Previous studies have shown that rodent
neurofilaments (NF) are obligate heteropolymers requiring NF-L plus
either NF-M or NF-H for filament formation. We have assessed the
competence of human NF-L and NF-M to assemble and find that unlike rat
NF-L, human NF-L is capable of self-assembly. However, human NF-M
cannot form homopolymers and requires the presence of NF-L for
incorporation into filaments. To investigate the stage at which
filament formation is blocked, the rod domains or the full-length
subunits of human NF-L, human NF-M, and rodent NF-L were analyzed in
the yeast "interaction trap" system. These studies demonstrated
that the fundamental block to filament formation in those
neurofilaments that do not form homopolymers is at the level of dimer
formation. Based on theoretical biophysical considerations of the
requirements for the formation of coiled-coil structures, we predicted
which amino acid differences were likely to be responsible for the
differing dimerization potentials of the rat and human NF-L rod
domains. We tested these predictions using site-specific mutagenesis.
Interestingly, single amino acid changes in the rod domains designed to
restore or eliminate the coiled-coil propensity were found respectively to convert rat NF-L into a subunit capable of homopolymerization and
human NF-L into a protein that is no longer able to self-assemble. Our
results additionally suggest that the functional properties of the L12
linker region of human NF-L, generally thought to assume an extended
-sheet conformation, are consonant with an
-helix that positions
the heptad repeats before and after it in an orientation that allows
coiled-coil dimerization. These studies reveal an important difference
between the assembly properties of the human and rodent NF-L subunits
possibly suggesting that the initiating events in neurofilament
assembly may differ in the two species.
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INTRODUCTION |
Intermediate filaments
(IFs)1 are a heterogeneous
family of proteins sharing common structural features that can be
subdivided into six types (I-VI) based on sequence homology (1). IF
genes are expressed in a cell type-specific and developmentally
regulated manner with cells frequently containing only a single IF type at a particular stage of differentiation. Neurofilaments (NFs) are the
predominant IF in mature neurons but are preceded during neuronal
differentiation by a succession of other IFs including vimentin (2)
nestin (49),
-internexin (3, 4), and peripherin (5). Neurofilaments
are assemblies of three subunits, the NF-L (molecular mass, 68 kDa),
NF-M (150 kDa), and NF-H (200 kDa) (6). These three components form
heteropolymeric 10-nm filaments that run parallel along the length of
the axon with frequent cross-bridges between neighboring filaments.
Axonal neurofilaments are thought to serve a primarily structural
function. Evidence from a Japanese quail (quiverer) with a
spontaneous mutation in NF-L (7) and a line of transgenic mice
expressing an NF-H-
galactosidase fusion protein (8) suggest that a
loss of axonal neurofilaments results in a decreased axonal
diameter.
The first step in filament formation is the lateral associations of the
-helical rod domains via hydrophobic interactions to form a
coiled-coil dimer (9). The rod consists of an
-helix that is
interrupted by three short non-helical linker sequences (L1, L12, and
L2). Heptad repeats of hydrophobic amino acids confer an amphipathic
character to the
-helical domain that allows coiled-coil interactions between compatible IF molecules. This may result in
homodimer formation in the case of vimentin or obligate heterodimer formation in the case of type I and type II keratins (9).
Both in vitro and in vivo studies have probed the
ability of individual neurofilament proteins to form homo- and
heteropolymers. Purified bovine (10-16), porcine (17, 18), and murine
NF-L (19) and, to a lesser degree, NF-M and NF-H (10, 14) assemble in vitro into 10-nm homopolymers (20). In contrast, rodent
neurofilament proteins expressed individually in cells that lack an
endogenous intermediate filament network (SW13 vim
cells)
are unable to form homopolymers, yet can form 10-nm filaments when
coexpressed with NF-M or NF-H (21, 22) or when expressed in cells
containing an endogenous vimentin network, through assembly with
vimentin (21, 23, 24).
In the present work we examine the human NF-L and NF-M and derivatives
of them in SW13 (vim
) cells and in the yeast
"interaction trap" system. Our results demonstrate that human NF-L
is capable of self-assembly, an important distinction from the rodent
NF-L subunits which are obligate heteropolymers. Human NF-M or
truncations of it that contained the rod domain could not polymerize in
the absence of NF-L. Our analysis shows that two distinct structural
features of the human NF-L rod are important determinants of its
ability to homodimerize. First, human NF-L has more charged residues
next to the hydrophobic residues of the heptad repeats in its rod
domain. These charged residues are believed to stabilize coiled-coil
interaction by electrostatic attraction between the strands. Second,
the combination of the absence of a serine residue (compared with the
rodent NF-L) and the presence of a nearby proline residue allows the
L12 linker region of human NF-L to mimic an
-helix with a heptad
repeat. The same region of rat NF-L contains the additional serine, and its conformation is predicted to be an extended
-structure (25). Since dimer formation is likely to be the initiating event in filament
formation, our results suggest that this important intracellular event
may be different in human than in rodents.
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EXPERIMENTAL PROCEDURES |
Neurofilament Clones and Their Derivatives--
The plasmid
pNF-L contains a complete human NF-L gene plus 2.8 kilobase
pairs of upstream sequence. The plasmid pRSV-NF-L containing a
full-length rat NF-L cDNA driven by a Rous sarcoma virus promoter
was obtained from Dr. R. Liem (21). A plasmid containing a complete
human NF-M genomic clone (pNF-M) has been previously described (26). To
create a "tagged" human NF-M, a nucleotide sequence encoding an 11 amino acid epitope tag (Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg) was inserted at either amino acid 21 (amino-terminal tagged, designated pSS019) or amino acid 444 (internal tagged, designated pSS028) of the
plasmid pNF-M (numbering according to Ref. 50).
The internal tagged NF-M plasmids were used to prepare the following
series of deletions:
amino-terminal domain (amino acids 22-80
deleted);
glutamic acid-rich domain (aa 451-611 deleted);
glutamic acid and multiphosphorylation repeat (aa 551-790 deleted); and
carboxyl-terminal domain (aa 690-914 deleted). An
NF-M-L-M hybrid gene was constructed by removing amino acids
89-444 from pNF-M and replacing it with a polymerase chain
reaction-amplified NF-L rod domain (aa 83-414). The rod region of the
final product was verified by sequencing.
For yeast interaction trap experiments, fragments encoding NF rod
domains (human NF-L amino acids 85-410, human NF-M amino acids
98-421, and rat NF-L amino acids 86-415) or full-length proteins were
cloned into the vectors pGBT9 and pGAD42, (27) using oligonucleotides
to preserve open reading frames.
Site-specific Mutagenesis--
Amino acids were inserted,
deleted, or substituted using the Quikchange site-directed mutagenesis
kit (Stratagene). Changes were verified by sequencing on an Applied
Biosystems automated DNA sequencer.
Tissue Culture and DNA Transfection--
Human SW13
(vim
) cells were obtained from Dr. R. Evans and cultured
as described (28). Cells were transiently transfected by
electroporation (440 V, 500 microfarads) with 20 µg of DNA per 10-cm
dish based on the protocol of van den Hoff et al. (29). Following electroporation, cells were plated directly into Lab-Tek 4-well glass chamber slides (Nunc) and incubated for 48 h before immunostaining.
Immunofluorescent Staining and Microscopy--
Cells grown on
glass chamber slides were washed three times in phosphate-buffered
saline (PBS) and fixed with 2% paraformaldehyde in PBS for 15 min at
room temperature. The paraformaldehyde was removed with three rinses of
PBS, and nonspecific binding was blocked by treatment for 1 h with
PGBA (PBS, 0.1% gelatin, 1% bovine serum albumin), 2% goat serum,
and 0.1% saponin. Treated slides were incubated for 60 and 30 min,
respectively, with primary and secondary antibodies diluted in PGBA,
2% goat serum, and 0.1% saponin. Unbound primary and secondary
antibodies were removed with three 10-min washes in PBS at room
temperature. Nuclei were counterstained by inclusion of
4',6-diamidino-2-phenylindole hydrochloride with the secondary
antibody. Slides were mounted in 90% glycerol + diazabicyclo-octane
and analyzed using laser scanning confocal microscopy (Leica TCS
confocal microscope).
A rabbit polyclonal anti-NF-L (Dr. V. Lee, University of Pennsylvania),
mouse monoclonal anti-TAG (ascites), and a rabbit anti-vimentin (Sigma)
all diluted 1:100 were used as primary antibodies. Donkey anti-mouse
Texas Red-conjugated (Amersham Corp.) and donkey anti-rabbit
fluorescein-conjugated (Amersham Corp.) secondary antibodies were used
at a 1:200 dilution.
Yeast Interaction Trap--
The yeast vectors pGAD424 or pGBT9
containing cDNAs encoding the rod domains or full-length forms of
human NF-L, human NF-M, human NF-M/L/M, and rat NF-L were introduced
into the yeast strain Y526 (30) by electroporation. Cells were
permeabilized and
-galactosidase activity assayed according to the
protocol of Guarente (48).
Molecular Modeling--
Sequences of vimentins, desmins,
keratins, and neurofilament light chains were retrieved from the
GenBankTM data base of nucleotide sequences using
STRINGSEARCH in the GCG package (31), and complete genomic sequences
and mRNA sequences were translated to protein sequences. The
resulting sequences of 6 keratins, 4 desmins, 12 vimentins, and 3 NF-Ls, including human and rat, were aligned using the PILEUP module of
the GCG package (31). The sequence alignment was inspected to identify heptad repeats defining coiled-coils (32) and differences between rat
and human NF-L; a total of eight differences was identified in the rod
sequence.
Models of the coiled-coil portion of human and rat NF-L were
constructed using the crystal structure of the GCN4 leucine zipper (33)
as a template. The GCN4 sequence was mutated to the selected pieces of
human and rat NF-L containing differences considered most likely to be
responsible for disrupting dimerization. The residues were modeled in
allowed rotameric conformations, and the structures were searched
manually for allowed rotamers that avoid steric clashes in the model
structure. This low resolution model was used to explore possible
proximities of residues and to identify the nature of interactions in
the rat NF-L sequence that may destabilize the dimerization process.
The propensities of specific amino acids to occupy particular positions
in the heptad repeat (34) and the structural role of Pro residues in helices (32, 35, 36) were used as criteria for selecting likely
candidates for disruptive interactions. For the helical part of the
structure, the backbone and dihedral angles were obtained as average
dihedral angle values from the crystal structure of the GCN4 leucine
zipper, with values of
63.0 and
42.5, respectively. The Pro-kink
segment between position a in heptad 2 to position d in heptad 5 (Fig. 6) were modeled with the parameters
defined in Table I. The resulting
structure of this distorted
-helix in that region was close to a
310 helix turn.
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Table I
Backbone dihedral angle values used in the model structure of the
Pro-kink region in the L12 subunit
Note that the turn before the Pro-kink assumes a conformation close to
a 310 helix.
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 |
RESULTS |
The Human NF-L Subunit Is Capable of Homopolymerization in
Vivo--
The ability of human NF-L to self-assemble into filaments
was assessed in human SW13 (vim
) cells that lack an
endogenous intermediate filament network (28). These cells were
transfected with a plasmid (pNF-L) containing the entire human
NF-L gene plus 2.8 kilobase pairs of upstream sequence.
Under our conditions we obtain an average transfection efficiency of
15-20%. NF-L and vimentin expression was monitored by
immunofluorescence 48 h following transfection. Transfection of
human NF-L alone resulted in extensive filament formation throughout the cytoplasm in the absence of any other IF protein (Fig.
1A). Staining with
anti-vimentin antibodies revealed that 1-4% of the cells were
vimentin positive. Others (22) have reported comparable levels of
spontaneous reversion in this cell line. However, double labeling with
antibodies to NF-L and vimentin demonstrated that filament formation by
human NF-L was not dependent on the presence of vimentin (data not
shown). These results contrast sharply with those reported previously
for rodent NF subunits which showed that the rat and mouse NF-L
proteins do not assemble into filaments under similar conditions (21,
22). This difference between our results and those of others cannot be
attributed to systematic or technical differences in the experiments.
We have transfected SW13 (vim
) cells with the rat
nf-l gene used by Ching and Liem (21) and obtained results
that are in complete agreement with theirs: the rat NF-L protein
distributed uniformly throughout the cytoplasm and no filamentous
structures present (Fig. 1B). Thus, differences in the
ability of human and rodent NF-L to homopolymerize appear to reflect
potentially important and fundamental differences that are traceable to
the few differences in the amino acid sequences of the two
proteins.

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Fig. 1.
Immunofluorescent staining of SW13
(vim ) cells transfected with neurofilament expression
plasmids. Cells were transfected with either pNF-L encoding human
NF-L (A), pSS028 encoding a tagged human NF-M
(B), or pRSV-NF-L encoding rat NF-L (C).
Immunofluorescent staining was carried out using rabbit polyclonal
anti-NF-L primary antibody followed by donkey anti-rabbit
fluorescein-conjugated secondary antibody. Immunofluorescent staining
was analyzed by laser scanning confocal microscopy. Scale
bar, 10 µm.
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Human NF-M Is Unable to Homopolymerize in Vivo--
We carried out
similar transfection experiments using human NF-M to determine if the
ability to homopolymerize was present in other human NF subunits or was
restricted to NF-L. The plasmid pSS028 contains a modified human
NF-M gene that encodes an NF-M protein containing an
11-amino acid tag sequence
(Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly-Arg) inserted at amino acid
444 (internal tag). The inclusion of this epitope tag allowed the
simultaneous detection of both phosphorylated and non-phosphorylated
forms of the protein with an anti-tag monoclonal antibody. Our previous
studies have demonstrated that the tag does not interfere with
expression or polymerization of NF-M in transgenic mice or transfected
cells and that filaments containing the tag appear to function
normally (37).
Expression of the tagged human NF-M protein in SW13 (vim
)
cells did not result in the formation of filamentous networks.
Immunofluorescence staining using anti-tag antibody revealed that the
human NF-M protein was uniformly distributed throughout the cytoplasm
in a diffuse and at times granular pattern (Fig. 1C). In
this regard, the human NF-M behaves similarly to the rodent NF-L and
NF-M subunits.
Human NF-M and Rat NF-L Form Filaments in Vivo When Expressed
Together or with Human NF-L--
Interestingly, rat NF-L and human
NF-M can participate in filament formation through
heteropolymerization. Double transfection experiments were performed in
which human NF-M was coexpressed with either human or rat NF-L. Cells
transfected with human NF-L and human NF-M contained an extensive
filamentous network throughout the cytoplasm that contained both NF
subunits co-localized within filaments (Fig.
2, A and C). In
Fig. 2C, the finer filaments corresponding to those labeled
with antibodies directed at the human NF-L do not appear in the
photomicrograph because the gain of the photomultiplier was set so that
the main fibers reveal good detail. At higher gain setting the full
array of fine filaments was visible. Expression of rat NF-L with human
NF-M also resulted in extensive filament formation and co-localization
of both proteins (Fig. 2, B and D) indicating the
assembly competence of each subunit, but only as a heteropolymer.

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Fig. 2.
Co-assembly of human NF-L and rat NF-L
subunits with human NF-M subunit in vivo. SW13
(vim ) cells were co-transfected with either pNF-L and
pSS028 encoding a tagged human NF-M (A and C) or
pRSV-NF-L and pSS028 (B and D). Double
immunofluorescent staining was carried out using rabbit polyclonal
anti-NF-L primary antibody followed by donkey anti-rabbit fluorescein-conjugated secondary antibody and mouse monoclonal anti-tag
primary antibody, followed by donkey anti-mouse Texas Red-conjugated
secondary antibody. The vertical panel pairs (A and
C; B and D) are scans of the same cell
showing NF-L (A and B) or anti-tag staining
(C and D). Scale bar, 10 µm.
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Deleting the Amino- or Carboxyl-terminal Sequences Flanking the
Human NF-M Rod Domain Does Not Result in NF-M
Homopolymerization--
Inhibitory regions in parts of the molecule
not directly involved in homophilic interactions could explain the
incompetence of human NF-M to homopolymerize. To address this
possibility, NF-M genes encoding truncated forms of the
tagged human protein were prepared and transfected into SW13
(vim
) cells. The following series of deletions were
examined:
amino-terminal domain (amino acids 22-80 deleted);
glutamic acid-rich domain (aa 451-611);
glutamic acid-rich and
multiphosphorylation repeat (aa 551-790);
carboxyl-terminal domain
(aa 690-914). Expression of these truncated proteins was assessed
using anti-tag monoclonal antibodies and, where appropriate, the
anti-NF antibodies SMI-31 and SMI-32 which detect
phosphorylation-dependent and independent epitopes of the
human NF-M, respectively (38). In all cases staining was diffuse and
distributed throughout the cytoplasm without evidence of filament
formation (data not shown). However, none of the deletions affected the
ability of human NF-M to form heteropolymers since filaments formed
when the deletions were coexpressed with human NF-L (data not
shown).
The failure of human NF-M to form homopolymeric filaments might
indicate the incompetence of its rod domain to form dimers. If so, then
replacement of the NF-M rod with the human NF-L rod should allow
homopolymerization of the hybrid molecule. To test this hypothesis a
hybrid NF-M-L-M gene was created which encodes a protein
with the amino- and carboxyl-terminal domains of NF-M but the rod
domain of human NF-L. When this construct was expressed alone in SW13
(vim
) cells, a diffuse granular staining was apparent
throughout the cytoplasm, and no filament assembly was detected.
However, when the NF-M-L-M protein was coexpressed with human NF-L,
extensive filament formation occurred. These results indicate that the
amino- and carboxyl-terminal sequences of NF-M might interfere with
homopolymerization, but the failure of the native NF-M to
homopolymerize cannot be solely attributed to this interference since
the NF-M rod domain without flanking sequences does not exhibit
significant homophilic interactions in the yeast interaction trap
analyses.
The
-Helical Rod Domains of Human NF-M and Rat NF-L Are
Incapable of Homodimerization--
The immunocytochemical analyses
described above show that human NF-L, but not NF-M, is capable of
homopolymeric assembly into filaments of a dimension and complexity
that can be visualized by light microscopy. Filament assembly is a
multistep process, and the block to NF-M homofilament assembly might
occur at steps after the initial interactions between neurofilament
subunits. We investigated the earliest steps in NF subunit interactions using the yeast "two-hybrid" or interaction trap system, similar to
that used by Meng et al. (39) to investigate homodimer
formation by vimentin. In this system, expression of a reporter gene
(Escherichia coli lacZ gene) is controlled by a
transactivator complex consisting of two hybrid proteins. A functional
transactivating complex is achieved when a DNA binding domain, supplied
by one hybrid subunit, and an activation domain, supplied by the
second, are held in close proximity by the adhesive domains of two
hybrid proteins. We examined the interactions between various NF
subunits or their rod domains by incorporating them as the adhesive
components in the two-hybrid complex. cDNA sequences encoding human
NF-L, human NF-M, rat NF-L, or only the
-helical rod domains of each
were separately fused to the activation or DNA binding subunits of the
transactivating complex. All pairwise combinations of activation subunits with DNA binding subunits were tested for interactions by
double transfections into yeast. The results of these experiments are
summarized in Table II, where strongly
positive results are presented in bold type. The results shown in the
first two vertical columns indicate that full-length human NF-M or its
rod domain interact strongly with only full-length human NF-L or its
rod domain. All combinations of human NF-M (rod domain or whole) with itself or rat NF-L (rod domain or whole) failed to interact. These results suggest that the yeast two-hybrid assay is a more stringent test of interaction than the SW13 transfection assay, since the human
NF-M was able to form filaments with the rat NF-L in the latter assay
(Fig. 2, B and D). It is possible that the yeast two-hybrid system places steric constraints on the association that are
not necessary for filament formation. The third and fourth vertical
columns show that full-length human NF-L or its rod interact very
strongly with the whole subunits or the rod domains of human NF-L,
human NF-M, or rat NF-L. The last two columns indicate that rat NF-L or
its rod domain interact with human NF-L or its rod domain while failing
to interact with itself or human NF-M or its rod domain. The results
shown in Table II were obtained with permeabilized yeast cells and have
been normalized for differences in culture turbidity. It should be
noted that each NF domain was tested as a fusion partner in both the
activation and DNA binding subunits, and all pairwise combinations were
tested for transactivation. The results of these tests do not show any
significant bias between reciprocal pairs of hybrid proteins. We
conclude from these analyses that human NF-L but not rodent NF-L or
human NF-M is capable of significant homodimer formation.
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Table II
Interaction-trap analysis of various neurofilament subunits and
their rod domains
The values shown are the relative -galactosidase activities
expressed in doubly transfected yeast cultures after normalization for
cell number. Plasmids containing the transactivating domain begin with
T (e.g. T-hM), and plasmids containing the DNA binding domain begin with D (e.g. D-hM). Abbreviations: hM, human
NF-M; hM rod, human NF-M rod; hL, human NF-L; hL rod, human NF-L rod; rL, rat NF-L; rL rod, rat NF-L rod.
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The Charge Interaction Provided by Arg161 May Stabilize
the Coiled-coil Associations of Human NF-L--
Based on theoretical
biophysical considerations, we attempted to determine which of the
amino acid differences between human and rat NF-L could be responsible
for the differences in their homopolymerization ability. Fig.
3 shows alignments of the human and rat
NF-L rod domains and indicates the position of the
-helical (1A, 1B,
2A, and 2B) and linker (L1, L2, and L12) regions (25). The human and
rat NF-L rod sequences are highly homologous with only 8 amino acid
differences (indicated by a * in Fig. 3). Two regions in particular
were noted which could account for the difference in
homopolymerization. The first was at aa 161 in the human protein which
is an arginine and the corresponding aa position 162 in the rat which
is a glutamine. This difference occurs at position e of the
heptad, next to a hydrophobic surface created by the heptad repeat,
where it is likely to affect the corresponding electrostatic
attractions between two rod domains forming a coiled-coil. Such charged
residues (usually Arg, Lys, or Glu) are considered to be responsible
for additional attractive interactions between helices forming a
coiled-coil (34) and are also major contributors to the characteristic
stagger for a given protein.

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Fig. 3.
Alignment of human and rat NF-L rod
domains. Positions of -helical regions 1A, 1B, 2A, and 2B and
linker regions L1, L2, and L12 are indicated. Sequence differences
between rat and human (*) are indicated. The bold symbols
represent amino acids that are referred to in the text.
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To test this hypothesis an arginine was substituted for the glutamine
at position 162 in the rat rod. The reciprocal substitution of
Arg161 for Gln161 was carried out in the human
NF-L rod. When transfected into SW13 (vim
) cells, the
human NF-L (Gln161) did not form filaments (Fig.
4A), whereas the rat NF-L
(Arg162) formed filaments (Fig. 4B). We
investigated whether these changes in polymerization could be traced to
differences in the abilities of the rod domains to form dimers. The
mutated rod domains of both human and rat NF-L were expressed in the
yeast two-hybrid system and tested for interactiveness (Table
III). Productive interactions occurred
with the mutated rat NF-L (Arg162) rod domain but not with
the human NF-L (Gln161) rod domain. These results show that a single
amino acid change in the rod domain of either protein is sufficient to
convert rat NF-L into a subunit capable of homopolymerization and human
NF-L into a protein that is no longer able to self-assemble.

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Fig. 4.
Immunofluorescent assessment of the
homopolymerization of mutant rat and human rod domains. SW13 cells
were transfected with relevant NF-L genes and
immunofluorescent stained using rabbit polyclonal anti-NF-L primary
antibody followed by donkey anti-rabbit fluorescein-conjugated
secondary antibody. Immunofluorescent staining was analyzed by laser
scanning confocal microscopy. A, human NF-L Gln161; B, rat NF-L Arg162;
C, human NF-L + Ser252; D, rat NF-L
Ser252. Scale bar, 10 µm.
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Table III
Homodimer interaction trap analyses of mutant rat and human NF-L
rod domains
Values shown are the relative -galactosidase activities in yeast
cultures doubly transfected with plasmids encoding GAL4 DNA binding and
GAL4 activation domains, each fused to the same rat or human NF-L rod
domain. rL and huL are wild type domains; R-Q, Q-R substitution of R
for Q and Q for R; S, +S deletion and insertion serine.
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The L12 Linker Regions of the NF-L Subunits Affect
Homopolymerization--
The second difference identified as
potentially important in the altered dimerization properties of the two
proteins was in the L12 linker region. A comparison of rat and human
NF-L sequences in this region reveals a major difference between them
produced by a serine insertion in the rat sequence. To assess the
importance of this difference for filament formation, we prepared
reciprocal mutations in the two NF-L rod domains as follows:
Ser252 was deleted from rat NF-L, making it more human
like, and a serine residue was inserted into human NF-L (following
Thr251) to make it more rat like. Each mutant was
transfected into SW13 (vim
) cells. Human NF-L with the
inserted serine failed to homopolymerize (Fig. 4C), whereas
rat NF-L with the serine deleted formed filaments (Fig. 4D).
Analysis of these mutations in the yeast two-hybrid system supported
the conclusions of the transfection experiment and demonstrated that
rat NF-L with Ser252 deleted dimerizes, whereas human NF-L
with the added serine did not (Table III).
The L12 linker region is generally believed to assume an extended
-sheet conformation (25). However, structural and biophysical considerations led us to investigate whether the L12 region of the
human NF-L rod domain might behave like a coiled-coil due to the heptad
repeat-like sequence in this region (Fig.
5). One striking characteristic of human
NF-L and several other IFs capable of homopolymerization is the absence
of a serine at position 252 that is present in rat NF-L. Human NF-L and
other IFs also have a proline residue that is highly conserved at
position b in the following heptad 4 (Fig. 5). In
-helices, prolines are well known to disrupt the helical hydrogen
bonding pattern and form a "Pro-kink" (35, 40, 41). A
characteristic structural distortion produced by the Pro-kink is a
"face shift" that causes the helical portions before and after the
proline to rotate with respect to each other, thereby interfering with
local helical periodicity (32, 35, 41). Since the orientation of the
hydrophobic residues in a heptad is a key element in coiled-coil
formation, the face shift due to the Pro kink may interfere with the
appropriate helix-helix interface (i.e. the dimerization
surface). However, structural considerations indicate that the deletion
of a residue from the heptad preceding the proline in the human
sequence (compared with the rat sequence) would compensate for the face
shift by bringing the two sets of hydrophobic residues back to one face
that incorporates the Pro-kink. This is illustrated in Fig.
6 where purple and
orange spheres indicate the dimerization surfaces in the
mutated Pro-kinked helix compared with an ideal
-helix. We
hypothesized that the serine deletion is therefore necessary for the
human NF-L helices to dimerize, and we tested this hypothesis by
assessing the homopolymerization abilities of three different rat NF-L
mutants. In the first construct described above, we deleted
Ser252 in rat heptad 3 (Fig. 5) that precedes
Pro254 to bring the hydrophobic residues to the same face
of the two helices (Fig. 4D; Table III). However, in itself
a dimerization of this construct would not necessarily prove the
existence of a coiled-coil in the region since other structural
explanations are feasible. We probed the existence of a coiled-coil in
the region by making two more mutant NF-L proteins. The
Pro254 of rat NF-L was mutated to an Ala254 to
remove the kink and bring positions a and d onto
the same face of the helix. However, the heptad (heptad 3) preceding
Pro254 contains an aspartic acid at position d
in the rat replacing a hydrophobic residue commonly at this position of
a canonical heptad (34). Consequently, we also changed
Asp249 to Leu249 to produce a more regular
heptad. Note that in a Pro-kinked helix, as in the human NF-L,
Asp249 does not lie on the dimerization surface; it is
brought to the dimerization surface by elimination of the Pro-kink in
Pro254 to Ala254 mutants. The conformations of
these regions of the rat NF-L rod domain are modeled in Fig.
7, A, C, and
E. Our hypothesis was that the rat NF-L double mutant
(Pro254 to Ala254 and Asp249 to
Leu249) should recover regular coiled-coil capabilities and
homopolymerize, whereas each mutation individually should not.

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Fig. 6.
Representation of the sequence region from
position a in heptad 2 to position d in heptad
5 (Fig. 5), on an -helix backbone containing the Pro-kink
(right) compared with an ideal -helix (left). Spheres indicate C atoms
of residues at heptad positions a and d on the
coiled-coil interaction face of the helices. Purple spheres
indicate a and d positions in a canonical heptad and therefore show the dimerization surface in the ideal helix on the
left. Orange spheres indicate a and
d positions in the heptads after a deletion at the locus of
the horizontal line, corresponding to the deletion in human
NF-L. In the Pro-kinked helix the face of the helix outlined by the
purple spheres of the regular heptad is not continuous, as
in the ideal helix, due to the rotation of the helix face caused by the
Pro-kink. The deletion in the Pro-kinked helix compensates for the face
shift due to the Pro-kink, as indicated by the continuous face created by the Orange spheres before the deletion (amino terminus)
and the purple spheres after the deletion (carboxyl
terminus). Thus the deletion restores the heptad positions a
and d to the continuous helix face and creates a
dimerization surface similar to that of a regular heptad in the ideal
helix. Note, however, that by the same mechanism a deletion in an ideal
helix would distort the dimerization surface.
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Fig. 7.
Structural models and the assessment of the
polymerizing capability of the mutant rat NF-L proteins.
Representations of the sequence region from position a in
heptad 2 to position d in heptad 5 (Fig. 5), on an ideal
helix and Pro-kinked helix backbones. In all panels, purple
spheres indicate C atoms of residues at heptad
positions a and d of a regular heptad, and the
horizontal line denotes the position of the deletion in the human NF-L. A, the rat mutant Pro254 to
Ala254, which eliminates the Pro-kink, is represented by an
ideal -helix. Note that the Asp249 in the rat
Pro254 to Ala254 mutant appears at the position
d on the dimerization surface. However, in the human NF-L
structure the corresponding Asp249 is pointing away from
the dimerization surface and is not structurally at a place
corresponding to d position because of the deletion. C, the mutation Asp249 to Leu249
(shown) still retains the Pro-kink, and consequently, the dimerization faces above and below the line are rotated. Note that the mutation of
Asp249 to Leu249 eliminated the charged residue
from the dimerization surface and substituted it with a residue
commonly preferred at position d in coiled-coil heptad
repeats. E, the rat double mutant (Pro254 to
Ala254) and (Asp249 to Leu249) is
represented by an ideal -helix (right) since the mutation Pro254 to Ala254 eliminates the Pro-kink. SW13
cells were transfected with each of the mutated rat NF-L constructs and
stained using rabbit polyclonal anti-NF-L primary antibody followed by
donkey anti-rabbit fluorescein-conjugated secondary antibody.
Immunofluorescent staining was analyzed by laser scanning confocal
microscopy. B, Pro254 to Ala254;
D, Asp249 to Leu249; F,
the double mutant (Pro254 to Ala254) + (Asp249 to Leu249).
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The results of the transfection experiments shown in Fig. 7,
B, D, and F, bear out the predictions
completely. Transfection of SW13 (vim
) cells with rat
NF-L constructs that contained only single mutations (either
Ala254 or Leu249) resulted in no filament
production, although when either was co-transfected with human NF-M,
filaments containing both proteins were seen (data not shown). However,
transfection of the construct containing the double mutation
(Ala254 and Leu249) resulted in extensive
filament formation (Fig. 7F), consonant with the properties
expected of a coiled-coil structure in the mutated L12 segment.
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DISCUSSION |
In mature neurons, the three neurofilament proteins copolymerize
to form 10-nm filaments. These filaments form bundles consisting of
"core filaments" interconnected by cross-bridges not found in
filaments composed of other IF proteins. When neurofilaments are
assembled in vitro all three NF subunits are incorporated integrally into the filament cores, whereas the carboxyl-terminal tail
sequences of NF-M and NF-H extend away from the filament surface and
are the major constituents of cross-bridges (11, 42). In
vitro studies have long suggested that homopolymerization can be
achieved with NF-L alone while under most conditions NF-M and NF-H do
not assemble into homopolymers (10-15, 17, 18).
However, the relevance of in vitro to in vivo
filament assembly has recently been challenged. Two groups using the
SW13 (vim
) cell line have shown that unlike type III IFs
(such as vimentin), none of the rat or mouse NF subunits can form
filaments when transfected individually into cells lacking an
endogenous IF network (21, 22). Nevertheless, all three NF subunits can
co-assemble with
-internexin (21), and NF-L can combine with either
NF-M or NF-H to form filamentous networks. Transfection experiments in the insect cell line Sf9 which also lacks endogenous IFs have yielded similar results (42); when rat NF-L was expressed alone no
filamentous staining was observed but when expressed together with NF-M
it co-assembled to form bundles of 10-nm filaments with frequent
cross-bridges resembling axonal neurofilaments (21). These results
convincingly demonstrate that rodent neurofilaments are obligate
heteropolymers in vivo requiring NF-L plus either NF-M or
NF-H. The present study used a similar design to investigate the
assembly properties of the human NF-L and NF-M subunits and demonstrates the distinctive ability of human NF-L to homopolymerize, a
finding that distinguishes human NF-L from human NF-M and its rodent
NF-L counterpart.
To investigate the level at which the block to filament formation
occurs we utilized the yeast interaction trap system. Since the yeast
interaction trap requires direct protein-protein interactions for
activation of a reporter gene, it should measure the first step in
filament production, i.e. dimer formation (39). The inability of human NF-M or rat NF-L to self-interact productively in
this assay argues that homopolymerization of these subunits is blocked
at the first step of assembly. Conversely the positive interaction
between human NF-L fusion pairs is consistent with this subunit's
ability to form homopolymers in vivo in transfected cells. Our results suggest that neurofilaments found in the Triton X-100-insoluble fractions of SW13 (vim
) cells transfected
with rat NF-L (21) may not be true homopolymers of NF-L but rather
associations that are nonspecific or of insufficient strength to be
scored by the yeast system. However, it is not clear how to reconcile
our results with reports that a truncated rat nf-l gene
containing amino acids 24-542 can productively self-interact in the
yeast two-hybrid system (43). Conceivably the particular truncation is
important in conferring this ability.
Two obstacles likely exist to the homopoly merization of human NF-M.
First, the failure of human NF-M to self-interact in the yeast
two-hybrid system suggests that the NF-M rod domain itself is
incompetent to homodimerize. However, replacement of the NF-M rod
domain with one known to be competent to dimerize (human NF-L rod) did
not produce a protein (NF-M-L-M) that homopolymerizes. Yet when the
NF-M-L-M hybrid was coexpressed with normal human NF-L, there was
extensive filament formation. These findings indicate a strong
influence of flanking sequences on subunit association. Thus,
homopolymerization of NF-M is prevented by two factors, each of which
is strong enough by itself to prevent homoassociation, but nonetheless
the protein retains the ability to heteropolymerize. These observations
suggest a previously unsuspected role of the flanking sequences in
neurofilament assembly, preventing certain associations while allowing
others.
Additionally these studies make two observations relevant to the
general process of intermediate filament assembly. First they show that
a single amino acid change in one heptad repeat can drastically alter
the dimerization potential of a rod domain. The sequence similarities
in the rod domains of homodimerizing and non-homodimerizing NF subunits
is very high. In NF-L there are only 8 amino acid differences in the
310 residues of the human and rat rod domains, and we show that a
single change in the rat rod domain (Gln162 to
Arg162) is sufficient to allow robust homopolymerization.
This substitution appears at position e which is generally
occupied by charged residues (usually Arg, Lys, or Glu) in a regular
heptad characteristic of coiled-coils (25). The residues in this
position are responsible for additional attractive interactions between
the helices forming the coiled-coil (34). Thus replacing the rat Gln
with an Arg renders this heptad more "ideal" and was sufficient to
allow robust homopolymerization. The failure of the human NF-L to
homodimerize following the reciprocal substitution at
Arg161 in human with the Gln present in rat demonstrates
the importance of this minor change on the overall ability of the
-helical rods to dimerize.
Homodimerization appears to have more stringent requirements than
heterodimerization and may demand the most perfect set of heptad
repeats. If the ability of an IF rod domain to homodimerize reflects
the summed homodimerizing capacities of each individual heptad, then
human NF-L may be just above a threshold of compatibility needed for
homodimerization while the rat NF-L may be just below it. Thus making
even a single heptad more ideal in the rat converts it into a
homodimerizing subunit while making one heptad less perfect
converts the human into a subunit incapable of homodimerizing.
A second finding likely relevant to the assembly of many homopolymeric
IFs is the critical role of the L12 linker region. As illustrated in
Table IV, intermediate filament subunits
that homopolymerize have a characteristic L12 sequence that includes a
highly conserved proline and a spacing of 25 residues between the last
full heptad repeat of the 1B segment and the proline residue in the L12
region. Those subunits known to be obligate heteropolymers have
acquired an additional amino acid residue or have undergone significant
sequence divergence. The net effect of the observed insertion relative
to the human sequence is to rotate, with respect to one another, the
hydrophobic patches of the heptad before and after the conserved
proline, such that they are no longer on the same face of the helix
(Fig. 6). The change in dimerization surface resulting from this
rotation weakens the interactions between subunits due to the inability
to form a sufficiently stable coiled-coil association. We have
demonstrated that rotating the positions of the helix before and after
the conserved proline of rat NF-L relative to one another, either by a
serine deletion or by the creation of a canonical
-helical heptad
repeat by replacing the proline and preceding aspartic acid, results in
a protein that can homopolymerize. Thus, the L12 region of the human
NF-L exhibits the structural interaction properties expected of an
-helix with a heptad repeat and aligns the heptad repeat before and
after it, allowing homopolymerization of the subunit.
We do not know the biological significance, if any, of the human
NF-L's ability to homodimerize. The human subunit has evolved or
preserved the same primary sequence characteristics of other homopolymerizing intermediate filaments. The ability of subunits to
homo- or heteropolymerize may shape the course and character of
filament formation and turnover. If rodent NF-L subunits can only
heterodimerize and human NF-L can hetero- and homodimerize, the
composition of neurofilaments may differ between species. Such an
assembly model predicts that the mole fraction of NF-L in rodent
neurofilaments must be 1/2 (assuming that NF-M and NF-H do not
form dimers with each other or with themselves). Conversely human NF-L,
which is unconstrained in its choice of partners and can form homo- as
well as heterodimers, may have a mole fractions between 1/2 and
1. Unfortunately the molar compositions of filaments reported in the
literature varies widely (44-47) and cannot be used to verify these
predictions. Moreover, since distinct subpopulations of neurons may
possess filaments with different subunit compositions, species
differences may only be apparent when cognate subpopulations are
compared.
Yet whatever functional significance these findings have for
neurofilament assembly in vivo, our results clearly indicate that subtle alterations in the rod domain of an intermediate filament protein can drastically alter its assembly properties. Our results also
point to the importance of the L12 linker region in filament assembly.
We thank Dr. R. Evans for the gift of SW13
(vim
) cells; Dr. R. Liem for the gift of the rat NF-L
cDNA expression vector (pRSVNF-L); and Dr. V. Friedrich and Dr. R. Hardy for assistance with confocal microscopy.