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INTRODUCTION |
Accurate chromosome segregation during mitosis into two daughter
cells is one of the requirements for stable genome maintenance. Until
recently, the molecular mechanisms regulating chromatid cohesion had
not been well understood. However, recent studies, mostly conducted in
budding yeast, have outlined the process generally (reviewed in Ref.
1). Concurrent with DNA replication, the sister chromatids become
connected along their entire length. Several distinct groups of
proteins are involved in establishing and maintaining chromatid
cohesion. The most intensively investigated protein complex, budding
yeast cohesin, consists of four subunit proteins, Scc1p/Mcd1p, Scc3p,
Smc1p, and Smc3p (2-4), and serves as a physical glue between sister
chromatids. Cohesin is phylogenetically conserved. Rad21p in fission
yeast and human Rad21 (hRad21) are homologues of Scc1p/Mcd1p (5, 6).
Smc1p and Smc3p are found in budding yeast, Xenopus, and
mammals (7-9). They are members of the SMC (structural
maintenance of chromosome) protein family that
is characterized by the presence of coiled-coil domains and ATPase
domains (reviewed in Ref. 10). Very recently, two Scc3p homologues, SA1
and SA2, have been found in Xenopus and human cells (11).
Interestingly, in Xenopus, two distinct classes of cohesin,
termed x-cohesinSA1 and x-cohesinSA2, are
present. These two complexes share Xenopus (X)SCC1, XSCC2, and XRAD21 and differ via containing either XSA1 or XSA2 (11). Immunodepletion of Xenopus cohesin from egg extracts led to
a failure of chromatid cohesion (8). Therefore, the cohesin complex is
likely to be conserved in all eukaryotes, including humans.
However, the precise roles of cohesin may be different between budding
yeast and other eukaryotes. In budding yeast, Scc1p/Mcd1p abruptly
dissociates from chromatin at the onset of anaphase (2, 4). In
contrast, ~95% of Xenopus cohesion molecules (XSMC1, XSMC3, and XRAD21) dissociate from chromatin at the entry of mitosis, much earlier than the metaphase-anaphase transition (8). Similarly, in
indirect immunofluorescence
(IF)1 experiments, it has
been found that human Smc1p and mouse Rad21 (called PW29) are mostly
excluded from mitotic chromosomes (9, 12). Therefore, most cohesins are
apparently absent on metaphase chromatids in higher eukaryotes. It is
not known what molecules or conditions are responsible for the
chromatid cohesion immediately before anaphase. Two models have been
proposed to explain the apparent inconsistency between the timings of
the cohesin-chromatin dissociation and the mitotic chromatid separation
(8). The first model proposes that cohesin molecules are responsible
for interphase-specific chromatid cohesion and that some yet
unidentified mitosis-specific cohesion machinery is responsible for
the chromatid cohesion from prophase until the onset of anaphase. The
second model hypothesizes that the same cohesin complex is required for both interphase- and mitosis-specific chromatid cohesions. However, in
this model, the complex dissociates from chromatin in two steps, whereby most cohesin is released from chromatin at the entry into mitosis. The remaining cohesin connects chromatids in metaphase and
dissociates from chromatin at the onset of anaphase. The recent discovery of Xenopus (X)SA proteins revealed that a small
population of XSA1 is associated with the metaphase chromosomes formed
in Xenopus cell-free extracts, thus supporting the second
model (11).
In this paper, we describe the biochemical and cytological behaviors of
hRad21. We aimed to better understand the roles of hRad21 particularly
in metaphase. We observed a small but significant population of hRad21
associated with colcemid-induced mitotic chromosomes. These results
suggest that the mitotic cohesion is mediated by cohesin, further
underscoring the conserved mechanisms regulating chromatid cohesion and
separation in eukaryotes.
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EXPERIMENTAL PROCEDURES |
Cloning of hRad21 cDNA and Preparation of Anti-hRad21
Antibodies--
Full-length hRad21 cDNA was isolated from a HeLa
cell cDNA library by reference to the published hRad21 cDNA
sequence (6). The cloned cDNA was completely sequenced. The
resulting sequence was identical to the published clone. The
full-length hRad21 cDNA was then subcloned into pGEX5X-1 (Amersham
Pharmacia Biotech). glutathione S-transferase-fused hRad21
was expressed in Escherichia coli. The recombinant protein
contained in the inclusion body was denatured and purified using Prep
Cell Model 491 (Bio-Rad). The purified protein was mixed with Freund's
complete adjuvant and injected into rabbits to obtain the anti-hRad21
antisera. To generate the anti-C-hRad21 antibodies, an oligopeptide
possessing a C-terminal amino acid sequence of hRad21 (QQAIELTQEEPYSD,
amino acids 606-619) was conjugated with keyhole limpet hemocyanine and was used to immunize the rabbits. The resulting antibodies were
purified by affinity chromatography. Mouse anti-
-tubulin monoclonal
antibody was purchased from Sigma. Mouse anti-PCNA, anti-lamin B, and
anti-cyclin B1 monoclonal antibodies were purchased from Santa Cruz
Biotechnology. Anti-CENP-B antibody was a kind gift from Dr. H. Masumoto (Nagoya University).
Western Blotting--
Western blotting was performed according
to Ref. 13. Briefly, membranes were first pretreated for 1 h in
Block-Ace solution (Dai Nippon Pharmaceuticals). Then all subsequent
incubations and washes were carried out in 1× TNT buffer (20× TNT:
0.4 M Tris-HCl, 2.8 M NaCl, 1.0% Tween 20).
Membranes were incubated for 1 h with primary antibodies at room
temperature, followed by three washes. Then membranes were incubated
for 30 min with horseradish peroxidase-conjugated anti-rabbit
antibodies (Amersham Pharmacia Biotech), followed by three washes.
Signals were detected using an ECL kit (Amersham Pharmacia Biotech).
In Vitro Transcription and Translation--
In vitro
transcription and translation experiments were performed using a TnT
Quick Coupled Transcription/Translation System (Promega). 1 µg of
template plasmid DNA was used in each reaction. For autoradiography,
proteins were labeled by addition of [35S]methionine
(1000 Ci/mmol at 10 mCi/ml; Amersham Pharmacia Biotech) to the reaction
mixture. For the Western analyses, cold methionine (0.02 mM) was used during synthesis of the protein.
Indirect Immunofluorescence Experiments--
Cells were grown on
coverslips. Two fixation protocols were used, producing essentially
similar results. In the first protocol, cells were fixed with 100%
methanol for 20 min at
20 °C. In the second protocol, cells were
fixed with 4% paraformaldehyde at room temperature for 10 min, and
then permeabilized with 0.1% Triton X-100 for 10 min. Fixed cells were
pretreated with PBS containing 0.1% bovine serum albumin and
0.1% skim milk and then incubated with primary antibodies for 1.0 h at 37 °C. Cells were washed by PBS three times and incubated with
secondary antibodies for 1 h at 37 °C. Coverslips were washed
as above, mounted in mounting solution containing 0.25 µg/ml
propidium iodide (PI) or TOTO3, and examined by laser confocal
microscopy (Zeiss).
Cell Cycle Analyses and Metabolic Labeling--
HeLa cells were
synchronized at early S phase by a thymidine and aphidicolin
double-block protocol as described in (14). In the
32P-metabolic labeling experiments, cells were incubated in
a phosphate-free Dulbecco's modified Eagle's medium supplemented with
[32P]orthophosphate to a final concentration of 0.5 mCi/ml for 1 h prior to harvesting. Cells were lysed by the
modified RIPA buffer. Cell lysates were pretreated with normal rabbit
IgG. Then the precleared cell lysates were incubated with primary
antibodies, and the antigen-antibody complex was immunoprecipitated by
protein A-Sepharose (Amersham Pharmacia Biotech). Obtained proteins
were fractionated on 6% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. The membrane was first subjected to
autoradiography. Then the same membrane was analyzed for hRad21 by
Western blotting using anti-yhRad21 antibodies. The signals were
detected by a peroxidase immunostaining kit (Wako).
Cell Fractionation Analyses--
1 × 107 HeLa
cells were washed with cold PBS, and collected. Cell pellets were
suspended in five volumes of hypotonic buffer (10 mM HEPES,
pH 7.5, 5 mM KCl, 1.5 mM MgCl2, and
1 mM dithiothreitol). Low speed cell pellets were
resuspended in original cell volumes of hypotonic buffer and
homogenized with a Dounce homogenizer using the tight pestle. Cells
were examined with a light microscope to confirm that most cells had
been disrupted. Cytoplasmic and nuclear fractions were separated by
centrifugation at 1,000 × g. Cytosolic fractions were
obtained from the cytoplasmic fractions following high speed
centrifugation at 15,000 × g. Nuclear pellet fractions
were washed twice with the hypotonic buffer and extracted by suspending
the nuclei in hypotonic buffer containing either salt or detergents for
30 min on ice. Nuclear extract was separated from insoluble materials
by high speed centrifugation. DNase I digestion of the crude nuclei was
done according to Ref. 15.
Mitotic Spread Chromosome Analyses--
HeLa cells were
synchronized at early S phase following a thymidine and aphidicolin
double-block protocol. To obtain metaphase-like chromosomes, cells were
released from the block and cultured for 8 h, followed by an
additional culture for 3 h in the presence of 0.5 µg/ml
colcemid. To obtain prometaphase-like chromosomes, cells were released
from the block and cultured for 8.5 or 9 h, followed by an
additional culture for 10 min in the presence of 0.5 µg/ml colcemid.
The cells were harvested, treated with a hypotonic buffer (10 mM Tris, 10 mM NaCl, 5 mM
MgCl2) for 15 min, and attached to a glass slides by
Cytospin. The cells were fixed using cold methanol for 20 min,
permeabilized with 0.1% Triton X-100 for 10 min, pretreated by
blocking solution (0.1% bovine serum albumin and 0.1% skimmilk in
PBS), and finally incubated with rabbit anti-hRad21 antibodies and
mouse anti-CENP-B monoclonal antibodies. These antibodies were detected
by Alexia Fluor 488-conjugated anti-rabbit Ig antibodies (Molecular
Probes) and Cy3-conjugated anti-mouse Ig antibodies (Amersham Pharmacia
Biotech), respectively. DNA was stained with TOTO3 (Molecular Probes).
Images were captured by a lazer confocal microscope (Zeiss).
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RESULTS |
hRad21 Protein Does Not Change Its Abundance during Cell
Cycle--
Budding yeast Scc1p/Mcd1p is a nuclear protein that
associates with chromatin from late G1 through the metaphase-anaphase transition (2, 3). Scc1p/Mcd1p abundance is strictly regulated in a
cell cycle-dependent manner. The protein is absent in early G1, accumulates in S, G2, and metaphase, and
declines in anaphase (2, 3). In addition, SCC1/MCD1 mRNA
is absent in early G1 and most abundant in late
G1/S (2, 3). hRad21 mRNA is most abundant in late S
through G2 (6). Therefore, we first investigated hRad21
protein levels throughout the cell cycle.
Full-length human Rad21 (hRad21) cDNA was isolated from a HeLa cell
cDNA library. Nucleotide sequencing of the obtained cDNA yielded a sequence of complete identity to the published one (6), thus
having an ORF that potentially encodes a protein of 631 amino acid
residues with a calculated molecular mass of 72 kDa.
Two different rabbit antisera were raised, one (anti-hRad21 antibody)
against the glutathione S-transferase-fused recombinant full-length hRad21 protein expressed in E. coli and the
other (anti-C-hRad21 antibody) against a C-terminal synthetic
oligopeptide (see "Experimental Procedures"). These antibodies were
purified by affinity chromatography using the respective cognate
antigens. Following hRad21 cDNA transfection both antibodies
recognized the recombinant hRad21 protein that was expressed in the
293T cells (human kidney cells transfected with SV40 T antigen) (Fig. 1A). Both antibodies also
recognized an endogenous protein in untransfected 293T cells that
showed the same mobility in SDS-PAGE with that of the overexpressed
hRad21 (Fig. 1B). These reactions were specific because
preincubating the antibodies with the antigens prior to Western
blotting prevented detection of hRad21 (Fig. 1, A and
B).

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Fig. 1.
Specificities of anti-hRad21 antibodies used
in this study. A, anti-hRad21 and anti-C-hRad21 antibodies
were raised in rabbits, using the full-length hRad21 protein and a
C-terminal oligopeptide as antigens, respectively. After the antibodies
were affinity-purified, the extract derived from 293T cells that had
been transfected with the hRad21 expression vector, was examined by
Western with (+) or without ( ) preabsorption of the antibodies with
the cognate antigen. B, anti-hRad21 and anti-C-hRad21
antibodies recognize the endogenous hRad21 protein derived from
untransfected 293T cells. 293T whole cell extracts were analyzed by
Western blotting with (+) or without ( ) preabsorption of the
antibodies to the cognate antigens. C, anti-hRad21 and
anti-C-hRad21 antibodies specifically recognize the recombinant hRad21
protein synthesized in rabbit reticulocyte lysates. Protein synthesis
in rabbit reticulocyte lysates was done in the absence of exogeneous
DNA ( ) or in the presence of pcDNA3 (Mock) or the
full-length hRad21 cDNA expression vector (hRad21).
Proteins synthesized in the presence of cold methionine were analyzed
by Western blotting with anti-hRad21 and anti-C-hRad21 antibodies
(left and middle panels). Proteins synthesized in
the presence of [35S]methionine were separated by
SDS-PAGE and visualized by autoradiography (right
panel).
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The apparent molecular mass of the hRad21 protein (about 120 kDa), as estimated using SDS-PAGE electrophoresis, was greater than
that predicted from its supposed amino acid sequence. Similar observations were made of budding yeast Scc1p (2), fission yeast rad21
protein (16), and Xenopus Rad21 homologue, XRAD21 (8). To
confirm that the 120-kDa band indeed represents hRad21 protein, we
transcribed and translated hRad21 cDNA in vitro using a
rabbit reticulocyte lysate system. Recombinant proteins were synthesized in the presence of either [35S]methionine or
cold methionine and then analyzed by SDS-PAGE. Labeled proteins were
detected by autoradiography, whereas unlabeled proteins were subjected
to Western blotting with anti-hRad21 and anti-C-hRad21 antibodies. As
shown in Fig. 1C, the [35S]methionine-labeled
hRad21 protein synthesized in vitro was detected as a
120-kDa protein band after SDS-PAGE electrophoretic migration. Because
this 120-kDa protein was specifically recognized by both anti-hRad21
and anti-C-hRad21 antibodies (Fig. 1C), we concluded that it
is indeed hRad21 protein.
HeLa cells were synchronized using a thymidine and aphidicolin
double-block protocol (Fig. 2). The cells
were harvested at intervals after release from the block and first
extracted by 1% Triton X (Fractions T), followed by 0.5 M
NaCl (Fractions N). The insoluble pellet fractions (Fractions P) were
also examined. These fractions were analyzed using Western blotting.
-Tubulin served as the control cytoplasmic protein and was
efficiently extracted to the T fractions, indicating that soluble
proteins were successfully extracted with the Triton X-100 treatment
(Fig. 2B). It is known that PCNA is insoluble during S phase
(17). PCNA was detected in Fractions P most strongly at 0-4 h and to a
lesser level at 8 h (Fig. 2C), indicating that most of
the cells were in S phase at 0-4 h. In contrast, Cyclin B1 was most
abundant at 8 and 10 h and suddenly disappeared at 12 and 14 h (Fig. 2B). These results indicate that the cells were in
G2/M phase at 8 to 10 h and exited from M phase at
12-14 h. FACscan analysis strongly supported the interpretation of the
cell cycle progression inferred from the marker proteins (Fig.
2A). In these cells, the abundance of hRad21, as detected by
anti-C-hRad21 antibody, remained essentially unchanged during the cell
cycle. It is known that Scc1p/Mcd1p is proteolytically cleaved at
the onset of anaphase (18). We reproducibly observed some more quickly
migrating proteins in hRad21 Western blots. At this moment, we do not
know whether these fragments were derived from hRad21 or whether they
had any physiological relevance. However, we did not see an increase of
these small fragments in M phase, suggesting that they were not related
to the chromatid separation event.

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Fig. 2.
hRad21 protein levels do not change during
cell cycle. HeLa cells were synchronized at early S phase
following a thymidine and aphidicolin double-block protocol. The cells
were harvested at intervals after release from the block. A,
FACscan analysis. The cells were stained with PI, and the DNA contents
were analyzed by FACscan. B, Western blotting analyses for
hRad21, cyclin B1, and -tubulin. The cells harvested from the
synchronized culture (indicated by the harvest time after block
release), as well as an asynchronized exponentially growing culture
(Expo), were first extracted with 1% Triton X-100
(T) and then with 0.5 M NaCl (N). The
proteins were run in a SDS-PAGE gel and analyzed in Western blotting
experiments using anti-C-hRad21, anti-cyclin B1, and anti- tubulin
antibodies. Cell cycle stages deduced from A and
C, and the cyclin B1 abundance are shown above.
C, the insoluble fractions (P) were analyzed for
PCNA by Western blotting with anti-PCNA antibodies.
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We found that hRad21 was adequately extracted in 0.5 M NaCl
but not in 1% Triton X-100 at all examined stages of the cell cycle
(Fig. 2B). We also found that hRad21 was not present in Fractions P (data not shown). Furthermore, hRad21 was not extracted by
DNase I (data not shown). These results indicate that hRad21 is not a
soluble protein or a protein loosely binding to chromatin. hRad21 may
be associated with nuclear structures at all cell cycle stages
including M phase.
hRad 21 Is Hyperphosphorylated in M Phase--
It has been shown
that fission yeast Rad21p and budding yeast Scc1p are phosphorylated
from S phase to anaphase (16). We next examined whether hRad21 is
phosphorylated in a cell cycle-dependent manner. For this
purpose, HeLa cells were synchronized following a thymidine and
aphidicolin double-block protocol. The cells were harvested at
intervals after being released from the block. Prior to each harvest,
the cells were cultured for 1 h in the presence of
[32P]orthophosphate. A parallel culture was used for
determining the cell cycle by FACscan analysis. hRad21 was
immunoprecipitated using anti-hRad21 antibodies from cell extracts
containing approximately the same number of cells. The
immunoprecipitates were separated in an SDS-PAGE gel, and the proteins
were blotted onto a membrane. First, the membrane was subjected to
autoradiography (Fig. 3B). Then the same membrane was analyzed for total hRad21 levels using a
Western analysis with anti-C-hRad21 antibodies (Fig. 3C).
From these experiments, it was found that hRad21 is phosphorylated most
intensely at 10, 12, and 14 h after release from the block. Because the FACscan analysis revealed that these samples were derived
mostly from M phase cells, these results imply that hRad21 becomes
hyperphosphorylated in M phase.

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Fig. 3.
hRad21 and associated proteins are
hyperphosphorylated in M phase. HeLa cells were synchronized at
early S phase following a thymidine and aphidicolin double-block
protocol, and the cells were harvested at intervals after release from
the block. The cells were cultured for 1 h in the presence of
[32P]orthophosphate prior to each harvest. A,
FACscan analysis. Synchronized cells (indicated by harvest times) and
exponentially growing cells (Expo) were stained with PI, and
the DNA contents were analyzed by FACscan. Because the samples were
fixed at different times, absolute values for the PI signals are not
consistent between the samples. B, autoradiography of
labeled proteins. Harvests containing approximately the same number of
synchronized and exponentially growing (Expo) cells were
subjected to immunoprecipitation either with anti-hRad21 antibodies
(I) or preimmune normal rabbit immunoglobulin
(P). The immunoprecipitates were fractionated in an SDS-PAGE
gel and blotted onto a membrane. The membrane was exposed to an x-ray
film to visualize the labeled proteins. The position of hRad21 as
identified by Western blotting in C is shown. The positions
of four additional labeled proteins, p150, p140, p95, and p80, are also
indicated. C, Western blotting of hRad21 with the same
membrane analyzed as in B.
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To test the hypothesis that hRad21 is hyperphosphorylated in M phase
more vigorously, we repeated the experiment in a quantitative manner.
HeLa cells were arrested at G1/S following a thymidine and
aphidicolin double-block protocol. A portion of the cells were released
from the block and cultured for 8 h and then arrested at the
following metaphase by incubating the cells with colcemid for 4 h.
These G1/S-arrested and metaphase-arrested cells, along with the control exponentially growing cells, were labeled with [32P]orthophosphate for 2 h prior to each harvest.
FACscan analysis of parallel cultures indicated that the two
populations of cells were indeed arrested at G1/S and
metaphase, respectively (Fig. 4A). hRad21 was
immunoprecipitated from the exponentially growing, G1/S-arrested and metaphase-arrested cell lysates using
anti-hRad21 antibodies. Three samples containing different protein
amounts (1×, 3×, and 9×; where 3× and 9× samples contained 3- and
9-fold more protein than 1× samples, respectively) were analyzed for each type of immunoprecipitate using SDS-PAGE and blotted onto a
membrane. The membrane was subsequently subjected to autoradiography (Fig. 4B), followed by Western blotting with anti-hRad21
antibodies (Fig. 4C). When the anti-hRad21-positive signals
in the Western analysis were quantified, the 3× and 9× samples showed
an almost 3-fold difference in their signal intensities, whereas the
1× samples were undetectable. This apparently linear dose-response relationship observed in the Western blotting data for 3× and 9×
samples indicates that the intensities reliably reflect protein abundance in this range of protein amounts. In contrast,
autoradiography intensities of the 9× samples displayed more than a
3-fold intensity difference relative to the 3× samples and therefore
seemed to lack a linear dose-response relationship. Nevertheless, when
we calculated relative phosphorylation levels by dividing the
autoradiography intensities by the Western blot intensities, we saw
consistent cell cycle-specific changes in hRad21 phosphorylation levels
both among the 3× samples and among the 9× samples (Fig.
4D). In both comparisons, the colcemid-arrested cells showed
3-fold increases in the specific phosphorylation levels of hRad21
compared with the G1/S-arrested cells. Therefore, we
concluded that hRad21 is hyperphosphorylated in M phase.

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Fig. 4.
hRad21 and its putative associated
proteins are hyperphosphorylated in M phase. A,
exponentially growing (Expo), G1/S-arrested
(G1/S), and colcemid-arrested (Meta) HeLa cells
were analyzed for their DNA contents by FACscan. B, extracts
containing the same 32P counts of cells were subjected to
immunoprecipitation with either anti-hRad21 antibodies (1×, 3×, and
9×), or with preimmune normal rabbit immunoglobulin (P).
Three different amounts of protein (1×, 3×, and 9×) were analyzed
for each type of immunoprecipitates. Immunoprecipitates were separated
by SDS-PAGE and blotted onto a membrane. The membrane was then exposed
to an x-ray film to visualize the labeled proteins. The position of
hRad21, as identified by Western blotting in C, as well as
the positions of six additional proteins (p180, p150, p140, p95, p85,
and p80) are indicated. Relative signal intensities of labeled hRad21
are shown at the bottom of the figure. Values were
normalized such that the value of the 3× exponentially growing cells
equaled one unit. C, Western blotting of hRad21 with the
same membrane that was analyzed in B. Relative signal
intensities of the total hRad21 are shown at the bottom of
the figure. Values were normalized to the 3× exponentially growing
cells. The slight discrepancy in the positioning of hRad21 from
left to right in the gel was caused by a
horizontal difference in protein migration rates in the gel.
D, specific labeling efficiencies of hRad21. Intensities of
the labeled hRad21 protein observed in 3× and 9× samples of
B were divided by the total hRad21 intensities observed in
the 3× and 9× samples of C. Calculated values were
normalized to the exponentially growing cells in the 9× sample.
Expo, exponentially growing cells; G1/S,
G1/S-arrested cells; M, metaphase-arrested
cells.
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In the autoradiographs of interphase immunoprecipitates, we observed
three major and several minor 32P-labeled bands additional
to hRad21 (Figs. 3B and 4B). The apparent molecular masses of the major bands as determined from SDS-PAGE were
about 150, 140, and 80 kDa (Fig. 3B). Because these three bands were not reactive with anti-hRad21 antibodies in the Western blotting analysis, we concluded that these three bands are not hRad21.
We interpret them as being hRad21-associated phosphorylated proteins
and have designated them p150, p140, and p80. Interestingly, the
phosphorylation levels of hRad21 and its putative associated proteins
(p150, p140, and p80) in the metaphase-arrested cells were
significantly higher than those found with the interphase cells (Figs.
3B and 4B). Furthermore, additional
phosphorylated proteins, with apparent molecular masses of 180, 95, and
85 kDa, were specifically found in the hRad21 immunoprecipitate derived from the metaphase-arrested cells. Again, these three proteins were
interpreted as putative hRad21-associated proteins and designated p180,
p95, and p85. Although we do not know the identity of these bands, the
results suggest that cohesin components may be coordinately modified
and/or regulated by phosphorylation during M phase.
hRad21 Is a Nuclear Protein Mostly Excluded from Chromosomes in
Mitosis--
We next examined the subcellular localization of hRad21
in the asynchronous HeLa cells by IF experiments using anti-hRad21 antibodies. HeLa cells were fixed with methanol, with or without being
permeabilized in 0.1% Triton X-100. The samples were stained with
TOTO3 to stain the DNA and examined by IF using anti-hRad21 and
anti-
-tubulin antibodies. hRad21 was detected in interphase nuclei
and was not present in nucleoli (Fig. 5).
When the cells were pretreated with Triton X-100 prior to fixation,
most of the
-tubulin was extracted to disappearance, whereas hRad21
remained. These results confirm the biochemical results described above and indicate that hRad21 is a nuclear protein associated with nuclear
structures.

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Fig. 5.
hRad21 is a nuclear protein associated with
nuclear strucutres. Asynchronous HeLa cells were stained with
TOTO3 (red) and analyzed by IF using anti-hRad21
(green) and anti- -tubulin antibodies
(blue). Before cold methanol fixation, cells were
pretreated (+) or not ( ) with 0.1% Triton X-100.
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If hRad21 is involved in sister chromatid cohesion, as has been
proposed, one would expect the subcellular localization of hRad21 to
change in M phase. However, it has been reported that most
Xenopus Rad21 is dissociated from prophase-like chromosomes (8). We therefore were interested in the intracellular distribution of
hRad21 in metaphase cells. Two fixation methods, one employing cold
methanol and the other involving paraformaldehyde (see "Experimental Procedures"), were used. We found these two protocols essentially gave rise to the same results, and the results obtained from
methanol-fixed cells are shown in Fig. 6
and described below. Asynchronous HeLa cells stained with DNA dye TOTO3
were examined by IF using anti-
-tubulin and anti-hRad21 antibodies
(Fig. 6). Although some hRad21 seemed to be associated with chromatin
during early prophase when the two centrosomes are not yet separated, a
significant amount was already dissociated from chromatin (Fig. 6,
row a). hRad21 was quite heterogeneously distributed in late
prophase cells, with some fraction of it still apparently bound to
chromatin (Fig. 6, row b). However, in metaphase and
anaphase, it seemed that most hRad21 had completely dissociated from
the chromatin and was associated with the spindles (Fig. 6, rows
c-e). The microtubule-hRad21 association was particularly
obvious in telophase and cytokinesis, because part of the hRad21 was
colocalized at midbody. hRad21-chromatin association resumed during
cytokinesis and completed in nascent two daughter cells.

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Fig. 6.
hRad21 localization in M phase.
Asynchronous HeLa cells were stained with DNA dye TOTO3
(red) and examined by IF using rabbit anti-hRad21
(green) and mouse monoclonal anti- -tubulin antibodies
(purple). a, prophase; b,
prometaphase; c, metaphase; d, anaphase A;
e, anaphase B; f, telophase; g,
cytokinesis; h, nascent cells. The scale bar
shown in h represents 10 µm.
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To understand the relationship of the nuclear envelope breakdown and
hRad21 dissociation from chromatin, we examined asynchronous cells by
IF using anti-hRad21 and anti-lamin B antibodies (Fig. 7). At entry to mitosis, a major
population of hRad21 was already excluded from chromatin of cells that
had been already undergone nuclear envelope breakdown (Fig. 7,
row a). It was not possible to determine whether this
fraction of hRad21 had once associated with the chromatin and then
dissociated from it in prophase or whether the fraction had never
associated with the chromatin. However, some additional fraction of
hRad21 apparently dissociated as the chromatin further condensed (Fig.
7, row b). At the exit of M phase, a fraction of hRad21
remained in the cytoplasm even when the nuclear envelope had apparently
reformed (Fig. 7, row c). Therefore, it is suggested that
hRad21 might be imported to nucleus actively. Eventually, nearly all
hRad21 was localized inside the nuclei (Fig. 7, row d).

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Fig. 7.
hRad21 localization at the entry and exit of
M phase. Asynchronous HeLa cells were stained with DNA dye TOTO3
(red) and examined by IF using anti-hRad21
(green) and anti-lamin B antibodies (purple).
a, a prometaphase cell with the nuclear envelope
disassembled; b, a prometaphase cell with further condensed
chromosomes; c, two nascent daughter cells in which the
nuclear envelopes apparently reformed; d, two nascent
daughter cells in which most hRad21 is localized in nuclei. The
scale bar shown in d represents 10 µm.
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Significant Fractions of hRad21 Are Associated with Prometaphase
Chromosomes and Metaphase Centromeres--
Because of the background
signals produced by the chromatin-unbound hRad21 in mitotic cells, it
was difficult to determine whether hRad21 associates with condensed
chromosomes in M phase or not. We therefore examined spread
prometaphase-like and metaphase-like chromosomes to clarify whether
hRad21 is present on condensed chromosomes. To this end, synchronized
HeLa cells were treated with colcemid, and the spread chromosomes were
analyzed by IF. We found that significant amounts of hRad21 associated
along the axes of the less condensed prometaphase-like chromosomes
(Fig. 8A, row a).
These signals were not observed when the antibody was preabsorbed by
the antigen and when chromosomes were stained by normal rabbit IgG,
indicating that the signals were specific (data not shown). The hRad21
signals appeared heterogeneous along the length of chromosomes. To
better understand the relative distribution of hRad21 on the
chromosomes, we also localized centromeres using antibodies recognizing
CENP-B, a protein that binds to centromeric alphoid DNAs. We found that
hRad21 was localized at both the centromeres and the arm regions on
prometaphase-like chromosomes (Fig. 8A, row
a).

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Fig. 8.
Dynamic changes of hRad21 distribution on
colcemid-induced prometaphase-like and metaphase-like chromosomes.
A, the spread HeLa cell chromosomes were obtained as
described under "Experimental Procedures" and classified into
prometaphase-like (panel a) or metaphase-like (panel
b) based on their chromosome lengths. The chromosomes were stained
with DNA dye TOTO3 (red) and analyzed by IF using
anti-hRad21 (green) and anti-CENP-B (blue)
antibodies. Colocalization of DNA and hRad21 is indicated by a yellow
color in the merged image. The scale bars represent 10 µm.
B, single metaphase-like chromosomes were examined under a
higher magnification. DNA, hRad21, and CENP-B are pseudo-colored in
blue, red, and green, respectively, to
better visualize the overlapping (yellow) between hRad21
signals and CENP-B signals. Two pairs of chromatids are shown in
a, whereas one pair of chromatids is shown in b
and c. The scale bars represent 10 µm.
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In contrast, when we analyzed further condensed metaphase-like
chromosomes, we found that hRad21 became localized at centromeres (Fig.
8A, row b). Images under a high magnification
revealed that hRad21 mostly disappeared from the arm regions and
specifically localized at centromeres of metaphase-like chromosomes
(Fig. 8B). Interestingly, when CENP-B signals were
recognized as two spots, hRad21 was detected not only at
CENP-B-positive regions but also at inner regions between the two
sister CENP-B signals (Fig. 8B, rows a and
c). Taken together, these observations strongly suggest that
hRad21 in M phase is dissociated from chromatin in two steps. The arm
region-associated hRad21 is dissociated during the prophase to
metaphase transition, whereas the centromere-bound hRad21 remains associated with metaphase-like chromosomes.
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DISCUSSION |
It is estimated that about 70% of total Scc1p/Mcd1p is associated
with chromatin in budding yeast during metaphase (18), whereas
vertebrate cohesin molecules are mostly excluded from chromatin in
prophase (8, 9). Therefore, it has not been determined whether cohesin
plays a role in the mitotic chromatid cohesion in higher eukaryotes. In
this study, we have found that a small but significant fraction of
hRad21 is localized at centromeres of colcemid-induced metaphase-like
chromosomes. Moreover, hRad21 and its potential associated proteins are
hyperphosphorylated in M phase. These results highly suggest that
hRad21 has a critical role in the chromatid cohesion and separation in
M phase, as has been demonstrated in budding yeast.
In human and mouse cells, an electron microscopy study revealed that
sister chromatid arm regions are morphologically distinguishable with
each other prior to metaphase, whereas the centromeric regions are
connected until the onset of chromatid segregation in anaphase (19).
This implies that chromatids may be separated in a two-step process in
mammalian cells. In the first step, the arm regions are disjoined
during prophase to metaphase. At this stage, the remaining cohesion at
centromeres serves as a hinge connecting two sister chromatids. In the
second step that happens at the metaphase-anaphase transition, the
centromere cohesion is dissolved, and the two liberated chromatids
separate and move toward centrosomes by pulling forces exerted by
spindles. Such a two-step cohesion-separation event has been suggested
cytologically in a variety of animals such as Drosophila,
chicken, muntjac, mouse, and human (reviewed in Ref. 20).
The changes of hRad21 distribution that we have found with mitotic
chromosomes are remarkably well correlated with these cytological observations. We have found that hRad21 is present along the entire length of chromosomes during prophase to prometaphase. However, the
arm-associated hRad21 disappeared and only the centromere-associated hRad21 remains on the colcemid-induced metaphase-like chromosomes. Therefore, the dynamics of hRad21 distribution on mitotic chromosomes perfectly corresponds to the morphological changes of cohesion between
mitotic chromatids. These results suggest that cohesin containing
hRad21 is responsible for the chromatid cohesion in M phase. After this
paper was submitted for publication, it was reported that a small
population of SA1 protein is associated with metaphase chromosomes both
in Xenopus egg cell-free extracts and Chinese hamster ovary
cells (21). Even more recently, mouse Scc1/Rad21 protein expressed
ectopically in HeLa cells was shown to be specifically associated with
centromeric regions in metaphase chromosomes (22). Our study indicates
that the endogenous hRad21 protein behaves as the ectopically expressed
protein and further substantiates the likelihood of a highly regulated
distribution of cohesin complex on vertebrate mitotic chromosomes as
revealed in the above-mentioned studies.
Scc1p/Mcd1p has been shown to associate with specific regions along the
chromosome arms, especially at centromere-proximal regions (23-25).
Especially, yeast cohesion becomes further preferentially localized at
centromere-proximal regions on metaphase-like chromosomes induced by a
microtubule inhibitor methyl 2-benzimidazolecarbamate (23). Therefore,
in both yeast and humans, cohesin is enriched at centromere-proximal
regions just prior to the onset of anaphase. It is tempting to
speculate that these particular cohesin fractions associated with
centromeres are the target of the APC (anaphase promoting
complex)-dependent pathway that triggers anaphase. The recent study by Waizenegger et al. (22) supports this
hypothesis by analyzing the biochemical and cytological behaviors of
HeLa cells cohesin and mitotic processes in Xenopus
cell-free extract.
Interestingly, we found that the centromere-associated hRad21 on the
metaphase-like chromosomes appeared to be present between two sister
CENP-B signals in some cases. Molecular cloning of the alphoid repeats
on human chromosome 21 identified two different classes of alphoid
repeats,
21-I and
21-II (26).
21-I contains the CENP-B box to
which CENP-B binds, but
21-II does not. Correspondingly, it has been
reported that
21-I is precisely colocalized with CENP-B signals on
spread metaphase-like chromosomes, but
21-II distributes over a
broader area. Our observation that hRad21 distributed in broader
regions that included the sister CENP-B signals and the inner paring
domain located between the two CENP-B signals, suggests that a subset
of alphoid DNAs similar to
21-II may be involved in sister chromatid
cohesion at centromeres.
Several aspects of the behavior of hRad21 are different from that of
yeast Scc1p/Mcd1p. We found that hRad21 levels do not change during the
cell cycle. This finding is different from that of budding yeast,
wherein Scc1p/Mcd1p levels have been found to fluctuate throughout the
cell cycle. We found that only a small fraction of hRad21 associated
with prophase chromatin. The remaining and major population of hRad21
did not locate to chromatin in the earliest prometaphase cells, wherein
the nuclear envelope breakdown had already occurred. We could not
determine whether the major fraction of hRad21 we observed had once
bound with chromatin and dissociated in early prophase or whether it
had been unbound to chromatin throughout the cell cycle. Nevertheless,
we conclude that only a minor fraction of hRad21 contributes to
chromatid cohesion in M phase, a situation different from that of yeast Scc1p/Mcd1p (18). This observation suggests that hRad21 may play a role
additional to chromatid cohesion. In support of this notion, the
mitotic hRad21 protein, the majority of which is dissociated from
chromatin (Figs. 6 and 7), was resistant to detergent extraction (Fig.
2B). Therefore, it is suggested that this "free" form of hRad21 is associated with other nuclear structures, such as nuclear matrix and spindles. We noted that a fraction of hRad21 appeared to
closely associate with spindle midzones and midbody in anaphase and
telophase, respectively. This behavior is reminiscent of that of the
chromosome passenger proteins INCENP (inner
cen/tromere proteins) (27). It has been
recently proposed that a primary function of INCENP involves chromosome
segregation processes (28). It may be interesting to investigate the
possible roles of hRad21 in later stages of mitosis.
It is not known how the dissociation of hRad21 from mitotic chromatin
is regulated. In budding yeast, Scc1p/Mcd1p is cleaved proteolytically
in a manner dependent on Esp1p at the onset of anaphase. Similarly, it
has been reported very recently that a small fraction of mammalian
Scc1/Rad21 is proteolytically cleaved at the metaphase-to-anaphase
transition (22). In this study, we could not demonstrate a similar
proteolysis that specifically happened to anaphase hRad21. However, we
have found that hRad21 is phosphorylated in a cell
cycle-dependent manner. Significantly, hRad21 and its
potentially associated proteins, p180, p150, p140, p95, p85, and p80,
were correspondingly phosphorylated most heavily in M phase. Recently,
it has been reported that Xenopus Scc3 homologues (XSA1 and
2) are phosphorylated by Cdc2-cyclin B in vitro.
Phosphorylated SA proteins show low affinities for chromatin, and thus
it has been proposed that this protein modification might prevent
unbound SA proteins in M phase from reassociating with chromatin (11). Interestingly, p140 and p150, which were coimmunoprecipitated with
anti-hRad21 in this study, showed apparent molecular masses in SDS-PAGE
similar to those of human SA proteins (140 kDa) (11), suggesting that
p140 and p150 may be human SA proteins. Xenopus cohesin
contains a fifth unidentified component having an apparent molecular
mass of 95 kDa in SDS-PAGE (8). p95 that was also coimmunoprecipitated
with anti-hRad21 in this study may be the human counterpart of this
Xenopus cohesin component. Identities of the other
phosphorylated proteins (p180, p85, and p80) revealed in this study are
not known. p180, p95, and p85 are particularly interesting because
these phosphorylated proteins were not detected in the interphase
hRad21 immunoprecipitates (Fig. 4B). These proteins may be
phosphorylated strictly in mitosis or associated with the cohesin
complex specifically in mitosis. Taken together, these results suggest
that numerous components of human cohesin are coordinately modified by
phosphorylation in M phase, which may regulate the function of cohesin.
Future investigations of hRad21 should undoubtedly be aimed at
understanding how arm-associated and centromere-associated fractions of
hRad21 are differentially regulated.