A High Molecular Weight Intermediate Filament-associated Protein in BHK-21 Cells Is Nestin, a Type VI Intermediate Filament Protein
LIMITED CO-ASSEMBLY IN VITRO TO FORM HETEROPOLYMERS WITH TYPE III VIMENTIN AND TYPE IV alpha -INTERNEXIN*

Peter M. SteinertDagger §, Ying-Hao Chou, Veena Prahlad, David A. D. Parryparallel , Lyuben N. MarekovDagger , Kenneth C. WuDagger , Shyh-Ing JangDagger , and Robert D. Goldman**

From the Dagger  Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892-2752,  Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611-3072, and parallel  Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand

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BHK-21 fibroblasts contain type III vimentin/desmin intermediate filament (IF) proteins that typically co-isolate and co-cycle in in vitro experiments with certain high molecular weight proteins. Here, we report purification of one of these and demonstrate that it is in fact the type VI IF protein nestin. Nestin is expressed in several fibroblastic but not epithelioid cell lines. We show that nestin forms homodimers and homotetramers but does not form IF by itself in vitro. In mixtures, nestin preferentially co-assembles with purified vimentin or the type IV IF protein alpha -internexin to form heterodimer coiled-coil molecules. These molecules may co-assemble into 10 nm IF provided that the total amount of nestin does not exceed about 25%. However, nestin does not dimerize with types I/II keratin IF chains. The bulk of the nestin protein consists of a long carboxyl-terminal tail composed of various highly charged peptide repeats. By analogy with the larger neurofilament chains, we postulate that these sequences serve as cross-bridgers or spacers between IF and/or other cytoskeletal constituents. In this way, we propose that direct incorporation of modest amounts of nestin into the backbone of cytoplasmic types III and IV IFs affords a simple yet flexible method for the regulation of their dynamic supramolecular organization and function in cells.

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Most animal cells possess complex cytoskeletons consisting of the following three principal classes of proteins: microtubules, microfilaments, and intermediate filaments (IF).1 In addition to the core structural proteins that comprise these classes, each is attended by a complex array of accessory proteins which in general serve to modulate their structures, states of assembly, supramolecular organizations, and functions. Of these three, IF are perhaps the most complex since they consist of more than 50 distinct proteins capable of forming morphologically similar filaments in different cell types (1-3). Currently, six different types of IF have been described including the types I/II keratins (40 or more protein chains) generally expressed only in epithelia; four known type III proteins that are widely expressed in tissues and cells; four known type IV chains usually expressed only in neuronal tissues; several type V nuclear lamins expressed in many nucleated cells; and nestin, a single type VI protein expressed primarily in a variety of embryonic cells including those of the nervous system. Nevertheless, all known IF chains are built according to a common plan of a highly conserved rod domain consisting of four segments that form two-chain coiled-coils with another compatible chain, enabling assembly of polymerized IF, and end domains of variable size and chemistry, which provide IF with a wide variety of unique binding and regulating domains. This nomenclature system of the six IF types has been based largely on the exon/intron structures of their genes as well as subtle sequence differences of the rod domain segments: members of a given sequence type generally show high degrees of homology, whereas those of different types show lesser degrees of homology (1-3). In this regard the status of the single protein nestin as a distinct type has been uncertain because its gene structure and protein sequence homology are of intermediate similarity to type IV IF chains (3). Furthermore, its rod domain is built with minor differences from that of the other cytoplasmic IFs because it does not possess an L2 linker region, and there are less regular distributions of ionic charges on the 1B and 2B segments.

The assembly proclivities of most IF chains are now well understood (1-3). Some chains are capable of assembly into IF by themselves, that is they are homopolymeric; examples include all type III chains and the type IV alpha -internexin and the neurofilament light chains (4-7). Many other chains require a partner for co-assembly; all keratin IF are obligate copolymers of virtually any type I chain with virtually any type II chain (8, 9); the larger type IV neurofilament medium and heavy chains can only participate in IF structures by co-assembly with the neurofilament light chain (10-12). Most if not all type III chains can form facultative copolymers with other type III proteins (4, 13-15). Furthermore, developmental in vivo as well as recent in vitro data have documented that some type III chains are capable of inter-type co-assembly with the type IV alpha -internexin chain (Refs. 7 and 16-21 and reviewed in Ref. 22). However, the status of the type VI nestin chain is less clear. Several reports have documented the co-expression of nestin with type III chains in vivo in developing neuronal and other embryonic cells (23-34), thereby raising the possibility that nestin may be capable of co-assembly, if not self-assembly (22, 35).

We have studied the function and dynamic properties of vimentin/desmin copolymer IF in baby hamster kidney (BHK-21) cells. In earlier studies we have documented that these IF co-cycle in in vitro assembly/disassembly experiments with certain high molecular weight proteins, suggestive of co-assembly (36-41), which we have termed intermediate filament-associated proteins (IFAPs). However, the nature of these proteins and details of their interactions with vimentin/desmin have not been explored. In this paper, we have made the surprising observation that one of these proteins is nestin. In vitro assembly and biochemical experiments document that whereas nestin cannot form typical IF by itself, it can form homodimer and heterodimer coiled-coil molecules with purified vimentin and alpha -internexin which may participate by co-assembly into typical IF.

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Isolation and Purification of an IFAP from BHK-21 Cells That Co-cycle with Vimentin-- BHK-21 cells were grown to 80% confluency in 850-cm2 roller bottles (Corning Glass Works, Corning, NY) in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% calf serum (Life Technologies, Inc.), 10% tryptose/phosphate broth (Difco), 100 units/ml each of penicillin and streptomycin. IF were then prepared from these cells with modifications from an earlier procedure (36). Specifically, cells were rinsed rapidly in phosphate-buffered saline (PBS) and then lysed in PBS containing 0.1% Triton X-100, 0.6 M KCl, 5 mM EDTA, 5 mM EGTA, and the protease inhibitors 1 mM phenylmethylsulfonyl fluoride, 1 mM p-tosyl-L-arginine methyl ester, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mg/ml aprotinin (all from Sigma). The extract was centrifuged at 15,000 × g at 4 °C for 30 min to pellet the IF-enriched cytoskeleton. Chromatin and actin in the pellet were solubilized by treatment with 5 mg/ml DNase for 30 min at 4 °C. Following recentrifugation at 15,000 × g for 30 min, the pellet of native IF and associated proteins was washed twice with PBS containing 5 mM EDTA, 5 mM EGTA, and protease inhibitors as above. EGTA was present throughout the purification procedures in order to prevent apparent Ca2+-dependent proteolysis of proteins.

This native IF preparation was solubilized into dissociation buffer containing 7.2 M urea, 50 mM Tris-HCl (pH 7.2), 5 mM EDTA, 5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 1 mM p-tosyl-L-arginine methyl ester (1 ml/roller bottle of cells). Following centrifugation at 14,000 × g for 30 min at 4 °C, it was dialyzed into two changes of 500 volumes each of PBS containing 5 mM EDTA, 5 mM EGTA, and protease inhibitors as above. Reassembled vimentin IF and associated proteins were recovered by centrifugation at 100,000 × g for 30 min. This disassembly/reassembly procedure could be repeated several times.

Further fractionation of the IF preparation was conducted by gel filtration. The pellet from the first recycling procedure as above was solubilized for 2 h in 1 ml/roller bottle of buffer containing 7.2 M urea, 50 mM Tris-HCl (pH 7.2), 5 mM EDTA, 5 mM EGTA, 2% ethylene glycol, 0.2% 2-mercaptoethanol, and protease inhibitors for, and clarified by, centrifugation at 100,000 × g. The supernatant was applied to a 100 × 2-cm column of Sepharose CL-400 (Amersham Pharmacia Biotech) pre-equilibrated in the same buffer. The flow rate was maintained at 6 ml/h. One-ml fractions were collected and analyzed by SDS-PAGE (42). Those containing the high molecular weight protein were pooled for ion exchange chromatography using a 5 × 1-cm column of Sepharose MonoQ FPLC (Amersham Pharmacia Biotech) equilibrated in the same buffer. The high molecular weight protein was eluted at 0.3 M NaCl as a single sharp peak using a 0-1 M gradient (see Fig. 2).

Similar Triton X-100/high salt extracts were made from a variety of other cultured cells using the same buffers and procedures. These included HeLa, NIH3T3, PtK2, and Pam212 cells. Each of these was obtained from ATCC and grown to near-confluency according to specifications. HaCaT cells were a kind gift of Dr. N. Fusenig and were grown as described (43). We also used normal primary human skin fibroblasts isolated from freshly excised neonatal foreskins (44) and normal human epidermal keratinocytes (Clonetics) (43). Hair follicles were isolated as described from 3-day-old newborn BALB/c mice (45).

Cloning and Sequencing of Nestin-- Hamster nestin clones were identified from a lambda 22a cDNA expression library constructed using poly(A)-enriched mRNA harvested from BHK-21 cells (46, 47) and a commercial cDNA synthesis kit (Life Technologies, Inc.). The library was screened first with a monoclonal antibody against rat nestin (28) (PharMigen, San Diego, CA) and subsequently using two polyclonal antibodies raised, respectively, against the IFAP material of HeLa (38) or BHK-21 (39-41) cells, the epitopes of which were heretofore unknown. The four cDNA clones that were positive for all three antibodies were subcloned into pBluescript pKS vector (Stratagene, La Jolla, CA) and sequenced. All four clones possessed identical 3'-ends, and the longest (2,969 bp) encompassed the three smaller clones. The sequence was highly homologous only to rat (Ref. 28; GenBankTM accession number M34384) and human (Ref. 29; GenBankTM accession number X65964) nestins. As the hamster nestin clone identified an mRNA species of about 6.5 kilobase pairs by Northern blot analyses, reverse transcriptase-PCR methods were employed to extend the available sequence information. A first strand cDNA synthesis reaction was performed using poly(A)-enriched mRNA from BHK-21 cells and the hamster-specific primer 5'-CGTTGTCTCTCTAGTCACTT located toward the 5'-end of the longest lambda 22a clone. The resulting cDNA was then used for PCR reactions with primer pairs consisting of the hamster-specific primer 5'-TTCCGATGCCATCTG-CTCAT nested within the above first strand primer, and a series of primers of sequences that are conserved between rat and human nestins near their 5'-ends. For example, two such primers 5'-CTACCAGGAGCGTGGTC (rat nestin, from nt 706) and 5'-AAGTTCCAGCTGGCTGTGGAA (rat nestin, from nt 905) located in sequences encoding the 2B rod domain segment, yielded PCR products of 2,983 and 2,784 bp. They were sequenced following subcloning into the pCR2.1 vector (Invitrogen, San Diego, CA).

Dot matrix comparisons of the amino acid sequences were performed with the programs COMPARE and DOTPLOT using a software package from the University of Wisconsin Genetics Computer Group (UWGCG, Madison, WI). Secondary structure predictions were undertaken using the AASAP package, which incorporates the methods of Chou and Fasman (48) and Garnier et al. (49). Fourier transform analyses to examine the possibilities of periodic distributions of residues or residue types were done as described (50).

In Vitro Assembly Experiments-- Purified recombinant human vimentin (51) and the keratin 5 and 14 chains (52) were prepared as described previously. An expression vector clone for human alpha -internexin was a generous gift of Dr. R. Liem. Following transfection into BL-21 Escherichia coli cells, protein was expressed in high yield and purified as described (21). The isolated hamster nestin and the above recombinant proteins were dissolved in a buffer of 9.5 M urea, 0.1 M Tris-HCl (pH 7.6), 1 mM EDTA, and 1 mM dithiothreitol, mixed (final protein concentrations in urea of 0.5-1 mg/ml) at the desired molar ratios, and then dialyzed into two changes of 1000 volumes of either L buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, and 1 mM dithiothreitol or H buffer which is the same but contains 0.15 M NaCl as well. IF were examined by electron microscopy following negative staining with 0.7% uranyl acetate (37). IF were pelleted (in 20-50-µl aliquots) at 100,000 × g in an Airfuge (Beckman instruments, Palo Alto, CA) for 30 min. Yields of protein in pellets were determined by protein assay (53) or spectrophotometrically.

Samples of IF assembly reactions in either L or H buffers were examined following cross-linking with 2 mM 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) exactly as described previously (54, 55), and the products were examined on 3.75-7.5% gradient SDS-PAGE gels. Gels were either stained with Coomassie Blue dye or electroblotted onto Nytran for Western analyses, using either anti-vimentin or the anti-nestin monoclonal antibodies, and then processed for development by enhanced chemiluminescence (Amersham Pharmacia Biotech) and quantitation as described previously (56).

Assay of Coiled-coil Molecular Stabilities-- Similar mixtures of proteins in 9.5 M urea solution were dialyzed into L buffer containing 2-9.5 M urea for 6 h and cross-linked with 0.1 M DTSSP. The higher concentration was needed to overcome the trace NH4+ ions in the solutions. The products were then resolved by 3.75-7.5% SDS-PAGE gels as above.

Isolation of alpha -Helix-enriched Particles from Nestin-Vimentin and Nestin-alpha -Internexin Copolymer IF-- Pellets of IF assembled from 1:4 molar mixtures of nestin and either vimentin or alpha -internexin (in which virtually all nestin was incorporated) were dissolved in 0.1 M sodium citrate buffer (pH 3.6) at about 1 mg/ml and then pipetted over a 30-s interval into a solution of trypsin (Sigma, sequencing grade) in 50 mM N-ethylmorpholine acetate buffer (pH 8.3) to give a final enzyme:protein ratio of 1:100 and final protein concentration of about 0.5 mg/ml. After 10 or 15 min digestion at 23 °C, a 1.5-fold molar excess of soybean trypsin inhibitor (Sigma) was added to terminate the digestion, and a one-tenth volume of 3 M sodium acetate (pH 5.2) was added to precipitate the alpha -helix-enriched products that were then pelleted at 15,000 × g. The pellets were redissolved in 50 mM sodium tetraborate containing 50 mM NaCl and chromatographed on a 5 × 1 cm FPLC column of Sepharose CL-4B (54). Protein material from peaks 2 and 3 was collected by precipitation at pH 5.2 as above, redissolved in borate buffer, cross-linked by use of DTSSP, and resolved on 3.75-7.5% SDS-PAGE gels. Peak 3 material was further resolved on a MonoQ column in the same buffer using a 0-0.15 M gradient of NaCl. The resulting three peaks of protein were collected, pelleted, redissolved in 50% aqueous acetonitrile, bound to a solid support, and examined by 10 Edman degradation cycles of protein sequencing (21). Some protein peaks from either the Sepharose or MonoQ columns in borate buffer were also used for estimates of alpha -helix contents by circular dichroism (57).

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High Molecular Weight IFAP Proteins Co-cycle with Isolated BHK-21 IF Preparations-- We have known for many years that native IF preparations isolated from BHK-21 fibroblasts as perinuclear "caps" following colchicine treatment contain numerous high molecular weight proteins in addition to the principal IF chains of desmin and vimentin (36-39) (Fig. 1). These IF preparations can be dispersed by dissolution in concentrated urea solutions or by resuspension in low ionic strength salt buffers, after which only traces of protein remain pelletable at 100,000 × g. When the urea is removed and or the ionic strength is subsequently raised to 0.15 M NaCl, the proteins in solution rapidly reassemble into morphologically native-like IF and >90% becomes pelletable (36, 37) (Fig. 1, lane 2); essentially only actin remains in solution (Fig. 1, lane 3). Moreover, the bulk of the high molecular weight proteins initially present are co-pelleted (arrow). This process may be repeated several times. For these reasons, we have always referred to these proteins as IFAPs.


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Fig. 1.   High molecular weight IFAPs co-cycle with vimentin/desmin IF from BHK-21 cells. Lane 1, colchicine-induced filament caps dispersed in L buffer. This solution was reassembled by raising the ionic strength to 0.15 M (=H buffer), and pelleted at 100,000 × g; lane 2, first pellet; lane 3, first supernatant. The pellet was then dispersed in L buffer, reassembled a second time as above, and repelleted; lane 4, second pellet; lane 5, second supernatant. The positions of migration of actin, desmin, vimentin and nuclear lamins are shown; the arrow denotes the high molecular weight proteins that co-cycle with vimentin/desmin. Positions of migration of standards are shown.

In this study, we have purified one of these major co-assembling proteins. We first used size-exclusion gel filtration chromatography on a Sepharose CL-400 column, from which two closely spaced bands were recovered (Fig. 2A). The upper of these was then purified to near-homogeneity by FPLC on a MonoQ column (Fig. 2B), with a net yield of 1-2 µg/roller bottle. The lower band, which may be a degradation product or another distinct protein species, could not be separated by this purification scheme.


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Fig. 2.   Purification of a high molecular weight protein from BHK-21 cells. The dispersed colchicine pellet of Fig. 1, lane 1, was chromatographed on Sepharose CL-400, and the fractions eluted near the void volume containing two high molecular weight proteins were recovered (A). These were rechromatographed by FPLC on a MonoQ column (B). Fractions 11 and 12 from the main peak eluted at about 0.3 M NaCl contained highly purified protein corresponding to the upper high molecular weight band. B, L = loaded sample.

The Co-assembling Protein Is Nestin-like-- Aliquots of this protein were digested with trypsin, and the products were resolved by fractionation using high pressure liquid chromatography (data not shown). A total of seven well resolved peptide peaks were sequenced, all of which displayed identity or high sequence homology only to rat (28) or human (29) nestins (Table I).

                              
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Table I
Peptides of purified IFAP reveal high homology to nestin
In each pair, the first sequence is the hamster peptide deduced in the present work and the second is the aligned homologous sequence of rat nestin (Swiss-Prot accession number P21263). Residue numbers of the hamster peptides were assigned from the available incomplete sequence; the accession number is AF110498. Notes that for peptides 4-6, the sequences occur multiple times.

Confirmation and Near-complete Sequence of Hamster Nestin-- The anti-rat nestin monoclonal antibody was used to screen a BHK-21 cell cDNA library configured in lambda gt22. A clone of 2,340 bp was identified, plaque-purified, and subcloned to facilitate sequencing. Its sequence is highly homologous only to those of rat (28) and human (29) nestins. During the course of this work, we discovered that two other polyclonal antibodies previously raised to high molecular weight proteins from HeLa (38) or BHK-21 (39-41) cells and of unknown specificity also identified the same cDNA clone as well as three other overlapping clones, the longest of which was 2,969 bp. In comparison to the rat and human nestins, this sequence extended from the middle of the carboxyl-terminal tail to the end of the coding region. Attempts were then made to obtain the full-length cDNA sequence by reverse transcriptase-PCR methods, and we were able to obtain an additional 2,886 bp of information (total of 5855 bp). However, we were unable to obtain the full-length sequence up to a potential initiation codon, perhaps due to mRNA instability or secondary structure interference. In comparison to the rat and human nestins, the available hamster data (GenBankTM accession number AF110498) starts from the nt encoding residue 186, or residue position 1 of the 2B rod domain segment of nestins. The available 120 residues of the rod domain sequences display 98% sequence homology. However, we do not possess an estimated 573 nt of additional sequences encoding about 6 residues of the head domain and the first 185 residues of the nestin rod domain. Thus the full-length hamster nestin mRNA would be expected to be about 6.5 kilobase pairs long, which corresponds closely to the size detected on Northern blots of BHK-21 cell RNA using the available cDNA clones as probes (data not shown), as well as rat and human nestins.

Most of the nestin sequences reside in long carboxyl-terminal tails containing 1,683 (hamster), 1,491 (rat), or 1,306 (human) residues. Using the COMPARE and DOTPLOT software packages, sequence homologies have been sought both within hamster nestin (data not shown) and between hamster and human nestins (Fig. 3). This approach illustrates that the first 200 residues in the carboxyl-terminal domains of the two proteins are very similar to one another as are the last 700 residues of both sequences. The intervening region (about 800 residues in hamster nestin and 300 residues for human nestin) displays an exceptionally high level of sequence identity in the form of 44-residue repeats in hamster (Table II) and 22-residue repeats in human nestin. In rat nestin the repeats are 44 residues in length. Some of the 44-residue repeats in hamster nestin, however, lack the decapeptide EGQESLSSPE. The 44-residue repeats consist of two very closely related halves, and the human repeat has a marginally higher identity to one of them and a relatively high identity to the other. In addition to this, even the 22-residue repeats or quasi-repeats can be further subdivided into a pair of 11 residue quasi-repeats. Interestingly, the DOTPLOT in Fig. 3 shows that parts of the sequences beyond the region characterized by the exceptionally well defined repeats also have some homology with the repeats themselves. In particular, the (longer) 44-residue repeats in hamster nestin display limited homology with the (shorter) 22-residue repeats in human nestin. This is especially evident over the carboxyl-terminal 350 residues in human nestin. The conclusion, therefore, is that vestiges of the 44- and 22-residue repeats exist in much of the carboxyl-terminal domains of both proteins. The secondary structure predictions for these repeats and quasi-repeats indicate very high alpha -helical content.


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Fig. 3.   Searches for homology between the sequences of hamster and human nestins using the COMPARE and DOTPLOT programs. The comparison uses a window of 30 amino acids and a stringency of 11 amino acids. Inclined lines, almost continuous, illustrate high homology between the sequences for segment 2B of the coiled-coil rod domain and for the first 200 and the last 700 residues of the carboxyl-terminal end domain of the chains. The intervening region (about 800 residues for hamster nestin and 300 residues for human nestin) shows highly conserved sequence repeats 44 and 22 residues long, respectively (see text for details), which are also found, albeit imperfectly, in the terminal 350 residues.

                              
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Table II
Hamster nestin peptide repeats
These are set in rows of 22 residues. Although the principal repeat motif of hamster nestin is 44 residues long, "harmonics" of 22 and even 11 residues are commonly observed. Also shown are the homologous equivalent rat nestin sequences where the principal repeat is also 44 residues and human nestin sequences where the principal repeat is 22 residues instead (and see Fig. 3). The dots indicate no consensus residue.

Nestin Is Widely Expressed in Fibroblastic but Not Keratinocyte Cell Lines-- We used a polyclonal anti-nestin antibody (38) to probe for the presence of nestin or nestin-like antigens in Triton X-100 extracts of a variety of cell types. In addition to BHK-21 cells, nestin was present in fibroblastic cells such as NIH3T3 and primary human foreskin fibroblasts, as well as the epithelioid HeLa cell line (Fig. 4, lanes 1-4). Each of these expresses abundant amounts of vimentin. However, nestin was only weakly present in PtK2 cells that make minor amounts of vimentin (lane 5) and was absent from cell types that do not make vimentin such as HaCaT- or PAM212-immortalized keratinocyte cell lines (lanes 6 and 7), primary human epidermal foreskin keratinocytes (lane 8), or neonatal mouse hair follicles (lane 9).


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Fig. 4.   Nestin is expressed in a variety of fibroblastic cell types but not keratinocytes or epithelioid cells. Triton X-100-insoluble cytoskeletal pellets were recovered from the following: lane 1, BHK-21 cells; lane 2, NIH3T3 fibroblasts; lane 3, primary normal human foreskin fibroblasts; lane 4, HeLa cells; lane 5, PtK2 cells; lane 6, HaCaT keratinocytes; lane 7, Pam212 keratinocytes; lane 8, primary normal human foreskin epidermal keratinocytes; and lane 9, neonatal mouse skin hair follicles. The 3.75-7.5% SDS-PAGE gels were then developed for Western blotting using an anti-nestin polyclonal antibody by enhanced chemiluminescence.

Co-polymerization of Isolated Hamster Nestin and Recombinant Human Type III Vimentin in Vitro-- Recombinant bacterially expressed human vimentin, isolated hamster nestin, or approximately 2:1 molar mixtures of both were mixed in the 9.5 M urea buffer and then dialyzed into either the low (L) or high (H) ionic strength assembly buffers. The products were then subjected to cross-linking by 2 mM DTSSP, resolved on 3.75 to 7.5% PAGE gels, and stained with Coomassie Blue dye (Fig. 5A), or transferred onto Nytran for Western blotting with either the monoclonal vimentin antibody (Fig. 5B) or monoclonal nestin antibody (Fig. 5C), and developed by use of enhanced chemiluminescence. Mobilities of the high molecular weight cross-linked products were compared with a ladder of cross-linked keratin 10 chains. As expected (41), vimentin formed primarily tetramers and some hexamer-octamers in L buffer, and mostly very high molecular weight aggregates in H buffer. In this gel system, purified monomeric nestin has an apparent size slightly less than that of the vimentin tetramer. In L buffer it formed primarily dimers and tetramers and higher molecular weight species in H buffer. When vimentin and nestin were mixed together in about 2:1 molar ratios, notable new bands appeared of apparent size of about 300 and 600 kDa (arrowheads), which the Western blots show contained both nestin and vimentin (Fig. 5, B and C). These intermediate sizes and the compositions of these bands suggest that nestin-vimentin heterodimers and heterotetramers, respectively, have been formed.


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Fig. 5.   Purified nestin and recombinant human vimentin co-assemble in vitro. Nestin and vimentin singly or mixed in 1:2 molar ratios in 9.5 M urea were assembled in either L or H buffer as shown and resolved on 3.75-7.5% SDS-PAGE gels and stained with Coomassie dye (A) or transferred for Western blotting by either vimentin antibody (B) or nestin antibody (C). The arrows at right denote new dimer and tetramer bands of intermediate size that contain both vimentin and nestin. The sizes of a ladder of cross-linked keratin 10 chain (44) are shown.

Isolated Hamster Nestin Assembles into IF with Recombinant Type III Vimentin-- Next we mixed varying amounts of bacterially expressed vimentin and isolated hamster nestin in 9.5 M urea buffer, dialyzed into H buffer, and then examined the structures formed by electron microscopy following negative staining. In molar ratios of nestin:vimentin of 2:8 and 4:6 (Fig. 6, b and c), IF were formed that were generally very similar to vimentin alone (Fig. 6a), although increasing amounts of fine subfilamentous particles became evident in the fields examined. At a 6:4 ratio, the IF were much shorter and thinner and appeared somewhat unraveled. At a 8:2 ratio, many fewer IF were present and were much shorter and largely unraveled (Fig. 6e). For nestin alone, no IF formed although there was an extensive background of particulate matter (Fig. 6f).


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Fig. 6.   Electron microscopy of copolymer IF formed in mixtures of vimentin and nestin. Recombinant vimentin and nestin were mixed in 9.5 M urea solution and dialyzed into H buffer as described. The mixtures contained the following: vimentin only (a); 2:8, 4:6, 6:4, and 8:2 molar ratios of nestin:vimentin, respectively (b-e); nestin only (f). Bar is 100 nm.

Samples of similar assembly mixtures were pelleted in an Airfuge, and the composition of the pelletable IF material was determined (Fig. 7A) and then quantitated by scanning densitometry (Fig. 7B). Nestin alone did not form oligomeric complexes that could be pelleted under these conditions. In the mixtures, the total amount of protein pelleted increased to >90% only as the relative amount of vimentin increased. In all cases, 90-95% of the vimentin pelleted, but only 2-20% of the nestin could be pelleted, which, interestingly, increased as the relative molar amounts of vimentin increased. Significantly however, the molar ratio of nestin present in all pellets remained constant at about 1:4.


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Fig. 7.   Stoichiometry of co-assembly of nestin and vimentin. A and B, nestin (N) and vimentin (V) in 9.5 M urea solutions were mixed in a range of molar ratios, assembled into H buffer, and then portions were pelleted in an Airfuge. Aliquots of the mixtures (M) and pellets (P) were resolved on 3.75-7.5% SDS-PAGE gels and stained with Coomassie dye (A). These gels were then scanned to determine the amounts of vimentin or nestin in the pellets, from which the molar ratio of nestin:vimentin was calculated. Also, the total amount of protein pelletable in each mixture was determined. C and D, identical experiments were performed but with nestin and vimentin which had been equilibrated in L buffer prior to mixing, followed by conversion to H buffer.

We tested a variety of buffers of pH ranging between 6 and 9, ionic strengths between 0.005 to 0.2, with a variety of added salts such as Mg2+, Ca2+, and Zn2+. However, we were unable to find conditions under which the isolated nestin alone could assemble into IF-like structures or form particles large enough to be pelletable at 100,000 × g in the Airfuge in 30 min.

In a related set of experiments, we prepared nestin and vimentin homo-oligomers in L buffer, mixed them in varying molar ratios, and then raised the ionic strength by addition of 1 M NaCl to 0.15 M (=H buffer). In this case, the molar amount of nestin incorporated into pelletable IF was only about 15% (Fig. 7, C and D).

Together, these data indicate that although nestin homo-oligomers alone cannot form IF-like structures in vitro, modest amounts are capable of participating in IF assembly with vimentin. As the nestin formed mostly hetero-oligomers with vimentin when assembled from 9.5 M urea solutions (Fig. 5), it appears that somewhat more nestin may be assimilated when in the form of a heterodimer with vimentin. Furthermore, the data suggest that typical 10 nm IF can form only when the amount of nestin hetero-oligomers is <50%. Indeed, it appears that larger amounts of nestin may directly interfere with the normal assembly of vimentin into IF.

Isolated Hamster Nestin Co-assembles with Type IV alpha -Internexin-- Similarly, we mixed recombinant human alpha -internexin and isolated hamster nestin in 9.5 M urea, and following dialysis into H buffer, pelletable IF were formed (Fig. 8A), provided the amount of nestin did not exceed about 25% (quantitations not shown).


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Fig. 8.   Nestin readily co-assembles with type IV alpha -internexin (A), but not with types I/II keratin 5/keratin 14 (B). Experiments were similar to those described in Fig. 7A.

Hamster Nestin Does Not Co-assemble with Types I/II Recombinant Keratin 5/Keratin 14 IF-- In contrast, in mixtures of nestin with recombinant expressed keratins 5 or 14, IF were formed in L but not H buffer, as demonstrated by negative staining and electron microscopy (data not shown). Analyses of the L buffer pellets of these IF revealed the presence only of the keratins and a complete absence of nestin (Fig. 8B). These data suggest that nestin is incapable of copolymerization with types I/II keratin IF.

Isolation of Stable alpha -Helix-enriched Particles, Confirmation of the Formation of Nestin-Vimentin or Nestin-alpha -Internexin Heterodimers-- We next performed a series of experiments to demonstrate more precisely that nestin could form heterodimers with vimentin or alpha -internexin, as suggested by the cross-linking experiments of Figs. 5 and 8. We have used a method established previously that involves examining the compositions of alpha -helical fragments containing portions of the rod domain segments 1 and 2 which arise from limited proteolysis of IF (54, 55, 58, 59). IF were assembled from 1:5 molar mixtures of isolated hamster nestin and recombinant vimentin from 9.5 M urea into H buffer mixtures. This ratio was chosen since under the conditions used in this work virtually all nestin could be incorporated into pelletable IF (Fig. 7, A and B). The assembled IF were dripped into trypsin solution and digested for 10 and 15 min, after which time about 20% of the starting IF protein could be precipitated at pH 5.2. When resolved by size exclusion chromatography on Sepharose CL-400, three peaks were recovered (Fig. 9A). The protein material from peaks 2 and 3 was recovered for further analysis. First, the approximate alpha -helical contents as measured by circular dichroism were 40 and 80%, respectively, as compared with an estimated alpha -helix content of about 30% for the undigested IF. Very similar results were obtained for nestin/alpha -internexin copolymer IF (data not shown). These recovery yields are consistent with the removal of large portions of the end domains of the nestin and vimentin or alpha -internexin chains due to their acute sensitivities to limited trypsin digestion (58, 59). Second, we then cross-linked material from peaks 2 and 3 with 2 mM DTSSP (Fig. 9B). Peak 2 contained a family of peptides of 10-13 kDa that were present as dimers and tetramers, whereas in peak 3 material, two peptides of 10 and 12 kDa were present as dimers. Similar data were obtained for nestin-alpha -internexin IF (not shown). Third, the material from peaks 2 and 3 was also used for amino acid sequencing. In the case of peak 2 material, multiple sequences were present that precluded identification. In the case of peak 3, two peptide sequences were identified as follows: one was from the beginning of the 2B rod domain region of hamster nestin, and the other was also from the beginning of the 2B rod domain region of either vimentin (see Fig. 9E) or alpha -internexin (see Fig. 9F). Fourth, we found that the 2B dimers of the peak 3 material could be resolved by FPLC chromatography on a MonoQ column into three peaks (Fig. 9, C and D). Sequencing analyses revealed that the minor basic peak A consisted only of nestin sequences, the more acidic peak C contained only vimentin (Fig. 9E) or alpha -internexin (Fig. 9F) 2B sequences, and the middle peak B contained equimolar amounts of both nestin and vimentin or nestin and alpha -internexin sequences, that is the middle peak could only have arisen from 2B heterodimers. These data confirm that the coiled-coil molecules formed by nestin and either vimentin or alpha -internexin copolymer IF are indeed heterodimers.


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Fig. 9.   Nestin forms heterodimer coiled-coil molecules with the type III vimentin and type IV alpha -internexin chains. Co-polymer IF formed in 1:4 mixtures with vimentin (A, C, and E) or alpha -internexin (B, D, and F) were digested with trypsin for 10 and 15 min, and the alpha -helical-enriched fragments were resolved by chromatography on a Sepharose column (A). Samples of peaks 2 and 3 were cross-linked with 2 mM DTSSP and resolved on 7.5% SDS-PAGE gels (B). The positions of migration of cross-linked dimers and tetramers are shown. As a control, dithiothreitol (DTT) cleaves the cross-linker. Samples of the peak 3 2B dimers were further resolved into three peaks by FPLC on a MonoQ column (C and D), from which sequence information was generated (E and F).

Nestin-Vimentin and Nestin-alpha -Internexin Heterodimer Molecules Are of Intermediate Stability-- The above data show that when mixed together, nestin prefers to form heterodimers with vimentin or alpha -internexin since only trace amounts of it form homodimers. A possible explanation for this is that nestin-vimentin or nestin-alpha -internexin heterodimers are more stable than homodimers. To confirm this, we set up assembly reactions in L buffer in the presence of a range of urea concentrations, performed cross-linking with 0.1 M DTSSP, and then resolved the products by SDS-PAGE. The data showed that nestin homotetramers were dissociated in 2-4 M urea to homodimers, which in turn were dissociated to monomers by 6 M urea (Fig. 10A). In contrast, vimentin exists largely as tetramers in the absence of urea, which are dissociated to dimers by 6.5 M urea, and to monomers by about M urea (Fig. 10B). However, when these experiments were performed with copolymer IF formed from a 1:4 molar mixture of nestin:vimentin, about 6 M urea was required to dissociate the nestin/vimentin tetramer and >7 M urea to dissociate the heterodimer (Fig. 10C). Similar data were obtained for nestin/alpha -internexin copolymers (data not shown). These data imply that the stability of the heterodimers and heterotetramers are intermediate between those of nestin and vimentin or alpha -internexin homopolymers.


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Fig. 10.   Nestin-vimentin heterodimers and heterotetramers are more stable than nestin homo-oligomers. Nestin (A), vimentin (B), or 1:4 mixtures of both (C) in 9.5 M urea were dialyzed into H buffer in the absence or presence of a series of increasing urea concentrations as shown and then cross-linked with 0.1 M DTSSP. The sizes and likely chain compositions of the various bands are illustrated. The products were resolved on 3.75-7.5% SDS-PAGE gels and stained with Coomassie dye. N, nestin; V, vimentin.

In support of these observations, we calculated the numbers of potential ionic interactions between e-g, a-g, and d-e pairs of charged residues across the dimer molecules. When the two chains are exactly aligned (as in all IF molecules so far examined in detail; Ref. 60), maximal ionic scores are as follows: 12 for nestin homodimers, 14 for vimentin homodimers, 18 for alpha -internexin homodimers, 13 for the nestin/vimentin heterodimer, and 15 for the nestin/alpha -internexin heterodimer.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nestin Is a Novel High Molecular Weight IF Protein of BHK-21 Cells and Perhaps of Fibroblasts in General-- We have been interested in the IFAPs present in and which co-assemble in vitro with vimentin-containing fibroblastic cells. In this study, we have isolated one of these from BHK-21 cells, and we have determined that it is the type VI IF protein nestin, heretofore known to be present only in neuroectodermal stem cells or early developing cell types of brain, muscle, and testis tissues. Furthermore, nestin or antigenically related proteins are abundantly expressed in several other types of fibroblastic cell lines, as well as in primary skin fibroblasts. However, using immunoblotting analyses, such antigens appear to be absent from epithelial cell types that do not express vimentin.

We show here that nestin co-assembles with the type III vimentin/desmin IF of BHK-21 cells through repeated cycles of assembly-disassembly-reassembly in vitro. Although we were unable to define conditions in which purified nestin could assemble into IF structures by itself, it readily co-assembled with purified recombinant human vimentin, providing the molar ratio of nestin:vimentin did not exceed about 1:4. Furthermore, we show in a variety of experiments that nestin formed heterodimer molecules with vimentin that are measurably more stable than nestin homo-oligomers. Similarly, nestin homo-oligomers could co-assemble to a very limited extent with vimentin. Thus both homo- and hetero-dimers can participate in IF co-assembly. In addition, we show that nestin formed heterodimer molecules and copolymer IF with about the same facility with the type IV alpha -internexin chain in vitro. Based on our sedimentation assay, the maximal "carrying" load of nestin in copolymer IF is about 1:4 (Fig. 7B), which implies an approximate 1:1 stoichiometry of one vimentin or alpha -internexin homodimer molecule:one nestin/vimentin or nestin/alpha -internexin heterodimer molecule; alternatively, assembly could only proceed to pelletable IF structures with about two vimentin or alpha -internexin homodimers for each nestin homodimer (Fig. 7D).

The most unusual features of the available nestin sequences are the peptide repeat motifs of the long carboxyl-terminal domains, which display marked degrees of homology between species, while varying somewhat in the number, organization, and precise sequence of the repeats (Fig. 3 and Table II). In addition, we predict these motifs form an extended flexible alpha -helical conformation, although we have no information as to how these elements are packed together in the tertiary structure of the carboxyl-terminal domain. Interestingly, the overall high charge properties of these sequences are reminiscent of those of the neurofilament triplet proteins NF-M and NF-H (reviewed in Ref. 22). Assuming nestin forms copolymers in vivo with a variety of types III and IV IF, as seems likely from our present in vitro experiments, then it is possible these terminal domains protrude from the IF core, perhaps in a manner analogous to those of the neurofilament proteins (61). Therefore, it is possible these sequences may serve as cross-bridging elements or "spacers" between IF, microtubules, and microfilaments (62). Like the neurofilament proteins, the occurrence of serine residues in the repeat motifs of nestin (Table II) may serve as sites for phosphorylation. Therefore, as is the case with NF-H, phosphorylation could modulate the configuration of the side arms and the formation of IF-IF cross-bridges as well as the connections between IF and other cytoskeletal components and/or organelles. In this way, we propose that nestin copolymerized into the core types III or IV IF may be able to participate directly in the dynamic interactions of these IF in cells.

Is Nestin a Member of a Growing Group of High Molecular Weight IF Proteins Formerly Thought to Be IFAPs?-- Current dogma suggests that IFAPs are involved in the supramolecular organization of IFs in cells (1-3). This is thought to be accomplished in at least three distinct ways as follows: (i) high molecular weight IFAPs that tend to organize the IFs into loose arrays; (ii) some smaller molecular weight IFAPs that bind the IFs tightly; and (iii) some IFAPs that bind at or near the end of IFs, that is act as "capping" proteins. Notably, known IFAPs of the second and third classes do not possess IF rod domain-like sequences. Their association with IFs may occur by ionic interactions of domains having complementary ionic charge distributions to the IF chains; an example of the second class is filaggrin (63); examples of the third class are desmoplakin (64), plectin (65), bullous pemphigoid antigens (66), etc.

Interestingly, extant examples thought to belong to the first type do possess rod domain motifs common to IF chains. Synemin (67) and paranemin (68) are high molecular weight proteins of muscle cells that appear to associate with vimentin and desmin IF. Like nestin, they co-localize with desmin by double immunofluorescence and co-pellet with desmin IF from cell extracts (69). Comparisons of their rod domain sequences reveal only modest (approx 30%) homologies with types I-IV cytoplasmic IF chains, but interestingly, the chick paranemin displays highest homology with rat and human type VI nestins (approx 50%) and frog tanabin (approx 65%) (70). Such high homologies are more typical of interspecies comparisons of members of the same sequence type of IF chain (1-3). Furthermore, their chains are organized the same way as follows: a short head domain, the conventional approx 310-residue rod domain, followed by a long tail domain. We note, however, that synemin, paranemin, and tanabin, in contrast to nestin, do contain an L2 segment in their rod domains. Whereas indirect data suggest that these chains may in fact participate in assembly in vivo with types III or IV IF proteins, there is clear evidence from transfection experiments that they are unable to form IF by themselves. Similarly, the type IV NF-M and NF-H chains which also possess typical rod domains (1-3) can only co-assemble with preformed neurofilaments containing other smaller chains (10-12). Thus it is possible that the long highly charged carboxyl-terminal tails on the neurofilament, nestin, synemin, and paranemin proteins may interfere with self-assembly. Recent data have established unequivocally that the NF-M and NF-H chains participate directly in IF assembly by formation with other neurofilament chains of a heterodimer coiled-coil molecule (13, 20). However, it remains to be determined whether synemin and paranemin simply associate with preformed desmin/vimentin IF or in fact directly co-assemble with them at the heterodimer level of IF hierarchy as demonstrated here for nestin. If so, then their previous imprimatur as IFAP proteins should be withdrawn. Instead, we favor the view that paranemin, synemin, and tanabin could be considered together with nestin as members of an enlarged type VI IF family. Further data will be needed to confirm this point.

Together, these four proteins may constitute a growing group of high molecular weight IF proteins that have evolved to fulfill functions related more to the supramolecular organization of cytoplasmic IFs containing mainly chains of lower molecular weight. Physically, this may be accomplished by incorporation in modest amounts directly into the packed coiled-coil backbone of the IF, from which their long tails may protrude to interact with or separate other cytoskeletal elements, including neighboring IF, as has been demonstrated for the larger neurofilament chains (10, 11, 54).

IF frequently form parallel arrays in cells (71). Thus our future experiments will address the questions as to whether and how nestin may potentially function as a cross-bridging or spacer protein in fibroblastic cells.

    ACKNOWLEDGEMENT

We thank Dr. R. Liem for the gift of the alpha -internexin bacterial expression vector and advice on its expression and purification.

    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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF110498

The amino acid sequence of this protein can be accessed through the Swiss Protein Database (Siss-Prot P21263).

§ To whom correspondence should be addressed: National Institutes of Health, Bldg, 6, Rm. 425, 9000 Rockville Pike, Bethesda, MD 20892-2752. Tel.: 301-496-1578; Fax: 301-402-2886; E-mail: pemast{at}helix.nih.gov.

** Supported by National Institutes of Health Grant GM36806-4.

    ABBREVIATIONS

The abbreviations used are: IF, intermediate filament(s); BHK-21, baby hamster kidney cells, clone 21; DTSSP, 3,3'-dithiobis(sulfosuccinimidyl propionate); IFAP, intermediate filament-associated protein(s); PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; PCR, polymerase chain reaction; nt, nucleotide; bp, base pair.

    REFERENCES
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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