Molecular Parameters of Type IV alpha -Internexin and Type IV-Type III alpha -Internexin-Vimentin Copolymer Intermediate Filaments*

Peter M. SteinertDagger §, Lyuben N. MarekovDagger , and David A. D. Parry

From the Dagger  Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892-2752 and  Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

During neuronal development, a dynamic replacement mechanism occurs in which the type VI nestin and type III vimentin intermediate filament proteins are replaced by a series of type IV proteins beginning with alpha -internexin. We have explored molecular details of how the type III to type IV replacement process may occur. First, we have demonstrated by cross-linking experiments that bacterially expressed forms of alpha -internexin and vimentin form heterodimer molecules in vitro that assemble into copolymer intermediate filaments. We show using a urea disassembly assay that alpha -internexin molecules are likely to be more stable than those of vimentin. Second, by analyses of the induced cross-links, we have determined the axial lengths of alpha -internexin homodimer and alpha -internexin-vimentin heterodimer molecules and their modes of alignments in filaments. We report that these dimensions are the same as those reported earlier for vimentin homopolymer molecules and, by implication, are also the same for the other neuronal type IV proteins. These data suggest that during neuronal development, alpha -internexin molecules are readily assimilated onto the pre-existing vimentin cytoskeletal intermediate filament network because the axial lengths and axial alignments of their molecules are the same. Furthermore, the dynamic replacement process may be driven by a positive equilibrium due to the increased stability of the alpha -internexin network.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Intermediate filaments (IF)1 are highly dynamic components of the cytoskeletons of most eukaryotic cells (1-3). Although a great deal is known about their protein complexity, composition, and expression characteristics during development and differentiation, comparatively less is known about IF structure and how this relates to their dynamism and functions in cells. Established data report that IF are built in an hierarchical manner. First, two compatible protein chains form a dimer molecule in which four well conserved alpha -helical sequences along a central rod domain of each chain associate in parallel and in register to make a segmented coiled-coil molecule (4-7). Second, a pair of molecules align in an antiparallel fashion (8, 9) in one of several basic modes (10-12) as follows: A11, half-staggered with the 1B segments largely overlapped; A22, half-staggered with the 2B segments largely overlapped; and A12, in which the entire molecules are largely overlapped. Determination of the exact alignments of the A11 and A22 modes has revealed a fourth mode termed ACN, in which the end of one molecule is overlapped by about 1 nm with the beginning of the next parallel molecule in the same axial row. Third, quantitative mass measurements suggest that 12-24 molecules, most commonly 16 in native IF (13-15), associate in three dimensions in as yet unknown ways to form an IF.

However, whereas most or all cytoplasmic IF appear morphologically similar in vivo or when reconstituted in vitro, there are complex variations in at least the first two of these hierarchical steps. In step one, the coiled-coil molecule may be either an obligatory heterodimer (all type I/II keratin chains) (6, 7), a homodimer (types III and some IV chains) (16-20), or a facultative heterodimer within a chain type, in which for example two different chains may participate (e.g. type III; Refs. 16 and 21-24) or be required (e.g. type IV; ref. 25, 26). In the second step, we have found that the exact axial alignments of the three nearest-neighbor assembly modes identified above vary between the different IF types. All cytokeratin molecules seem to be aligned the same way (10, 11) but differently from vimentin molecules (12). The consequence of this is that these chain types cannot and do not copolymerize in vitro, or in vivo, so that certain cells that co-express keratins and vimentin, for example, have elaborated different cytoplasmic IF networks (27). A related complexity arises in cells and tissues during development. Many embryonic or immature differentiating cell types express first one type of IF chain and then replace it in a dynamic exchange/replacement process (28) as differentiation proceeds. A cogent example of this are neuronal cells (reviewed in Ref. 29). Neuroectodermal stem cells initially express the type VI IF chain nestin (30) alone or with type III vimentin (central nervous system) (31, 32), or peripherin (peripheral or regenerating nerves) (33-35), or desmin (neuromuscular cells) (36). When the stem cells become postmitotic, transcription of types VI and III IF genes diminishes, and they are replaced by the first of a series of type IV neuronal IF chains, alpha -internexin (37-40), which in turn is largely replaced by the other type IV chains including the NF-L, NF-M, and finally the NF-H chains as mature neurons are formed (25, 41). To date there is considerable evidence from co-expression and co-transfection experiments for copolymerization of these different chain types onto the same IF networks or even individual IF in living cells (Refs. 21-26; reviewed in Ref. 30). However, certain molecular details of these changes in IF composition remain unclear. It has not been determined yet whether type III vimentin or peripherin and type IV alpha -internexin copolymerize into the same IF and, if so, at which level of IF hierarchy: does this occur at the dimer (that is homo- versus heterodimer) or tetramer (that is different pairs of homodimers) (4, 42-44), or higher (45)?

As a first step toward addressing the issue of type replacement during development, we have previously demonstrated that different keratin chain pairs can easily substitute for one another during differentiation because keratin molecules possess the same dimensions and align themselves in the same way (11). The related question thus arises as to how the type III vimentin network in neuronal cells can be substituted by the series of type IV chains during neuronal development. To address this question, we have explored in vitro the co-assembly properties of the type IV IF chain alpha -internexin with type III vimentin. We show by cross-linking experiments for the first time that these chain types can indeed co-assemble to form heterodimers and that heterodimers or homodimers of them may then participate to form IF. This then allowed us to perform further cross-linking experiments to establish that their precise molecular alignments are in fact the same.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Expression and Purification of Proteins-- A vector containing the full-length coding sequence of rat alpha -internexin in the pET11a system was a generous gift of Dr. Ron Liem (46). The vector expresses a product with an additional 14-amino acid sequence on the head domain of alpha -internexin derived from the T7 10 promoter. Following transformation into the host Escherichia coli B strain BL 21/DE3 pLys(S) (Novagen), cultures (0.5-1-liter volume) were grown in LB medium supplemented with 50 µg/ml ampicillin to A600 nm of 0.6, and protein expression was induced by the addition of 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 3 h. The alpha -internexin was recovered and purified from bacterial pellets (10,000 × g for 30 min) as described (47). The yield was about 20 mg/liter of bacterial growth. In one experiment, 35S-labeled methionine (1000 Ci/mmol) was added during protein expression (0.5 µCi/ml of medium) to yield protein of 86 dpm/µg. The purified protein was stored at -70 °C in a buffer containing 10 mM Tris-HCl (pH 7.6), 1 mM dithiothreitol, 1 mM EDTA, and 0.1% SDS, freed of SDS by ion-pair extraction (48), and redissolved in a desired buffer (see below) containing 9.5 M urea immediately prior to use.

Purified bacterially expressed human vimentin was a generous gift of Dr. Robert Goldman and was stored and prepared for use as above.

IF Assembly in Vitro-- The conditions used for assembly of alpha -internexin were essentially identical to those described previously (37, 39, 49), at 0.2-0.5 mg/ml using a buffer containing 10 mM triethanolamine HCl in the pH range of 6.7-8.0, 0.17 M NaCl, 1 mM dithiothreitol, and 1 mM EDTA. Vimentin assembly was done at 0.2-0.5 mg/ml in a similar triethanolamine HCl buffer but at pH 7.4-8.0 (16, 19). alpha -Internexin and vimentin co-assembly experiments were done in similar buffers at pH 7.0-8.0. Solutions of expressed proteins in 9.5 or 6 M urea either singly or mixed as required were dialyzed twice against 1000 volumes of the buffer of choice. In all assembly experiments, protein exposure to concentrated urea solutions was kept to a minimum (<= 4 h). Samples were examined by negative staining with uranyl acetate on glow-discharged carbon-coated grids (50).

Cross-linking Procedures-- Cross-linking was performed using the periodate-cleavable bifunctional cross-linking reagent disulfosuccinimidyl tartrate (DST) as described previously (10-12). Before reaction, the pH of the triethanolamine HCl buffers was raised to 8.0. Cleavage was done by making desired protein solutions to 0.1 M sodium periodate and reaction for 1 h at room temperature.

In trial cross-linking experiments to determine molecular alignments, the amount of DST used ranged from 0.1 to 1 mM, but we found that 0.15 mM gave the most reproducible results; at higher concentrations, it appeared that the numerous lysine residues of the tail domain of alpha -internexin produced multiple random and nonspecific cross-linked products with rod domain lysines. Using an iodoacetamide titration assay (11, 12), only about 5% of the epsilon -NH2 groups of lysines were modified under these conditions. Thus we were able to achieve a very high degree of cross-linking specificity, but the molar yield of cross-linked peptides was quite low. Reactions were done for 30 min, stopped by quenching in 0.1 M NH4HCO3 (final concentration), and dried. We performed cross-linking reactions on homopolymeric alpha -internexin IF, oligomers of alpha -internexin (primarily homodimers and homopolymers estimated to be 16-mers (49)) formed in the absence of 0.17 M NaCl, and IF formed in mixtures of 70% alpha -internexin and 30% vimentin in 0.17 M NaCl. Following cross-linking, the products were resolved on 3-mm thick slab gels. The homodimer and homopolymer bands were eluted, freed of glycine salts, and dried.

In cross-linking experiments performed in concentrated urea buffers to examine molecular stabilities, we used 0.1 M DST instead to overcome the trace amounts of NH4+ ions in the urea solutions. In this case, to minimize cross-linking of collision complexes, the protein concentrations used were reduced 10-fold to 25-40 µg/ml. In trial experiments, we found that the extensive degree of protein modification that occurred under these conditions changed the mobilities of the monomer and dimer bands by <10%. alpha -Internexin or vimentin were equilibrated in the pH 8.0 buffer (50 µl) of desired urea concentration for 1 h at room temperature before addition of the DST, reacted and terminated as above, and made to 2% SDS in sample buffer before electrophoresis on 3.75-7.5% gradient PAGE gels.

To examine formation of heterodimers in mixing experiments, copolymer IF were assembled as described above, pelleted at 100,000 × g for 30 min in a Beckman Airfuge, and amounts in pellets, that is assembly efficiencies, were determined either spectrophotometrically or by amino acid analysis after acid hydrolysis. The pellets were redissolved in 10 mM triethanolamine HCl buffer (pH 8.0) containing M urea (10-40 µg/ml) for 1 h at room temperature before addition of the DST to 0.1 M. Alternatively, mixtures in 9.5 M urea were dialyzed into 6 M urea buffer for 2 h at room temperature before cross-linking as above. In these two cases, the terminated reactions were resolved on shallow 5-7.5% gradient SDS-PAGE gels. Coomassie-stained gels were scanned with a densitometer. Quantitative data were obtained after normalization of staining intensities of 0.1-1-µg amounts of alpha -internexin and vimentin on control gels. Alternatively, gels were examined after Western blotting using monoclonal antibodies against either vimentin (Boehringer Mannhiem) or alpha -internexin (a kind gift of Dr. Liem) and developed by enhanced chemiluminescence (Amersham Pharmacia Biotech).

Analysis of DST Cross-linked Peptides-- The dried DST cross-linked products and untreated alpha -internexin or vimentin were redissolved (1 mg/ml) in 70% aqueous formic acid and reacted with a 1:1 weight of protein:CNBr overnight. Following drying, the reactions were digested to completion with trypsin (Sigma, bovine, sequencing grade) using 2% enzyme for 6 h at 37 °C, at which time another 1% was added for a further 12 h. Peptides were resolved by reverse-phase HPLC as before (10-12). Potential DST cross-linked peaks were identified by comparisons of profiles before and after cross-linking and then harvested preparatively. When necessary, peaks were purified by a second run. An aliquot of each candidate peak was cleaved with periodate, and the products resolved by HPLC. When two peaks were obtained, they were each subjected to Edman sequencing in a Porton LF-3000 sequencer. When only one peak was obtained, another uncleaved aliquot was used to measure the amount by amino acid analysis following acid hydrolysis, and a third aliquot was used for sequencing. In this way, it could be determined whether the single-cleaved product peak contained only one uncross-linked peptide, or two peptides that were cross-linked together but could not be resolved by HPLC, or contained a peptide cross-linked to itself. By a combination of these methods, the lysine residues involved in cross-links could be unequivocally identified.

Isolation and Characterization of Stable alpha -Helix-enriched Particles-- Copolymer IF (0.2-0.4 mg/ml) were digested by dripping during a 30-s period into a solution of trypsin (Sigma, sequencing grade) in a buffer of 50 mM triethanolamine HCl (pH 8.0) to achieve a net protein:enzyme ratio of 100:1, and the digestion continued for 10-17 min at room temperature (8, 9, 51, 52). Tetramer and dimer alpha -helix-enriched particles were recovered by precipitation at pH 5.2 with 0.1 volume of 3 M sodium acetate, collected by centrifugation, and resolved by chromatography on a 20 × 1-cm column of Sepharose 6B equilibrated in a buffer of 50 mM sodium tetraborate (pH 9.2). The fractions eluted in peak 3 consisting of 2B dimers were collected by reprecipitation at pH 5.2. Aliquots were cross-linked with 0.1 M DST as above, and the products were resolved on 5-10% gradient SDS-PAGE gels. Aliquots were also chromatographed by FPLC on a 5 × 1-cm column of MonoQ equilibrated in the borate buffer and eluted with 50 ml of a 0-0.15 M KCl gradient. Samples of each of the three peaks were sequenced.

Determination of Axial Parameters in alpha -Internexin and alpha -Internexin/Vimentin Copolymer IF-- Each of the cross-links could be assigned by simple inspection to one of four modes (see Figs. 6 and 7); one group arose from intramolecular cross-links across the alpha -internexin homodimer or alpha -internexin-vimentin heterodimer. Each of the others could be assigned to one of three groups that arose from intermolecular cross-links established previously for epidermal keratin (10, 11) and vimentin (12) IF. These data were used in a least squares analysis to refine the six parameters (A12, A11, and A22, and the linker lengths L1, L12, and L2) that define the axial lattice structure of the alpha -internexin IF (10-12).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Assembly of alpha -Internexin in IF in Vitro and Copolymerization with Vimentin-- Vimentin IF assembled in vitro in buffers of pH 7.4 to 8.0 were long (>20 µm) smooth-walled structures uniformly 10 nm in diameter (Fig. 1A). Optimal assembly of alpha -internexin occurred at pH 6.7, but IF were much shorter (0.5-2 µm) and somewhat irregular in width (8-15 nm) and appearance (Fig. 1B). Also, irrespective of the protein concentration used for assembly, some particles were only 60-100 nm long, described previously as "half-thickness unit-length" molecular aggregates that contain 16 chains (49). Above pH 7.0, such particles predominated (Fig. 1C, at pH 8.0). These characteristics for alpha -internexin IF have been reported previously (37, 39, 49). Mixtures of alpha -internexin with 25 (Fig. 1D), 50, or 75% vimentin formed IF that were longer at pH 7.0 (Fig. 1D) than at pH 8.0 (Fig. 1E) and appeared more like vimentin IF alone, since there were very few short particles and most were regularly shaped. Similar images for alpha -internexin and vimentin IF were obtained when assembled from either 9.5 or 6 M urea (data not shown). Together, these studies indicate that the bacterially expressed proteins are indeed co-assembly capable and, moreover, encouraged us to explore further the hierarchical level of IF structure at which the type III vimentin and type IV alpha -internexin co-assembled.


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Fig. 1.   Morphology of IF assembled in vitro. Recombinant human vimentin assembled at pH 8.0 (A). Recombinant rat alpha -internexin assembled at pH 6.7 (B) or pH 8.0 (C). A 25/75% mixture of vimentin/alpha -internexin assembled at pH 7.0 (D) or pH 8.0 (E). Bar, 100 nm.

Cross-linking with DST Suggests alpha -Internexin Forms Stable Heterodimers with Vimentin-- Two high molecular weight bands of protein were observed when vimentin (Fig. 2A) or alpha -internexin (Fig. 2B) at 25 or 250 µg/ml (data not shown) in pH 8.0 buffer were assembled in the presence of 0.17 M NaCl, followed by cross-linking with 0.1 M DST. Irrespective of the protein concentration, in the absence of salt, vimentin formed primarily tetramers and some higher oligomers, as expected (16, 19, 22, 53, 54) (Fig. 2A). In contrast, alpha -internexin formed a high molecular weight band, corresponding to the smaller molecular weight band seen in the presence of salt, that consists of a 16-chain oligomer (49) (Fig. 2B, arrow), as well as dimers and only traces of tetramers. When the two assembly reactions were performed at 25-40 µg/ml in the presence of various urea concentrations and then cross-linked after 1 h, we observed that vimentin tetramers were dissociated to dimers above 4 M urea and then to monomeric chains by 8.75 M urea (approximate 50% dissociation point) (Fig. 2A). However, the alpha -internexin dimers were not dissociated to monomers until about 9.25 M urea instead (Fig. 2B).


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Fig. 2.   Cross-linking reveals that vimentin (A) and alpha -internexin (B) oligomers possess different stabilities in concentrated urea solutions. The two proteins (25-40 µg/ml) were separately equilibrated from 9.5 M urea into pH 8.0 triethanolamine buffer in the presence or absence of 0.17 M NaCl for 16 h or in the presence of urea of the concentrations shown with no NaCl for 1 h. After cross-linking with 0.1 M DST, samples were resolved by SDS-PAGE on 3.75-7.5% gradient gels and developed with Coomassie Blue stain. The sizes of the major products, including the alpha -internexin 16-mer (arrow), are marked.

To assess further these different properties, alpha -internexin and vimentin in 9.5 M urea buffer were mixed in proportions ranging between 0 and 100%. Following assembly in the presence of 0.17 M NaCl in pH 7.0 buffer, IF were pelleted in the Airfuge, redissolved in 6 M urea, under which conditions they are dissociated into dimers (Fig. 2), cross-linked with 0.1 M DST, and resolved by SDS-PAGE. The shallow gradient used allowed separation of three dimer bands of 100-120 kDa which, by Western blotting with specific antibodies, contained alpha -internexin-alpha -internexin homodimers (uppermost band), alpha -internexin-vimentin heterodimers, or vimentin-vimentin homodimers (Fig. 3A). Notably, quantitation of the amounts of each protein observed in Coomassie-stained gels (Fig. 3B) revealed that in the presence of molar excesses of alpha -internexin, significant amounts of alpha -internexin-vimentin heterodimers were formed; particularly in mixtures of 70% alpha -internexin and 30% vimentin, the resulting IF contained only traces of vimentin homodimers but about equal amounts of alpha -internexin-vimentin heterodimers and alpha -internexin homodimers. Note in the control experiments of Fig. 2 that the M urea solutions could not effectively dissociate either the alpha -internexin or vimentin homodimers to permit chain exchange in dimers. Also, in experiments using tracer amounts (0.1%) of 35S-labeled alpha -internexin, no label was incorporated into the central heterodimer band when mixed with vimentin in 6 M urea for up to 16 h (not shown). These experiments were repeated by dialysis of the initial protein mixtures in 9.5 to 6 M urea instead, followed by cross-linking and resolution by SDS-PAGE. Essentially identical data were obtained (not shown). Thus together, these data provide robust support for the notion that alpha -internexin and vimentin form heterodimers that are about as stable as alpha -internexin homodimers and measurably more stable than vimentin homodimers.


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Fig. 3.   Mixing experiments reveal that alpha -internexin forms homodimers or heterodimers with vimentin that are more stable than vimentin homodimers. Aliquots of alpha -internexin (Inx) and vimentin (Vim) in 9.5 M (A and B) or 6 M (C) urea were mixed in amounts ranging between 0 and 100%, assembled into IF, pelleted in the Airfuge, and redissolved in 6 M urea buffer. Assembly efficiencies were determined spectrophotometrically or by amino acid analysis after acid hydrolysis (green lines). The urea solutions were cross-linked with 0.1 M DST, resolved by 5-7.5% gradient SDS-PAGE, and either stained with Coomassie Blue stain (A) or developed for Western blotting using anti-alpha -internexin or anti-vimentin monoclonal antibodies (A, right lanes, respectively) to ascertain the dimer compositions. The stained gels were scanned with a densitometer to quantitate the amounts of each of the dimers, which are displayed in B and C. alpha -Internexin homodimers, blue lines; vimentin homodimers, black lines; alpha -internexin-vimentin heterodimers, red lines. Data are the averages ± S.D. of 2-4 separate experiments. A periodate (PER) control for DST cross-linking is shown in A.

As an independent analysis of these observations, we counted the numbers of potential ionic interactions between e-g, g---a, and d-e pairs of charged residues on the heptad repeats across these two homodimer and heterodimer molecules, as well as for other pairs of neuronally expressed IF chains (Table I). We determined the numbers for scenarios in which the two chains are aligned in exact axial register or offset by one or two heptads. The data reveal first that the largest number of favorable ion-pair interactions always occurs when the two chains are in exact axial register, as expected (55). Significantly, the highest two scores were found for alpha -internexin homodimers and alpha -internexin-vimentin heterodimers. Scores for alpha -internexin-neurofilament M and alpha -internexin-neurofilament H heterodimers were the next highest, followed by the score for vimentin homodimers. These analyses offer a potential explanation for the observed urea stability data seen in Figs. 2 and 3.

                              
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Table I
Summary of ionic interaction scores between various neuronally expressed IF chains

As a further control for these mixing experiments, we individually dialyzed the solutions of alpha -internexin or vimentin in 9.5 M urea to 6 M urea for 1 h, then prepared 0-100% mixtures, and assembled them into IF. Following cross-linking as above, we did not find the intermediate alpha -internexin-vimentin heterodimer band by SDS-PAGE (data not shown), but instead, the copolymer IF contained amounts of the two proteins in the same proportions as included in the mixtures (Fig. 3C).

Limited Proteolysis Methods to Isolate Stable alpha -Helix-enriched Particles Confirmed That alpha -Internexin and Vimentin Can Form Copolymer IF from Either Heterodimers or Homodimers-- We have demonstrated previously that it is possible to isolate stable alpha -helix-enriched oligomeric particles from keratin (8, 9, 51) and vimentin/desmin (54) IF by limited proteolysis procedures. These particles are derived from the longer 1B and/or 2B coiled-coil rod domain segments of the molecules because the head and tail domains, and linker segments of the rod domain, are considerably more sensitive to cleavage by proteolysis. When IF co-assembled in a 70:30% mixture of alpha -internexin and vimentin from 9.5 M urea were subjected to limited trypsin digestion, three time-dependent peaks of protein of different apparent sizes were recovered from a Sepharose 6B column (Fig. 4A). Previous studies have indicated that peak 3 material contains dimeric alpha -helix-enriched particles about 20 nm long that are derived from the 2B rod domain segments of the constituent chains (8, 9, 53, 54). Indeed, amino acid sequencing for 15 Edman degradation cycles of protein eluted in peak 3 yielded two sequences FANLNEQAARSTEAI and FADLSEAANRNNDAL that correspond exactly to residues 290-304 and 293-307 of rat alpha -internexin and human vimentin, respectively; these are equivalent to positions 5-19 of their 2B rod domains. When denatured and resolved on SDS-PAGE gels, two bands of about 12 and 13 kDa were obtained, thus indicating that this peak 3 peptide material consisted of most of the 2B rod domain regions of the two chains (data not shown). We further noted that peak 3 material could be resolved on a MonoQ FPLC column into three peaks labeled A, B, and C (Fig. 4B). Following sequencing again, peak A contains homodimeric 2B rod domain sequences of alpha -internexin only; the minor peak C contained the more acidic homodimeric 2B sequences from vimentin only; and the central peak B contained equimolar amounts from both chains. These peaks resolved because of significant charge differences between the 2B rod domain sequences of alpha -internexin and vimentin. When copolymer IF were assembled from mixtures made in 6 M urea instead, followed by digestion and fractionation on the Sepharose column, an identical elution profile was obtained (data not shown). However, when the peak 3 material was resolved by FPLC on the MonoQ column, only peaks A and C were obtained (Fig. 4C), containing homodimeric alpha -internexin and vimentin 2B fragments, respectively.


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Fig. 4.   Recovery of alpha -helix-enriched particles from alpha -internexin-vimentin heterodimers. Separation of particles on Sepharose 6B after limited trypsin digestion of copolymer alpha -internexin-vimentin IF assembled in a 70/30% mixture. Protein from peak 3 containing 2B dimer particles from a 9.5 M (B) or 6 M (C) urea assembly experiment was further resolved by FPLC on a MonoQ column. By amino acid sequencing the peaks contained the following: A, alpha -internexin 2B homodimers; B, alpha -internexin-vimentin 2B heterodimers; and C, vimentin 2B homodimers.

Cross-linking of alpha -Internexin and alpha -Internexin-Vimentin Copolymer IF with DST-- We then undertook cross-linking reactions in an effort to determine the exact alignments of nearest neighbor molecules in alpha -internexin homopolymeric and alpha -internexin-vimentin copolymer IF. In this case, cross-linking was done with 0.15 mM DST in order to maximize specificity and avoid interference between rod and tail domain lysines.

In the first set of experiments with alpha -internexin homopolymeric IF, Fig. 5 shows HPLC chromatograms of CNBr/tryptic peptides recovered from cross-linking reactions of the dimer formed in M urea (Fig. 5B) and 16-mer species formed in low salt buffer (Fig. 5C). Although the molar yields of cross-links were low, a total of 4 and 14 peaks, respectively, which disappeared on treatment with periodate and thus were candidate cross-linked species, were isolated and characterized by a combination of amino acid analysis and protein sequencing (Table II). The four peaks found in Fig. 5B were also present in Fig. 5C and clearly resulted from intramolecular cross-links between the two chains of the coiled-coil alpha -internexin dimer molecule. All 11 lysines of the rod domain of alpha -internexin participated in cross-links, and most were used multiple times with different partners. Interestingly, a number of these occupy exactly the same positions as were found in previous cross-linking studies of K1/K10, K5/K14, and vimentin IF (10-12), which adds validity to the significance to the present data set. Moreover, all 11 (10 unique species) seen in Fig. 5C could be confidently assigned to one of three modes of intermolecular alignment seen previously for these other types of IF as follows: the A11 (two cross-links, and see Fig. 6, second model from right) and A22 (three cross-links; Fig. 6, second model from left) modes which suggest staggered antiparallel alignments; and A12, in which the two molecules are antiparallel and in near registration (five cross-links, Fig. 6, center model).


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Fig. 5.   HPLC fractionation of DST cross-linked CNBr/tryptic peptides. The profiles are as follows: A, unreacted alpha -internexin; B, alpha -internexin dimers formed in 2 M urea solutions; and C, alpha -internexin 16-mer formed at pH 6.7 in low salt buffer. Comparisons of profile A with profiles B and C revealed shifted peaks that were chosen as candidate cross-linked peptides that were recovered for analyses. Numbers correspond to those of Table II. The broken line denotes the acetonitrile gradient.

                              
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Table II
Cross-links in alpha -internexin homopolymer and alpha -internexin-vimentin copolymer IF


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Fig. 6.   Molecular alignments adduced in alpha -internexin by DST cross-linking. Data are from Table II. Left model, four cross-links were induced between two parallel in-register chains in the fundamental homodimer molecule. Model A22, three cross-links joined two antiparallel molecules offset so that their 2B rod domain segments were largely overlapped; model A12, five cross-links joined two antiparallel molecules that were essentially completely overlapped; model A11, two cross-links joined two antiparallel molecules offset so that the 1B rod domain segments were overlapped; and model ACN arises on consideration of the A11 and A22 models from which it becomes clear that the end of the 2B rod domain segment of one molecule overlaps with the beginning of the 1A rod domain segment of a parallel molecule (shaded orange).

We then repeated these experiments with intact copolymer IF assembled from a 70:30 mixture of alpha -internexin:vimentin from 9.5 M urea. Under these conditions, about half of the molecules are alpha -internexin homodimers, and another half are alpha -internexin-vimentin heterodimers (Figs. 3 and 4). Since the amount of vimentin-vimentin cross-links was expected to be very low, and the positions of the cross-links on the HPLC profile in homopolymeric alpha -internexin were already known (Fig. 5), it was possible to identify new peaks that were candidates for cross-links between heterodimers. In this way, 29 peaks were obtained by HPLC (not shown) and sequenced (Table II), of which 3 were between alpha -internexin molecules, 5 were intramolecular across the heterodimer, and 4, 7, and 10 could be assigned to the A11, A22, and A12 modes, respectively (Fig. 7).


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Fig. 7.   Molecular alignments in alpha -internexin-vimentin copolymer IF by DST cross-linking. The data from Table II are summarized as in Fig. 6. In this case green designates the vimentin chain in the alpha -internexin-vimentin heterodimer in each of the alignment modes.

Calculation of Rod Axial Alignments and Linker Segments-- These data were used in a least squares analysis to refine the six parameters (A12, A11, and A22, and the linker lengths L1, L12, and L2) that define the axial lattice structure of the alpha -internexin. In principle, the actual positions of the cross-links determine the number of unique equations that can be derived. Providing that there are enough of these, the parameters can be refined successfully. In the case of alpha -internexin homopolymers, only four parameters refined satisfactorily (A12, A11, L1, and L2) and two did not (A22 and L12) (Table III). Interestingly, however, the nature of the equations is such that the difference between A22 and L12 is refinable and has a value similar to that seen previously for vimentin. Using the 16 unique data elements from the copolymer IF, however, all six parameters refined successfully (Table IV). Significantly, there is a high correspondence in these refined parameters and those previously determined for homodimeric vimentin. They differ from those determined for vimentin only in those parameters which were previously relatively poorly defined in vimentin, because of the nature and disposition of the limited cross-link data then available.

                              
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Table III
Unique alpha -internexin cross-links and the equations used in the least squares analysis
The equations are derived for example as follows: 1A-17/2B-112 is given by (1 × A12) + (0 × A11) + (0 × A22- (0 × L1) + (0 × L12) + (0 × L2) = 7 residues.

                              
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Table IV
Unique alpha -internexin-vimentin copolymer cross-links and the equations used in the least squares analysis


    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

alpha -Internexin and Vimentin Promiscuously Form Copolymer IF-- In this paper, we have demonstrated by a variety of biochemical experiments that type III vimentin and type IV alpha -internexin protein chains can form copolymer IF in vitro under a wide range of mixing conditions. Moreover, our data show that this co-assembly may occur at different levels of IF structural hierarchy. When assembled from mixtures made in 9.5 M urea solutions, wherein the proteins exist almost exclusively as single chains (Fig. 2), heterodimer formation is favored (Figs. 3 and 4) and IF are formed in high yield. In 6 M urea solutions, the chains form homodimers instead (Figs. 3 and 4), which also favored the subsequent formation of IF. Control experiments revealed that once homodimers are assembled in 6 M urea, little or no chain exchange can occur, until or unless the urea concentration is raised to approx 9-9.5 M. Similar data were established previously for keratin IF (8, 9). Accordingly, these data mean that copolymer alpha -internexin-vimentin IF may be assembled from either heterodimer or homodimer molecules. In mixtures formed in either 6 or 9.5 M urea containing <= 90% alpha -internexin, long (>5 µm) smooth-walled IF of uniform width are formed (Fig. 1), that is by all available criteria, the IF are morphologically identical. Thus there is no a priori reason why normal appearing copolymer IF could not consist of mixtures of both homodimers and heterodimers in vitro and in vivo.

These data complement and extend earlier in vitro studies on the assembly proclivities of types III and IV proteins (43, 44). In co-assembly experiments with the type III protein peripherin and alpha -internexin, it was not possible to ascertain whether the two proteins engaged in heterodimer formation because the authors were unable to resolve homodimers from heterodimers on the SDS gel systems employed (44). Furthermore, the cross-linking conditions with the copper-phenanthroline reagent could not provide evidence for heterodimeric interactions (44). However, the SDS gels and cross-linking experiments did reveal heterodimer formation of peripherin with the other larger type IV NF-M or NF-H chains (44). Accordingly, combinations of our present work and the studies with peripherin allow the general conclusion that all type III and all type IV IF chains are capable of heterodimer formation and co-assembly into IF.

Molecular Packing of Coiled-coil Molecules in Type IV IF Is the Same as Type III-- The only way by which such promiscuous co-assembly into IF could occur is if in fact each of the alpha -internexin or vimentin homodimer and alpha -internexin-vimentin heterodimer molecules has the same dimensions that permit co-alignment through several levels of structural hierarchy to form IF. Indeed, extensive cross-linking studies performed here all point to the conclusion that the molecular alignment parameters are all the same within experimental error. The limitations of the present data sets are imposed on us primarily by the availability of numbers of informative cross-links, which in turn is a reflection of the juxtaposition of available lysines for cross-linking. In the case of alpha -internexin homodimers, it was not possible to refine adequately all six parameters clearly (Table III), but robust data were obtained in the case of the alpha -internexin-vimentin copolymer IF (Table IV).

As the alignments are likely to be the same, we then re-evaluated the unique cross-link data (22 cross-links) from vimentin, alpha -internexin, and the alpha -internexin-vimentin copolymer IF in a comprehensive analysis to refine the axial parameters relating to the molecular disposition for all type III and all type IV IF (Table V). The values are determined with small standard errors (0.13-0.34) consistent with all of the data originating from a common set. Values of the parameters determined for the epidermal keratins using cross-link data, as well as those modeled for hard alpha -keratin from x-ray and other data, are given for purposes of comparison.

                              
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Table V
Summary of axial parameters of alpha -internexin and alpha -internexin/vimentin copolymer cross-link data adduced in this work, and comparison of similar data for other IF types
Axial data are measured in multiples of the mean axial rise in a coiled-coil conformation, hcc (=0.1485 nm).

It is interesting to compare these parameters and to assess common and differing features. First, the parameters for epidermal keratin, vimentin, and alpha -internexin lead to a head-to-tail overlap of about 7-10 residues between the beginning of segment 1A and the end of segment 2B of molecules aligned in the same axial row. These regions are the most highly conserved ones in IF chains as a whole. They also correspond to the regions in which many mutations leading to keratinopathies are located or closely juxtaposed (2, 3, 56). It has been predicted, nonetheless, that in hard alpha -keratin this head-to-tail overlap does not exist in vivo after disulfide bond formation occurs (57). Second, a feature common to IF (except type V), however, is the predicted molecular length, i.e. 308 residues in a coiled-coil conformation, a value equivalent to 45.6 nm. Likewise, the combined length of the linkers L1, L12, and L2 is equivalent to about 32 residues in a coiled-coil conformation (i.e. about 4.75 nm). Third, another common feature is the parallel in-register chain arrangement in all IF coiled-coil molecules whether they be homodimers or heterodimers. Fourth, key differences exist, however, between the various types of IF related to their A11 and A22 alignments of two neighboring antiparallel molecules. Hard alpha -keratin differs from the epidermal keratins in only one major respect, its value of A11. Likewise, hard alpha -keratin differs from the type III and type IV IF in only one parameter, its value of A22. The epidermal keratins thus differ from the type III and type IV IF in two parameters, A11 and A22. The axial periods of these structures are predicted to differ slightly but nonetheless significantly (hard alpha -keratin, 47.0 nm; epidermal keratins, 45.2 nm; types III and IV IFs, 44.2 nm).

In summary, it appears there may be three methods by which coiled-coil molecules are packed in cytoplasmic IF: (i) types I/II cytokeratins; (ii) types III and IV IF; and (iii) perhaps types I/II hard alpha -keratins. Type V lamins do not assemble into typical IF, and their molecules are longer and perhaps are packed differently (58, 59). It remains to be determined how molecules containing type VI nestin chains fit into this scheme.

alpha -Internexin Homodimers and alpha -Internexin-Vimentin Heterodimers Are More Stable Than Those of Vimentin, Implications for Neuronal Development-- Although alpha -internexin and vimentin chains appear to be able to co-assemble into morphologically identical IF and in equivalent yields, we have detected subtle but significant differences in the apparent stabilities of homo- and heterodimer molecules and in their states of assembly in low ionic strength buffers (Fig. 2). First, in low ionic strength buffer at pH 8.0 where efficient and specific cross-linking experiments are possible, vimentin forms primarily tetramers (Fig. 2A; Refs. 19, 22, 53, and 54), but alpha -internexin forms primarily a 16-mer oligomer (Fig. 2B; Ref. 49). However, by ultracentrifugation methods, alpha -internexin forms a more heterogeneous population of species as the pH is lowered toward known optimal IF assembly conditions near 7.0 (49).2 Such pH-dependent association phenomena have not been reported for vimentin (19, 22, 54). These data therefore allow the speculation that in living cells the state of assembly of an alpha -internexin-containing IF may be significantly affected by the micro-environment. Second, we noted that alpha -internexin homodimers and alpha -internexin-vimentin heterodimers are measurably more resistant to urea dissolution than vimentin homodimers (Fig. 2), and indeed, when mixed together, alpha -internexin prefers to form heterodimers with vimentin (Fig. 3). A proximal explanation for this may be the ability to form a larger number of pairs of ionic charges along the rod domains of the dimers (Table I). The potential cell biological consequences of this in vivo are significant. A variety of data have revealed that vimentin expression precedes that of alpha -internexin in developing neuronal cells (31, 32). Therefore it could be argued that the proposed equilibrium dynamic exchange/replacement of type III vimentin by type IV alpha -internexin (28) may be driven by a net increase in the stability of alpha -internexin-containing molecules and in this way more assertively drive the gradual replacement process during development. Moreover, this transition may result in a more stable IF network in the axons of maturing neurons.

    ACKNOWLEDGEMENTS

We thank Dr. Ron Liem for the generous gift of the protein expression vector for and monoclonal antibody against rat alpha -internexin; Dr. Robert Goldman and staff for a generous gift of purified bacterially expressed human vimentin; and Will Idler for expression and purification work.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Bldg. 6, Rm. 425, NIAMS, National Institutes of Health, Bethesda, MD 20892-2752. Tel.: 301-496-1578; Fax: 301-402-2886; E-mail: pemast{at}helix.nih.gov.

The abbreviations used are: IF, intermediate filament(s); DST, disulfosuccinimidyl tartrate; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography; FPLC, fast protein liquid chromatography.

2 J. Cohlberg, personal communication.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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