Characterization of Early Assembly Intermediates of Recombinant Human Keratins

Journal of Structural Biology 137, 82–96 (2002) doi:10.1006/jsbi.2002.4466

Characterization of Early Assembly Intermediates of Recombinant Human Keratins Harald Herrmann,*,1 Tatjana Wedig,* Rebecca M. Porter,† E. Birgitte Lane,† and Ueli Aebi‡ *Division for Cell Biology, German Cancer Research Center, 69120 Heidelberg, Germany; †Cancer Research Campaign Cell Structure Research Group, School of Life Sciences, The University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom; and ‡M.E. Mu¨ller Institute, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland Received January 29, 2002, and in revised form February 26, 2002

Key Words: assembly; intermediate filaments; keratins; electron microscopy; epidermolysis bullosa simplex (EBS).

The intermediate filaments (IFs) form major structural elements of the cytoskeleton. In vitro analyses of these fibrous proteins reveal very different assembly properties for the nuclear and cytoplasmic IF proteins. However, keratins in particular, the largest and most heterogenous group of cytoplasmic IF proteins, have been difficult to analyze due to their rapid assembly dynamics under the near-physiological conditions used for other IF proteins. We show here that keratins, like other cytoplasmic IF proteins, go through a stage of assembling into full-width soluble complexes, i.e., “unit-length filaments” (ULFs). In contrast to other IF proteins, however, longitudinal annealing of keratin ULFs into long filaments quasi-coincides with their formation. In vitro assembly of IF proteins into filaments can be initiated by an increase of the ionic strength and/or lowering of the pH of the assembly buffer. We now document that 23-mer peptides from the head domains of various IF proteins can induce filament formation even under conditions of low salt and high pH. This suggests that the “heads” are involved in the formation and longitudinal association of the ULFs. Using a Tris-buffering protocol that causes formation of soluble oligomers at pH 9, the epidermal keratins K5/14 form less regular filaments and less efficiently than the simple epithelial keratins K8/18. In sodium phosphate buffers (pH 7.5), however, K5/14 were able to form long partially unraveled filaments which compacted into extended, regular filaments upon addition of 20 mM KCl. Applying the same assembly regimen to mutant K14 R125H demonstrated that mutations causing a severe disease phenotype and morphological filament abnormalities can form long, regular filaments with surprising efficiency in vitro. © 2002 Elsevier Science (USA)

INTRODUCTION

Of the three filament systems constituting the cytoskeleton, the intermediate filaments are uniquely heterogeneous and differentiation-specific. A search of the human genome sequence database has revealed at least 65 intermediate filament genes, 49 of which are keratins (Hesse et al., 2001). Of these keratins, 22 are expressed in various epithelial cells and are also referred to as cytokeratins. Another 20 are used to form hair and nails and are often called trichocyte or “hard” keratins. The expression pattern of the other 7 is not yet known. The general structure of all intermediate filament proteins is highly conserved and consists of a central ␣-helical domain interrupted by three non-␣-helical linker domains and flanked by non-␣-helical head and tail domains (for review see Conway and Parry, 1988; Aebi et al., 1988; Fuchs and Weber, 1994; Parry and Steinert, 1995). Apart from two highly conserved sequence motifs at either end of the ␣-helical rod domain, the primary sequences of individual intermediate filament proteins can differ greatly, and they have been grouped into six sequence homology classes. These six homology classes can be divided, according to their member’s ability to coassemble with one another, into three assembly groups: (1) nuclear lamins (type V); (2) vimentin, desmin, and neurofilaments (types III, IV, and nestin, i.e., type VI); and (3) keratins (types I and II) (Herrmann and Aebi, 2000). Hence, keratins form IFs only with keratins; i.e., they do not copolymerize with nuclear lamins (Bader et al., 1991) nor with other cytoplasmic IF proteins (Herrmann et al., 1993; Steinert et al., 1993; Monteiro et al., 1994). And vice versa the pro-

1 To whom correspondence should be addressed. E-mail: [email protected].

1047-8477/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

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teins of sequence classes III and IV do form IFs only with representatives of these two classes and with nestin which, in fact, may better fit class IV rather than constituting its own single-membered class (for functional aspects, see Steinert et al., 1999). Although intermediate filaments are predominantly heteropolymers in vivo, some (e.g., desmin, vimentin, and nuclear lamins) can assemble into homopolymers. In contrast, keratins must form a heterodimer between one type I and one type II keratin in order to form a filament (Hatzfeld and Weber, 1990; Steinert, 1990). Their assembly characteristics have been conserved through evolution such that the assembly potential of vertebrate intermediate filaments (e.g., human keratins) can be used to determine the class type of nonvertebrate intermediate filament proteins (Karabinos et al., 2000; Wang et al., 2000). For assembly experiments, keratins were first isolated from skin and denatured with urea for column purification and then renatured into various buffers (Steinert et al., 1976; Franke et al., 1982; Steven et al., 1982, 1985; Eichner et al., 1985). In any case, the ionic strength was kept very low, and particularly in studies aimed at elucidating early assembly intermediates, the experimental conditions were chosen closer to inhibiting assembly rather than trying to mimic physiological conditions (Steinert, 1991; Aebi et al., 1983). However, the in vitro assembly properties of keratins, nuclear lamins, and vimentin have been shown to be very different (reviewed in Herrmann and Aebi, 2000). The assembly of nuclear lamins is similar to that of invertebrate intermediate filament proteins: tetramers anneal longitudinally to form long protofilaments, which then further associate laterally to eventually yield mature filaments (Geisler et al., 1998). In contrast, vimentin and desmin filament assembly can be dissected into three distinct phases: (I) lateral association of tetramers to form a unit-length filament (ULF); (II) longitudinal annealing of ULFs into loosely packed filaments several hundred nanometers long; and (III) radial compaction into mature filaments (Herrmann et al., 1996; Herrmann and Aebi, 1998a,b). It was shown that under assembly conditions optimized for vimentin, keratins also form ULFs, but assembly was much more rapid; i.e., long filaments were observed even at 1 s after initiation of assembly. However, these filaments were clearly thinner, suggesting that the assembly process was suboptimal (Herrmann et al., 1999). To date, it has not been possible to dissect keratin filament assembly as thoroughly as that of vimentin. In the present study we have examined the in vitro assembly of keratins employing several different buffer systems. We have studied filament assem-

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bly of the simple epithelial keratin pair K8 and K18 and that of the predominant keratin pair of the basal cells of the epidermis K5 and K14. We show that these keratins form 50- to 60-nm-long fullwidth ULFs that then anneal longitudinally, thus distinguishing them from invertebrate intermediate filament proteins. In addition, we have analyzed the assembly of K5 with a mutated K14 carrying an arginine to histidine change at amino acid position 125, termed K14 R125H. The mutation is localized in the highly conserved domain at the beginning of the central ␣-helical rod domain (helix initiation motif). It is particularly interesting, since it causes keratin aggregation in keratinocytes and cytolysis in a skin blistering disorder called epidermolysis bullosa simplex (EBS). It has been assumed that keratin filament aggregation was a consequence of the mutation disrupting a molecular interaction of the helix initiation motif critical for filament assembly (Letai et al., 1993). We now show that this mutation has in fact no effect on filament assembly. Hence the formation of keratin aggregates in EBS may require a different mechanistic explanation. MATERIALS AND METHODS Recombinant Proteins Human K8 and K18 were expressed and purified as described (Herrmann et al., 1999). The human epidermal keratin cDNA clones coding for K5, K14, and K14 R125H were generated by PCR from cDNA derived from human keratinocyte RNA and subcloned into the polylinker of pET-23b, i.e., NdeI–EcoRI for K5 and NdeI–XhoI for K14 and K14 R125H. These clones were sequenced on both strands and found to correspond to the published sequences (for K5 see Lersch et al., 1989; for K14 Marchuk et al., 1985; see also the Image clone with Accession No. BC019097). In our K5 clone amino acids 8 to 11 are FRS instead of SGA, and 37 and 38 are RS instead of GP. Inspection of the nucleotide sequences makes minor errors in the original sequence probable. Amino acid 197 is a D instead of an E, and 261 is an E instead of a Q, which may represent a polymorphism in the sequence. The K14 sequence is identical except for amino acids 26, G instead of A, and 34, S instead of N. The clone coding for K14 R125H is identical with our K14 clone except for amino acid 125. Proteins were isolated from inclusion bodies of transformed BL21 Codon Plus cells (Stratagene, La Jolla, CA) without induction and purified as described using Q- and SP-Sepharose for K5 and DEAEand CM-Sepharose for K14 and K14 R125H (Herrmann et al., 1992, 1999). Preparation of Intermediate Filament Peptides The 23-mer peptides corresponding to the N-terminal sequences of IF proteins (see Table I) were chemically synthesized with a cysteine on the C-terminus and dissolved in 5 mM Tris– HCl (pH 8.4). Dimers of these peptides were created by oxidative crosslinking of the cysteine residues using H2O2 as described (Rogers et al., 1996). The human vimentin coil 2B peptide, amino acids 355 to 412, was prepared as described recently (2B2 in Strelkov et al., 2001). Keratin Assembly Protocols Four assembly protocols were used. In all cases the purified keratins were mixed first in 8 M urea in a 1:1 ratio, dialyzed into

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10 mM Tris–HCl (pH 8.5), 2 mM DTT, 6 M urea (“column buffer”), and applied to a Q-Sepharose column (Pharmacia, Freiburg i. Br., Germany) in the same buffer. Heterodimers were isolated by elution with a gradient of 0 – 0.2 M guanidinium chloride in column buffer (Coulombe and Fuchs, 1990). The day before use, proteins were dialyzed into the corresponding dialysis buffer at room temperature by lowering the urea concentration in a stepwise (4 and 2 M) fashion. Dialysis was continued overnight at 4°C into fresh dialysis buffer. Assembly was then initiated by the addition of an assembly buffer. The protocols were as follows: (1) Assembly method routinely used for vimentin. Heterodimers of K8 and K18 (0.2 mg/ml) were dialyzed into 5 mM Tris–HCl (pH 8.4), 1 mM EDTA, O.1 mM EGTA, and 1 mM DTT and diluted to a concentration of 45 ␮g/ml with this buffer. Assembly was initiated by the addition of an equal volume of assembly buffer (100 mM NaCl in 40 mM Tris–HCl, pH 7.0). (2) Peptide-induced assembly of keratins. Heterodimers of K8 and K18, dissolved in column buffer, were dialyzed into 5 mM Tris–HCl (pH 8.4). Filament assembly was promoted by the addition of monomeric or dimeric N-terminal peptides (Table I), dissolved in 5 mM Tris–HCl (pH 8.4), to implement a 20-fold or 10-fold molar excess, respectively. (3) Low-Tris-buffer method for keratin assembly. Keratins were dialyzed into 2 mM Tris–HCl (pH 9.0), 1 mM DTT at 4°C overnight. Type I and type II keratins were mixed 1:1 at a concentration of 0.2 mg/ml and assembly was initiated by the addition of an equal volume of 2⫻ assembly buffer (20 mM Tris–HCl, pH 7) at room temperature. (4) Sodium phosphate keratin assembly method. Column-purified keratins K5 and K14 were mixed 1:1 at a concentration of 0.3– 0.4 mg/ml, and heterodimers were isolated by Q-Sepharose column chromatography and dialyzed into 0.7 mM sodium phosphate (pH 7.5), 1 mM DTT. Filament assembly was initiated by adding an equal volume of 40 mM KCl in 0.7 mM sodium phosphate (pH 7.5) at 37°C to increase the ionic strength. Filament assembly was terminated at various time points, e.g., 1 s, 10 s, 10 min, and 1 h, by the addition of stop buffer (0.2% glutaraldehyde in 20 mM KCl, 0.7 mM sodium phosphate, pH 7.5, for 3–5 min). Then aliquots of the keratin assembly reactions were immediately applied to carbon-coated grids and negatively stained for visualization using a Zeiss Model 900 electron microscope (Carl Zeiss, Oberkochen, Germany). Images were captured at a magnification of 40 000 to 50 000 on Kodak 4489 film. Unraveling experiments were essentially done as described in Aebi et al. (1983). The concentration used for assembly of authentic epidermal keratins was 1.1 mg/ml, and that of the recombinant K5, K14, and K5, K14 R125H was 0.2 mg/ml. RESULTS

Assembly of Human Keratins 8 and 18 under Standard Conditions We studied early assembly intermediates of recombinant human K8 and K18 using different types of regimens. In particular, we investigated whether keratins start to assemble from ULFs when subjected to a sudden increase of ionic strength to medium-high values. Under conditions routinely used for vimentin, i.e., adding an equal volume of 100 mM NaCl buffered with 40 mM Tris–HCl (pH 7.0) to protein kept soluble in 5 mM Tris–HCl (pH 8.4), long

FIG. 1. Electron microscopic analysis of human recombinant keratin 8 and 18 assembled for 10 s at (a) 45 ␮g/ml in 25 mM Tris–HCl, 50 mM NaCl (pH 7.5) and at (b) 200 ␮g/ml in 10 mM Tris–HCl (pH 7.5). Bar, 100 nm.

filaments were generated even at a protein concentration (45 ␮g/ml) close to the critical concentration as early as 10 s after initiation of assembly (Fig. 1a). In contrast, with the low Tris-buffer assembly mode, i.e., keeping the protein soluble in 2 mM Tris–HCl (pH 9.0) and adding an equal volume of 20 mM Tris–HCl (pH 7.0), filaments were on the average much shorter (Fig. 1b). In addition, by the latter assembly mode many short full-width filaments, called ULFs, were encountered that could be sorted into four groups with lengths centered around 50, 75, 100, and 125 nm (Fig. 2). These short filaments often exhibited pointed or frayed ends, indicating that they did not contain the full complement of molecules at their ends and remained open to connect to the next ULF (Fig. 3, arrows). No protofibrils were revealed using this kind of assembly regimen, indicating that the lateral association of soluble keratin assembly intermediates (i.e., tetramers and higher oligomers) is very fast. The association rate for longitudinal growth of keratin ULFs is therefore obviously much higher than that for vimentin.

ASSEMBLY INTERMEDIATES OF HUMAN KERATINS

FIG. 2. Length distribution of keratin 8/18 filaments obtained 10 s after initiation of assembly and measured from exposures taken from a negatively stained specimen (see Figs. 3a–3w for some of the corresponding fibers). The brackets numbered I to IV indicate major length classes peaking at approximately 50, 75, 100, and 120 nm.

Assembly of Human K8 and K18 in the Presence of IF Head Peptides Since part of the non-␣-helical head domain is essential for the formation of tetramers and intermediate filament assembly (Hatzfeld and Burba, 1994; Herrmann et al., 1996), we reasoned that an increase in ionic strength could cause activation of the head domains for interactions mediating the lateral association of tetramers into ULFs, as well as longitudinal annealing of ULFs into mature filaments (Herrmann and Aebi, 1998b). We had previously used short 10-mer head peptides of vimentin to compete with interactions by these domains in the absence of salt and shown that these peptides induced formation of tactoidal structures resembling ULFs (Hofmann and Herrmann, 1992; Herrmann et al., 1996). Even 23-mer Xenopus vimentin head peptides induced the formation of human K8/18 polymers (Herrmann and Aebi, 1998a). Therefore, we now investigated the effect of “mimetic” K8 or K18 head peptides on the assembly of these keratins to test whether IF head peptides in general cause aggregation of soluble IF proteins. In order to be able to compare the results directly with those obtained previously for vimentin, we dialyzed the keratins into 5 mM Tris–HCl (pH 8.4) buffer. Rodlets of approximately 50 nm in length were readily visualized in addition to some irregular, extended filaments (Fig. 4a). Upon addition of K8 or K18 23-mer head peptides, formation of longer filaments was enhanced significantly, although these were more regular with the K8 peptides (compare Fig. 4b with 4c). In addition, the K8 peptide induced the formation of fibers 20 to 40 nm in diameter, probably as a result of bundling of individual IF-like structures (Fig. 4c). Next, we investigated the effect of fusing two head

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peptides by C-terminal cysteines to mimic the “twoheaded” nature of naturally occurring intermediate filament dimers. Longer and more regular filaments were observed with K18-derived dimeric peptides. An even more pronounced bundling of filaments was observed with C-terminally crosslinked 23-mer head peptides of human K8, vimentin, and NF-L (Figs. 5b to 5d). Such thick bundles were indeed generated by lateral association of individual IFs as demonstrated with a sample of C-terminally crosslinked 23-mer peptides of Xenopus vimentin (Fig. 5e). Note the sequential association of three filaments (arrows 1 and 2) into a bundle, and a corresponding reduction in the diameter of the fiber, presumably reflecting the eventual termination of two of the filaments (filled arrowhead). Arrows 3 and 4 indicate the occurrence of additional lateral filament association events, and the open arrowhead marks a second termination site. The peptides by themselves did not form any type of filament. This was an important control, since various desmin peptides, 20 to 43 amino acids long, were shown to form extensive filament arrays, albeit at neutral pH and high salt concentration (Geisler et al., 1993). Assembly of Epidermal Keratins Human keratins K5 and K14 exhibit the remarkable ability to form heterodimers even in the presence of 9.5 M urea (Coulombe and Fuchs, 1990). Nevertheless, using the low-Tris-buffer protocol little association of dimers occurs before the addition of assembly buffer (Fig. 6a). ULFs were present 10 s after initiation of assembly with many of them having annealed longitudinally and associated laterally to yield some sort of “meshed” fiber arrays (Fig. 6b). After 10 min, longitudinal annealing into comparatively short filaments of variable diameter prevailed (Fig. 6c). These filaments appeared rather “poor” compared to those formed by K8/18 when following the same protocol. In contrast, dialyzing K5/14 dimers into 0.7 mM sodium phosphate buffer (pH 7.5) resulted in the polymerization of long IFs even before the addition of assembly buffer. Under identical buffer conditions vimentin remains tetrameric (Muecke et al., 2001, and manuscript in preparation). Addition of assembly buffer to these K5/14 filaments led to their further compaction and elongation (Figs. 6d to 6f). With such a promising assembly system at hands, we wondered how a new keratin carrying an amino acid mutation in the highly conserved helix initiation motif would respond to these assembly conditions. To this end the mutation K14 R125H is of particular relevance since it is the most frequent mutation found in patients with a severe EBS phenotype. The prominent hallmark of

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FIG. 3. Collection of keratin 8/18 filaments attributed to classes I–IV depicted in Fig. 2. (a–f) I; (g–l) II; (m– q) III; (r–w) IV. Arrowheads point to pointed ends; arrows point to frayed ends. The bars represent 25 nm and are positioned next to filament segments showing some kind of independent structural organization from the remainder of the filament. Open arrowhead in (o): filament of exceptionally high diameter.

the disease is the aggregation of the keratin filaments in the cytoplasm of keratinocytes carrying this mutation. Interestingly, in vitro experiments did not yield “clumping” of filaments but instead the formation of short and rather open IFs (Letai et al., 1993). Therefore we investigated the assembly of K14 R125H in combination with K5, and we were surprised to find that K14 R125H assembled into long and even more regular filaments than wild-type K14, both in the absence and in the presence of salt. Since this result was so perplexing to us, we reexpressed the plasmid carrying the CGC to CAC mutation leading to the R125H amino acid change after transformation of Escherichia coli and picking a single colony. We divided the bacterial mass culture, as usual, into one part for protein isolation and another part for plasmid preparation. Sequencing verified that indeed the plasmid with the mutated cDNA had driven the expression of the recombinant protein (Fig. 7A). We then repeated the assembly experiments with K5 and K14 R125H, now with no wildtype K14 control run in parallel to ensure that no

fortuitous mix-up with K14 was possible. Using the low-Tris/high pH regimen, few short fibers, but no extended IFs, were found after dialysis of the K5/14 dimers, formed in 6 M urea, into 2 mM Tris–HCl (pH 9.0), 1 mM DTT (Fig. 7B, a). After addition of Tris– HCl to 10 mM and pH 7.5, long filaments were observed already after 10 s of assembly (Fig. 7B, b). These filaments were much longer than those formed by vimentin or desmin under comparable conditions (data not shown). After 10 min, the filaments grew even longer and “more regular,” i.e., radially more compact than after 10 s (Fig. 7B, c). Notably, these long filaments did not exhibit any pronounced tendency for bundle formation or any other kind of aggregation except for the typical entanglement as a result of reptation movements seen with any kind of very long filaments or fibers (for a theoretical treatment, see Grosberg and Khokhlov, 1997). This aspect was most pronounced in lowmagnification overview micrographs exhibiting extensive filament crowdings (data not shown). With the phosphate buffer system, filaments were already

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chloride to 20 mM, essentially as observed with the first preparation of K14 R125H (Figs. 7B, d to f). We have now documented that the R125H amino acid change in K14 has no obvious effect on in vitro filament assembly, hence the phenotype observed in living cells may have to do with the processing and/or structural integration of the polymer into the IF cytoskeleton. A hallmark feature of epidermal keratin IFs (Fig. 8a) is their behavior toward incubation with phosphate buffers, which causes them to unravel (Fig. 8b). Most interestingly, this behavior is also exhibited by filaments made from recombinant K5 and K14 (Fig. 8c). The mutated K14 R125H, coassembled with K5, does unravel just the same, indicating that Arg125 is not involved in this distinct filament dissociation process (Fig. 8d). The large arrows indicate IFs in the process of unraveling, some exhibiting a beaded structure as depicted in Fig. 8d. Some IFs are still in a compacted form side by side and partly unraveled (arrowhead in Fig. 8d), whereas many are already entirely unraveled (small arrows in Figs. 8c and 8d). Taken together, our data clearly indicate that the dramatic reorganization of the keratin cytoskeleton in the basal epidermal cells of EBS patients requires the application of mechanical stress and is not simply the result of an assembly defect. Epidermal Keratin IFs Are Dynamically Reorganized by Vimentin Coil 2B Peptides

FIG. 4. Induction of filament formation of keratin 8/18 by 23-mer keratin “head” peptides. (a) Structures obtained after overnight dialysis into 5 mM Tris–HCl (pH 8.4), 1 mM EDTA, 0.1 mM EGTA, 1 mM DTT. Note that in addition to tetramer-like fibers, some irregular aggregates are seen. (b) With a 20-fold molar excess of the 23-mer K18-peptide (see Table I), extended fibrillar structures are abundantly present after 1 h of incubation at room temperature. (c) Following addition of the K8 peptide, long regular, IF-type filaments are present, some of which have apparently further associated into thick bundles. The structures obtained were fixed in solution with 1.1% glutaraldehyde for 5 min, applied to carbon-coated grids, and negatively stained with 1% uranyl acetate. Bar, 200 nm.

present after dialysis into 0.7 mM sodium phosphate, pH 7.5 (Fig. 7B, d), and they further reorganized into bona fide IFs after addition of potassium

We have recently shown that a 60-amino-acidlong helix 2B peptide of human vimentin causes the structural transformation of mature vimentin IFs into irregular fiber arrays resembling those obtained with the temperature-sensitive amphibian vimentin when assembled at the nonpermissive temperature, i.e., 37°C (Strelkov et al., 2002; see also Herrmann et al., 1993). We now wondered if this peptide, representing the evolutionarily conserved C-terminal rod segment of IF proteins, would also affect K5/14 IFs. To our surprise, at a 10-fold molar excess, the vimentin peptide destroyed the IF morphology of K5/14 filaments (Fig. 9a) within seconds (Figs. 9b and 9c). This dramatic structural reorganization was not readily reversible, since by 5 min the beadlike morphology of the filament remnants persisted (Fig. 9d). These data document that epidermal keratin K5/14 IFs are intrinsically open and dynamic structures that are directly accessible by external interaction domains such as the helix 2B peptide of human vimentin. More importantly, although the primary sequence of vimentin, K5 and K14 deviate to some extent even in the highly conserved coil 2B end segment, the critical functional amino acid side chains have been evidently conserved to the extent

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FIG. 5. Assembly of keratin 8/18 (0.2 mg/ml) as mediated by incubation with a 10-fold molar excess of C-terminally crosslinked peptides (see Table I) in 5 mM Tris–HCl (pH 8.4), 1 mM EDTA, 0.1 mM EGTA, 1 mM DTT for 1 h at room temperature. (a) (K18 –23-mer)2; (b) (K8 –23-mer)2; (c) (HuVim-23-mer)2; (d) (Hu NF-L-23-mer)2; (e) (XlVim-23-mer)2. Note that the filaments in (a) and (d) are approximately 10 nm in diameter. Bar, 200 nm.

that mimetic coil 2B peptides can interact with mature keratin IFs, thereby causing a drastic reorganization of their structure. This behavior of keratin filaments and IFs more generally may be of principal physiological importance. DISCUSSION

The Effects of Employing Different Assembly Regimes Previously, in vitro assembly of keratins has been carried out following a number of different regimes using stepwise dialysis from high-urea buffers into either moderately concentrated Tris solutions (5 to 50 mM) of physiological pH or into buffers of very low ionic strength and high pH followed by a neutralization step using Tris buffers of neutral pH. The resulting filaments were analyzed by EM both morphologically and in terms of their mass-per-length distribution. In addition, kinetic and rheological studies were undertaken with the low ionic strength/high pH system (Steinert et al., 1976; Steven et al., 1982; Franke et al., 1982; Aebi et al., 1983; Coulombe and Fuchs, 1990; Hatzfeld and Weber, 1992; Hofmann and Franke, 1997; Herrmann et al., 1999; Ma et al., 2001). By now it has become evident that keratins, very

much like the other major cytoplasmic intermediate filaments (vimentin, desmin, and NF-L), form ULFs as a distinct assembly intermediate (Herrmann et al., 1999). In the present study we have further investigated the assembly of keratins and have shown that with K8 and K18 longitudinal filament growth occurs almost simultaneously with ULF formation. At early time points, short full-width filaments were observed that varied in length roughly by 25-nm increments similar to those observed with mouse epidermal keratins in pioneering studies by Steinert (1991). Accordingly, over the first 20 min these epidermal keratins (consisting of mostly K1 and K10) assembled into IFs very slowly, i.e., by the association of short IF-like particles that we would now call ULFs (Steinert, 1991). In contrast, K8/18 form ULFs by 2 s, suggesting very different assembly kinetics for these two pairs of keratins (Herrmann et al., 1999). Hair keratins, in turn, are different again in that they cannot assemble under low-Tris-buffer conditions, but instead require vimentin-like assembly conditions for efficient filament formation (Winter et al., 1997). Moreover, we observed that K5 and K14 also assemble extremely rapidly, i.e., within seconds, into IFs from ULF-like

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FIG. 6. Electron microscopy of negatively stained structures obtained with human recombinant K5 and K14. (a) Proteins were dialyzed from 6 M urea into 2 mM Tris–HCl (pH 9.0) overnight and directly applied to a carbon-coated grid. (b) After addition of Tris–HCl to 10 mM (pH 7.5), ULF-like structures were obtained by 10 s. (c) By 10 min filaments looked more regular but still far from normal IFs. (d) Dialysis from 6 M urea into 0.7 mM sodium phosphate (pH 7.5) reveals filaments already in the absence of salt which apparently elongate and have a smoother surface by 10 s (e) and 10 min (f) after addition of KCl to 20 mM. The insert in (a) demonstrates by gel electrophoresis that K5/14 are quantitatively retained in the supernatant (S) after centrifugation of a mixture of K5 and 14 (M) for 30 min at 10 psi in an Airfuge. No material was obtained in a pellatable form (P). This indicates that no major filamentous or aggregated structures were lost from the grid during preparation for electron microscopy. Bar, 100 nm.

particles, even at low ionic strength and low protein concentration. In low-Tris buffers, however, the K5/14 filaments were shorter and of more variable diameter than those formed by K8 and K18. A longstanding challenge for intermediate filament biologists has been to determine why there is a need for so many intermediate filament proteins, in particular, epithelial keratins. For example, the big differences in assembly kinetics as documented in the present investigation hint at one area in which these proteins may differ functionally and thereby could have important implications for their distinct properties in tissues. Similarly, large differences in assembly kinetics were noted by us for vimentin and desmin, two filament proteins of high sequence identity that are often assumed to be functionally more or less interchangeable (Herrmann et al., 1999). An alternative model for intermediate filament assembly has assumed the formation of full-width

filaments by lateral association of protofilaments, rather than longitudinal annealing of ULFs. The distinct protofibrillar substructure of intermediate filaments manifested itself through experiments that caused unraveling of keratin intermediate filaments (Aebi et al., 1983). More specifically, when intermediate filaments that were assembled in 5 to 10 mM Tris–HCl were adsorbed to a glow-discharged carbon support film on an EM grid and then incubated with 10 mM sodium phosphate for 10 min, they started to unravel into distinct subfilamentous strands. This subfilamentous organization of keratin IFs is particularly well demonstrated in unidirectionally metal-shadowed and negatively stained specimens (see Figs. 8b to 8d). In the absence of more conclusive evidence for any other assembly model, this unraveling of IFs was interpreted by a number of experts in the field as an indication of a distinct substructure that, in turn, might reflect

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their mode of assembly (cf. Parry and Steinert, 1995, for an overview). Unraveling of IFs can be observed not only with keratins (Franke et al., 1982; Aebi et al., 1983; Eichner et al., 1985; for K8/18, see Fig. 2d in Hofmann and Franke, 1997); it also occurs just as efficiently with vimentin (Herrmann and Aebi, 1998a) and the neurofilament triplet protein NF-L (Heins et al., 1993). Although IFs may be unraveled for their entire length, there are never longer segments of autonomous protofibrils depicted, unlike microtubules where individual protofilaments can form long stable structures (for a historical micrograph where a microtubule splits up into protofilaments, see Ballowitz, 1888, in Schliwa, 1986). We concluded that the protofibrillar appearance within unraveling IFs may reflect their tendency to bind or exchange oligomers such as, e.g., tetramers rather than catching them in the act of assembly (Herrmann and Aebi, 1998b). Taken together, it has now been well documented that, except for the “long-rod” intermediate filament proteins, i.e., the nuclear lamins (Stuurman et al., 1998) and the invertebrate filaments (Geisler et al., 1998) that undergo longitudinal polymerization before lateral association, intermediate filament polymerization proceeds via a ULF-like assembly stage.

(Herrmann and Aebi, 1998a). When mimetic peptides are dimerized by C-terminal cross-linking, in addition to “unlocking” the IF head domains these peptides also enhance filament–filament interactions so as to yield filament bundles. On the one hand, the extent of peptide-mediated filament formation appears to correlate with the number of basic residues residing within the mimetic peptide (see Figs. 4 and 5, and Table I). On the other hand, the specific amino acid sequence of a given mimetic peptide is not critical since peptides and fragments derived from other proteins, such as filaggrin and plakophilin 1, may exert a similar effect on in vitro IF assembly and bundling (Mack et al., 1993; Hofmann et al., 2000). In contrast to the basic head peptides, the 60residue coil 2B peptide, considerably longer than those used in the past by Hatzfeld and Weber (1992) and Geisler et al. (1993), causes a drastic, instantaneous reorganization of the IFs. Just as in the case of vimentin, we did not observe any significant bundling of keratin IFs. Bundling of vimentin has previously been reported to occur with a vimentin consensus motif peptide; however, that peptide was much shorter and used at a very high molar excess (Kouklis et al., 1992).

Basic Mimetic IF Head Peptides Induce Filament Formation, whereas a Helix 2B Peptide Abolishes Normal IF Organization

The EBS-type Keratin K14 R125H Mutation Does Not Inhibit in Vitro Filament Assembly

Dialysis of both vimentin and keratins into solutions of neutral pH and moderately high ionic strength yields abundant filaments (for example, K5/14 at 0.7 mM sodium phosphate; see Figs. 6 and 7). In contrast, at high pH no IF assembly occurs. The primary reason for this pH dependence may be the “locking” of the very basic head domains into an unproductive conformation that, in turn, may be “unlocked” by raising the salt concentration. In support of this hypothesis, a number of IF mimetic peptides are able to “force” keratin tetramers and higher-order oligomers into filaments. In fact, a similar behavior has also been shown for vimentin

Investigation of keratin assembly dynamics is of particular interest since single amino acid changes have been shown to represent the primary cause of a variety of genodermatoses (Irvine and McLean, 1999; Lane, 1994; Fuchs, 1995). The first such disease to be discovered was EBS, which is a disease characterized by severe skin blistering (Coulombe et al., 1991) and, in severe cases, by pronounced keratin clumps found in the cytoplasm of the affected cells. The severity of the disease is usually correlated with the exact position of the point mutation in the keratin, with the most severe mutations residing in the highly conserved motifs at either end of the ␣-helical rod domain. In several cases a highly con-

FIG. 7. Analysis of the assembly properties of the EBS mutant K14 R125H. (A) Sequencing profile with translation into the single-letter amino acid code below the DNA sequence. The mutated nucleotide is indicated by arrows above and below the sequence profile. (B) Electron microscopy of negatively stained structures obtained with human recombinant K14 R125H and wild-type K5. (a– c) Filament formation was initiated by adding an equal volume of 20 mM Tris–HCl (pH 7.0) to proteins (a) dialyzed into 2 mM Tris–HCl (pH 9.0), (b) leading to long open IFs after 10 s, and (c) even longer, more compact IFs after 10 min. (d–f) The sample was dialyzed into phosphate buffer (d) and analyzed 10 s (e) and 10 min (f) after addition of KCl to 20 mM (for details and comparison with wild-type K5 and 14, see Fig. 6). Bar, 100 nm. FIG. 10. Hypothetical view of how a 60-nm-long antiparallel, half-staggered tetrameric subunit might orient within a cylindrical structure of 10-nm diameter. The numbers indicate how far the tetramers may extend along the axis of the tube. We assume that the two coiled-coil chains are rod-like and are flexible at the hinges (which would correspond to linkers L12 in an A11 orientation; for details see Parry and Steinert, 1995).

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FIG. 8. (a, b) Unidirectionally shadowed keratin IF obtained from cultured human epidermal cells (a) reconstituted in 5 mM Tris–HCl (pH 7.5). (b) After incubation in 10 mM sodium phosphate (pH 7.5) for 10 min individual filaments start to unravel (arrows), thereby presenting a fibrillar substructure (for details see Aebi et al., 1983). (c, d) Electron microscopic analysis of negatively stained IFs. After the identical phosphate buffer treatment, recombinant K5/14 (c) and (d) K5/14 R125H IFs unravel extensively with only few IF segments left intact. Sometimes filaments appear to be caught during unraveling (arrowhead in d). The small arrows indicate fully unraveled IFs. Bar, 100 nm.

served arginine (R125) has been found to be mutated to either a cysteine or a histidine (Coulombe et al., 1991; Irvine and McLean, 1999). In this context, we compared the in vitro assembly into filaments of K5 and K14 R125H with wild-type K5 and K14. To our surprise, we found that the mutant keratin pair formed completely normal IFs. In fact, the mutant filaments were even longer and more regular than the corresponding wild-type filaments. This is a rather unexpected finding because other evidence suggested that keratin mutations in either the helix initiation or the helix termination motif are detrimental to filament assembly. For ex-

ample, K14 R125H caused disruption of the keratin intermediate filament network when transfected into SCC-13 cells (Letai et al., 1993). However, these experiments were performed by transient transfection of plasmids carrying strong promoters that may lead to heavy overexpression of the respective protein so that there is not enough partner keratin in the affected cells. Moreover, both the transfections and in vitro assembly assays were performed with tagged proteins, which may cause additional problems. Letai and co-workers also carried out in vitro assembly experiments documenting that shorter and more irregular filaments were formed with K14

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FIG. 9. Electron microscopic analysis of negatively stained K5/14 IFs (a) before and (b– d) after addition of a 10-fold molar excess of the human vimentin coil 2B peptide. Already after 10 s (b and c) the filament structure was heavily affected, such that only a few ULF-type fibers were left (arrows in b) but most of the IFs were converted into beaded structures (open arrowhead in b). By 5 min (d) only arrays of lined-up beads were present, indicating that these structures prevailed over time. Bar, 100 nm.

R125H, although here low-molarity Tris buffers were used. Also, keratins isolated from epidermal cultures of EBS–Dowling-Meara patients assembled into normal IFs, albeit appearing somewhat shorter. It is conceivable that these rather subtle morphological differences may indeed have to do with the exact experimental setup used by the different research groups. Interestingly, chemical crosslinking has also suggested a role for the R125 residue in the correct molecular registration of the tetramer stage (Mehrani et al., 2001), a role that would predict this mutation to cause a substantial impediment to normal filament formation. Nevertheless immortalized keratinocyte cell lines derived from patients with EBS can incorporate mutant keratins into their IF network with no significant aberration unless the cells are stressed (Morley et al., 1995; and our unpublished data).

Our experimental findings document that early filament assembly stages are not inhibited by the R125H mutation in K14. Hence, we may have to look further downstream for the actual pathogenic mechanism eventually causing EBS. For example, rather than interfering with filament assembly, some of the EBS mutations may affect filament bundling (Ma et al., 2001) and/or inhibit interactions with other cytoskeletal components (see Wiche, 1998; Clubb et al., 2000). General Concepts Governing Intermediate Filament Substructure One fundamental problem associated with intermediate filament structural biology is that, as yet, we do not know exactly how the dimers, i.e., the basic building blocks of IFs, are spatially oriented within an intermediate filament. Assuming a high

TABLE I Amino Acid Sequence of the First 22 Amino Acids Excluding the Initial Methionine of the N-Terminal Domain of Various Human IF Proteinsa Protein

Amino-terminal sequence (including an additional C-terminal cysteine)

Number of basic residues

Number of acidic residues

huK18 huK8 huVim huNF-L XlVim

SFTTRSTFSTNYRSMGSVQAPSC SIRVTQKSYKVSTSGPRAFSSRC STRSVSSSSYRRMFGGPGTASRC SSFSYEPYYSTSYKRRYVETPRC ATTKSSYRRIFGGNPRSSSSGNC

2 5 4 4 4

— — — 2 —

a Basic residues are underlined; acidic residues are indicated by boldface type. Xl, Xenopus laevis; hu, human; Vim, vimentin, NF-L, low-molecular-weight neurofilament-triplet protein.

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flexibility of the linkers joining the different rod segments (cf., Fig. 2b in Herrmann et al., 1996) and the mechanical properties of the coiled coil itself, an approximately 60-nm-long antiparallel and halfstaggered tetramer may pack in various ways within a 10-nm-wide filament (for a possible model, see Fig. 10). The orientation of the different tetramer segments relative to the filament axis determines the axial extent of the tetramer that, according to our model, may vary between 60 and 32 nm (see Fig. 10). The latter value and the depicted orientation and bending of the tetramer about linker L12 are as yet arbitrary and meant to illustrate the principle. Such spatial information, however, should be able to explain the typical 21- to 22-nm axial repeat seen in glycerol-sprayed, rotary-metal-shadowed specimens (Henderson et al., 1982), as well as the 46-nm axial periodicity depicted in negatively stained intermediate filaments (Steven et al., 1982). Last but not least, we do not even know the exact tetramer geometry in cross sections and if or how this may change during assembly (Herrmann and Aebi, 1998b). Therefore, whether oligomeric complexes run in continuation for a significant length of an IF, i.e., whether protofibrils are authentic building blocks of IFs, remains to be shown in the future. A preferred neighborhood of certain groups of tetramers/octamers in cross section, compatible with a protofibril-type organization, has been suggested by Parry et al. (2001) as an outcome of extensive analyses of chemically crosslinked IFs. Definitely, more information is needed in order to eventually understand how tetramers associate into octameric or higher-order complexes and to discern these distinct oligomeric species during assembly as well as in the mature filament. From our extensive assembly studies with vimentin it has become evident that the lateral association of dimers or tetramers into ULFs is highly preferred over their longitudinal association into protofilaments or protofibrils. The different stages of assembly probably involve unique reorganization events within the filament, i.e., longitudinal annealing of ULFs first involves a torsional “lock-in-type” event, followed by a radial compaction process that is probably mediated by a set of coordinated rearrangements of dimers, tetramers, and possibly higherorder oligomers relative to one another. We are currently undertaking crystallographic studies (Strelkov et al., 2001; 2002) aimed at solving the 3D structures of the different types of intermediate filaments at atomic detail in order to eventually arrive at a structure-based understanding of the differences between nuclear, epidermal, and vimentintype IFs and to begin to understand their distinct functional properties in the cellular content.

We thank Monika Brettel for expert technical assistance in the early phases of this work. Anne Wohlfahrt competently assembled the manuscript and Jutta Osterholt helped with the figures. We also thank Andreas Hunziker for DNA sequencing and Ralf Zimbelmann for computer work. We acknowledge support by the Deutsche Forschungsgemeinschaft and the “Fo¨ rderprogramm der Gemeinsamen Forschungskommission der Medizinischen Fakulta¨t Heidelberg (Projekt No. 158/96)” (both to H.H.), by the Tumorzentrum Heidelberg/Mannheim (to T.W.), as well as by the Swiss National Science Foundation, the Canton Basel-Stadt, and the M. E. Mu¨ ller Foundation of Switzerland (to U.A.). Support from the Cancer Research Campaign (SP2060/0103) is also gratefully acknowledged (R.M.P.; E.B.L.). Last but not least, we thank David A. D. Parry and John Squire for their patience and their interest in this paper. REFERENCES Aebi, U., Fowler, W. W., Rew, P., and Sun, T.-T. (1983) The fibrillar substructure of keratin filaments unravelled. J. Cell Biol. 97, 1131–1143. Aebi, U., Ha¨ ner, M., Troncoso, J., Eichner, R., and Engel, A. (1988) Unifying principles in intermediate filament (IF) structure and assembly. Protoplasma 145, 73– 81. Bader, B., Magin, T. M., Freudenmann, M., Stumpp, S., and Franke, W. W. (1991) Intermediate filaments formed de novo from tailless cytokeratin in the cytoplasm and in the nucleus. J. Cell Biol. 115, 1293–1307. Clubb, B. H., Chou, Y.-H., Herrmann, H., Svitkina, T. M., Borisy, G. G., and Goldman, R. D. (2000) The 300-kDa intermediate filament-associated protein (IFAP300) is a hamster plectin ortholog. Biochem. Biophys. Res. Commun. 273, 183–187. Conway, E. Y., and Parry D. A. D. (1988) Intermediate filament structure. 3. Analysis of sequence homologies. Int. J. Biol. Macromol. 10, 79 –98. Coulombe, P. A., and Fuchs, E. (1990) Elucidating the early stages of keratin filament assembly. J. Cell Biol. 111, 153–169. Coulombe, P. A., Hutton, M. E., Letai, A., Hebert, A., Paller, A. S., and Fuchs, E. (1991) Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: Genetic and functional analyses. Cell 66, 1301–1311. Eichner, R., Rew, P., Engel, A., and Aebi, U. (1985) Human epidermal keratin filaments: Studies on their structure and assembly. Ann. N.Y. Acad. Sci. 455, 381– 402. Franke, W. W., Schiller, D. L., and Grund, C. (1982) Protofilamentous and annular structures as intermediates during reconstitution of cytokeratin filaments in vitro. Biol. Cell 46, 257–268. Fuchs, E. (1995) Keratins and the skin. Annu. Rev. Cell Dev. Biol. 11, 1123–1153. Fuchs, E., and Weber, K., (1994) Intermediate filaments: Structure, dynamics, function, and disease. Annu. Rev. Biochem. 63, 345–382. Geisler, N., Heimburg, T., Schu¨ nemann, J., and Weber, K. (1993) Peptides from the conserved ends of the rod domain of desmin disassemble intermediate filaments and reveal unexpected structural features: A circular dichroism Fourier transform infrared, and electron microscopic study. J. Struct. Biol. 110, 205–214. Geisler, N., Schu¨ nemann, J., Weber, K., Ha¨ ner, M., and Aebi, U. (1998) Assembly and architecture of invertebrate cytoplasmic intermediate filaments reconcile features of vertebrate cytoplasmic and nuclear lamin-type intermediate filaments. J. Mol. Biol. 282, 601– 617. Grosberg, A. Y., and Khokhlov, A. R. (1997) Giant Molecules, Academic Press, San Diego, CA.

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P. M. (1985) Conformity and diversity in the structures of intermediate filaments. Ann. N.Y. Acad. Sci. 455, 371–380. Strelkov, S. V., Herrmann, H., Geisler, N., Lustig, A., Ivaniskii, S., Zimbelmann, R., Burkhard, P., and Aebi, U. (2001) Divideand-conquer crystallographic approach towards an atomic structure of intermediate filaments. J. Mol. Biol. 306, 773–781. Strelkov, S. V., Herrmann, H., Geisler, N., Wedig, T., Zimbelmann, R., Aebi, U., and Burkhard, P. Segments 1A and 2B of the intermediate filament dimer: Their atomic structures and role in filament assembly. EMBO J. 21, 1255–1266. Stuurman, N., Heins, S., and Aebi, U. (1998) Nuclear lamins: Their structure, assembly, and interactions. J. Struct. Biol. 122, 42– 66. Wang, J., Karabinos, A., Shunemann, J., Riemer, D., and Weber, K. (2000) The epidermal intermediate filalament proteins of tunicates are distant keratins; a polymerisation-competent hetero coiled coil of the Styela D protein and Xenopus keratin 8. Eur. J. Cell Biol. 79, 478 – 487. Wiche, G. (1998) Role of plectin in cytoskeleton organization and dynamics. J. Cell Sci. 111, 2477–2486. Winter, H., Hofmann, I., Langbein, L., Rogers, M. A., and Schweizer, J. (1997) A splice site mutation in the gene of the human type 1 hair keratin hHa1 results in the expression of a tailless keratin isoform. J. Biol. Chem. 272, 32345–32352. Xu, J., Tseng, Y., and Wirtz, D. (2000) Strain hardening of actin filament networks. Regulation by the dynamic cross-linking protein alpha-actinin. J. Biol. Chem. 275, 35886 –35892.