From the Laboratory of Skin Biology, NIAMS, National
Institutes of Health, Bethesda, Maryland 20892-2752 and
¶ Institute of Fundamental Sciences, Massey University,
Palmerston North, New Zealand
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ABSTRACT |
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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 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 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, 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
Expression and Purification of Proteins--
A vector containing
the full-length coding sequence of rat
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
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
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%.
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 6 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 Analysis of DST Cross-linked Peptides--
The dried DST
cross-linked products and untreated Isolation and Characterization of Stable Determination of Axial Parameters in Assembly of Cross-linking with DST Suggests
To assess further these different properties,
As an independent analysis of these observations, we counted the
numbers of potential ionic interactions between e-g,
g
As a further control for these mixing experiments, we individually
dialyzed the solutions of Limited Proteolysis Methods to Isolate Stable Cross-linking of
In the first set of experiments with
We then repeated these experiments with intact copolymer IF assembled
from a 70:30 mixture of 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
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
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
As the alignments are likely to be the same, we then re-evaluated the
unique cross-link data (22 cross-links) from vimentin,
It is interesting to compare these parameters and to assess common and
differing features. First, the parameters for epidermal keratin,
vimentin, and
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
-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
-internexin and vimentin form heterodimer molecules in vitro that assemble into copolymer
intermediate filaments. We show using a urea disassembly assay
that
-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
-internexin homodimer and
-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,
-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
-internexin network.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-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.
-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
-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)?
-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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-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
-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-
-D-galactopyranoside for 3 h. The
-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.
-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).
-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).
-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
-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
-internexin IF, oligomers of
-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%
-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.
-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.
-internexin and vimentin on
control gels. Alternatively, gels were examined after Western blotting
using monoclonal antibodies against either vimentin (Boehringer
Mannhiem) or
-internexin (a kind gift of Dr. Liem) and developed by
enhanced chemiluminescence (Amersham Pharmacia Biotech).
-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.
-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
-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.
-Internexin and
-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
-internexin homodimer or
-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
-internexin
IF (10-12).
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-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
-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
-internexin IF have been reported previously (37, 39, 49). Mixtures of
-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
-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
-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 -internexin assembled at pH 6.7 (B) or pH 8.0 (C). A 25/75% mixture of
vimentin/
-internexin assembled at pH 7.0 (D) or pH 8.0 (E). Bar, 100 nm.
-Internexin Forms Stable
Heterodimers with Vimentin--
Two high molecular weight bands of
protein were observed when vimentin (Fig.
2A) or
-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,
-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
-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 -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
-internexin
16-mer (arrow), are marked.
-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
-internexin-
-internexin homodimers (uppermost band),
-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
-internexin, significant amounts of
-internexin-vimentin heterodimers were formed; particularly in
mixtures of 70%
-internexin and 30% vimentin, the resulting IF
contained only traces of vimentin homodimers but about equal amounts of
-internexin-vimentin heterodimers and
-internexin homodimers.
Note in the control experiments of Fig. 2 that the 6 M urea
solutions could not effectively dissociate either the
-internexin or
vimentin homodimers to permit chain exchange in dimers. Also, in
experiments using tracer amounts (0.1%) of 35S-labeled
-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
-internexin and vimentin
form heterodimers that are about as stable as
-internexin homodimers and measurably more stable than vimentin homodimers.
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Fig. 3.
Mixing experiments reveal that -internexin
forms homodimers or heterodimers with vimentin that are more stable
than vimentin homodimers. Aliquots of
-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-
-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.
-Internexin homodimers, blue
lines; vimentin homodimers, black lines;
-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.
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
-internexin homodimers and
-internexin-vimentin heterodimers. Scores for
-internexin-neurofilament M and
-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.
Summary of ionic interaction scores between various neuronally
expressed IF chains
-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
-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).
-Helix-enriched
Particles Confirmed That
-Internexin and Vimentin Can Form Copolymer
IF from Either Heterodimers or Homodimers--
We have demonstrated
previously that it is possible to isolate stable
-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
-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
-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
-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
-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
-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
-internexin and vimentin 2B fragments, respectively.
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Fig. 4.
Recovery of -helix-enriched particles from
-internexin-vimentin heterodimers. Separation of particles on
Sepharose 6B after limited trypsin digestion of copolymer
-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,
-internexin 2B homodimers; B,
-internexin-vimentin 2B
heterodimers; and C, vimentin 2B homodimers.
-Internexin and
-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
-internexin homopolymeric and
-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.
-internexin homopolymeric IF,
Fig. 5 shows HPLC chromatograms of
CNBr/tryptic peptides recovered from cross-linking reactions of the
dimer formed in 2 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
-internexin dimer molecule. All 11 lysines of the rod
domain of
-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 -internexin; B,
-internexin dimers formed in
2 M urea solutions; and C,
-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.
Cross-links in -internexin homopolymer and
-internexin-vimentin
copolymer IF
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Fig. 6.
Molecular alignments adduced in
-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).
-internexin:vimentin from 9.5 M
urea. Under these conditions, about half of the molecules are
-internexin homodimers, and another half are
-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
-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
-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
-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
-internexin-vimentin heterodimer in each of the alignment
modes.
-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
-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.
Unique -internexin cross-links and the equations used in the least
squares analysis
(0 × L1) + (0 × L12) + (0 × L2) = 7 residues.
Unique -internexin-vimentin copolymer cross-links and the equations
used in the least squares analysis
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-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
-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
9-9.5 M. Similar data were established previously for
keratin IF (8, 9). Accordingly, these data mean that copolymer
-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%
-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.
-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.
-internexin or vimentin homodimer and
-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
-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
-internexin-vimentin copolymer IF (Table IV).
-internexin,
and the
-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
-keratin from x-ray and other data, are given for purposes
of comparison.
Summary of axial parameters of -internexin and
-internexin/vimentin copolymer cross-link data adduced in this work,
and comparison of similar data for other IF types
-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
-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
-keratin differs from the epidermal
keratins in only one major respect, its value of A11.
Likewise, hard
-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
-keratin, 47.0 nm; epidermal keratins, 45.2 nm;
types III and IV IFs, 44.2 nm).
-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.
-Internexin Homodimers and
-Internexin-Vimentin Heterodimers
Are More Stable Than Those of Vimentin, Implications for Neuronal
Development--
Although
-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
-internexin forms primarily a 16-mer oligomer (Fig. 2B;
Ref. 49). However, by ultracentrifugation methods,
-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
-internexin-containing IF may be significantly affected by the
micro-environment. Second, we noted that
-internexin homodimers and
-internexin-vimentin heterodimers are measurably more resistant to
urea dissolution than vimentin homodimers (Fig. 2), and indeed, when
mixed together,
-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
-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
-internexin (28) may be driven by a net increase
in the stability of
-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.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ron Liem for the generous gift
of the protein expression vector for and monoclonal antibody against
rat -internexin; Dr. Robert Goldman and staff for a generous gift of
purified bacterially expressed human vimentin; and Will Idler for
expression and purification work.
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FOOTNOTES |
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* 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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REFERENCES |
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