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Vol. 14, Issue 6, 2399-2409, June 2003
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* Department of Biophysics and Biochemistry, Graduate School of Science,
University of Tokyo, Tokyo 113-0033, Japan;
Laboratory for Developmental Genomics, RIKEN Center for Developmental Biology,
Hyogo 650-0047, Japan
Submitted September 20, 2002;
Revised December 8, 2002;
Accepted January 30, 2003
Monitoring Editor: Douglas Koshland
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Lee and Orr-Weaver,
2001). In budding yeast Saccharomyces cerevisiae, cohesin
is composed of four proteins, namely, Scc1 (also called Mcd1), Scc3, Smc1, and
Smc3 (Guacci et al.,
1997; Michaelis et
al., 1997; Toth et
al., 1999). Equivalent complexes have been identified in
fission yeast Schizosaccharomyces pombe, where Rad21 corresponds to
budding yeast Scc1, and frog Xenopus laevis
(Losada et al., 1998;
Tomonaga et al.,
2000). In addition, homologs of each cohesin component have been
found in other organisms based on sequence similarities, although their
function has not been clarified extensively
(Hirano, 1999;
Parisi et al., 1999;
Toth et al., 1999;
Dong et al.,
2001).
Among the four kinds of cohesin subunits, Scc1/Rad21 plays a crucial role
for the onset of anaphase. Scc1 is cleaved by an endopeptidase called
separase, and this triggers the separation of sister chromatids
(Ciosk et al., 1998;
Uhlmann et al.,
1999). Rad21 undergoes similar cleavage
(Tomonaga et al.,
2000). In budding and fission yeast meiosis, Scc1/Rad21 is
replaced with its meiosis-specific counterpart called Rec8
(Klein et al., 1999;
Watanabe and Nurse, 1999).
Like Scc1/Rad21, Rec8 is cleaved by separase and gives a cue for the onset of
meiotic anaphase I in budding yeast
(Buonomo et al.,
2000). Because the other three subunits of cohesin apparently
function in both mitosis and meiosis, the replacement of Scc1/Rad21 with Rec8
seems to be crucial for establishing the reductional pattern of chromosome
segregation specific to meiosis I.
In the nematode Caenorhabditis elegans, four Scc1/Rad21 family
members have been reported (Parisi et
al., 1999; Pasierbek
et al., 2001). One of them, REC-8, was found to localize
to synaptonemal complexes at the pachytene stage and to chromosomal axes at
the diakinesis stage in meiotic prophase I
(Pasierbek et al.,
2001). In addition, depletion of REC-8 by RNA interference (RNAi)
resulted in splitting of the chromosomes into sister chromatids and appearance
of univalents at the diakinesis stage
(Pasierbek et al.,
2001). Thus, REC-8 seems to be the functional homolog of both
budding and fission yeast Rec8. Two Scc1/Rad21 homologs, called COH-1 and
COH-2, were found, respectively, to be essential for the viability of the
worm, whereas no apparent phenotype was observed in an animal depleted of the
fourth homolog, COH-3 (Pasierbek et
al., 2001). The question has not been critically answered,
however, whether they are involved in mitotic chromosomal cohesion.
Furthermore, homologs of the other three kinds of cohesin subunits remain
largely uncharacterized in C. elegans. Under these circumstances, we
set out to explore possible function of cohesin component homologs of C.
elegans during its development. Herein, we show that COH-2 and the
homologs of Scc3, Smc1 and Smc3, are involved in proper chromosome segregation
during mitosis, but COH-1 seems to have novel function necessary for
development but unrelated to mitosis. Because the use of scc-1
instead of coh-2 as the main registered gene name has been recently
agreed (Hodgkin, Meyer, and Loidl, unpublished data), we hereafter denote the
gene product as SCC-1/COH-2 in this article.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The wild-type C. elegans var. Bristol strain N2
and AZ212 (unc-110(ed3) ruIs32[pAZ132:pie- 1/GFP/histone
H2B] III) were used. N2 was maintained at 20°C and AZ212 was maintained at
25°C.
RNA Interference
As the templates to prepare double-stranded RNA (dsRNA), the following cDNA
clones were used: yk226d1 (coh-1), yk256h5
(scc1/coh-2), yk97g6 (scc-3), yk52h10
(him-1/smc-1), yk72e8 (coh-3), and yk295d12
(smc-3). These templates were polymerase chain reaction amplified
using either a pair of T7 and T3 primers (T7: 5'-GTA ATA CGA CTC ACT ATA
GGG C-3'; T3: 5'-AAT TAA CCC TCA CTA TTG GG-3'), or a pair
of M13-forward and M13-reverse primers (forward: 5'-GTA AAA CGA CGG CCA
GT-3'; reverse: 5'-GGA AAC AGC TAT GAC CAT G-3'). RNA was
synthesized in vitro with T7 and T3 RNA polymerases, and complementary strands
were mixed to generate dsRNA to be used for RNAi
(Fire et al., 1998).
Delivery of dsRNA into worms was performed by either microinjection or
soaking. In the case of microinjection, 1–3 µg/µl dsRNA was
injected into either the gonad or the intestine of young adult hermaphrodites,
and phenotypes of F1 progeny laid 12–30 h after the injection were
characterized. In the case of RNAi by soaking
(Maeda et al., 2001),
L4 larvae were treated in a dsRNA solution (1 µg/µl in soaking buffer)
for 24 h, and phenotypes of the F1 generation were observed. To examine
postembryonic phenotypes caused by RNAi, L1 larvae were soaked in a dsRNA
solution similarly, and abnormalities of the recovered worms were observed. In
addition, worms that hatched from the embryos laid soon after dsRNA injection
(6–12 h) were also examined to assess the postembryonic phenotypes.
Generation of COH-1 and SCC-1/COH-2 Antibodies
Rabbit and rat polyclonal antibodies against COH-1 and SCC-1/COH-2 were
prepared as follows. The relatively unique region in each open reading frame
(amino acids 173–559 for COH-1; amino acids 118–544 for
SCC-1/COH-2) was polymerase chain reaction amplified from a cDNA clone and
cloned into pGEX-KG vector (Guan and
Dixon, 1991). Each glutathione S-transferase (GST) fusion
protein was expressed from the resulting plasmid in Escherichia coli
and used as an antigen after purification. COH-1- and
SCC-1/COH-2–specific antibodies raised in rabbit and rat were affinity
purified using histidine (His)-tagged COH-1 and SCC-1/COH-2 proteins,
respectively. To create a His-tagged fusion construct, the same cDNA fragments
as used above were cloned into pET19b vector (Novagen, Madison, WI). The
affinity-purified antibodies, respectively, recognized COH-1 and SCC-1/COH-2
specifically. Both rabbit and rat antibodies were used in immunofluorescence
analysis, and they gave the same staining patterns.
Immnofluorescence and DNA Staining
Embryos were processed for staining as described previously
(Miller and Shakes, 1995). In
brief, embryos permeabilized by the freezecrack method were fixed by placing
in methanol for 2 min at -20°C and then in acetone for 4 min at -20°C,
and rehydrated at room temperature. Rehydrated embryos were treated with a
blocking solution (3% bovine serum albumin in phosphate-buffered saline
containing 0.5% Tween 20) for 30 min at room temperature. They were then
incubated with the primary antibody at 4°C overnight and then with the
secondary antibody at room temperature for 1–2 h.
4,6-Diamidino-2-phenylindole was added to the final concentration of 2
µg/ml, and the sample was mounted for epifluorescence microscopy. To
visualize DNA under confocal laser microscopy, fixed samples were treated with
1/10,000 diluted Sytox Green (Molecular Probes, Eugene, OR) for 10 min. The
following antibodies were used: primary antibodies, anti-COH-1(this study),
anti-SCC-1/COH-2 (this study), and anti-nuclear pore complexes mouse
monoclonal antibody (MAb414) (Babco, Richmond, CA); and secondary antibodies,
fluorescein isothiocyanate-conjugated sheep anti-mouse IgG antibody (Cappel
Laboratories, Durham, NC), Cy3-labeled goat anti-rabbit IgG antibody (Chemicon
International, Temecula, CA), and rhodamin-labeled anti-rat IgG antibody
(Cappel Laboratories). To reduce nonspecific background signals, the secondary
antibodies were treated with the C. elegans acetone powder before
use.
For confocal imaging, the LSM510 system attached to an Axioplan 2 microscope (Carl Zeiss, Jena, Germany) was used. Other images were taken digitally by either of the following combinations: an AxioCam charge-coupled device camera attached to an Axioplan 2 microscope with the AxioVision software (Carl Zeiss); or a cooled charge-coupled device camera C4742–95-10NR (Hamamatsu Photonics) attached to a Zeiss Axioplan 2 microscope with the FISH Imaging Software (Hamamatsu Photonics, Bridgewater, NJ).
Live Observation of Embryos and Four-Dimensional Recording
Young adult hermaphrodites were dissected in M9 buffer and the collected
embryos were mounted on a 2% agar pad under a coverslip. Four-dimensional
recording of green fluorescent protein (GFP)-fluorescence and differential
interference contrast (DIC) images was performed using the LSM510 system
attached to an Axioplan 2 microscope (Carl Zeiss). Images were taken every 40
s, at five different focal planes at least.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Comparison of their amino acid sequences has shown that
COH-1 and SCC-1/COH-2 are highly similar to each other and both belong to the
Scc1/Rad21 subfamily, as opposed to the Rec8 subfamily. REC-8 is the ortholog
of yeast Rec8 and is specifically involved in meiotic chromosome cohesion,
whereas the homology of COH-3 with the Scc1/Rad21 family members was less
significant (Pasierbek et al.,
2001). To examine whether these putative cohesin components in
C. elegans were involved in chromosomal cohesion during mitosis, we
depleted each protein by RNAi and monitored mitosis in embryos.
We first examined the RNAi phenotypes for the scc-3,
him-1/smc-1, and smc-3 genes. Depletion of any of these
gene products resulted in embryonic lethality with complete penetrance. To
characterize the process of chromosome segregation in RNAi animals,
chromosomes were visualized by GFP-tagged histone H2B, and time-lapse images
of the fluorescence of H2B-GFP as well as DIC images of the embryos were
collected using a confocal microscope. For each of these three genes,
RNAi-affected embryos were all arrested during embryogenesis, and 
30% of
them displayed defective mitosis in early cell cycles, as described below.
In wild-type embryos, condensed chromosomes aligned on the metaphase plate
and then they separated at once in anaphase, giving a view of splitting two
parallel discs (Figure 1,
A–D, movie WT.mov). After cytokinesis, chromosomes were
decondensed and nuclear membrane was reassembled around them. In contrast,
embryos depleted of either SCC-3, or HIM-1/SMC-1, or SMC-3 behaved differently
from the wild-type, with these three kinds of RNAi embryos showing phenotypes
indistinguishable from each other (Figure
1, E–H, movie scc-3.mov; our unpublished data). Chromosomes
were condensed at prometaphase in these embryos, but the metaphase plate often
looked diffuse, or was completely missing in some cases. Then, a mass of
chromosomes staying in the middle of the bipolar spindle frequently seemed to
be divided by a cleavage furrow (Figure
1F, arrows). This might mean the lack of anaphase, or
alternatively, the progression of aberrant anaphase with extensive chromosome
bridging, which were not distinguishable at the current resolution.
Chromosomes were decondensed after cytokinesis, as in wild-type embryos, but
multiple (mainly two to four) nuclei of variable size were often formed in
daughter cells (Figure 1H,
arrowheads). In the case of wild-type embryos, nuclear membrane has been shown
to reassemble around subsets of decondensed chromosomes at telophase, but they
fuse until they form a single nucleus that encloses the whole chromosome set
(Newport, 1987). We speculate
that nuclear membrane could reassemble around each chromosome or a subset of a
few chromosomes but failed to fuse to form a single nucleus in cells devoid of
either SCC-3, or HIM-1/SMC-1, or SMC-3. This is probably because chromosomes
were not separated synchronously in these cells and hence were not close
enough to each other to compose a unified nucleus. Subsequent cell cycles were
also aberrant similarly in the RNAi animals. Nuclear membrane of the multiple
small nuclei generated in the previous cell cycle broke down simultaneously
and chromosomes were condensed, but only a loose metaphase plate was formed
again, and a mass of chromosomes was divided irregularly by a cleavage furrow.
These observations suggest that the three homologs of cohesin components,
namely, SCC-3, HIM-1/SMC-1, and SMC-3, are essential for proper chromosome
segregation, but unlikely to be involved in other aspects of the cell
cycle.
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COH-1 and SCC-1/COH-2 Play Distinct Roles in Development
Among the four C. elegans Scc1/Rad21 homologs, REC-8 is involved
specifically in meiosis, and no detectable phenotype results from RNAi for
coh-3 (Pasierbek et al.,
2001). Therefore, we focused on the two remaining Scc1/Rad21
homologs, namely, COH-1 and SCC-1/COH-2, and examined their possible function
in mitosis. When we disrupted function of the scc-1/coh-2 gene by
RNAi, F1 embryos exhibited embryonic lethality with complete penetrance
(Figure 2A), as described
previously (Pasierbek et al.,
2001). We carefully observed the cell division process in
scc-1/coh-2(RNAi) embryos, and found similar chromosome
segregation defects in early cell cycles to the scc-3(RNAi),
him-1/smc1(RNAi), or smc-3(RNAi)
embryos, although the penetrance was relatively low
(Figure 1, I–L, movie
coh-2.mov). This low penetrance (10% of the RNAi-affected embryos) might
be due to either gene-specific inefficiency of RNAi reaction or relatively
high stability of the SCC-1/COH-2 protein compared with other cohesin
components. The scc-1/coh-2(RNAi) embryos showed loose
metaphase plates, asynchronous separation of chromosomes at anaphase, and
formation of multiple nuclei after decondensation of chromosomes. Thus, we
speculated that SCC-1/COH-2 was likely to function in the same process as the
three other cohesin component homologs described above, at least in early
embryogenesis.
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When we chased possible postembryonic phenotypes of scc-1/coh-2(RNAi) animals by examining F1 worms laid soon after the injection of dsRNA (see MATERIALS AND METHODS), they either arrested as larvae or became sterile adults. Some arrested larvae were thin at their posterior part and showed a severe Unc (uncoordinated) phenotype unable to move backward. Variable defects were seen in germ cells of the sterile adults. Some animals had very few germ cells with no mature gametes, whereas others had normal-looking sperm and >100 undifferentiated germ cells in the gonad. In addition, some sterile adults showed a Pvl (protruding vulva) phenotype (Figure 2B). Thus, the cell types affected in these worms, namely, ventral nerve cord, vulva, and germ cells, apparently corresponded to the ones that are known to divide postembryonically. These observations suggest that the SCC-1/COH-2 function is essential for normal cell division during larval development, in addition to embryogenesis.
We also disrupted function of the coh-1 gene by RNAi. Whereas
8% of the F1 progeny arrested as unhatched embryos, the majority of
progeny arrested as larvae in this case (37% L1-L2 arrest, and 22% L3-L4
arrest, n = 328). The variable phenotypes observed were probably due to
genedependent inefficacy inherent of RNAi experiments. These larvae showed no
apparent morphological abnormality (Figure
2C), but were affected severely in locomotion (our unpublished
data). Even F1 progeny that arrested as unhatched embryos seemed to have
completed normal morphogenesis. Because expression of COH-1 became detectable
at the mid-embryogenesis stage (see below), and the RNAi-arrested embryos
showed a significantly reduced level of COH-1, it seemed that the most
rigorous loss-of-function phenotype for coh-1 would be late embryonic
lethality after completion of morphogenesis. We next disrupted the function of
COH-1 postembryonically by soaking L1 worms to a dsRNA solution and found that
some coh-1(RNAi) adults were defective in egg laying
(Figure 2, D and E). The
observed defects in locomotion and egg laying implicated that the function of
COH-1 might be important for proper function of muscle and/or the nervous
system. In summary, the function of COH-1 seems to be dispensable for
embryonic development but essential for viability of the organism. So far, no
indication of defects in cell division has been observed in
coh-1(RNAi) worms.
We then performed double and triple RNAi experiments for coh-1, scc-1/coh-2, and coh-3. When the function of coh-1 and scc-1/coh-2 was simultaneously disrupted by RNAi, the F1 embryos displayed phenotypes similar to those of scc-1/coh-2(RNAi) worms, with no apparent enhancement in the phenotypes (our unpublished data). Two double RNAi experiments involving coh-3, namely, coh-1(RNAi); coh-3(RNAi), and scc-1/coh-2(RNAi); coh-3(RNAi), resulted in the same phenotypes as single RNAi for each of coh-1 and scc-1/coh-2, respectively (our unpublished data). In addition, the triple RNAi [coh-1(RNAi); scc-1/coh-2(RNAi); coh-3(RNAi)] caused essentially the same embryonic phenotypes as scc-1/coh-2(RNAi) (our unpublished data). Therefore, we speculate that the function of coh-1 and that of scc-1/coh-2 is neither synergistic nor redundant and that coh-3 may contribute little to the physiology of the worm.
Cell Cycle-dependent Localization of SCC-1/COH-2 in Early
Embryos
To visualize expression and localization of COH-1 and SCC-1/COH-2 during
worm development, we generated polyclonal antibodies against the central
region of each protein, because this region showed no significant similarity
between the two. We confirmed that the affinity-purified anti-COH-1 and
anti-SCC-1/COH-2 antibodies specifically recognized each protein, COH-1 as an
apparent 95-kDa band and SCC-1/COH-2 as an apparent 110-kDa band, in Western
blotting against the worm extract (our unpublished data). Both proteins
migrated slower than the expected size of 75 kDa. This abnormal migration of
the worm Scc1/Rad21 family members has been described previously, although the
observed apparent molecular masses are somewhat different from our estimation
(Pasierbek et al.,
2001). The specificity of the antibodies was further confirmed by
immunostaining of RNAi embryos depleted of either COH-1 or SCC-1/COH-2 (see
below).
We analyzed localization of SCC-1/COH-2 in early embryos by using the
specific antibodies (Figure
3A). SCC-1/COH-2 seemed to localize to the chromosomes in a cell
cycle-dependent manner (Figure
3B). In interphase, SCC-1/COH-2 was seen throughout the nucleus,
overlapping largely with DNA. At mitotic prophase, SCC-1/COH-2 started to
separate from condensing chromosomes, and it was not detected on the
chromosomes at prometaphase and metaphase. At metaphase, the SCC-1/COH-2
signal seemed as if surrounding the metaphase plate, although it was possible
that a small amount of SCC-1/COH-2 was remaining on the metaphase chromosomes
but escaped detection, because cohesin is reported to become detectable on
metaphase chromosomes only after detergent extraction of soluble background in
other metazoans (Warren et al.,
2000). The SCC-1/COH-2 signal was then dispersed in the cytoplasm
at anaphase. At telophase, the SCC-1/COH-2 protein began to reaccumulate on
the chromosomes.
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Whereas nuclear envelope breakdown normally occurs at prometaphase of
mitosis in higher eukaryotes, nuclear membrane persists until the beginning of
anaphase in early embryos in C. elegans
(Lee et al., 2000;
Liu et al., 2000).
Therefore, the SCC-1/COH-2 signal observed around the metaphase plate might
represent the protein molecules that were dissociated from the chromosomes and
trapped by the nuclear envelope, as has been demonstrated for small nuclear
ribonucleoprotein particles (Lee et
al., 2000). Alternatively, it could be the molecules adhering
to the side of condensed chromosomes. To distinguish these possibilities, we
stained the embryos with both anti-SCC-1/COH-2 antibodies and an antibody
against a component of the nuclear pore complexes. The SCC-1/COH-2 signal was
evenly distributed within the nuclear envelope except for the chromosomal
region (Figure 3C), suggesting
that SCC-1/COH-2 molecules dissociated from the chromosomes at metaphase were
trapped by the nuclear envelope. Consistently with this interpretation, the
SCC-1/COH-2 staining around the metaphase plate was no longer seen at later
stages of embryogenesis involving >30 cells, where nuclear envelope is
known to break down before metaphase (Lee
et al., 2000). SCC-1/COH-2 was dispersed into the whole
cytoplasm of metaphase cells at these stages (our unpublished data).
SCC-1/COH-2 Is Expressed in Dividing Cells throughout
Development
The developmental profile of SCC-1/COH-2 expression was examined by
staining embryos and worms at various stages with anti-SCC-1/COH-2 antibodies.
SCC-1/COH-2 was strongly expressed in virtually all cells in early embryos,
but its expression was gradually weakened, and the signal could hardly be
detected in late embryos, in which cell division was ceased almost completely
(our unpublished data). Strong nuclear signals of SCC-1/COH-2 reappeared in
larvae, though they were limited to a subset of cells
(Figure 4). The cell lineage
throughout the development has been elucidated in C. elegans, and the
timing as well as the number of cell divisions is known to be invariant
(Sulston and Horvitz, 1977).
This enabled us to identify which types of cells expressed SCC-1/COH-2
postembryonically. We found that SCC-1/COH-2 was detectable only in cells that
were going to divide. For example, in an L1 larva, intense SCC-1/COH-2 signals
were detected in the 14 hypodermal V lineage cells, which divide synchronously
(Figure 4A). Figure 4A illustrates the
SCC-1/COH-2 signal dispersed and not detectable on condensed chromosomes, as
observed in embryos of an intermediate stage. In a slightly older L1 larva,
expression of SCC-1/COH-2 was seen in 22 P lineage cells to constitute the
ventral nerve cord and in four Q lineage cells to produce posterior neuronal
cells, all of which divide at the same time
(Figure 4B). In this L1 larva,
no signal was detected in the V lineage cells
(Figure 4B), suggesting that
the SCC-1/COH-2 protein is present only for a short time in the cell cycle,
and likely to be degraded quickly after cell division. Larvae of later stages
also expressed SCC-1/COH-2 in dividing cells: in an L3 larva, SCC-1/COH-2 was
detected in four M lineage cells to produce the uterine and vulval muscle
cells and in 10 P lineage vulval precursor cells, which divide concurrently
(Figure 4C).
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SCC-1/COH-2 was expressed in germ cells throughout the development,
including the adult stage. Germ cells of C. elegans continue to
divide at the distal part of the gonad, from the L1 stage through adulthood,
and we detected SCC-1/COH-2 in virtually all mitotic germ nuclei. Similarly to
somatic cells in embryos, SCC-1/COH-2 was dispersed in the cytoplasm at
mitotic prometaphase and was absent from the condensed anaphase chromosomes in
germ cells (Figure 5A). In
female germ cells that entered meiotic prophase in adult hermaphrodites,
SCC-1/COH-2 was observed uniformly in the nuclei. It was unclear whether
SCC-1/COH-2 localized to the condensed meiotic chromosomes, because of the
strong SCC-1/COH-2 signal emitted from the nucleoplasm
(Figure 5B). The localization
of SCC-1/COH-2 we have assigned in female germ cells is somewhat different
from that of a previous report (Pasierbek
et al., 2001), in which SCC-1/COH-2 has been observed as
distinct spots in pachytene nuclei but not detected in diplotene or later
nuclei. The reason for this apparent difference is unclear. SCC-1/COH-2 was
detected also in male germ cells at mitosis and meiosis, but it was not
detectable in mature sperm (our unpublished data). Together, we conclude that
expression of SCC-1/COH-2 is coupled strictly with progression of the cell
division cycle throughout the development.
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Localization of COH-1 in Embryos and Larvae
The temporal and spatial expression pattern of COH-1 was totally different
from that of SCC-1/COH-2. Unlike SCC-1/COH-2, COH-1 was not detected in early
embryos (Figure 6A). The COH-1
protein emerged in virtually all cells in embryos after the 100-cell
stage and seemed to be expressed most strongly in embryos undergoing
morphogenesis. During larval development, COH-1 could be seen apparently in
all somatic cells, although the signal in the intestinal cells was somewhat
weaker compared with other cells. Interestingly, however, COH-1 was not
detected in germ cells throughout the larval development
(Figure 6B). Moreover, in
striking contrast to SCC-1/COH-2, which showed a cell cycle-dependent dynamic
change in subcellular localization, COH-1 was found in association with
chromosomes regardless of the stage of the cell cycle in all the cells
expressing it. We note that the COH-1 localization we describe herein is again
somewhat different from the previous observation
(Pasierbek et al.,
2001), in which COH-1 has been reported to be absent from the
condensed mitotic chromosomes. This disagreement may be due to the difference
of the antibody preparation and/or the staining procedures used. Together, we
conclude that COH-1 and SCC-1/COH-2 are likely to be regulated in distinct
manners with respect to their subcellular localization and developmental
expression.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
Losada et al., 1998,
2000;
Michaelis et al.,
1997; Tomonaga et
al., 2000; Sonoda et
al., 2001). Our study seems unique in that it characterizes
mitotic cohesin in a multicellular animal at the organismal level. The results
we obtained suggest that SCC-1/COH-2, SCC-3, HIM-1/SMC-1, and SMC-3 are
essential for proper segregation of chromosomes during mitosis. Depletion of
any of these four proteins caused similar deficiency in chromosome
segregation. The homologs of these proteins in yeast and frog have been shown
to form a cohesin complex (Losada et al.,
1998,
2000;
Toth et al., 1999;
Tomonaga et al.,
2000). Thus, it is presumable that these four proteins, which are
homologous with Scc1/Rad21, Scc3, Smc1, and Smc3, respectively, constitute a
mitotic cohesin complex and function in sister chromatid cohesion in C.
elegans, although it needs to be critically confirmed by biochemical
analysis. Our findings also suggest that the function of cohesin components
has been conserved among eukaryotic organisms.
In budding and fission yeast, mutants of cohesin components exhibit
premature sister chromatid separation, and they consequently arrest at
metaphase due to operation of the spindle checkpoint mechanism
(Guacci et al., 1997;
Michaelis et al.,
1997; Tomonaga et
al., 2000). Similarly, Scc1-deficient chicken DT40 cells
result in premature sister chromatid separation and show a mitotic delay at
prometaphase or metaphase, with only a small proportion of cells undergoing
anaphase (Sonoda et al.,
2001). In C. elegans embryos lacking any of the
aforementioned cohesin subunit homologs, condensed chromosomes failed to form
a tight metaphase plate, as in yeast and DT40-defective cells, but the
following cell cycle events could proceed in them, probably because the
spindle checkpoint is not operative in early embryos. This allows us to
evaluate the extent of the direct involvement of C. elegans cohesin
components in the cell cycle progression: SCC-1/COH-2, SCC-3, HIM-1/SMC-1, and
SMC-3 are all dispensable for cytokinesis, nuclear disassembly/reassembly, and
chromosome condensation/decondensation. It has been shown recently that
homologs of the SMC-type subunits of the condensin complex are involved in
chromosome condensation in C. elegans
(Hagstrom et al.,
2002). Thus, unlike budding yeast, in which the cohesin complex
affects both chromosome condensation and cohesion, the two chromosomal events
are likely to be regulated by distinct complexes in C. elegans, as in
vertebrates (Michaelis et al.,
1997; Sonoda et al.,
2001).
The cell cycle-dependent localization of Scc1/Rad21 homologs has been
reported in several organisms, though there seem to be considerable
differences from organism to organism. In budding yeast, Scc1 is localized to
the chromatin in late G1 and S phase. Scc1 dissociates from chromosomes as the
cell enters mitotic anaphase, triggered by its cleavage by separase, a
proteolytic enzyme activated by the anaphase-promoting complex
(Guacci et al., 1997;
Michaelis et al.,
1997). In contrast, the fission yeast Rad21 protein is detectable
on the chromosome throughout the cell cycle, and the cleavage of only a small
fraction of Rad21 seems to be sufficient for the onset of anaphase in this
organism (Tomonaga et al.,
2000). The situation is further different in vertebrates and
Drosophila. In these organisms, Scc1 is presumed to be removed from
chromosomes in a two-step mechanism: First, Scc1 is removed from the
chromosomal arms in a cleavage-independent manner during prophase, and then
the protein staying in the centromeric region is removed at
metaphase–anaphase transition, triggered by the cleavage by separase
(Losada et al., 1998;
Waizenegger et al.,
2000; Warren et al.,
2000). The chromosomes of C. elegans are holocentric,
that is, nonlocalized kinetochores spread along the length of each chromosome
(Albertson and Thomson, 1982).
Therefore, if SCC-1/COH-2 persists on the centromeric regions until the onset
of anaphase, as in higher animals, we can expect that it may constitute a
longitudinal band on each chromosome. However, we could not detect SCC-1/COH-2
on the condensed chromosomes from prophase through anaphase. Two types of
interpretation of the observation seem to be possible. Difficulties have been
reported in detecting vertebrate Scc1/Rad21 homologs on metaphase chromosomes
by immunofluorescence under certain conditions, probably because of low
antigen accessibility to the condensed chromosomes
(Losada et al.,
2000). Therefore, it is possible that we technically failed to
detect a small amount of SCC-1/COH-2 persisting at the centromeric regions.
Alternatively, the possibility may remain as well that SCC-1/COH-2 indeed
dissociates from chromosomes during prophase, and that another unidentified
protein may withhold the chromosomal cohesion until the onset of anaphase in
C. elegans.
The cleavage of Scc1/Rad21 by separase triggers metaphase–anaphase
transition in yeast and vertebrates
(Uhlmann et al.,
1999; Hauf et al.,
2001). In C. elegans, a homolog of separase, called
SEP-1, has been shown to be essential for sister chromatid separation
(Siomos et al.,
2001). Thus, the simplest hypothesis is that SCC-1/COH-2 is
cleaved by SEP-1 during mitosis. Indeed, there are several sequences that
match the separase cleavage consensus (E/DxxR) in the SCC-1/COH-2 protein.
Because we have not detected SCC-1/COH-2 on metaphase chromosomes, it seems
intriguing to address whether SCC-1/COH-2 is really a target of SEP-1, and
whether the cleavage of SCC-1/COH-2 is a trigger for the
metaphase–anaphase transition.
Distinct Roles for COH-1 and SCC-1/COH-2
Our study has indicated that COH-1 and SCC-1/COH-2 play distinct function
in the development of C. elegans. As described above, SCC-1/COH-2 is
most likely to function as a component of the mitotic cohesin complex, whereas
COH-1 does not seem to play any significant role in chromosomal cohesion,
judging from its loss-of-function phenotypes and subcellular localization.
COH-1 was present on chromosomes in all somatic cells, including nonmitotic
cells, from the stage of 100-cell embryos through adulthood, and its
depletion did not cause any cell cycle-related abnormalities. The
coh-1(RNAi) embryos and animals looked morphologically normal,
although they had a severe defect in locomotion, implying that COH-1 may be
unrelated to cell division and cell fate determination. It may be that COH-1
is essential for the function or maintenance of differentiated cells. For
example, because COH-1 is present on chromosomes of all somatic cells, it may
regulate general gene expression by affecting the chromatin organization.
Another possibility may be that COH-1 is involved in DNA repair, as has been
suggested for fission yeast Rad21
(Tatebayashi et al.,
1998).
The majority of organisms examined so far have two Scc1/Rad21-related
proteins, one of which is a member of the Scc1/Rad21 subfamily for mitosis and
the other is a member of the Rec8 subfamily for meiosis
(Parisi et al.,
1999). C. elegans and Arabidopsis are the only
exceptions thus far reported to have multiple Scc1/Rad21 subfamily members
(Bai et al., 1999;
Dong et al., 2001;
Pasierbek et al.,
2001). Arabidopsis has at least three Scc1/Rad21-related
proteins. One of them, SYN1, has been suggested to be the ortholog of Rec8
(Bai et al., 1999).
SYN2 and SYN3, whose function remains unspecified, are known to be transcribed
in tissues undergoing mitosis, although not limited to them
(Dong et al., 2001).
Therefore, C. elegans COH-1 may be currently the only Scc1/Rad21
family member that is implicated to have a function unrelated to chromosomal
cohesion. This may sound more interesting if we consider the fact that COH-1
and SCC-1/COH-2 are the most similar two among the worm Scc1/Rad21 family
members.
Two types of Scc3 subunit(s) have been identified in Xenopus and
human. These subunits, named SA1 and SA2, constitute separate cohesin
complexes in the cell, and it is suggested that association of
cohesinSA1 and cohesinSA2 to the chromatin may be
differently regulated, although their functional difference in vivo is as yet
unclear (Losada et al.,
2000). This apparently raises an intriguing possibility that some
organisms may have acquired multiple variants for a single cohesin subunit and
may carry distinct cohesin complexes, each of which plays a unique function.
It is therefore of interest to see whether COH-1 fulfills its function by
forming a complex with other cohesin subunits in C. elegans. The
unusual characteristics of COH-1 revealed in this study seem to make further
analysis of this protein promising.
|
ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
|---|
Online version of this article contains video material for some figures.
Online version available at
www.molbiolcell.org. 
Corresponding author. E-mail address:
myamamot{at}ims.u-tokyo.ac.jp.
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