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J Biol Chem, Vol. 274, Issue 14, 9881-9890, April 2, 1999
From the 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 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 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
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
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
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 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.
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.
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).
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
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 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).
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.
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).
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.
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
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 Nestin-Vimentin and Nestin-
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 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
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 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 (
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.
We thank Dr. R. Liem for the gift of the
*
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.
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.
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 
-INTERNEXIN*
§,
,,,, and 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
Institute of Fundamental Sciences, Massey University,
Palmerston North, New Zealand
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-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 -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).
-internexin which may participate by co-assembly into typical
IF.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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 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).
-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.
-Helix-enriched Particles from Nestin-Vimentin
and Nestin--Internexin Copolymer IF--
Pellets of IF assembled
from 1:4 molar mixtures of nestin and either vimentin or -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 -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 -helix contents by circular dichroism
(57).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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.
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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.
Peptides of purified IFAP reveal high homology to nestin
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.
-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.
Hamster nestin peptide repeats
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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.
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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.
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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.
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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.
-Internexin--
Similarly, we mixed recombinant human
-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
-internexin (A), but not with
types I/II keratin 5/keratin 14 (B). Experiments
were similar to those described in Fig. 7A.
-Helix-enriched Particles, Confirmation of
the Formation of Nestin-Vimentin or Nestin--Internexin
Heterodimers--
We next performed a series of experiments to
demonstrate more precisely that nestin could form heterodimers with
vimentin or -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 -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 -helical contents as measured by circular dichroism were
40 and 80%, respectively, as compared with an estimated -helix
content of about 30% for the undigested IF. Very similar results were obtained for nestin/-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 -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--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 -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 -internexin (Fig. 9F) 2B sequences, and
the middle peak B contained equimolar amounts of both nestin and
vimentin or nestin and -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 -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
-internexin chains. Co-polymer IF formed in
1:4 mixtures with vimentin (A, C, and E) or
-internexin (B, D, and F) were digested with
trypsin for 10 and 15 min, and the -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).
-Internexin Heterodimer Molecules Are
of Intermediate Stability--
The above data show that when mixed
together, nestin prefers to form heterodimers with vimentin or
-internexin since only trace amounts of it form homodimers. A
possible explanation for this is that nestin-vimentin or
nestin--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 9 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/-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 -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.
-internexin homodimers, 13 for the
nestin/vimentin heterodimer, and 15 for the nestin/-internexin heterodimer.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-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 -internexin homodimer molecule:one
nestin/vimentin or nestin/-internexin heterodimer molecule;
alternatively, assembly could only proceed to pelletable IF structures
with about two vimentin or -internexin homodimers for each nestin
homodimer (Fig. 7D).
-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.
30%) homologies with types I-IV
cytoplasmic IF chains, but interestingly, the chick paranemin displays
highest homology with rat and human type VI nestins (50%) and frog
tanabin (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 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.
ACKNOWLEDGEMENT
-internexin bacterial expression vector and advice on its
expression and purification.
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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