CNRS UPR 2228, Régulation de la Transcription et Maladies Génétiques, Université René Descartes, 45 rue des Saints-Pères, 75270 Paris Cedex 06, France
* Author for correspondence (e-mail: djian{at}biomedicale.univ-paris5.fr )
Accepted 21 November 2001
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Summary |
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Key words: Polyglutamine, Huntington's disease, Inclusions, Centrosomes, Cytoskeleton, Perikaryon
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Introduction |
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We show that huntingtin specifically interacts with ß-tubulin and
binds to microtubules. This explains why immunoelectron microscopy of neurons
had shown that huntingtin appeared to be colocalized with microtubules
(Gutekunst et al., 1995).
Huntingtin is concentrated in the perinuclear region, where it associates with
microtubules and in the centrosome. The perinuclear distribution of huntingtin
is likely to explain why the inclusions characteristic of Huntington's disease
are mostly nuclear or perinuclear.
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Materials and Methods |
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For immunocytochemistry we used five anti-huntingtin antibodies: two
polyclonal antibodies prepared by immunizing rabbits against the first 17
amino acid residues of human huntingtin and affinity purified using the
antigen (QCB, Hopkinton, MA), MAB 2166 and two polyclonal antibodies (HF1 and
HP1) generated by Persichetti et al.
(Persichetti et al., 1995).
One monoclonal anti-ß-tubulin (3F3G2) and one monoclonal
anti-
-tubulin (GTU 88, Sigma) were also used.
Preparation of extracts from lymphoblasts and brain
Protein extracts containing normal (Q10) and expanded (Q80) huntingtin were
prepared from lymphoblastoid line SALVAL 2050013. Cell suspension (50 ml) was
centrifuged at 800 g for 5 minutes at 4°C. The pellet was
washed twice with 30 ml of cold phosphate buffer saline (PBS) and resuspended
in 300 µl of hypotonic buffer containing 50 mM Tris-HCl pH 7.8, 10%
glycerol, 10 mM EDTA, 5 mM KCl, 1 mM phenylmethylsulfonyl fluoride (PMSF) and
0.1 mM leupeptin. After a 10 minute incubation on ice, cells were disrupted by
sonication.
For preparation of brain extracts, cortex (50 mg) was homogenized in 500 µl of the buffer described above, using a motor-driven Teflon pestle (13 strokes at 900 rpm). The protein concentration was determined with a Bio-Rad protein assay kit using IgG as standard.
Immunopurification with magnetic beads
The anti-huntingtin (MAB 2166, Chemicon) or the anti-ß-tubulin (3F3G2,
ICN) were crosslinked to an anti-IgG antibody, itself covalently coupled to
magnetic beads by the manufacturer (Dynal). Chemical crosslinking of the
anti-huntingtin or the anti-ß-tubulin antibody to the anti-IgG antibody
was necessary. In the absence of such crosslinking, the two antibodies used
for immunopurification elutes from the magnetic beads together with huntingtin
and are stained by the secondary anti-mouse antibody that is used in
conjunction with the monoclonal anti-tubulin antibody during the western blot.
Because the size of the IgG heavy chain is practically identical to that of
tubulin, the two proteins cannot be resolved by the electrophoresis.
Magnetic beads (2.67.107) were incubated overnight at 4°C in the presence of 16 µl of specific antibody (8-40 µg IgG/µl) in PBS containing 0.1 mM leupeptin, 1 mM PMSF and 0.1% Tween 20. The excess of specific antibody was removed by washing the beads six times in the same buffer using a magnetic particle concentrator (Dynal).
For crosslinking of the specific antibody to the anti-mouse IgG, the beads were washed twice in 0.2 M triethanolamine (TEA) pH 9.0, then incubated in the same buffer containing 20 mM dimethylpimelimidate dihydrochloride (Sigma) for 45 minutes at room temperature. Crosslinking was stopped by removing the beads from the buffer containing the crosslinker. The beads were then incubated for 10 minutes at 4°C in TEA buffer containing 1% Triton, and washed three times in PBS-Tween buffer.
For immunopurification, the lymphoblast or brain extracts (100 µg of protein) were incubated in the presence of the beads for 2 hours 30 minutes at 4°C. After six washes in the PBS-Tween buffer, the immunopurified product was eluted from the beads by vortexing in 35 µl of a solution containing 2 M NaI and 50 mM Tris-HCl pH 9 for 1 minute. The magnetic beads were concentrated with a magnet, the eluate was recovered and the few remaining beads were removed with the magnetic particle concentrator. The entire eluate derived from 100 µg of extract was then analyzed by western blotting.
Western blots
For analysis of huntingtin, proteins (100 µg) were subjected to
electrophoresis according to Laemmli
(Laemmli, 1970) using a 4%
acrylamide plus bisacrylamide stacking and resolving gels (ratio 29:1 for both
gels). Electrophoresis was at 18.2 V/cm for 2 hours. For analysis of tubulins,
proteins (50 µg) were subjected to electrophoresis using an 8%
polyacrylamide gel. Immunoblotting was carried out exactly as described
(Kahlem et al., 1998
). All
antibodies used were absolutely specific as they detected a single protein
with the appropriate molecular weight. Bands on films were quantitated with an
AGFA digital light sensing scanner and the Fotolook version 2.07 program;
images were analyzed with the NIH IMAGE version 1.61 program.
Association of huntingtin with paclitaxel-stabilized
microtubules
Rat brain (500 mg) was homogenized in 350 µl of cold PEM buffer (0.1 M
PIPES-NaOH (pH 6.6), 1 mM EGTA, 1 mM MgSO4, 1 mM PMSF and 0.1 mM
leupeptin), using a motor-driven teflon pestle (10 strokes at 1200 rpm). The
homogenate was clarified by centrifugation at 150,000 g for 1
hour at 4°C and the supernatant (S1) containing soluble tubulin
was collected. Half of S1 was then incubated in the presence of 20
µM paclitaxel (Sigma) and 1 mM GTP for 30 minutes at 37°C to allow
microtubules to form. Microtubules were then purified by centrifugation (at
48,000 g for 30 minutes at 37°C) in a 50 Ti rotor through
an equal volume of PEM buffer containing 10% sucrose. The resulting
supernatant (S2) was collected, whereas the pellet (P2)
was washed and resuspended in PEM buffer supplemented with paclitaxel and GTP.
The remaining half of S1 was sedimented by centrifugation at 48,000
g for 30 minutes at 4°C (depolymerization conditions); the
resulting supernatant (S2') and pellet (P2') were
collected. The presence of huntingtin and ß-tubulin in S2,
P2, S2' and P2' was then determined by
western blotting. Equal fractions of the pellets and supernatants were loaded
on the gels for western blot analysis.
Immunocytochemistry
For immunocytochemistry, Swiss 3T3 cells
(Todaro and Green, 1963) were
inoculated on glass coverslips. The next day, cells were washed twice with
PBS, then fixed in acetone-methanol (1:1) for 20 minutes at -20°C and air
dried. To block nonspecific binding, the coverslips were incubated in PBS
containing 5% bovine serum albumin (BSA) for 1 hour. For tubulin and
huntingtin staining, cells were incubated in 5% BSA/PBS containing both the
rabbit anti-huntingtin (1:100 dilution) and a monoclonal anti-tubulin (Sigma)
antibody (1:1000 dilution), then washed three times for 5 minutes with PBS
containing the nonionic detergent IGEPAL CA 630 (Sigma). Cells were then
incubated for 1 hour at room temperature in the presence of 5% BSA/PBS
containing both a goat anti-rabbit IgG linked to the fluorescent dye Alexa 488
(Molecular Probes) and a goat anti-mouse IgG linked to biotin (Jackson
Immunoresearch). The anti-rabbit IgG antibody used to detect huntingtin was
added at a 1:100 dilution and the anti-mouse IgG used to detect tubulin was
added at a 1:1000 dilution. At the end of the incubation, coverslips were
washed three times in PBS containing 0.1% IGEPAL and incubated for 1 hour with
streptavidin linked to cyanin-3 at a 1:1000 dilution to stain tubulin.
Coverslips were then washed at room temperature with PBS/IGEPAL before being
incubated for 5 minutes in the presence of PBS containing bisbenzimide H 33258
(1 µg/ml) and 0.1% IGEPAL. Coverslips were then washed once in PBS/IGEPAL,
then in water and mounted onto slides in mowiol containing 2.5%
1-4,diazobicyclo-(2.2.2.)-octane (DABCO). Cells were viewed using a Nikon E600
fluorescent microscope equipped with a Nikon digital camera. The images were
displayed with the accompanying software and Adobe Photoshop V5.5 For confocal
laser microscopy either a Zeiss or a Leica microscope were used. In all
immunocytochemistry experiments it was verified that there was no detectable
staining when the specific antibodies were omitted and that there was no
overlapping of either color (red or green) onto the opposite filter in
single-labeled cells.
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Results |
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Because the anti-huntingtin antibody used for immunopurification was
monoclonal and was therefore directed against a single huntingtin epitope, it
was necessary to verify that this epitope was not also present in
ß-tubulin. We therefore submitted tubulin purified to homogeneity by
phosphocellulose chromatography
(Weingarten et al., 1975;
Boucher et al., 1994
) to
immunopurification by the anti-huntingtin antibody. This tubulin preparation,
which was confirmed to be free of huntingtin by western blotting, could not
bind the anti-huntingtin antibody. This result confirmed that the
anti-huntingtin antibody purified ß-tubulin in cell and tissue extracts
because of its interaction with huntingtin.
We then carried out the reciprocal experiment of immunopurifying huntingtin
with the anti-ß-tubulin antibody. Lymphoblast and brain extracts were
incubated in the presence of an anti-ß-tubulin antibody (3F3G2), itself
crosslinked to the anti-IgG antibody coupled to magnetic beads. Western blot
analysis of the eluate revealed that equal amounts of normal and expanded
huntingtin were recovered from the lymphoblast extract by the
anti-ß-tubulin antibody. A small amount of -tubulin was also
recovered. In control experiments performed in the absence of
anti-ß-tubulin antibody, no huntingtin was bound. In rat brain also, the
anti-ß-tubulin antibody copurified ß-tubulin and huntingtin
(Fig. 1B). The yield of
huntingtin purification by the anti-ß-tubulin antibody was relatively low
because of the large excess of competing ß-tubulin, particularly in
brain.
To show further the specificity of the immunopurification, several abundant brain proteins were tested: MAP2, kinesin, neurofilaments M and H. None of these proteins was purified by the anti-huntingtin antibody: no signal was detected even when the blots were largely overexposed.
In summary, our results clearly establish with the use of two unrelated antibodies (one anti-huntingtin and one anti-ß-tubulin) that there exists a specific interaction between huntingtin and ß-tubulin.
In the cell, most ß-tubulin forms a heterodimer with -tubulin.
The purification of monomeric ß-tubulin but not
-tubulin by the
anti-ß-tubulin antibody (Fig.
1) could result either from dissociation of the tubulin
heterodimer by the antibody or binding of huntingtin to some free
ß-tubulin that might exist within the cell. To determine whether the
anti-ß-tubulin caused dissociation of the tubulin heterodimer, we
incubated the tubulin heterodimer purified by phosphocellulose chromatography
and known to contain no free monomeric ß-tubulin
(Weingarten et al., 1975
) in
the presence of the anti-ß-tubulin antibody bound to beads. The fact that
the anti-ß-tubulin antibody purified mostly monomeric ß-tubulin and
little
-tubulin confirmed dissociation of the tubulin heterodimer
during immunopurification (Fig.
2).
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Huntingtin binds to microtubules
Because huntingtin interacts with ß-tubulin in brain, it should be
associated with purified microtubules prepared from this tissue. Irreversibly
polymerized microtubules were prepared from rat brain in the presence of
paclitaxel and purified by centrifugation through a sucrose cushion
(Vallee, 1986). Proteins
present in the pellet and in the supernatant were analyzed by western
blotting. About 25-30% of huntingtin and 75% of MAP2 were reproducibly found
to be associated with the microtubules. Two proteins not known to be
associated with microtubules, actin and caspase-1, remained in the supernatant
(Fig. 3).
|
These experiments demonstrate the association of huntingtin with microtubules formed in vitro, but they do not show whether such an association exist in the cell. It has been very difficult to observe clear colocalization of huntingtin with microtubules by immunofluorescence microscopy. The density of the microtubular network in most cells and the fact that some huntingtin is not associated with microtubules render the staining rather diffuse. To circumvent these problems, we used Swiss 3T3 cells, which, because of their large size and flat morphology, have a sparse microtubular network at their periphery. 3T3 cells were double stained with a polyclonal anti-huntingtin and a monoclonal anti-tubulin antibody. Colocalization of huntingtin with individual microtubules was evident at the periphery of the cell. Some of the huntingtin staining was also diffuse within the cytoplasm (Fig. 4A). After exposure to nocodazole (10 µg/ml) for 20 hours, microtubules were disrupted and huntingtin also acquired a diffuse and patchy distribution. However, some huntingtin was still colocalized with fragments of microtubules that persisted after treament with the drug (Fig. 4B).
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Huntingtin has a perinuclear distribution and is found at the
centrosome
Microtubules converge around the nucleus and ultimately to the centrosome
where their minus-ends bind -tubulin
(Oakley and Oakley, 1989
;
Stearns et al., 1991
;
Li and Joshi, 1995
). We had
observed that most of the huntingtin staining was perinuclear and we
attributed this to the binding of the protein to microtubules
(Fig. 4A). However, this
apparent perinuclear localization could simply reflect the fact that in
cultured cells the cytoplasm is much thicker around the nucleus than at the
periphery of the cell. We therefore decided to use confocal microscopy in
order to analyze a thin section of the cell (0.8 µm). Staining with both a
polyclonal anti-huntingtin and a monoclonal anti-ß-tubulin antibody
revealed that most of huntingtin was indeed found around the nucleus where it
colocalized with the denser part of the microtubular network
(Fig. 5). Perinuclear
distribution overlapping with the microtubules was also observed with the
monoclonal anti-huntingtin antibody MAB 2166.
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To determine whether huntingtin was also present at the centrosome, 3T3
cells were stained with both a polyclonal anti-huntingtin and a monoclonal
anti--tubulin antibody. The anti-
-tubulin antibody strongly
stained the centrosome in which the two centrioles could often be
distinguished. It also stained the perinuclear region, probably because of the
existence of a large pool of soluble
-tubulin in this region
(Murphy et al., 1998
). In a
fraction of the cells (30-40%), the anti-huntingtin antibody stained the
centrosome and often the two centrioles
(Fig. 6A). The presence of
huntingtin at the centrosome was confirmed by laser confocal microscopy and
analysis of local image correlation with the appropriate software
(Demandolx and Davoust, 1997
)
(Fig. 6B). The presence of
huntingtin in the centrosomal region could also be detected with another
anti-N-terminal antibody (G.H. and P.D., unpublished), as well as with the HP1
and HF1 antibodies (Persichetti et al.,
1995
).
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Discussion |
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Under our conditions of immunopurification, both the anti-huntingtin and
the anti-ß-tubulin antibody caused dissociation of the tubulin
heterodimer (Figs 1,
2). This is indeed why we could
show that huntingtin was specifically associated with ß-tubulin.
Dissociation of the tubulin heterodimer under nondenaturing conditions is far
from unprecedented. Reversible dissociation of the tubulin dimer with a
Kd of about 10-6 M has been shown by a variety
of techniques (Detrich and Williams,
1978; Mejillano and Himes,
1989
; Sackett and Lippoldt,
1991
; Panda et al.,
1992
; Shearwin et al.,
1994
). Dissociation of the heterodimer also occurs after treatment
with lactoperoxidase, which forms complexes with the tubulin monomers
(Wolff and Knipling, 1995
) and
during immunopurification in Tris buffer
(Giraudel et al., 1998
). At
least three proteins, Rb12p-ß/cofactorA, Cin1p/cofactorD and the inner
centromere protein have been shown by immunoprecipitation to interact with
monomeric ß-tubulin, and not with
-tubulin
(Archer et al., 1995
;
Archer et al., 1998
;
Fleming et al., 2000
;
Wheatley et al., 2001
).
The fact that huntingtin appeared to associate with microtubules and
vesicles had suggested that huntingtin participated in vesicle trafficking and
that expansion of the polyglutamine sequence of huntingtin could lead to
disease by decreasing transport of vesicles
(Gutekunst et al., 1995;
Di Figlia et al., 1995
;
Gutekunst et al., 1998
).
However, this idea is not supported by the finding that expansion of the
polyglutamine sequence did not alter the affinity of huntingtin for
ß-tubulin (Fig. 1B).
Several proteins interacting with huntingtin have been found by affinity
methods: calmodulin, ubiquitin-conjugating enzyme
(Kalchman et al., 1996),
glyceraldehyde phosphate dehydrogenase
(Burke et al., 1996
), HAP 1
(Li et al., 1995
) and HIP 1
(Wanker et al., 1997
;
Kalchman et al., 1997
). HAP 1
and glyceraldehyde phosphate dehydrogenase are thought to associate with
microtubules (Martin et al.,
1999
; Kumagai and Sakai,
1983
). These proteins could therefore mediate the interaction of
huntingtin with ß-tubulin. However, the binding of huntingtin to these
two proteins has been shown to increase with the length of the polyglutamine
sequence (Burke et al., 1996
;
Li et al., 1995
), whereas
normal and expanded huntingtin bind ß-tubulin with no obvious difference
(Fig. 1B). The fraction of
huntingtin that is free in the cytoplasm of neurons could interact with
proteins such as HIP 1 or calmodulin independently of microtubules.
Although ß-tubulin is a ubiquitous protein, it is much more abundant
in neurons than in other cell types. Its participation in the aggregates
formed by the huntingtin bearing an expanded polyglutamine could therefore
lead to the more rapid formation of inclusions in neurons than in other cell
types. It would be of interest to determine by immunocytochemistry whether
ß-tubulin is present in the inclusions characteristic of Huntington's
disease (Davies et al., 1997).
Such experiments would require purification of the inclusions, as the large
amount of ß-tubulin present in neurons would render very difficult the
detection of ß-tubulin in inclusions by direct staining of tissue
sections.
The inclusions characteristic of Huntington's disease are mostly nuclear or
perinuclear (Di Figlia et al.,
1997; Meriin et al.,
2001
; Waelter et al.,
2001
). The high concentration of huntingtin around the nucleus and
at the centrosome would readily explain why aggregation is more likely to
develop in the perinuclear region than in the rest of the cytoplasm. An
N-terminal fragment of huntingtin with a molecular weight of about 40 kDa has
been identified in the nuclear fraction of the cerebral cortex of patients
with Huntington's disease (DiFiglia et
al., 1997
). The perinuclear distribution of huntingtin shown in
Fig. 5 would also explain why a
small fragment would tend to diffuse into the nucleus, although it lacks a
functional nuclear localization signal
(Hackam et al., 1999
. The
presence of an excessively long polyglutamine sequence would then promote its
aggregation in the nucleus.
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Acknowledgments |
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