A RT I C L E S
Unified mode of centromeric protection by shugoshin
in mammalian oocytes and somatic cells
Jibak Lee1,4, Tomoya S. Kitajima2,4, Yuji Tanno2, Kayo Yoshida3, Takashi Morita3, Takashi Miyano1, Masashi
Miyake1 and Yoshinori Watanabe2,5
Reductional chromosome segregation in germ cells, where sister chromatids are pulled to the same pole, accompanies the
protection of cohesin at centromeres from separase cleavage. Here, we show that mammalian shugoshin Sgo2 is expressed in
germ cells and is solely responsible for the centromeric localization of PP2A and the protection of cohesin Rec8 in oocytes,
proving conservation of the mechanism from yeast to mammals. However, this role of Sgo2 contrasts with its mitotic role in
protecting centromeric cohesin only from prophase dissociation, but never from anaphase cleavage. We demonstrate that, in
somatic cells, shugoshin colocalizes with cohesin in prophase or prometaphase, but their localizations become separate when
centromeres are pulled oppositely at metaphase. Remarkably, if tension is artificially removed from the centromeres at the
metaphase–anaphase transition, cohesin at the centromeres can be protected from separase cleavage even in somatic cells, as in
germ cells. These results argue for a unified view of centromeric protection by shugoshin in mitosis and meiosis.
During both mitosis and meiosis, sister chromatid cohesion of chromosomes is mediated by a multi-subunit complex called cohesin. In
mitosis, cohesion is dissolved by the cleavage of the Rad21 (also known
as Scc1) subunit of cohesin by separase, which is usually sequestered
by securin until metaphase, but activated by the anaphase promoting
complex (APC)-dependent degradation of securin1. In yeast meiosis,
separase-mediated cleavage of Rec8, a meiotic counterpart of Rad21,
first occurs along chromosome arms, triggering homologue separation,
whereas Rec8 is protected at centromeres throughout meiosis I until
metaphase II (refs 2, 3). For this protection, shugoshin plays a crucial
role by associating with protein phosphatase 2A (PP2A)4–8. In mammalian mitotic cells, most, but not all, cohesin dissociates from the chromosome arms prior to metaphase without cleavage through the prophase
pathway, whereas centromeric cohesin is largely retained to ensure that
sister kinetochores can be captured by spindle microtubules from opposite poles9. Mammalian Sgo1 and Sgo2 have an essential role in protecting centromeric cohesin, both also cooperate with PP2A and are likely
to counteract the phosphorylation of cohesin — a crucial prerequisite
of dissociation7,10–12. Importantly, however, they never protect cohesin
from cleavage at anaphase in mitosis, in contrast with the meiotic roles
of shugoshin suggested in yeast4,5. Thus, it is unclear whether these shugoshins are indeed involved in the centromeric protection of cohesin
from cleavage during meiosis I in mammals and, if so, what defines the
different mode of protection of centromeric cohesion between mitosis
and meiosis. Here, we describe the investigation of this fundamental
issue of chromosome segregation, which may be also related to human
birth defects13–16.
RESULTS
Sgo2 is highly expressed in germ cells
In mouse, Sgo1 and Sgo2 are ubiquitously expressed in proliferating
cells, whereas Sgo2 expression is markedly stronger in testis (Fig. 1a
and see Supplementary Information, Fig. S1). Consistent with this
result, the knockout of either Sgo1 or Sgo2 prevents the birth of
homozygous knockout mice (K.Y., T.S.K, Y.W. and T. Morita, unpublished observations). Oocytes cultured in vitro were used to study
meiotic roles of mammalian shugoshins. Immunostaining of oocytes
with shugoshin antibodies revealed that Sgo2 is highly expressed in
oocytes, whereas the expression of Sgo1 is moderate (data not shown);
both Sgo1 and Sgo2 localize around the inner kinetochore, directly
adjacent to the spindle checkpoint component Bub1, within centromeres in both meiosis I and meiosis II (Fig. 1b and see Supplementary
Information, Fig. S2).
Sgo2 is the centromeric protector in oocytes
To address the function of shugoshins in oocytes, oocytes were prepared
for culture and treated with small interfering RNAs (siRNAs) against
Sgo1 and Sgo2 . This treatment largely reduced the amount of mRNA for
Laboratory of Reproductive Biology and Biotechnology, Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan. 2Laboratory of
Chromosome Dynamics, Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Tokyo 113-0032, Japan. 3Department of Molecular Genetics,
Graduate School of Medicine, Osaka City University, Asahimachi, Abeno, Osaka 545-8585, Japan. 4These authors contributed equally to this work.
5
Correspondence should be addressed to Y.W. (email: [email protected])
1
Received 29 June 2007; accepted 29 November 2007; published online 16 December 2007; DOI: 10.1038/ncb1667
42
nature cell biology volume 10 | number 1 | JANUARY 2008
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L iv
er
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ym
us
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s
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A RT I C L E S
a
Sgo1
Sgo2
GAPDH
b
Metaphase I
Sgo1
Bub1
Metaphase I
Sgo2
Bub1
Metaphase II
DNA
Metaphase II
DNA
Figure 1 Sgo2 is highly expressed in germ cells. (a) Northern blot for Sgo1
and Sgo2 using total RNA prepared from the indicated organs. GAPDH
is used as a control. (b) Oocytes at metaphase I and metaphase II were
stained with anti-Sgo1 or anti-Sgo2 and anti-Bub1 antibodies. DNA was
counterstained with TP3. Magnifications of the boxed regions are shown. The
scale bar represents 10 µm.
Sgo1 and Sgo2 (Fig. 2a). Immunostaining indicated that approximately
80% of siRNA-treated oocytes showed almost completely abolished
centromeric Sgo signals at metaphase I (6 h after the start of maturation culture) or metaphase II (14 h; see Supplementary Information,
Fig. S2a, b), verifying that the signals detected by these antibodies derive
from endogenous shugoshin proteins. Depletion of either one or both
of Sgo1 and Sgo2 did not affect meiotic progression to metaphase I or
the alignment of bivalents (pairs of homologues; see Supplementary
Information, Fig. S2a, c), indicating that shugoshins are largely dispensable for monopolar attachment of sister chromatids and bipolar attachment of homologues. However, in metaphase II, Sgo2 and Sgo1–Sgo2
double RNA interference (RNAi) increased the frequency of oocytes
showing disordered chromosome alignment, whereas the frequency of
the misalignment phenotype was modest in Sgo1-depleted oocytes (see
Supplementary Information, Fig. S2b–d).
The chromosome misalignment phenotype at metaphase II could be
the consequence of premature separation of sister chromatids during
anaphase I, similarly to observations in the yeast shugoshin mutant. To
address this possibility, chromosome spreads from metaphase II oocytes
were examined. In control oocytes, univalents (pairs of sister chromatids)
were predominantly observed, albeit approximately 6% of chromatids
were separated, presumably by mechanical stress during chromosome
spreading. Sgo1-depleted oocytes similarly exhibited largely intact cohesion of sister chromatids. In striking contrast, however, separated single
chromatids were prevalent in Sgo2-depleted oocytes (Fig. 2b, c). The
residual population of univalents in Sgo2 siRNA-treated oocytes can
be accounted for by the inefficiency of the RNAi. As separation of sisters was already evident in anaphase I oocytes but not at metaphase I
(Fig. 2d), the single chromatids are likely to have originated during
anaphase I — the period when sister chromatid cohesion is usually dissociated only along chromosome arms, but is retained at centromeres.
Sgo2- and Sgo1–Sgo2-depleted oocytes exhibited approximately 40 single chromatids (or a mixture of single chromatids and univalents with a
total of 40 chromatids) in metaphase II nuclei (Fig. 2b), consistent with
the intact alignment of bivalents in these oocytes at metaphase I. These
results indicate that chromosome segregation at meiosis I is mostly, if
not entirely, intact in Sgo2-depleted oocytes, although the retention of
centromeric cohesion of sisters is abolished in anaphase I. We concluded
that Sgo2 alone plays a predominant role in protecting centromeric cohesion in meiosis I in oocytes, whereas Sgo1 is mostly, if not entirely, dispensable for this function. This result contrasts with what occurs during
mitotis, as Sgo1 is more important than Sgo2 for centromeric protection
during this type of division7,17.
Sgo2 cooperates with PP2A to protect Rec8
To determine the relationship between Sgo2 and the sister chromatid
cohesion complex, the localization of a meiosis-specific cohesin component, Rec8, was examined18,19. Rec8 localizes on metaphase I bivalents
to centromeres, as well as to the interchromatid axes (both proximal and
distal to chiasmata), but not at the chiasmata. This localization of Rec8 at
metaphase I is virtually the same in either Sgo1 or Sgo2-depleted oocytes
(Fig. 3a, b). Accordingly, ACA (anti-centromere antibodies) staining of
bivalents is indistinguishable between control and Sgo2-depleted oocytes
(see Supplementary Information, Fig. S3), verifying that sister chromatid
cohesion at centromeres is intact at this stage. By metaphase II, however,
Rec8 is removed from the chromosome arms and preserved only around
Sgo2-localizing regions in control univalents (Fig. 3b), confirming that
Rec8 is retained only near centromeres at metaphase II in oocytes20,21.
Consistent with the results obtained from chromosome spreads, Rec8
was preserved at centromeres in Sgo1-depleted oocytes, but had disappeared in Sgo2-depleted oocytes (Fig. 3a–c). Given that the removal of
Rec8 at the onset of anaphase I is dependent on the proteolytic activity
of separase in oocytes21, our results argue that Sgo2 has a crucial role in
protecting Rec8 at centromeres from cleavage by separase.
In mammalian somatic cells, Sgo2 has the ability to localize PP2A
to centromeres7. In agreement with these observations, the PP2Acatalytic subunit (PP2A-C) colocalized with Sgo2 on metaphase I
bivalents, as well as metaphase II univalents, in oocytes (Fig. 3d and
see Supplementary Information Fig. S4). This localization of PP2A
was not affected by Sgo1 depletion, but was markedly abolished
by Sgo2 depletion (Fig. 3d). Thus, the presence of PP2A at centromeres correlates well with the protection of centromeric cohesin
in meiosis I. Taken together with the observation that treatment of
oocytes with okadaic acid (a phosphatase inhibitor) induces premature separation of sister chromatids during meiosis I (ref. 22), our
results suggest that Sgo2 cooperates with PP2A to protect centromeric cohesin in oocytes, as it does in mammalian somatic cells or
yeast meiotic cells7,8.
nature cell biology volume 10 | number 1 | JANUARY 2008
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43
A RT I C L E S
100
d
828
Metaphase I
968 1040
1220
100
Percentage
Percentage
532
80
80
60
40
20
Metaphase II
612
600
**
517
**
60
40
20
0
siRNA: Control Sgo1
b
Single
Univalent
c
iRN
A
o2
s
Sg
siR
NA
o1
s
ol
Sgo2
Sg
ntr
Co
Sgo1
iRN
A
Bivalent
a
Sgo2
Sgo1
–Sgo2
0
siRNA: Control Sgo1 Sgo2
Metaphase I
Sgo1
–Sgo2
Metaphase II
Control
siRNA
Sgo1
siRNA
Control
siRNA
Sgo1
siRNA
Sgo2
siRNA
Sgo1–Sgo2
siRNA
Sgo2
siRNA
Sgo1–Sgo2
siRNA
Figure 2 Sgo2, but not Sgo1, is required for the protection of centromeric
cohesion in oocytes. (a) RT–PCR indicates that Sgo1 or Sgo2 mRNAs
are reduced in oocytes transfected with the corresponding siRNA. (b)
Chromosome spreads were prepared from oocytes that had been cultured
for 6 h (metaphase I) and 14 h (metaphase II) for maturation after
siRNA treatment. A typical chromosome in each chromosome spread is
magnified in the inset. Chromosome numbering refers to sequential order
and not karyotype nomenclature. (c) The spread chromosomes in b were
classified into bivalent, univalent and single (N = 4, 2 and 1; N represents
chromatid number). The bivalents observed in Sgo1 RNAi represent the
80 chromatids (20 bivalents) derived from only one metaphase I oocyte.
For further information about the effects of Sgo1 and Sgo2 depletion, see
Supplementary Fig. S2. n values are indicated above the bars. The double
asterisk indicates P <0.01. (d) A chromosome spread of an Sgo2 siRNAtreated oocyte at anaphase I. The sister chromatids in the oocyte were
precociously separated (arrow), showing 40 single chromatids instead of
20 univalents. The arrowhead indicates the chromosomes in the first polar
body. The scale bars represent 10 µm in b and d.
Sgo2 dissociates from Rec8 at metaphase II
The meiotic mode of Sgo2–PP2A function differs from the mitotic
one, in which cohesin is protected only from the prophase dissociation pathway, but not from cleavage at anaphase7. This difference
cannot be attributed simply to the variation in cohesin subunits
(that is, Rad21 versus Rec8), as centromeric Rec8 is protected from
cleavage only in meiosis I but not meiosis II, in spite of the presence of comparable levels of Sgo2 (and PP2A) at centromeres at both
stages (Fig. 1b and see Supplementary Information, Fig. S4). A similar discrepancy between presence and protection activity has been
observed for yeast and Drosophila shugoshin, as mere centromeric
localization of shugoshin at meiosis II or mitotic anaphase does not
impair separation of sister chromatids5,23,24. Possibly resolving this
discrepancy, recent observations of Sgo2 signals in spermatocytes
indicate a spatial change in Sgo2 localization around centromeres
between metaphase I and metaphase II25. We noticed a similar change
in Sgo2 distribution in oocytes: in metaphase I, the cohesion sites of
centromeres are embedded by the side-by-side configuration of sister
kinetochores, and Rec8 signals around centromeres always overlap
with Sgo2 (Fig. 3b). In contrast, in metaphase II, when sister kinetochores are pulled outward, centromeric Sgo2 localization often shifts
towards the kinetochores. Most importantly, at this stage, Rec8 is
44
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A RT I C L E S
Metaphase I
b
Metaphase II
Sgo1 Rec8 DNA
Rec8
Anaphase I
Sgo2 Rec8 DNA
Metaphase II
Sgo2 Rec8 DNA
Rec8
Control siRNA
Metaphase I
Sgo1 Rec8 DNA
Sgo2 siRNA
Sgo1 siRNA
Oocytes positive for
centromeric Rec8 at metaphase II
n = 36
n = 30
d
40
n = 32
**
20
0
RNAi:
Cont.
Sgo1
Sgo2
PP2A-C
Sgo1 DNA
PP2A-C
Sgo2 DNA
PP2A-C
PP2A-C
Control
siRNA
60
Control
siRNA
Percentage
80
Sgo2
siRNA
c
Sgo2
siRNA
Control siRNA
a
Figure 3 Inter-centromeric localization of Sgo2 and Rec8 at metaphase I
and metaphase II in oocytes. (a, b) Oocytes cultured for 6 h (metaphase I)
or 14 h (metaphase II) after siRNA treatment were fixed and stained with
anti-Rec8 antibody and either anti-Sgo1 (a) or anti-Sgo2 (b) antibodies.
The chromosomes aligned on the metaphase plate are viewed from the
lateral direction in general except in metaphase II plates in b. All images
were acquired by scanning one focus within the spherical oocytes so
that only centromeric Rec8 signals in focus could be detected, which
was necessary because of the low intensity. DNA was stained with TP3.
Magnifications of the boxed regions in b are shown. In anaphase 1, Rec8
is retained only at Sgo2 sites. (c) Metaphase II oocytes were evaluated
for the presence of Rec8 signals in one focus. The double asterisk
indicates P <0.01. (d) Oocytes treated with Sgo1 or Sgo2 siRNAs were
double-stained with anti-PP2A-C antibody and either anti-Sgo1 or antiSgo2 antibodies. DNA was stained with TP3. Representative stains of
metaphase I oocytes are shown. The scale bars represent 10 µm.
retained at the inner centromere where it presumably functions to
maintain centromeric cohesion (Fig. 4a), although previous observations in spermatocytes did not detect this Rec8 (ref. 25). The relocation of Sgo2 from the inner centromere towards the kinetochore
during metaphase II progression occurs in parallel with an increase
in sister kinetochore distance, an indicator of tension exerted across
centromeres (Fig. 4b). Provided that a close localization of shugoshin
with cohesin is required for its protection at the onset of anaphase,
these findings in oocytes, together with previous observations in
spermatocytes25, afford an excellent explanation for why Rec8 is protected only at the onset of anaphase I, but not anaphase II, in spite of
the presence of Sgo2 at both stages (Fig. 4c). This hypothesis predicts
that centromeric protection will be impaired even during meiosis I
if sister kinetochores are captured and pulled to opposite poles.
Consistently, a recent study of achiasmatic univalents in oocytes
reported that separation of sister chromatids frequently occurs once
they establish bipolar attachment26. Moreover, fission yeast mutants,
which break down monopolar attachment in metaphase I, fail to protect centromeric cohesion at anaphase I, although shugoshin and
meiotic cohesin are located and function at centromeres 27,28. Thus,
protection seems linked to mono-polar attachment.
division30. Therefore, the cleavage of cohesin by separase may have
a major role in triggering the separation of sister chromatids at the
onset of anaphase. Recognizing the change in shugoshin localization in oocytes at metaphase–anaphase II, we envisaged that the
relocation of shugoshin from cohesin may also occur prior to anaphase in mitosis, so as to end the action of protecting cohesin,
which could be a reason why shugoshins do not protect cohesin at
the onset of anaphase in mitosis. The redistribution of Sgo2 within
the mitotic centromere was recently reported7,17,25; however, its relative localization with cohesin, and the relevance to centromeric
protection, remain elusive. The distribution of shugoshins (not only
Sgo2, but also Sgo1 — a major protector in mitosis) and the cohesin
Rad21 in HeLa cells was examined by immunostaining. Both Sgo1
and Sgo2 mostly colocalized with cohesin at the inner centromere
during prometaphase, when they protect cohesin from the prophase
pathway (Fig. 5a, b). However, Sgo2 relocated towards kinetochores
in nearly half of the metaphase centromeres, whereas approximately 10% of centromeres exhibited Sgo1 relocation (Fig. 5c, d).
Importantly, shugoshins persisted at kinetochores in early anaphase
cells (Fig. 5a and data not shown), indicating that degradation or
extinction of shugoshins is not the primary reason for centromeric
deprotection. To focus on late metaphase or the end of metaphase,
mitotic cells were prepared and treated with the proteasome inhibitor MG132, which prevents anaphase onset. Consequently, these
cells were mostly arrested with chromosomes aligned at the spindle
equator. Remarkably, similarly to Sgo2, most Sgo1 signals relocated
Shugoshins relocate under tension in somatic cells
In somatic cells, cohesin is located throughout prophase and until
metaphase near centromeres, rather than along chromosome
arms9,29, and the expression of non-cleavable cohesin blocks nuclear
nature cell biology volume 10 | number 1 | JANUARY 2008
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A RT I C L E S
b
c
Metaphase–Anaphase I
Metaphase–Anaphase II
DNA
100
Ch
80
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Bub1
60
40
Rec8
20
Chromatid
0
Outer kinetochore
Sgo2–PP2A
**
**
**
Microtubules
eI
Me
(n = 67)
I
Me
An taph
ap
a
ha se–
se
II
Separase action
ha
s
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
ta
ma
ias
Metaphase –
Anaphase II
Metaphase
–Anaphase II
Sgo2 Bub1
I
Metaphase II
Metaphase II
tap
Early
metaphase II
Early metaphase II
me Ear
tap ly
ha
se
I
Sgo2 Rec8 DNA
Distance between
Pattern of Sgo2
sister kinetochores (µm) localization (percentage)
a
(n = 60)
(n = 69)
Figure 4 Sgo2 relocates towards kinetochores during metaphase II. (a) Oocytes
cultured for 12 h (early metaphase II) and 16 h (metaphase II), and those
further activated to anaphase II onset for 15 min (metaphase–anaphase II)
were stained for Sgo2 and Rec8 and typical images of metaphase II plates
viewed from the lateral direction are shown. (b) Similarly prepared oocytes
were stained for Sgo2 and Bub1. Spatial locations of Sgo2 were categorized as
centred between or separated close to kinetochores (pattern localization) and
the distance of Bub1 signals was measured in each categorized centromeres
(distance between sister kinetechores). The error bars represent s.e.m. The
double asterisk indicated P <0.01. Note that at either time point during
metaphase II, the kinetochore distance is significantly wider in centromeres
where Sgo2 signals are separated into kinetochore regions. (c) Schematic
representation of meiosis chromosome segregation and protein localization.
The scale bars represent 5 µm in a and b.
to the site adjacent to kinetochore (Fig. 5c and see Supplementary
Information, Fig. S5). Taken together, these data imply that both
Sgo1 and Sgo2 can relocate towards kinetochores until the end of
metaphase, yet before the onset of anaphase.
the dissociation of centromeric cohesion was impaired by the depletion
of Wapl (a protein that promotes the prophase pathway31,32), but not by
the depletion of separase (Fig. 6c). When nocodazole was added together
with MG132, cohesin localization (and tight cohesion) was preserved
at centromeres, as for treatment with nocodazole alone (Fig. 6a, b),
implying that the decrease of cohesin at centromeres was not the direct
consequence of MG132 treatment, but the consequence of the tension
across centromeres. Moreover, suppression of the spindle checkpoint by
Mad2 RNAi did not abrogate the nocodazole effect (see Supplementary
Information, Fig. S6). Taken together, these results argue that the tension
generated by spindle microtubules, rather than a time-dependent decay
of shugoshin localization or silencing of the spindle checkpoint, acts to
make shugoshins come apart from cohesin and thereby render inert the
protective function of shugoshins.
Deprotection of mitotic cohesin at metaphase
Given that both shugoshins are displaced from the inner centromere
during metaphase arrest, one could assume that cohesin is deprotected
and, therefore, would be targeted by the prophase pathway. This possibility was examined by staining cohesin together with centromeres.
Remarkably, cohesin at centromeres was largely diminished in MG132treated cells and the close association of sister kinetochores was impaired,
whereas cells treated with nocodazole (a microtubule-destabilizing drug)
showed preserved cohesin localization as well as tight centromeric cohesion, over the arrest period (Fig. 6a). As addition of nocodazole during
the last 1 h of MG132 treatment did not restore the close association
of centromeres, the enhanced centromeric distance in MG132-treated
cells is likely to result from looseness of cohesion, rather than merely
from the tension acting across centromeres attached to the spindle.
Sister chromatid cohesion was further examined by Giemsa staining of
spread chromosomes, which revealed impaired centromeric cohesion
in MG132-treated cells (Fig. 6b and see Supplementary Information,
Note S1). Given that MG132 treatment should prevent the biochemical
initiation of anaphase (including separase activation), this dissociation
of cohesin may be mainly mediated by the prophase pathway. In fact,
46
Tension-less anaphase preserves centromeric cohesin
The tension-dependent deprotection model described above makes
a key prediction that centromeric cohesin in mitosis will also be protected from separase cleavage if anaphase begins without tension
across sister centromeres, as in meiosis I. To examine this possibility,
cells were cultured in the presence of nocodazole, which prevents the
formation of the spindle and thus the tension across centromeres in
mitosis (Figs 5d and 6a). Under these conditions, although cells usually arrest at prometaphase because of the activation of the spindle
checkpoint, the depletion of Mad2 should override this arrest and
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A RT I C L E S
b
Rad21 Sgo1 ACA
Rad21 Sgo2 DNA
Rad21 Sgo2 ACA
Rad21 Sgo2 ACA
Metaphase
Rad21 Sgo1 ACA
Prometaphase
Rad21 Sgo1 DNA
Early
anaphase
Metaphase
Prometaphase
a
d
Sgo1 Sgo2 ACA
Sgo ACA
Sgo1 Sgo2 ACA
Sgo1
100
Percentage
Sgo2
80
60
40
20
13
2
MG
co
da
zo
Pro
le
me
tap
ha
se
Me
tap
ha
se
No
13
2
MG
MG132
Metaphase
No
co
da
zo
le
me
tap
ha
se
Me
tap
ha
se
0
Pro
Sgo1 Sgo2 DNA
Prometaphase
Nocodazole
c
Figure 5 Shugoshins relocate towards kinetochores during mitotic
metaphase. (a, b) Myc–Rad21-expressing HeLa cells were immunostained
with anti-Sgo1 (a) or anti-Sgo2 (b), anti-Myc and ACA antibodies. DNA
was counterstained with Hoechst 33342. Representative prometaphase
and metaphase images are shown. Enlarged views of representative sister
kinetochore pairs (boxed) are shown on the right. (c) HeLa cells were
mock-treated or treated with nocodazole or MG132 for 5 h, and then
immunostained with anti-Sgo1, anti-Sgo2 and anti-ACA antibodies. DNA
was counterstained with Hoechst 33342. Enlarged views of representative
sister kinetochore pairs (boxed) are shown on the right. (d) Spatial location
of Sgo1 or Sgo2 in c was categorized as centred between or separated
close to kinetochores (n = 45).
instead activate separase. We thus examined whether centromeric
cohesin is preserved in this ‘tension-less anaphase’. To identify the
anaphase stage of cells subjected to chromosome spread, Cyclin B
signals associated with centromeres were measured, which culminate during prometaphase or metaphase but decline at anaphase onset
similarly to cellular Cyclin B signals (Fig. 7a). Nocodazole-treated
control RNAi cells mostly arrested at prometaphase with preserved
cohesin at the centromeres, as well as Cyclin B (Fig. 7b). In contrast,
substantial amounts of Mad2 RNAi cells were in anaphase under the
same conditions, as judged from the extinction of Cyclin B signals,
but, remarkably, most of these cells preserved both Rad21 cohesin
signals and shugoshin signals between paired centromeres (Fig. 7b,
c). We concluded that, if tension does not act on centromeres and
therefore the colocalization of shugoshin and cohesin is preserved,
cohesin can be protected from separase cleavage at the centromeres,
even in mitosis.
DISCUSSiON
Our study reveals that the mammalian shugoshin Sgo2, which is essential
for protection from the prophase pathway in mitosis, also plays a crucial
role in oocytes to preserve centromeric cohesion throughout meiosis I,
a hallmark of reductional division. Together with previous observations
in yeast, flies and plants3, we have established that the mechanisms to
protect centromeric cohesion in meiosis are essentially conserved across
nature cell biology volume 10 | number 1 | JANUARY 2008
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A RT I C L E S
a
MG132+
nocodazole
Nocodazole
MG132
MG132+
1h nocodazole
b Control
Nocodazole
Rad21
ACA
DNA
MG132+nocodazole
No.of separated
centromeres
40
1–5
20
6–10
13
2
1–5
20
6–10
0
siRNA:
Wapl
Tubulin
MG
Co
Wapl
Cells with separated centromeres
60
No.of separated
centromeres
40
Separase
ntr
ol
Wa
pl
Co
Se
ntr
o
siRNA:
Wapl siRNA +MG132
Control
Tubulin
Separase siRNA +MG132
Percentage
Separase
Co
siRNA:
pa
ras
e
Control siRNA +MG132
l
c
ole
MG
no
co 132+
da
zo
le
>11
az
co
d
Cells with separated centromeres
0
No
No
60
20
0
co
d
az
ole
M
no G13
co
da 2+
zo
le
MG
13
2
1h MG1
no 32
co +
da
zo
le
0
40
l
20
60
ntr
o
40
80
No
co
d
60
Percentage
Percentage
Percentage
80
ACA separation
100
az
ole
M
no G13
co
da 2+
zo
le
MG
13
2
1h MG1
no 32
co +
da
zo
le
Cohesin-positive
kinetochores
100
MG132
>11
+MG132
Figure 6 The tension on bi-oriented mitotic chromosomes displaces
shugoshins from cohesin sites within the centromeres. (a) Mitotic cells were
treated with either or both nocadozole and MG132 for 5 h and spun onto
glass slides before immunostaining with anti-Rad21 (green) and ACA (red).
For MG132 + 1 h nocodazole, nocodazole was added in the last 1 h of the
MG132 treatment. DNA was counterstained with Hoechst 33342 (blue).
Representative pairs of sister kinetochores (boxed) are enlarged and shown
above each panel. The frequency of cohesin-positive kinetochores (n = 5
cells, >80 kinetochores in each cell) and of separated ACA signals (distance
>1.1 µm; n = 5 cells, 10 kinetochores in each cell) are also shown. The error
bars represent s.e.m. (b) Mitotic cells were treated with the indicated drugs
for 5 h and chromosome spreads were prepared. Cells were categorized
according to the number of separated centromeres and the frequency of
each category is presented (n = 30 cells). (c) HeLa cells treated with control,
separase and Wapl siRNA were synchronized at mitosis and treated with
MG132 for 5 h to prepare chromosome spreads. Cells were categorized as in
b (n = 30 cells). The efficiency of RNAi was confirmed by western blot. The
scale bars represent 10 µm in a–c.
eukaryotes, including mammals. Similarly to Sgo1, the centromeric
localization of Sgo2 is under the regulation of Bub1 (ref. 17; data not
shown), the expression of which reportedly decreases in aged human
oocytes33. Therefore, our findings raise the possibility that impairment
of Sgo2 function, which leads to precocious sister separation, could contribute to the birth defects that correlate with maternal age.
48
nature cell biology volume 10 | number 1 | JANUARY 2008
© 2008 Nature Publishing Group
Myc–Rad21 positive
15
Prometaphase–Metaphase
ACAMyc–Rad21 DNA
ACA
Myc-Rad21
Cyclin B
10
ACA
5
Myc–Rad21
0
Cyclin B
50
Number of cells
Myc–Rad21 negative
a
Number of cells
A RT I C L E S
Anaphase
40
30
ACA
20
Myc–Rad21
10
Cyclin B
0–
0 .2
0 .2
–0
.4
0. 4
–0
.6
0 .6
–0
.8
0. 8
–1
.0
1 .0
–1
.2
1. 2
–1
.4
1 .4
–1
.6
0
Intensity of Cyclin B signals (AU)
20
ACA Myc–Rad21 DNA
Control siRNA + nocodazole
ACA
Myc-Rad21
Cyclin B
15
ACA
10
Myc–Rad21
5
0
15
Cyclin B
Mad2 siRNA + nocodazole
10
ACA
5
Cyclin B
–0
.4
0.4
–0
.6
0.6
–0
.8
0.8
–1
.0
1.0
–1
.2
1.2
–1
.4
1.4
–1
.6
0.2
0–
0
Myc–Rad21
0.2
Myc–Rad21 positive
Number of cells
Number of cells
Myc–Rad21 negative
b
Anaphase
Prometaphase–Metaphase
c ACA Sgo1 DNA
ACA
Intensity of CyclinB signals (AU)
Sgo1
Cyclin B
ACA
Mad2 siRNA
+nocodazole
Sgo1
Cyclin B
Figure 7 Cohesin can be protected at the centromeres from separase
cleavage, even in somatic cells if tension does not act on the centromeres.
(a) HeLa cells expressing Myc–Rad21 were released from the thymidine
block, cultured for 12 h and spun onto glass slides before immunostaining
with ACA, anti-Myc and anti-Cyclin B antibodies. DNA was counterstained
with Hoechst 33342. Prometaphase, metaphase and anaphase cells were
examined for the intensity of Cyclin B signals relative to ACA signals at
centromeres. AU, arbitrary unit. Histograms show the frequency of Myc–
Rad21-staining positive or negative cells with the indicated intensities of
Cyclin B signals. Note that ~30% of cells do not express Myc–Rad21 in this
cell line. Representative prometaphase and anaphase images are shown.
Enlarged view of representative sister kinetochore pair or single kinetochore
(boxed) are shown on the right. (b) HeLa cells expressing Myc–Rad21 were
treated with control or Mad2 siRNA, released from thymidine-block and
cultured for 12 h (with addition of nocodazole in the last 1 h). Mitotic cells
with condensed chromosomes were examined as in a. Note that the Mad2
siRNA-treated culture contained Cyclin B-negative but Myc–Rad21-positive
mitotic cells, indicating that centromeric Myc–Rad21 was preserved in
anaphase. (c) Mad2 siRNA-treated Cyclin B-negative cells retain Sgo1 at
centromeres. The scale bars represent 10 µm in a–c.
Moreover, our analysis revealed that the dissociation of Sgo1 and
Sgo2 from cohesin occurs at centromeres under tension in mitotic
metaphase, as in meiotic metaphase II, which is crucial for the separation of chromatids at the following anaphase. To date, we do not know
how tension causes the relocation of shugoshins; however, it should
be noted that another inner-centromere protein, mitotic centromereassociated kinesin (MCAK), redistributes to the kinetochore region
under the regulation of tension and Aurora B phosphorylation34,35. This
raises the possibility that a similar regulation may act on shugoshins,
which are also in vitro substrates of Aurora B36 (data not shown). The
relative timing of relocation is different for the two shugoshins; Sgo1
comes apart later than Sgo2 at mitosis (Fig. 5c). This retardation of
Sgo1 relocation would be important in normal metaphase to prevent
premature separation of centromeres through the prophase pathway.
It is possible that the Sgo1 relocation requires much spindle force or
duration that culminates at the onset of anaphase; the prolonged metaphase arrest might mimic this situation. It is also possible that Sgo2
is solely responsible for the protection of cohesin from cleavage, as
in meiosis, whereas Sgo1 protects cohesin mainly from the prophase
pathway. Regardless, our findings clarify the previously unexplained
observation that centromere cohesion is substantially released at anaphase in APC-inactive cells, or even in cells expressing non-cleavable
nature cell biology volume 10 | number 1 | JANUARY 2008
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49
A RT I C L E S
cohesin37–39. Moreover, our findings explain why centromeric separation is so efficient at the metaphase–anaphase transition and why
some centromeres separate even earlier than the arms in mammalian
somatic cells39. Also, in budding yeast, the dissociation of centromeric
cohesin is triggered in a tension-dependent manner at metaphase40. As
centromeric cohesion is crucial for establishing bipolar attachment, but
deleterious at the following anaphase, the tension-dependent deprotection or dissociation of cohesin could be a fundamental mechanism to
reconcile this contradiction.
In summary, refining a previous hypothesis25, our analyses in
oocytes, combined with those in somatic cells, substantiate a unified view of the ability of mammalian shugoshin to protect cohesin.
Protection may be regulated by the intra-centromeric geometry of shugoshin relative to cohesin, which changes depending on the tension
between sister kinetochores. In either mitosis or meiosis, cohesin is
protected whenever kinetochores are not pulled in opposite directions;
thus, protection is largely released by the end of metaphase in mitosis
and only by the end of metaphase II, but not metaphase I, in meiosis
(Fig. 8). This view is consistent with the fact that centromeric cohesion
in mitosis and meiosis is regulated by essentially the same molecules
and mechanisms, at the centre of which stands the phosphorylation of
cohesin and its counteraction by shugoshin-associated PP2A7,8,11,41,42.
We propose that centromeric protection during reductional division
may have developed by preserving the tension-less situation observed
in mitotic prometaphase.
Methods
Northern-blot analysis. Total RNAs were isolated from adult mouse (C57BL/6)
organs using RNeasy kit (Qiagen, Hilden, Germany). Total RNAs (10 µg) were
separated in 1.2% agarose–2.2 M formaldehyde gel and blotted onto a nitrocellulose membrane filter (NitroPure; Osmonics, Minnetonka, MN). The filter was
prehybridized for 1 h and then hybridized at 42 °C for 16 h in 6× SSC, 50% formamide, 5× Denhardt’s solution, 120 µg sheared salmon sperm DNA per ml,
and 0.1% SDS with γ-32P dCTP-labelled Sgo1, Sgo2 or human glyceraldehyde-3
phosphate dehydrogenase (GAPDH) cDNAs. The filter was washed at 55 °C in
0.2× SSC buffer containing 0.1% SDS.
Culture of mouse oocytes. Collection and culture of mouse oocytes were
performed as previously described 20. Cell-cycle stage was judged by the
DNA-staining pattern. This study was approved by the Institutional Animal
Care and Use Committee (Permission number: 18-04-16) and carried out
according to the Guidelines of Animal Experimentation of Kobe University
(Kobe, Japan).
siRNA treatment of oocytes. As shugoshins were expressed already in germinal
vesicle oocytes, oocytes in were arrested in germinal vesicle stage by treating
with dibutyryl cyclic AMP (dbcAMP; Sigma, St Louis, MO) and 3-isobutyl-1methylxanthine (IBMX; Sigma) for 45 h before initiating oocyte maturation,
and siRNA was applied for the first 6 h of the arrest. Stealth siRNAs (Invitrogen,
Carsbad, CA) were used for the depletion of Sgo1 and Sgo2 (Sgo1-1 siRNA, CCC
TACTACACTGGATGGAGGTATT; Sgo2-1 siRNA, GGATAAAGACTTCCCA
GGAACTTTA). Virtually identical phenotypes (data not shown) were obtained
using another set of siRNAs (Sgo1-2 siRNA, GGGATTGGGAAACGCAAGT
CCTTTA; Sgo2-2 siRNA GAAGAGGAGGCAAACATATACGACA). Stealth
RNAi negative control (Medium GC, #45-2001) was used for control RNAi.
For the transfection of siRNA, the denuded oocytes were incubated in acid
Tyrode’s solution (137 mM NaCl, 2.7 mM KCl, 1.4 mM CaCl2·2H2O, 0.5 mM
MgCl2·6 H2O, 5.6 mM d-glucose and 0.1% polyvinylalcohol at pH 2.5, adjusted
with HCl) for several seconds to dissolve the zona pellucida. The oocytes were
then incubated with 200 nM siRNA and 0.6% (v/v) Lipofectoamine 2000
(Invitrogen) in Opti-MEM1 (Invitrogen) containing 0.1% PVA, 100 µg ml–1
dbcAMP and 100 µM IBMX for 6 h. After washing, the oocytes were cultured
50
Mitosis
Metaphase–Anaphase
Prometaphase
Metaphase–Anaphase I
Metaphase–Anaphase II
Meiosis
Outer kinetochore
Sgo1/Sgo2/PP2A
Chromatid
Cohesin
Microtubules
Figure 8 Schematic representation of a unified view of centromeric
protection of cohesin in mitosis and meiosis. Tension-dependent relocation
of shugoshin ends the protection of cohesin, which occurs at metaphase
in mitosis and metaphase II, but not metaphase I, in meiosis. This view
provides insight into the evolution of centromeric protection in meiosis;
the co-orientation of sister kinetochores at meiosis I extends the mitotic
prometaphase-like (tension-less) situation of sister kinetochores, thereby
enabling shugoshin to protect cohesin beyond meiosis I until bipolar
attachment at meiosis II.
for 39 h in mKSOM supplemented with 10% FCS instead of 3 mg ml–1 BSA,
200 µg ml–1 dbcAMP and 100 µM IBMX. The germinal vesicle state-arrested
oocytes were then further cultured until 14 h in mKSOM containing 10% FCS
for oocyte maturation.
Quantification of RNAi effects in oocytes by RT–PCR. Total RNA was
extracted from 30 oocytes that had been cultured for 45 h in the presence of
dbcAMP and IBMX, including 6 h of transfection of 200 nM of either control, Sgo1 or Sgo2 siRNAs using Absolutely RNA Microprep Kit (Stratagene,
La Jolla, CA). Single-stranded cDNAs were generated from the RNAs with
RETRO script (Ambion, Austin, TX) according to the manufacturer’s
instruction. The resultant cDNAs were used as templates for RT–PCR. cDNA
fragments of Sgo1 and Sgo2 were amplified for 32 and 30 cycles, respectively,
using specific primer sets. The PCR products were run on 1% agarose gels
and visualized by staining with ethidium bromide. The following primer
sets were used for the RT–PCR: Sgo1, 5´-ATGGCTAAGGAAAGGTGTCAG3´ (forward) and 5´-CTGTGTTTGCTTGGTTCTTCT-3´ (reverse);
Sgo2, 5´-TAGACTCTGGCTTAAGACAC-3´ (forward) and 5´TCTCCTCATCTTGCTTCTAAG-3´ (reverse).
Fixation and immunocytochemistry of oocytes. After culture, the oocytes
were fixed in 2% paraformaldehyde in KB buffer (20 mM Tris–HCl at pH 7.5,
150 mM NaCl). After treatment with 0.2% Triton X-100 in KB with 0.1%
BSA), the oocytes were washed in KB with BSA twice and stored for one
or two days at 4 °C. For detection of PP2A-C, the zona-free oocytes were
first transiently fixed in 2% paraformaldehyde in KB (1 min), followed by
immediate extraction and fixation of the oocytes in KB containing 2% paraformaldehyde and 0.2% Triton X-100 for 29 min. The fixed oocytes were
then incubated with primary antibodies at appropriate dilutions. After washing three times in KB with BSA, Alexa Fluor-labelled secondary antibodies
were used for the detection of signals. For the double labelling of Sgo1 and
Sgo2, the anti-Sgo2 antibody was conjugated with HiLyte Fluor 555 using
a labelling kit (Dojindo Molecular Technologies, Gaithersburg, MD. DNA
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© 2008 Nature Publishing Group
A RT I C L E S
was counterstained with either propidium iodide or TO-PRO-3 iodide. The
images were acquired by a Bio-Rad MicroRadiance confocal microscope 1024
system. Immunofluorescence staining of oocytes was performed using antiSgo1 (1:2,000), anti-Sgo2 (1:100,000), anti-Rec8 (1:100)20, anti-ACA (1:1,000;
gift from Y. Takasaki, Juntendo University School of Medicine, Tokyo, Japan),
anti-Bub1 (1:10; gift from S. S. Taylor) and anti-PP2A-C (1D6, 1:200; Upstate,
Lake Placid, NY) antibodies. Note that 50 times more diluted anti-Sgo2 antibody was used than anti-Sgo1 antibody, as Sgo2 is highly expressed in oocytes.
Secondary antibodies used were Alexa Fluor 488 anti-rabbit and anti-mouse
antibodies, Alexa Fluor 568 anti-mouse and anti-human antibodies, and
Alexa Fluor 555 anti-rabbit antibody (1:400; Invitrogen).
Antibodies. For production of polyclonal antibodies against mouse Sgo1, a
cDNA fragment encoding amino acids 1–248 of Sgo1 was inserted in frame
into the plasmid pGEX4T-1 (Amersham, Piscataway, NJ) to produce GST-fused
Sgo1. GST–Sgo1 was expressed in Escherichia coli BL21 and purified by glutathione–Sepharose 4B (Amersham), according to the manufacturer’s instructions.
GST–Sgo1 was used for immunization of a rabbit. Anti-Sgo1 antibodies were
affinity purified from the immunized serum by GST–Sgo1-coupled CNBractivated Sepaharose (Amersham) after removal of anti-GST by GST-coupled
Sepharose. For production of antibodies against mouse Sgo2, a cDNA fragment encoding amino acids 813–1164 was inserted in frame into the plasmids
pGEX4T-2 and pET19b (Novagen, Madison, WI) to produce GST- and Histagged Sgo2, respectively. Production of the recombinant Sgo2 proteins and
immunization of a rabbit with GST–Sgo2 were performed similarly. Antibodies
were affinity purified by His–Sgo2-coupled sepharose.
Preparation of chromosome spreads from mouse oocytes. Preparation of chromosome spreads from mouse oocytes was perfromed as previously described43.
Immunostaining, chromosome spreads and RNAi in HeLa cells.
Immunostaining of HeLa cells in Fig. 5a–c was performed after pre-extraction, as previously described29. For the immunostaining of HeLa cells in
Fig. 6a and Fig. 7, cells were spun onto glass slides by cytospin (Thermo electron Corporation, Waltham, MA) for 5 min at 254g before pre-extraction and
immunostaining. Primary antibodies were anti-hSgo1 (1:1000), anti-hSgo1
13G1 (gift from H. Suzuki, The Jikei University School of Medicine, Tokyo,
Japan; 1:100)44, anti-hSgo2 (1:2000), anti-Myc 9E10 (1:330; Santa Cruz, CA)
or anti-Myc (1:1000; Gramsch Laboratories, Schwabhausen, Germany), antiRad21 53A303 (1:100; Upstate,), anti-Cyclin B (1:1000; Santa Cruz) and ACA
(1:3000). Secondary antibodies were donkey anti-rabbit Alexa Fluor 488, donkey anti-mouse Alexa Fluor 488, goat anti-human Alexa Fluor 647 (Invitrogen)
and goat anti-human Cy3 (Jackson ImmunoResearch, West Grove, PA). DNA
was counterstained with 3 µg ml–1 Hoechst 33342. Giemsa staining of chromosome spreads was performed as previously described45. Nocodazole (Sigma) and
MG-132 (Calbiochem, La Jolla, CA) were used at 330 nM and 20 µM, respectively. RNAi against Mad2, separase or Wapl was performed as described previously31,32,46,47. For western blots, anti-Mad2 antibody (Covance, Princeton, NJ),
anti-separase antibody (XJ11-1B12; MBL, Nagoya, Japan) or anti-Wapl antibody
(gift from T. Hirano, RIKEN, Saitama, Japan) were used.
Note: Supplementary Information is available on the Nature Cell Biology website.
ACKNOWLEDGMENTS
We thank: S. Hauf for critical reading of the manuscript; T. Hirota for personal
communication; S. S. Taylor for the anti-mBub1 antibody; H. Suzuki for the antihSgo1 13G1 antibody; T. Hirano for the anti-Wapl antibody; and J.-M. Peters for
Myc–Rad21-expressing HeLa cells. This work was supported in part by: a Grantin-Aid for Scientific Research from the Japan Society for the Promotion of Science
to J.L. and T.S.K.; by 21st Century COE Programs to J.L., T. Miyano and M.M.; by
Ground-based Research Program for Space Utilization from Japan Space Forum
to K.Y. and T. Morita; and by the Toray Science Foundation and a Grant-in-Aid
for Specially Promoted Research from the Ministry of Education, Culture, Sports,
Science and Technology of Japan to Y.W.
Published online at http://www.nature.com/naturecellbiology/
Reprints and permissions information is available online at http://npg.nature.com/
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S U P P L E M E N TA R Y I N F O R M AT I O N
a
Intestine
Control
IgG
c
Sgo1
Testis
Sgo2
Sgo1
Sgo2
b
Thymus
1
Sgo1
3
S
2
2
S
1
3
Sgo2
4
4
Figure S1 The wild-type mouse (C57BL/6J) organs (intestine, thymus and
testis) were frozen in liquid nitrogen and sectioned at 5 µm on a cryostat.
Immunostaining was carried out using a polyclonal rabbit antibody that was
raised against Sgo1 and Sgo2 proteins. The primary antibody was used at
1:5,000 dilution, except Sgo1 staining of testis at 1:1,000 dilution. The
mixture was incubated at 4°C for 16 hr or overnight. Staining was developed by
using the Vectastain Elite ABC kit (Vector Laboratories, Burliname, CA) using
Diaminobenzidine. For counter staining, hematoxylin was used. (a) In the small
intestine, both Sgo1 and Sgo2 proteins are expressed in the crypts at the base
of the villi, which contain stem cells that continuously divide by mitosis. The
controls were stained without the first antibodies against Sgo1 and Sgo2. Bar
= 100 µm. (b) In thymus, both Sgo1 and Sgo2 proteins are expressed in the
immature T stem cells concentrated under the capsule in the outer cortex (1,
4). They are also expressed in a small number of round cells both in the inner
cortex (2) and medulla of thymus (3). Squares are enlarged at the right. Bar
= 100 µm. (c) Immunostaining of testis. Squares are enlarged at the bottom.
The cells in the outer layer of the seminiferous tubules are spermatogonia,
undergoing mitotic division (open arrow heads), whereas sperm heads are
observed near the centre of the tubules (S). Spermatocytes between these cells
are in meiotic stage (black arrow head). Bar = 50 µm.
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1
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S U P P L E M E N TA R Y I N F O R M AT I O N
a
b
6 h of maturation
Sgo2
14 h of maturation
Sgo1
Sgo2
Sgo1 Sgo2
DNA
Sgo2
RNAi
Metaphase II misalignment
Sgo1-Sgo2
RNAi
Sgo2
RNAi
Metaphase I
(%)
6 h of maturation
100
90
80
70
60
50
40
30
20
10
0
Sgo2 Sgo1-Sgo2
RNAi: Control Sgo1
(n = 38) (n = 31) (n = 25) (n = 25)
(%)
14 h of maturation
100
90
*
**
**
80
70
60
50
40
30
20
10
0
Control Sgo1 Sgo2 Sgo1-Sgo2
(n = 70) (n = 55) (n = 33) (n = 33)
Sgo1-Sgo2
RNAi
Sgo1/Sgo2
RNAi
c
Sgo1
RNAi
Sgo1
RNAi
Metaphase II
Control
RNAi
Control
RNAi
Sgo1
Sgo1 Sgo2
DNA
d
Metaphase I
Ana/Telophase I
Metaphase II
Mis-alignment
(*p < 0.05, **p < 0.01)
(%) Oocytes with metaphase II misalignment
100
90
80
70
60
50
40
30
20
10
0
+
Ĺ
Ĺ
2+
o1
o2
o1
go
g
S
Sg
S
Sg
(n = 10) (n = 44) (n =7) (n = 24)
Figure S2
Sgo1 RNAi
Figure S2 Mouse oocytes transfected with Sgo1, Sgo2 or Sgo1-Sgo2 siRNAs
were cultured for 6 h (a) or 14h (b) for maturation, fixed and labeled with
anti-Sgo1 and anti-Sgo2 antibodies. Sgo1 siRNA treatment diminished only
Sgo1 signals but not Sgo2 signals and vice versa, indicating that Sgo1 and
Sgo2 localize to centromeres independently. DNA was stained with TP3.
Bar = 10 µm. (c) The frequencies of oocytes at various cell cycle stages
and metaphase II chromosome misalignment are counted. Note that only
Sgo2 RNAi
metaphase I/II, but not prometaphase I/II, chromosomes are counted as
proper alignment. (d) The rate of metaphase II misalignment in Sgo1- or
Sgo2-depleted oocytes in c were classified by immunostaining into the Sgo
signal-positive (+) or negative (-) groups, and the rate of oocytes showing
metaphase II misalignment in each group is shown. In this assay, one
oocyte of Sgo1 RNAi and 2 of Sgo2 RNAi were still during meiosis I and
therefore excluded.
2
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S U P P L E M E N TA R Y I N F O R M AT I O N
Sgo2 RNAi
control RNAi
Sgo2 ACA DNA
Sgo2 ACA DNA
Metaphase I
Figure S3 Oocytes with or without depletion of Sgo2 were cultured for 6 h, fixed and stained with anti-Sgo2 antibody and ACA. DNA was counterstained with TP3 iodide. Bar = 10 µm.
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S U P P L E M E N TA R Y I N F O R M AT I O N
PP2A-C
Sgo2 DNA
PP2A-C
Sgo2
Figure S4 Oocytes cultured for 14 h were fixed and stained by anti-PP2A-C and anti-Sgo2 antibodies. DNA was counter-stained with TP3 iodide. Bar =
10 µm.
4
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© 2008 Nature Publishing Group
S U P P L E M E N TA R Y I N F O R M AT I O N
Sgo1
Sgo1 Sgo2 DNA
Sgo2
ACA
Transient
Sgo1 Sgo2 ACA
Complete
Figure S5 HeLa cells were treated with MG132 and immunostained with
anti-Sgo1, anti-Sgo2 and ACA antibodies, as in Figure 5c. This metaphase
cell shows an intermediate separation of centromeres, frequently showing
transient redistribution as well as complete redistribution of Sgo1 (enlarged
picturesat the right). This suggests that Sgo1 redistribution is the reason,
rather thanthe consequence, of centromeric separation. Bar = 10 µm.
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S U P P L E M E N TA R Y I N F O R M AT I O N
c
Mad2 RNAi
(%)
100
Separated ACA
80
60
40
20
Tubulin
0
MG
Mad2
13
2
M
+ n G1
oc 32
od
az
ole
Ma
d2
R
M
+n G1 NAi
oc 32
od
az
ole
control
a
b
MG132
MG132+nocodazole
Mad2 RNAi
MG132+nocodazole
ACA DNA
Figure S6 Cells were mock-treated or treated with Mad2 siRNA, incubated for
6 h, treated with2 mM thymidine for 24 hr, and released for 12 hr. Mitotic
cells were harvested byshake-off and treated with the indicated drugs for 5
h. (a) Cells were analyzed byWestern blot with anti-Mad2 and anti-tubulin
antibodies. (b) Cells were spun ontoglass slides and immunostained with ACA
(red). DNA was counterstained withHoechst 33342 (blue). Bar = 10 µm. (c)
The frequency of separated ACA signals(distance > 1.0 µm) is shown. Error
bars indicate s.e.m. (n = 5 cells, 10 kinetochoreswere scored in each cell).
6
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Note S1
It is notable that chromosomes in MG132-arrested cells stayed side-by-side even though
the prophase pathway may act on the whole chromosome length. This can be explained
by the recent finding that prophase pathway is not sufficient to completely remove
chromosomal cohesin on the arm region but requires basal activation of separase, which
can be blocked by MG132 treatment1). It is also notable that, in contrast to mitotic
metaphase arrest, centromeres do not separate at metaphase II arrest in spite of the
efficient relocation of shugoshins. In mouse oocytes, however, cohesin Rec8 is not
released from chromosome arms during the prolonged metaphase I arrest caused by the
inactivation of separase2), implying that the prophase pathway is already absent at the
stage of metaphase I and presumably metaphase II as well. This fact can explain why
centromeric Rec8 persists throughout metaphase II in spite of the relocation of Sgo2;
separase must be activated to remove chromosomal Rec8 in either metaphase I or
metaphase II stage.
References:
1) Nakajima, M., Kumada, K., Hatakeyama, K., Noda, T., Peters, JM. and Hirota, T., The
complete removal of cohesin from chromosome arms depends on separase J Cell Sci.
doi: 10.1242/jcs.011528 (2007)
2) Kudo, N et al., Resolution of chiasmata in oocytes requires separase-mediated
proteolysis Cell 126, 135-146 (2006)
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