1 Groupe Structure Dynamique de la Chromatine, CNRS, UMR 6061, Faculte de
Medicine, 35043, Rennes, France
2 Cell Cycle Group, Division of Electron Microscopy, A. N. Belozersky Institute
of Physico-Chemical Biology, Moscow State University, 119899, Moscow,
Russia
3 Institute of Agricultural Biotechnology, 127550 Moscow, Russia
4 Division of Electron Microscopy, A. N. Belozersky Institute of
Physico-Chemical Biology, Moscow State University, 119899, Moscow,
Russia
5 Department of Cell and Structural Biology, University of Illinois at
Urbana-Champaign, Urbana 61801, USA
Author for correspondence (e-mail:
ikireev{at}life.uiuc.edu)
Accepted 6 December 2002
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Summary |
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Key words: Chromosome condensation, Condensin, Chromatin, Nucleolus
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Introduction |
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The high level of compactness is achieved by ordered folding of DNA through
its interaction with chromosomal proteins. Interaction of DNA with histones
gives rise to nucleosomes and a 30 nm chromatin fiber
(Wigler and Axel, 1976). The
mode of DNA folding at higher levels of compaction and molecular mechanisms
involved in formation and maintaining of higher order chromatin structures
remain largely elusive. Recent studies have led to the identification of a new
class of chromosomal proteins, which take part in mitotic chromosome
compaction (Strunnikov et al.,
1993
; Hirano and Mitchison,
1994
; Saitoh et al.,
1994
). These proteins, termed SMC (structural maintenance of
chromosomes), participate in multiple chromosomal activities, including
mitotic chromosome compaction and segregation
(Strunnikov et al., 1993
),
sister chromatid cohesion (Guacci et al.,
1997
; Michaelis et al.,
1997
; Losada et al.,
1998
), recombination and repair
(Jessberger et al., 1996
;
Stursberg et al., 1999
) and
dosage compensation (Lieb et al.,
1998
; Meyer,
2000
).
Isolation of condensed chromosomes from cell-free extracts enabled
biochemical studies of the proteins associated with DNA during mitosis.
Proteins identified by this approach using Xenopus laevis cell-free
extract (Hirano and Mitchison,
1994; Hirano et al.,
1997
) form complexes sedimenting at 8S and 13S. The former
consists of a heterodimer of SMC proteins belonging to the SMC2/4 subfamily
(XCAP-E and XCAP-C), whereas the latter contains three additional subunits,
XCAP-D2, XCAP-H and XCAP-G. XCAP-D2 was simultaneously identified as pEg7 by
differential screening of the Xenopus egg cDNA library for genes
expressed during oocyte maturation
(Cubizolles et al., 1998
). On
the basis of their chromosome condensation activity, which was demonstrated in
immunodepletion/rescue experiments, these complexes were termed condensins
(Hirano et al., 1997
). It is
believed that 13S condensin is involved in active reconfiguration of DNA
(Kimura and Hirano, 1997
;
Kimura et al., 1999
). Non-SMC
proteins can act as regulators of condensin activity
(Kimura and Hirano, 2000
).
Initiation of complex assembly and/or modulation of its activity during
mitosis may be controlled by cell cycle dependent phosphorylation of XCAP-D2,
XCAP-H and XCAP-G (Hirano et al.,
1997
; Kimura et al.,
1998
).
Recently it became clear that condensin subunits have more than one
function in the cell. To date, the only clear example of such a dual function
is given by the Caenorhabditis elegans protein MIX-1, which is
homologous to XCAP-E and plays an essential role in gene dosage compensation
(Lieb et al., 1998). MIX-1
forms a complex with another SMC-protein, DPY-27, and several other proteins.
During mitosis, MIX-1 participates in chromosome compaction by interacting
with a yet unidentified SMC protein (Lieb
et al., 1998
). The interphase behavior of other condensin subunits
is yet to be determined.
In the present work, we studied ultrastructural localization of two subunits of the Xenopus laevis condensin complex, XCAP-E and pEg7, and the level of their expression during the cell cycle.
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Materials and Methods |
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Xenopus cultured cells
The embryonic Xenopus laevis cell line XL2
(Anizet et al., 1981) was a
gift from J. Tata (Mill Hill-NIMR Laboratory, London). Cells were grown at
25°C in L-15 medium supplemented with 10% fetal calf serum and
antibiotic-antimycotic solution (Gibco-BRL).
Cell synchronization
XL2 cells were synchronized according to the protocol of Uzbekov et al.
(Uzbekov et al., 1999) with
modifications. After serum starvation for 24 hours, cells were incubated 30
hours in complete medium with 2 µg/ml aphidicolin, then released from the
block by several washes with fresh complete medium. Fractions enriched with S
phase (max S) and G2 phase cells (max G2) were collected 2 and 10 hours after
the last wash, respectively. For preparation of a fraction of mitotic cells
(max M), 8 hours after washing out aphidicolin, cells were incubated for 3
hours in complete medium with 0.5 µg/ml nocodazole and then for 4 hours in
complete medium with 0.5 µg/ml nocodazole and 40 µg/ml calpain inhibitor
I (ALLN). Mitotic cells were collected for 20 minutes after washing off
nocodazole and ALLN with fresh medium. The fraction enriched with G1 phase
cells (max G1) was collected 11 hours after removing the
nocodazole/ALLN mixture. The fraction of cells in G0 (max G0) was obtained by
cultivating cells for 7 days in complete medium at 90°C and then 24 hours
in the medium without serum at 25°C.
The composition of all fractions was controlled for by BrdU labeling (see
below). The fraction of cells in G0 was estimated by prolonged BrdU labeling
(30 hours); the fraction of cells in S phase was assessed by impulse BrdU
labeling (30 minutes). The fraction of mitotic cells was estimated by direct
counting in a phase contrast microscope. The percentage of cells in G2 and G1
was calculated as described elsewhere
(Uzbekov et al., 1999). Data
from more than 22,000 cells were used for estimation of cell fraction
composition.
Antibodies
The generation and purification of anti-Eg7G polyclonal antibodies were
reported in our previous paper (Cubizolles
et al., 1998). Polyclonal anti-XCAP-E antibodies were raised
against the last 14 amino acids of XCAP-E and affinity purified on CNBr
Sepharose column (Amersham Pharmacia Biotech) coupled to the same peptide.
Human autoimmune sera to UBF and fibrillarin were kindly provided by D.
Hernandez-Verdun (I. Jacques Monod, France), and antibodies to B23 were a gift
from T. I. Bulycheva (National Center for Hematology RAMS, Moscow, Russia)
(Bulycheva et al., 2000).
Polyclonal anti-topoisomerase II (anti-topoII) antibodies were provided by D.
F. Bogenhagen (State University of New York, Stony Brook, USA)
(Luke and Bogenhagen, 1989
);
monoclonal anti-human topoII antibodies were obtained from Calbiochem;
monoclonal anti-Pleurodeles topoII antibodies were a gift from R. Hock
(University of Wurzburg) (Hock et al.,
1996
).
Indirect immunofluorescence microscopy
Xenopus laevis XL2 cells were grown on round glass coverslips in
12-well plates (Corning Inc., Acton, USA) for 48 hours, washed with
phosphate-buffered saline (PBS: 120 mM NaCl, 2.7 mM KCl, 10 mM
phosphate-buffer, pH 7.2) before fixation. The following fixation protocols
were tested: (1) 100% methanol for 6 minutes at -20°C; (2) 100% methanol
with subsequent post-fixation with 100% acetone for 6-20 minutes at -20°C;
(3) 1:1 mixture of methanol and acetone for 6-20 minutes at -20°C; (4) 3%
formaldehyde in PBS for 10-30 minutes at room temperature; (5) mixture of 3%
formaldehyde and 0.1% glutaraldehyde for 30 minutes at room temperature with
subsequent `quenching' of free aldehyde groups by two 10 minute washes with 2
mg/ml NaBH4 in PBS. In some cases, cells were additionally
permeabilized for 3 minutes with 1% Triton X-100 in PBS at room temperature,
either prior to or after fixation. Subsequent immunostaining was essentially
the same for all fixation protocols. Following three washes in PBS, cells were
blocked in PBS containing 3% BSA for 30 minutes and then incubated with a
mixture of primary antibodies: rabbit antibodies to pEg7 or XCAP-E (1:50) and
either mouse anti-topoII monoclonal antibody or anti-B23 antibody (dilution
1/100), or human antisera to fibrillarin or UBF (1:100). The antibodies were
subsequently revealed by fluorescein isothiocyanate (FITC)-conjugated goat
anti-rabbit IgG (dilution 1/100) and Texas-Red-conjugated goat anti-mouse IgG
(dilution 1/70) or Texas-Red-conjugated goat anti-human IgG (dilution 1/100).
All antibody reagents were diluted in PBS containing 1% BSA, and incubations
were performed at room temperature for 60 minutes. Cells were rinsed three
times in PBS containing 1% BSA after each incubation.
For anti-BrdU labeling, cells grown on glass coverslips were incubated in complete medium with 40 µM BrdU either for 30 minutes (pulse-labeling) or longer. Then cells were briefly washed in warm (25°C) PBS and fixed with 70% ethanol for 30 minutes. The coverslips were rinsed in PBS and immersed in 4 M HCl for 20 minutes, then washed 5 times in PBS and incubated for 60 minutes at room temperature with mouse anti-BrdU (Sigma) and then anti-mouse Texas-Red-conjugated antibodies for 60 minutes at room temperature.
After immunolabeling, cells on coverslips were rinsed in PBS and mounted in Mowiol (Calbiochem). Samples were observed using a Zeiss Axiolab microscope (AXIOVERT 35) equipped with phase contrast and epifluorescence, using 40x/0.65 NA and 100x/1.25 NA achroplan objectives, and photographed using a Nikon 601 camera. For quantitative analysis, images were captured using a CH-250 CCD camera (Photometrics, Tuscon, USA) mounted on a Zeiss Axioscope microscope with a 100x/1.25 NA objective. Digital image processing was performed using Scion Image software (Scion Corp., Frederick, USA).
Western blot analysis
Electrophoreses on SDS-polyacrylamide gel were performed according to the
protocol of Laemmli (Laemmli,
1970) and transferred onto nitrocellulose membranes, as described
previously (Towbin et al.,
1979
). Membranes were blocked in TBST (Tris buffer saline with
0.05% Tween-20, pH 7.5) containing 5% skimmed milk for 2 hours at room
temperature and incubated for 3 hours with antibodies diluted in TBST
containing 5% skimmed milk. Immuno-complexes were revealed with antibodies
coupled with peroxidase or alkaline phosphatase (Sigma) by using either
NBT/BCIP (Sigma) or an ECL kit (NEN, Boston, MA) according to the
manufacturer's instructions.
Immunoelectron microscopy
Cells were rinsed in PBS permeabilized by incubation in buffer containing
50 mM imidazole, pH 6.8, 50 mM KCl, 0.1 mM EDTA, 1 mM EGTA, 5 mM
MgCl2, 0.1 mM beta-mercaptoethanol and 1% Triton X-100 for 3
minutes at room temperature and fixed in cold absolute methanol (6 minutes,
-20°C). After three washes in PBS, preparations were blocked for 30
minutes in PBS containing 3% BSA. Cells were then incubated with a mixture of
polyclonal purified antibodies against pEg7 or XCAP-E and anti-topoII
monoclonal antibodies, washed and incubated in a mixture of secondary
antirabbit 5 nm gold and anti-mouse 10 nm gold-conjugated antibodies. Antibody
reagents were diluted in PBS containing 1% BSA, 3% temperature inactivated
(56°C, 30 minutes) goat normal serum, 0.1% Tween 20 and incubated at room
temperature for 60 minutes. Cells were then washed with PBS and fixed for 90
minutes in 0.1 M phosphate buffer at pH 7.2
(KH2PO4-Na2HPO4) containing 2.5%
glutaraldehyde. After being rinsed in 0.1 M phosphate buffer, cells were
post-fixed with 1% osmium tetraoxide in 0.1 M phosphate buffer, stained with
uranium acetate, dehydrated and embedded in an Epon 812 mixture. Serial
ultrathin (70 nm) sections were obtained parallel to the substrate plane using
an LKB-III ultramicrotome and mounted on single slot grids. The sections were
examined using Hitachi-11 and Hitachi-12 electron microscopes operating at 80
kV and photographed.
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Results |
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Two proteins with known behavior during the cell cycle were used as
standards: ß-tubulin, whose level remains constant during the cell cycle,
and pEg2, whose concentration changes cyclically, rising at the beginning of
mitosis (Roghi et al., 1998;
Arlot-Bonnemains et al.,
2001
).
All used antibodies, except monoclonal anti-topoII, detected one single
specific band on western blots; purified Eg7G polyclonal antibodies recognized
a band of 150 kDa [Fig. 1A, see
also Fig. 2C (Cubizolles et al., 1998)],
purified XCAP-E antibodies a band of 125 kDa
(Fig. 1B), monoclonal
antibodies against pEg2 [clone 1C1, see
Fig. 4B
(Roghi et al., 1998
)] a band
of 46 kDa, monoclonal antibodies against ß-tubulin (clone no. TUB 2.1,
Sigma) a band near 50 kDa (Fig.
1Aa). Monoclonal antibodies against human Topoisomerase-II
(Clone SWT3D1 Calbiochem) detect one major specific band near 180 kDa and one
additional small band near 50 kDa (Fig.
1B).
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The synchrony of the cell population and its progression through the cell
cycle were monitored by BrdU incorporation and immunofluorescent microscopy
(Uzbekov et al., 1998;
Uzbekov et al., 1999
). This
approach, although rather time-consuming, has some advantages over FACS
analysis, particularly in discriminating between late G1 and early S cells,
and seems to be especially useful when working with partially aneuploid or
polyploid cells. Table 1 shows
the percentage of cells in different phases of the cell cycle in synchronized
cell populations, which were used for western blot analysis.
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For quantification of protein levels at different cell cycle stages,
densitometric data were normalized to that of ß-tubulin. In
Fig. 2, cell cycle dependent
changes in protein level is shown (OD ratio=1 in G1). From G1 to M, the
quantity of pEg2 increased more than 15-fold. TopoII level was maximal in G2
(3.5 G1 level) and remained practically the same during mitosis, which is in
good agreement with published data for topoII
(Heck et al., 1988
;
Drake et al., 1989
;
Woessner et al., 1991
).
In contrast, levels of pEg7 and XCAP-E increased slowly as cells progressed
from G1 to M and became less than two-fold higher in the mitotic fraction when
compared to the G1 fraction (Fig.
1b). Similar results were obtained for human orthologs of XCAP-E
and XCAP-C (Schmiesing et al.,
1998).
The ratio of pEg7: ß-tubulin and XCAP-E: ß-tubulin in the fraction `max G0' (G0, 86.7%; G1, 6.7%; S, 5.9%; G2, 0.8%, M, 0.3%) was even higher than in G1: 1.38 for pEg7 and 1.33 for XCAP-E. This increase apparently correlates with the low level of ß-tubulin brought about by cultivation of cells at low temperature, but in any case, these data show that both pEg7 and XCAP-E are continuously expressed in non-dividing cells.
Taken together, the data suggest that not only SMC proteins, representing core subunits of the 13S-condensin complex, but also some non-SMC subunits are expressed throughout the cell cycle.
The effect of fixation on immunolocalization of pEg7 and XCAP-E
In our previous paper (Cubizolles et
al., 1998), we reported chromosomal localization of pEg7 during
mitosis. With the formaldehyde-glutaraldehyde fixation protocol used in these
experiments, immunostaining with anti-pEg7 antibodies produced only weak
diffuse nuclear labeling in interphase cells. However, under these conditions,
nuclear antigens could be partially masked. In order to test this possibility,
in the current work we used some other fixation protocols for both anti-pEg7
and anti-XCAP-E antibodies. Several antibodies were tested after methanol,
Triton-methanol, Triton-formaldehyde and Triton-glutaraldehyde fixation
protocols (see Materials and Methods). The intensity of mitotic chromosome
staining was used as a reference point for estimation of labeling efficiency.
Since Eg7G and XCAP E1 antibodies gave the best results on mitotic
chromosomes, we used these antibodies for further experiments.
The results of the effect of fixation protocols on pEg7 immunostaining are summarized in Table 2. It appeared that mitotic chromosome staining for pEg7 demonstrates little sensitivity to variations in fixation conditions. Both crosslinking-type and precipitation-type fixatives gave similar results. In contrast, interphase staining was notably different when various protocols were compared.
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While most methanol- or aldehyde-based protocols gave fine granular staining of the cytoplasm and weak diffuse staining of the nucleoplasm, pretreatment of cells with detergent prior to fixation revealed the concentration of antigen in the nucleolus. Introduction of glutaraldehyde in fixation mixture completely abolished nucleolar staining despite detergent pretreatment. The data suggest that the inability to visualize nuclear localization of pEg7 in previous reports may be caused by the inaccessibility of antigen to antibodies. Similar experiments performed using antibodies against XCAP-E (Table 3 and Fig. 3) demonstrate that same tendency. However, in contrast to anti-pEg7, antibodies XCAP-E1 labeled the nucleolus also after the formaldehyde-Triton fixation procedure.
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These results partially explain the inconsistencies between previous
reports, where condensins were immunolocalized to nuclear
(Saitoh et al., 1994;
Schmiesing et al., 2000
) or
cytoplasmic compartments of interphase cells
(Steen et al., 2000
).
pEg7 and XCAP-E are associated with nucleolus in interphase
Comparison of the effects of the fixation protocol on condensin
immunolocalization permitted us to establish the most reliable conditions for
studies of nucleolar localization of pEg7 and XCAP-E in cultured
Xenopus cells. Although some recent data suggested localization of
condensin subunits to the nucleolus, no detailed data on subnucleolar
compartmentalization of these proteins have been presented. However, our
analysis could provide a clue to the possible role of condensin subunits in
the interphase nucleus. In the present study, we addressed the function of the
condensin subunits by performing colocalization of pEg7 and XCAP-E with marker
proteins of subnucleolar domains. We used UBF for labeling fibrillar centers;
fibrillarin for the dense fibrillar component, and B23 for the granular
component [see (Scheer and Hock,
1999; Olson et al.,
2000
) and references therein]. We also studied the colocalization
of condensins with topoisomerase II
. It had been shown previously, in
Drosophila, that topoII interacts with Barren, a homologue of another
member of condensin complex XCAP-H
(Bhat et al., 1996
) and
also partially colocalized with condensins during mitosis. By contrast,
nucleolar localization of topoII has been demonstrated in a number of studies
(Zini et al., 1994
;
Mo, Beck, 1999
;
Christensen et al., 2002
).
The nucleolar labeling by anti-pEg7 antibodies was not homogeneous; rather, the peripheral part of the nucleoli were preferentially stained, leaving the central part unstained. Depending on the orientation of the nucleolus in different cells, single or double cap-like regions could be observed (Fig. 3). This type of labeling was especially visible in cells with large nucleoli. In small nucleoli or in nucleolus-like bodies, which are often seen in some nuclei, homogeneous staining prevailed.
Anti-XCAP-E antibodies also stained the nucleolus (Fig. 4), but, in contrast to anti-pEg7, labeling was more intense and less sensitive to the fixation protocol used. In interphase cells, besides nucleolar staining, bright spots in the cytoplasm and fine granular staining of nucleoplasm were also observed with anti-XCAP-E antibodies (Fig. 4).
Fig. 3C-E shows double staining of interphase XL2 cells with anti-pEg7 and anti-topoII. The distribution of topoII at the nucleolar periphery perfectly matches that of pEg7. Double staining of topoII and XCAP-E also revealed obvious colocalization of these two proteins at the nucleolar surface, although XCAP-E was localized more towards the inside of the nucleolus (Fig. 4D,E).
For studying the ultrastructural localization of pEg7 and XCAP-E proteins in interphase cells, we performed immunogold labeling using affinity-purified antibodies against these proteins, in combination with monoclonal antibodies against topoII. Fig. 3F-H and Fig. 4F-H show typical staining patterns of interphase nuclei. For all three antibodies, the most intense labeling was detected at the nucleolar surface. The nucleoplasmic and cytoplasmic labeling was very weak. These observations are in agreement with our immunofluorescence data, with the only exception that, at the electron microscopic level, localization of all three proteins is restricted to the surface of the nucleolus, whereas immunofluorescent labeling shows deeper penetration into the nucleolus. This is possibly because of some sterical constraints that limit the diffusion of gold-conjugated antibodies into the densely packed nucleolus in the case of a pre-embedded immunogold staining procedure.
Counterstaining with DAPI shows no apparent colocalization of DNA with condensin subunits, indicating that these proteins are localized in the nucleolus itself, rather than in surrounding perinucleolar heterochromatin. In order to localize the condensins in the nucleolus precisely, double immunostaining with antibodies to UBF, fibrillarin and B23 was performed. As shown on Fig. 5, UBF is located in series of small dots that reside outside the condensin-positive zone. XCAP-E and fibrillarin also occupy mutually exclusive regions in the nucleoli (Fig. 6A,B). The same distribution of these proteins was seen in smaller nucleolus-like bodies, where distinct domains occupied by XCAP-E and fibrillarin were also visible (Fig. 6). pEg7 labeling was the same as that of XCAP-E (data not shown). By contrast, the distributions of B23 and XCAPE are practically identical (Fig. 7). These data strongly indicate that both SMC-protein XCAP-E and the non-SMC component of condensin are localized to granular component of the nucleolus. However, some differences in intranucleolar distribution of these proteins suggest the existence of a subpopulation of nucleolar XCAP-E that does not interact with pEg7.
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|
Taken together, these observations indicate that, during interphase, two of the condensin subunits, pEg7 and XCAP-E, are targeted to the nucleolus, where they are localized in the granular component.
The effect of rRNA transcription inhibition on the nucleolar
localization of condensins
In order to address the question of what is the functional meaning of
nucleolar localization of condensins during interphase, we determined the
distribution of pEg7 and XCAP-E in cells treated with actinomycin D at a
concentration of 5 µg/ml for 2, 4 and 6 hours. Under these conditions, the
transcription by RNApol I is completely blocked
(Schofer et al., 1996).
Indeed, morphological changes in nucleoli in actinomycin-D-treated cells,
which result in nucleolar segregation, clearly indicate that rRNA synthesis is
severely affected (Figs
5,6,7,8,9).
After 6 hours of treatment, there was a notable decrease in the nucleolar size
(from 4.5 to 1.8 µm in diameter) as judged by phase contrast microscopy. At
the electron microscopic level, segregation of nucleolar material into two
distinct compartments was clearly seen (data not shown). These segregated
nucleoli were often in close contact with small blocks of condensed chromatin,
which were clearly visible after DAPI staining.
|
|
Immunolocalization of pEg7 and XCAP-E showed that both proteins were found in the segregated nucleoli as homogeneously stained spherical regions. These regions occupy only a part of the segregated nucleolus, since they are smaller than the entire nucleolus. Again, comparison of the localization of pEg7 and XCAP-E and DNA demonstrates that condensins do not reside in condensed perinucleolar chromatin (Figs 5,6,7,8,9). Double staining for XCAP-E and UBF and fibrillarin does not show any colocalization of these proteins (Fig. 5). As in control nucleoli, these proteins occupy distinct spatially separated nucleolar domains. By contrast, B23 staining displays perfect overlap with XCAP-E in segregated nucleoli (Fig. 7).
After 6 hours of treatment with actinomycin D, the size of the nucleolar compartment containing these proteins decreased several-fold, on average, in treated cells, compared with control untreated cells. The local concentration of pEg7 and XCAP-E did not change significantly. Thus, the overall decrease in the nucleolar level of pEg7 and XCAP-E occurs under these conditions.
In actinomycin-D-treated cells, nucleolus-like bodies also decrease in size, but, in contrast to large nucleoli, fibrillarin staining is undetectable, whereas XCAP-E remains detectable (Fig. 6).
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Discussion |
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Condensin function during interphase is not so obvious. Taking into account
the fact that intracellular levels of condensin subunits remain practically
constant throughout the cell cycle [this study; (Schmeising et al., 1998;
Cabello et al., 2001)], one
should expect condensins to exert some yet unknown function(s) during
interphase. It is a plausible hypothesis that condensins act as regulators of
gene expression by controlling the accessibility of chromatin loci to
transcription factors via local condensation/decondensation of interphase
chromosomes. The only example, so far, of dual function of SMC proteins in
mitosis and interphase is the participation of C. elegans protein
MIX-1 in both mitotic chromosome compaction and dosage compensation of the X
chromosome in XX hermaphrodite worms (Lieb
et al., 1998
). Recently it was shown that Cnd2, a non-SMC subunit
of fission yeast condensin, the analog Drosophila Barren
(Lavoie et al., 2000
), is
required for mitotic chromatin condensation and is important for correct DNA
reparation in interphase (Aono et al.,
2002
). Thus, the participation of condensin subunits in some
related activities in interphase cannot be ruled out.
It is generally agreed that condensins are nuclear proteins, but some
controversial observations were made concerning their subnuclear localization.
Most authors demonstrated diffuse distribution of SMC proteins throughout the
nucleus (Saitoh et al., 1994;
Hirano and Mitchison, 1994
),
whereas others reported concentration of these proteins in discrete subnuclear
domains of an unknown nature (Schmiesing
et al., 1998
). Recently, nucleolar localization of the human
condensins hCAP-H has been reported
(Cabello et al., 1999
;
Cabello et al., 2001
). Similar
results were obtained for S. cerevisiae, where GFP-tagged pSmc4 was
shown to accumulate in the nucleolus prior to entry into mitosis
(Freeman et al., 2000
).
In the present study, we demonstrate, for the first time, a nucleolar
localization of both a SMC protein (XCAP-E) and a non-SMC member of the X.
laevis condensin complex (pEg7) throughout interphase. In our previous
work (Cubizolles et al.,
1998), a diffuse signal was detected in the interphase nucleus
when using more robust formaldehyde-glutaraldehyde fixation. Careful
comparison of various fixation protocols showed that interphase staining is
indeed very sensitive to fixation procedure (Tables
2 and
3). So, it could be
hypothesized that the differences in interphase localization of condensins,
reported by other authors, is based on variations of antigen preservation
and/or accessibility, depending on the protocol of fixation. This explanation
seems most acceptable when comparing our data on XCAP-E localization with
those of Hirano and Mitchison (Hirano and
Mitchison, 1994
) and for its ortholog proteins hCAP-E (Schmeising
et al., 1998) and ScII (Saitoh et al.,
1994
). The same holds true for the immunolocalization of pEg7 or
its homologs (Cubizolles et al., 1997;
Steen et al., 2000
;
Schmiesing et al., 2000
).
However, immunoblot analysis of subcellular fractions indicated that human
orthologs of XCAP-E and pEg7 are predominantly cytoplasmic during interphase
(Schmiesing et al., 2000
;
Steen et al., 2000
). This
inconsistency may be explained by the `leakiness' of nucleolar condensins
(Saitoh et al., 1994
), which
exit the nucleus during fractionation procedure.
The functions, if any, of condensins in the nucleolus during interphase
remain unclear. As was shown previously by many authors, the 13S condensin
complex and its core subunits SMC proteins display DNA-binding
activity in vitro (Akhmedov et al.,
1998; Sutani and Yanagida,
1997
; Kimura and Hirano,
1997
) and interaction with condensing chromosomes both in vivo and
in vitro (Hirano and Mitchison,
1994
; Freemen et al., 2000). Therefore, in agreement with the
generally accepted hypothesis, it may be speculated that condensin subunits
bind to perinucleolar or intranucleolar chromatin. In the X. laevis
nucleolus, two related explanations can be proposed. On the one hand, in
X. laevis cells about 500 rRNA cistrons per haploid set
(Wallace and Birnstiel, 1966
)
are located on the short arm of chromosome 12
(Kahn, 1962
), close to the
centromere (Graf and Kobel,
1991
), so that during interphase, the centromeric region of
chromosome 12 appears in close proximity to the nucleolus. Non-random
association of centromeres with the nucleolus has also demonstrated in other
cell types (Stahl et al.,
1976
; Cerda et al.,
1999
; Chou and DeBoni, 1996;
Haaf and Schmid, 1989
;
Lee et al., 1999
;
Leger et al., 1994
;
Park and DeBoni, 1992
). These
associations were thought to be important for spatial arrangement of the
genome or regulation of rDNA transcription. On the other hand, during mitosis,
accumulation of condensins in the centromeric regions was detected. Since
blocks of heterochromatin maintain their highly compacted state throughout the
cell cycle, one should expect condensins to keep interacting with these loci
during interphase. However, such interaction has not been clearly demonstrated
so far.
Alternatively, the nucleolar localization of condensins could reflect a
direct and specific interaction with rDNA in interphase. In this case,
condensin-driven compaction of ribosomal gene loci might serve as a mechanism
of controlling rRNA synthesis at the level of higher-order chromatin
structure. In situ hybridization with specific rDNA probes often demonstrates
the presence of rDNA in clumps of nucleolus-associated chromatin both at
microscopic and ultrastructural levels
(Thiry et al., 1988;
Thiry and Thiry-Blaise, 1991
;
Kaplan et al., 1993
). The
retraction of rDNA out of the nucleolus into perinucleolar chromatin upon
inhibition of transcription by actinomycin D
(Schofer et al., 1996
)
confirms the idea that the balance between compacted (perinucleolar) and
extended (intranucleolar) rDNA is correlated with the level of rRNA synthesis.
Strong correlation between condensin and topoII localization, observed in
nucleoli, implies the cooperative action of these proteins in
transcription-related reconfiguration of the rDNA template
(Kimura et al., 1999
).
However, treatment with teniposide (stabilizing covalent catalytic DNA
intermediates of topoII) causes relocation of topoII (both
and ß)
from the nucleoli to nucleoplasmic granules
(Christensen et al., 2002
),
thus indicating that nucleolar subpopulation of this proteins seems to be
catalytically inactive.
In the present work no colocalization of XCAP-E and pEg7 with DNA was
detected during interphase. The lack of colocalization was especially clear
after actinomycin treatment, when nucleolar DNA and XCAP-E occupied different
non-overlapping domains. Moreover, XCAP-E is distributed throughout the
granular component of nucleolus, where little or no DNA was detected
(Thiry and Thiry-Blaise, 1989;
Derenzini et al., 1990
;
Wachtler et al., 1992
;
Jimenez-Garcia et al.,
1993
).
Recent findings on the essential role of condensins in rDNA segregation
during mitosis in S. cerevisiae
(Freeman et al., 2000) provide
another clue to the function of condensins in the nucleolus. The authors
demonstrated accumulation of condensins in the nucleolus at the G2/M
transition and blocked segregation of rDNA loci in smc2 and
smc4 mutants. These observations were considered as evidence for a
special role of condensins in proper segregation and/or folding up the arrays
of repetitive DNA. This idea is further supported by the apparent
concentration of condensins in centromeric and telomeric regions of mitotic
chromosomes. In Xenopus cells, however, nucleolar localization of
condensins is cell cycle independent and does not correspond to localization
of rDNA during interphase, which is indicative of some additional function for
condensin subunits. Although the existence of a minor subfraction of
condensins bound to nucleolar DNA cannot be completely ruled out, nucleolar
localization of the majority of XCAP-E and pEg7 cannot be explained
exclusively by their association with DNA.
Another possible role of condensins in the nucleolus might be related to
rRNA synthesis and/or processing. Localization of condensins in the granular
component and their behavior upon the inhibition of transcription by
actinomycin D indicate that condensins could be associated with released rRNA
transcripts. This association is apparently maintained in hypotonically
treated cells, where the granular component is redistributed to aggregates of
RNP particles containing nucleolar proteins scattered throughout the
nucleoplasm (Kireev et al.,
1988; Dudnik and Zatsepina,
1995
; Zatsepina et al.,
1997
). Under the same conditions, XCAP-E displays dynamics similar
to that of B23 (Timirbulatova et al.,
2002
). The mechanisms of condensin action in nucleolar function
remain largely unknown. Since heterodimers of cut3/cut14 (S. pombe homologs of
XCAP-E and XCAP-C) possess a DNA-renaturing activity
(Sutani and Yanagida, 1997
),
it seems possible that condensins (or the XCAP-E/C dimer only) facilitates the
process of adoption of specific secondary structure in rRNA.
Condensins may also play a scaffolding role in spatial organization of the
nucleolus. Structural motifs of SMC protein rod domain, reminiscent of those
in intermediate filament proteins, may favor cooperative self-association and
filament formation. This speculative mechanism of condensin-driven chromosome
compaction (Strunnikov, 1998)
may also take place during interphase.
![]() |
Acknowledgments |
---|
We are grateful to O. Zatsepina (Moscow State University), G. Giudice
(University of Palermo) and M. Bellini (University of Illinois at
Urbana-Champaign) for helpful discussion and critical reading of the
manuscript. We also gratefully acknowledge the generous gifts of polyclonal
anti-topoII antibodies from D. F. Bogenhagen (State University of New
York, Stony Brook, USA), monoclonal anti-Pleurodeles topoII antibodies from R.
Hock (University of Wurzburg, Germany), human autoimmune sera to fibrillarin
and UBF fromD. Hernandez-Verdun (Institut Jacques Monod, Paris, France) and
XL2 cells from J. Tata (Mill Hill-NIMR Laboratory, London). We also give
thanks to V. V. Kruglyakov for the invaluable help with the electron
microscopy and to S. A. Grabeklis for technical assistance. This work was
supported by ARC grant 9808 and RFBR grants 99-04-49128 and 01-04-49287.
![]() |
Footnotes |
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References |
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