than 6 M (Herrling and Sparrow, 1991). Combined, these expression of K35 and K85 (Langbein et al., 1999, 2001; results suggest that the heterotypic human hair keratin com- Langbein and Schweizer, 2005; Rogers et al., 2004, 2005; plexes are less stable in urea solutions than those of cytoker- Schweizer et al., 2007). K36 and K81 as well as other atins, although the biological significance of this is unknown. human hair keratins including K33a, K33b, K83 and K86 Two-dimensional gel electrophoresis indicated that the are expressed in the mid- to upper cortex during the stabilities of the heterotypic complexes in urea differ advanced stage of differentiation (Langbein et al., 1999, depending on the combination of type I and II hair keratins 2001; Langbein and Schweizer, 2005; Rogers et al., 2004, (Fig. 3), as observed for various cytokeratin combinations 2005; Schweizer et al., 2007). The recombinant human hair (Eichner et al., 1986; Franke et al., 1983). Among the com- keratins prepared here were used to investigate protein- binations examined here, K35-K85 (Fig. 3j–l) and K36- protein interactions with an emphasis on the specificity of K81 (Fig. 3d–f) formed relatively stable complexes interactions between particular type I and II hair keratins. compared with the other combinations. These results are The proteins were also used to explore the characteristics of consistent with the findings of the SPR measurements in in vitro IF formation by changing the subunit combinations which soluble K35 bound preferentially to immobilized of the type I and II hair keratins. K85 rather than to K81 (Fig. 2b and c), although the bind- In the SPR measurements, the heterotypic binding of ing between K36 and K81 could not be characterized by soluble K35 to immobilized K85 or K81 apparently predo- SPR because of the insolubility of K36 in the buffer used. minated over the homotypic binding of either soluble K85 As described above, K35 and K85 are co-expressed in the to immobilized K85, or soluble K81 to immobilized K81 matrix/pre-cortex during the initial stage of differentiation, (Fig. 2). These results support the idea that both type I and while K36 and K81 are co-expressed in the mid- to upper II hair keratins need to form coiled-coil dimers that then cortex during the advanced stage of differentiation lead to IFs (Hofmann et al., 2002; Schweizer et al., 2007; (Langbein et al., 1999, 2001; Langbein and Schweizer, Wang et al., 2000). Interestingly, soluble K35 displayed a 2005; Rogers et al., 2004, 2005; Schweizer et al., 2007). signal upon binding to immobilized K85 that was twice as Although heterotypic complex formation for other hair ker- intense as that of binding to immobilized K81. This result atin combinations remains to be analyzed, the relatively suggests that there is a heterotypic binding specificity high affinity observed for the combinations of K35-K85 between the type I and II hair keratins. In the SPR meas- and K36-K81 might have biological importance when dif- ured for non-epidermal cytokeratins, soluble type I cytoker- ferent hair keratin proteins exist in the same cell. These atins such as K13 and K18 display much higher binding results provide useful information on the structural signifi- than K19 and K20 to the immobilized type II cytokeratin cance of type I and II hair keratins that are co-expressed in K8, and homotypic binding of soluble K8 to immobilized the matrix or cortex at the same stage of differentiation. K8 was hardly detected (Hofmann and Franke, 1997). K8 Interestingly, K38 formed a heterotypic complex at a char- and K18 are the only cytokeratin pair present in some sim- acteristically low urea concentration (4–5 M urea) (Fig. 3g– ple epithelial cells such as early embryonic epithelia and i and p–r), indicating a particularly low affinity of K38 for hepatocytes (Moll et al., 1982). K8 and K13 are expressed either K81 or K85. Because K38 is produced in the lower together in tracheal epithelium and urothelium, while K8, cortex following the expression of the first pair K35-K85 K18, K19 and K20 are found in intestinal mucosa. Thus, and simultaneously with K31 (Langbein et al., 1999; some specific pairs of type I and II cytokeratins that are co- Langbein and Schweizer, 2005; Yu et al., 2009), the low expressed in certain cell types and growth stages have affinity of K38 for K85 seems to be a disadvantage when intrinsic mutual high affinities. The SPR results obtained competing with K35 and K31 for heterotypic complex for- here suggest that K35 and K85 have mutually compatible mation with K85 in a cell. structures as the first hair keratin pair expressed in the IF assembly experiments carried out in this study matrix/pre-cortex of human hair follicles. revealed the assembly properties of human hair keratins. The heterotypic interactions between the type I and II hair First, while the human hair keratins formed ULFs under keratins were further characterized using two-dimensional low salt conditions (25 mM Tris-HCl and 0 M NaCl) (Fig. gel electrophoresis in urea (Fig. 3). All combinations exam- 4a inset, Fig. 5a and b), 180 mM NaCl was required to con- ined formed heterotypic complexes at lower urea concen- nect ULFs longitudinally into IFs (Fig. 4a–c and Fig. 5c). trations (4–6 M) than that of the urea concentration (7 M) Because the head domains regulate IF assembly both in at which human cytokeratins K14 and K5 form a complex. vitro and in vivo (Fuchs and Weber, 1994; Herrmann and The results were in good agreement with previous findings Aebi, 2004; Inagaki et al., 1996), it is also likely that the that the combinations of human hair keratins K31-K83 and head domain structure determines the optimal ionic K31-K86 afforded the corresponding heterotypic complexes at strength for in vitro IF formation. In our previous study on

40 Assembly of Hair Keratins the type III IF protein , the aromatic residues in the residues) and aromatic residues (3 residues) but low in cys- motif (-Tyr-Arg-Arg-X-Phe-) near the amino terminus of teine (6 residues) compared with the tail domains of K81, the head domain were substituted with hydrophilic residues K83 and K86 (total of 69–88 residues; 3–4 arginine and no Asn and Ser (Gohara et al., 2008). The resulting vimentin aromatic residue, and 8-14 cysteine residues). mutants were very sensitive to ionic strength and formed Third, K38 appeared to lack the ability of de novo IF for- mature IFs even at 50 mM NaCl, while the wild type mation through copolymerization with the type II hair kera- required 150 mM NaCl to form IFs. The aromatic residues, tin K81 or K85 (Fig. 4d and Fig. 5j). Analysis of the especially the Tyr residue, in the motifs appeared to regu- molecular evolution based on the rod domain sequences of late the intermolecular interactions involved in IF assembly human type I hair keratins has revealed that K38 is posi- and determine an optimal ionic strength for IF assembly. tioned a relatively long distance from other type I hair kera- Substitution of the two Arg residues with other amino acids tins including K35 and K36, both of which are located resulted in the loss of the ability to form IFs (Gohara et al., close to each other (Rogers et al., 2004). In addition, K38, 2008). These motifs can be found near the amino termini in K35 and K36 lack homology in the head and tail domain the head domains of both type III and IV IF proteins sequences. It has been reported that the expression of K38 (Herrmann et al., 1992), but not in the head domains of occurs in distinct cells that are randomly scattered through- cytokeratins. Although human type I and II hair keratins do out the entire cortex (Langbein et al., 1999). It has been not have the same motif in the head domains, type II hair also reported that K38 is asymmetrically expressed in the keratins such as K81, K83, K85 and K86 all possess several cortex of crimped human hair and wool (Thibaut et al., -Tyr-Arg- sequences and a unique -Arg-X-Phe-X-Tyr-Arg- 2007; Yu et al., 2009). Further work is required to under- sequence in the head domain. It would be interesting to stand the biological significance of K38. examine the effects of substitution of these Tyr residues on In this study, it is noteworthy that the combination of in vitro IF formation of hair keratins. K35-K85 formed tightly packed, short IF bundles and large Second, the in vitro assembly properties of type II hair paracrystalline assemblies (Fig. 5d–h), rather than forming keratins K81 and K85 differed markedly. K81 afforded IFs ordinary IFs in vitro. In human hair follicles, K35 and K85 by copolymerization with the type I hair keratins except are the first pair expressed in the matrix/pre-cortex during K38 (Fig. 4a–d), although the IFs were slightly narrower the initial stages of differentiation (Langbein et al., 1999, and shorter than the IFs formed by cytokeratin K14 and K5 2001; Langbein and Schweizer, 2005; Schweizer et al., (Fig. 4e). In contrast, K85 formed various types of assem- 2007). They are expressed before hair keratin-associated bly products, such as short straight IFs (Fig. 5c), IF bundles proteins (KAPs), which are matrix proteins that embed hair (Fig. 5d), large paracrystalline assemblies (Fig. 5e–h), and keratin IFs in macrofibrils (MFs) (Rogers et al., 2006). IF tangles (Fig. 5i), depending on the type I protein that Among the human KAPs, KAP8 and KAP11, which are was copolymerized with. In particular, the combination of high in glycine-tyrosine and sulfur, respectively, are K35-K85 resulted short IF bundles with widths of 79±37 expressed first in the lower cortex (just above the papilla) nm and lengths of 300–400 nm (Fig. 5d). These IF bundles (Rogers et al., 2006). Many other KAPs as well as other further polymerized into large paracrystalline assemblies type I and II hair keratins are expressed primarily in the (Fig. 5e–h). Thus, K85 promoted lateral association rather mid- to upper cortex during the advanced stage of differen- than longitudinal association of short IFs. The promoted tiation (Rogers et al., 2006). TEM studies of follicles have lateral association is not caused by formation of disulfide revealed the appearance of hair keratin IF bundles, i.e., bonds between K85 and the type I hair keratins because proto-macrofibrils (proto-MFs), in the lower cortex (Jones K85 has fewer cysteine residues (27 residues) than K81 (34 and Pope, 1985; Marshall et al., 1991; Morioka, 2009; residues). Unfavorable oxidation of the cysteine residues Orwin, 1979; Orwin and Woods, 1982). Small proto-MFs during IF assembly experiments was suppressed suffi- reported to date are around 80 nm wide and 300–700 nm ciently under N2 atmosphere and by the use of an effective long (Marshall et al., 1991; Orwin, 1979), so they are simi- reducing reagent such as 10 mM DTT or 5 mM TCEP lar in size to the short IF bundles formed by the combina- (Hofmann et al., 2002). Although the structural elements tion of K35-K85 in this study (Fig. 5d). Some TEM images that control the assembly of K85 remain to be clarified, the of the cross-sections of early proto-MFs showed that dis- terminal domains of K85 might be responsible, because the tinctly separated IFs are arranged on a hexagonal basal lat- rod domain sequence of K85 did not contain any remarka- tice like a columnar hexagonal liquid crystal, with no ble structural features when compared with those of other matrix proteins interpolated between the filaments type II hair keratins. The head domain of K85 is relatively (McKinnon, 2006). Proto-MFs coalesce with each other long (122 residues), and rich in both arginine (11 residues) into MFs that are around 400 nm wide and several micro- and aliphatic residues (27 residues), compared with the meters long in the mid- to upper cortex (Jones and Pope, head domains of K81, K83 and K86 (total of 105–110 resi- 1985; McKinnon, 2006; McKinnon and Harland, 2011; dues; 8 arginine and 21 aliphatic residues). The tail domain Orwin, 1979). It is more efficient to form proto-MFs com- (totally 73 residues) of K85 is relatively rich in arginine (5 posed of short IFs and then connect them longitudinally

41 Y. Honda et al. and laterally into MFs than to bundle long IFs into MFs. Acknowledgments. We acknowledge Prof. Y. Takasaki of Saga This study revealed that the pair K35-K85 formed short IF University and Prof. K. Takamune of Kumamoto University for their bundles even in the absence of KAPs (Fig. 5d). The intrin- pertinent comments and discussion. We also thank T. Tabata, H. Sato, R. Ishimatsu and K. Kamihara for their technical assistance. sic assembly property of K85 that promotes lateral associa- tion rather than longitudinal elongation of short IFs seems References to be important for formation of proto-MFs. K85 is abun- Ando, S., Nakao, K., Gohara, R., Takasaki, Y., Suehiro, K., and Oishi, Y. dantly expressed in the matrix, pre-cortex, cortex and cuti- 2004. Morphological analysis of glutaraldehyde-fixed vimentin inter- cle of the hair root, as well as in the nail matrix (Langbein mediate filaments and assembly-intermediates by atomic force micro- et al., 2001; Langbein and Schweizer, 2005; Perrin et al., scopy. Biochim. Biophys. Acta, 1702: 53–65. 2004; Schweizer et al., 2007). The characteristic assembly Bryson, W.G., Harland, D.P., Caldwell, J.P., Vernon, J.A., Walls, R.J., of K85 is probably crucial for hair and nail formation, Woods, J.L., Nagase, S., Itou, T., and Koike, K. 2009. Cortical cell types and intermediate filament arrangements correlate with fiber because mutations of K85 result in severe phenotypes curvature in Japanese human hair. J. Struct. Biol., 166: 46–58. (Naeem et al., 2006; Shimomura et al., 2010). Burns, J.A., Butler, J.C., Moran, J., and Whitesides, G.M. 1991. Selectve In this study, the combination of K36-K81 formed a rela- reduction of disulfies by tris(2-carboxyethyl)phosphine. J. Org. Chem., tively stable complex in the two-dimensional gel electro- 56: 2648–2650. phoresis (Fig. 3d–f) and gave IFs with lengths of 200–500 Chapman, R.E. and Gemmell, R.T. 1971. Stages in the formation and nm in vitro (Fig. 4b). In hair follicles, the expression of keratinization of the cortex of the wool fiber. J. Ultrastruct. Res., 36: both K36 and K81 starts in the mid- to upper cortex, where 342–354. Coulombe, P.A. and Omary, M.B. 2002. ‘Hard’ and ‘soft’ principles the expression of K35 has been completed (Langbein et al., defining the structure, function and regulation of keratin intermediate 1999, 2001; Langbein and Schweizer, 2005; Schweizer et filaments. Curr. Opin. Cell Biol., 14: 110–122. al., 2007). It may be speculated that IFs formed by the Eichner, R., Sun, T.T., and Aebi, U. 1986. The role of keratin subfamilies combination of K36-K81 play an important role in the and keratin pairs in the formation of human epidermal intermediate fila- growth of MFs, e.g., by connecting proto-MFs composed of ments. J. Cell Biol., 102: 1767–1777. K35-K85 laterally and longitudinally. It has been reported Franke, W.W., Schiller, D.L., Hatzfeld, M., and Winter, S. 1983. Protein that the initiation of proto-MFs is limited to the lower cor- complexes of intermediate-sized filaments: melting of cytokeratin com- plexes in urea reveals different polypeptide separation characteristics. tex, and that the increases in keratin content in the mid- to Proc. Natl. Acad. Sci. USA, 80: 7113–7117. upper cortex result from increases in the size of existing Fraser, R.D.B., Fogers, G.E., and Parry, D.A.D. 2003. Nucleation and proto-MFs, rather than through initiation of new ones growth of macrofibrils in trichocyte (hard-alpha) keratins. J. Struct. (Marshall et al., 1991). 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Biochem., 144: 675–684. or waviness of a fiber (Bryson et al., 2009; Jones and Pope, Herrling, J. and Sparrow, L.G. 1991. Interactions of intermediate filament 1985; Marshall et al., 1991; McKinnon, 2006; McKinnon proteins from wool. Int. J. Biol. Macromol., 13: 115–119. Herrmann, H., Hofmann, I., and Franke, W.W. 1992. Identification of a and Harland, 2011; Morioka, 2009; Orwin, 1979). The nonapeptide motif in the vimentin head domain involved in intermedi- molecular mechanism inducing the formation of these three ate filament assembly. J. Mol. Biol., 223: 637–650. types of MFs has not yet been clarified (Fraser et al., 2003; Herrmann, H., Wedig, T., Porter, R.M., Lane, E.B., and Aebi, U. 2002. McKinnon, 2006; McKinnon and Harland, 2011). Differen- Characterization of early assembly intermediates of recombinant human ces in both the composition and content of KAPs in the keratins. J. Struct. Biol., 137: 82–96. three types of MFs suggest that they play an important role Herrmann, H. and Aebi, U. 2004. Intermediate filaments: molecular struc- in determining the precise mode of MF packing (Fujikawa ture, assembly mechanism, and integration into functionally distinct intracellular scaffolds. Annu. Rev. Biochem., 73: 749–789. et al., 2011; Matsunaga et al., 2013; Plowman et al., 2007; Hisanaga, S. and Hirokawa, N. 1990. Molecular architecture of the neuro- Rogers et al., 2006). In addition, an unknown characteristic filament. II. Reassembly process of L protein in vitro. J. surface structure of hair keratin IFs, probably constructed Mol. Biol., 211: 871–882. by the head or tail domain, may also contribute to the IF-IF Hofmann, I. and Franke, W.W. 1997. Heterotypic interactions and fila- and IF-KAP interactions and the development of the three ment assembly of type I and type II cytokeratins in vitro: viscometry types of MFs. Further studies on the molecular interactions and determinations of relative affinities. Eur. J. Cell Biol., 72: 122–132. Hofmann, I., Winter, H., Mucke, N., Langowski, J., and Schweizer, J. between hair keratins and KAPs are required to understand 2002. The in vitro assembly of hair follicle keratins: comparison of the development of MFs leading to the formation of hair cortex and companion layer keratins. Biol. Chem., 383: 1373–1381. fibers with variable waviness and physical strength, and Inagaki, M., Matsuoka, Y., Tsujimura, K., Ando, S., Tokui, T., Takahashi, also hereditary hair anomalies. T., and Inagaki, N. 1996. Dynamic property of intermediate filaments:

42 Assembly of Hair Keratins

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