Regulation of sororin by Cdk1-mediated
Megan R. Dreier, Michael E. Bekier 2nd and William R. Taylor*
Department of Biological Sciences, University of Toledo, 2801 W. Bancroft Street, MS 601, Toledo, OH 43606, USA
*Author for correspondence (firstname.lastname@example.org)
Accepted 17 May 2011
Journal of Cell Science 124, 2976–2987
? 2011. Published by The Company of Biologists Ltd
Tumor cells are commonly aneuploid, a condition contributing to cancer progression and drug resistance. Understanding how
chromatids are linked and separated at the appropriate time will help uncover the basis of aneuploidy and will shed light on the behavior
of tumor cells. Cohesion of sister chromatids is maintained by the multi-protein complex cohesin, consisting of Smc1, Smc3, Scc1 and
Scc3. Sororin associates with the cohesin complex and regulates the segregation of sister chromatids. Sororin is phosphorylated in
mitosis; however, the role of this modification is unclear. Here we show that mutation of potential cyclin-dependent kinase 1 (Cdk1)
phosphorylation sites leaves sororin stranded on chromosomes and bound to cohesin throughout mitosis. Sororin can be precipitated
from cell lysates with DNA–cellulose, and only the hypophosphorylated form of sororin shows this association. These results suggest
that phosphorylation of sororin causes its release from chromatin in mitosis. Also, the hypophosphorylated form of sororin increases
cohesion between sister chromatids, suggesting that phosphorylation of sororin by Cdk1 influences sister chromatid cohesion. Finally,
phosphorylation-deficient sororin can alleviate the mitotic block that occurs upon knockdown of endogenous sororin. This mitotic block
is abolished by ZM447439, an Aurora kinase inhibitor, suggesting that prematurely separated sister chromatids activate the spindle
assembly checkpoint through an Aurora kinase-dependent pathway.
Key words: Cell cycle, Cohesion, Sister chromatids, Spindle assembly checkpoint, Aneuploid
Cell cycle control mechanisms are altered in cancer cells, often
leadingto chromosomal instability
chromosome number. Several processes normally ensure that
chromosomes are segregated with high fidelity. For example, the
spindle assembly checkpoint (SAC), allows anaphase entry only
after all chromosomes acquire a bipolar attachment to the spindle
(Maresca and Salmon, 2010). Equal chromosome segregation to
daughter cells also strictly depends upon cohesion between sister
chromatids, allowing them to travel together and migrate to the
cell equator upon bipolar attachment to the spindle. The timing of
dissolution of cohesion at the metaphase to anaphase transition is
essential to ensure that chromosome segregation is temporally
coupled to mitotic exit and spatially coupled to cytokinesis
(Michaelis et al., 1997).
Cohesion of the sister chromatids is maintained in part by a
four-subunit complex, which consists of structural maintenance
of chromosomes 1 and 3 (Smc1, Smc3), the kleisin family protein
sister chromatid cohesin (Scc1) and the accessory subunit Scc3
(Haering et al., 2002; Michaelis et al., 1997). Pds5 and Wap1 are
weakly associated with the cohesin complex and regulate the
interaction of cohesin with chromatin (Panizza et al., 2000;
Shintomi and Hirano, 2009). Smc1 and Smc3 have ATP-binding
cassette (ABC)-like ATPases at one end of an extended coiled
coil. At the other end are the hinge domains that interact to create
V-shaped Smc1–Smc3 heterodimers (Haering et al., 2002). Scc1
binds to the Smc3 and Smc1 ATPase heads, creating a tripartite
ring that is 45 nm in diameter (Onn et al., 2008). Cohesin is
thought to associate with chromosomes by trapping DNA within
a monomeric ring (Haering et al., 2008). In vertebrates, cohesin
binds to DNA in telophase and continues to bind until anaphase
(Guacci et al., 1997; Sumara et al., 2000). Cohesin-bound
chromatin is found in two states: noncohesive and cohesive.
Because cohesin associates with chromatin before DNA
replication, cohesin first binds to chromatin in a noncohesive
During prophase and prometaphase, the majority of cohesin
dissociates from the chromosome arms in a process referred to as
the prophase pathway (Waizenegger et al., 2000). Polo-like
kinase 1 (Plk1) phosphorylates an isoform of Scc3 (SA1 or SA2),
causing release of cohesion from the arms (Hauf et al., 2005;
Sumara et al., 2002). Interestingly, some cohesin along the arms
is protected by shugoshin 1 (Sgo1) and is cleaved by separase
(Nakajima et al., 2007; Shintomi and Hirano, 2009). The Sgo1–
protein phosphatase 2A (PP2A) complex protects centromeric
cohesin by counteracting Plk1 until the kinetochores are captured
by the spindle microtubules (Kitajima et al., 2006). At the
complex/cyclosome (APC/C) causes the degradation of securin,
releasing separase. Plk1 phosphorylates Scc1 to facilitate its
cleavage by separase, which leads to the dissolution of cohesion
(Onn et al., 2008; Sumara et al., 2002).
Sororin was originally identified using a small pool expression
screen for proteins degraded by the APC/C in Xenopus laevis
extracts (Rankin et al., 2005). Suppression of sororin with small
interfering RNAs causes loss of sister chromatid cohesin,
implicating sororin in the maintenance of cohesion (Diaz-
Martinez et al., 2007; Rankin et al., 2005; Schmitz et al.,
Journal of Cell Science
2007). In fact, sororin is recruited to the cohesin complex during
S phase as a result of concurrent acetylation of SMC protein by
Eco2 (Lafont et al., 2010; Nishiyama et al., 2010). Once at the
cohesin complex, sororin displaces Wapl from Pds5 (Nishiyama
et al., 2010). Because Wapl is a cohesion destabilizer, the
recruitment of sororin to DNA during S phase helps to solidify
sister chromatid cohesion until mitosis. It is not clear how this
stabilizing effect is overcome during prophase to allow removal
of cohesin from chromosome arms. Here we demonstrate that
sororin is phosphorylated in response to cyclin-dependent kinase
1 (Cdk1) in vitro and in vivo. Interestingly, mutation of
potential Cdk1 phosphorylation sites in sororin creates a protein
that is unable to dissociate from chromosomes in mitosis.
sororin increases cohesion yet is still able to rescue a mitotic
arrest triggered by knockdown of the endogenous protein. These
observations suggest that phosphorylation of sororin by Cdk1
inhibits the ability of sororin to stabilize cohesion upon entry
Cdk1 phosphorylates sororin
Sororin extracted during mitosis exhibits a slower electrophoretic
mobility as a result of phosphorylation (Rankin et al., 2005). We
investigated the role of Cdk1, a kinase that is highly active in
mitosis, in this phosphorylation event. Two truncated forms of
sororin were fused to glutathione S-transferase (GST) and used
for in vitro kinase reactions. We identified a potential cyclin
interaction motif (CIM) in the sororin sequence and retained it in
our truncated forms of the protein. In other Cdk substrates, cyclin
binds to the CIM and recruits Cdk allowing more efficient
phosphorylation. Full-length, as well as both truncated forms of
sororin were phosphorylated by recombinant Cdk–cyclin B1 in
vitro (Fig. 1A,B). The full consensus for Cdk1 phosphorylation is
[S/T]Px[K/R], although some substrates simply contain a serine
or threonine followed by proline (Ubersax et al., 2003). Sororin
contains three sites that conform to the full consensus and six
others that are serines or threonines followed by prolines
(Fig. 1A). Mutation of one of the full-consensus sites (S21) to
alanine had no effect on phosphorylation. Phosphoproteomic
mapping data indicate that all nine of the potential Cdk1 sites
could be phosphorylated in vivo (Beausoleil et al., 2006; Cantin
et al., 2008; Chen et al., 2009; Dephoure et al., 2008; Gnad et al.,
2007; Olsen et al., 2006; Van Hoof et al., 2009). Therefore, we
mutated all nine serines/threonines followed by prolines to
alanines to further investigate the role of phosphorylation in
sororin function. (Throughout this paper, superscripts are used to
indicate which form of sororin is used, for example sororinWTfor
wild-type sororin and sororin9Afor the mutant in which all nine
serines or threonines followed by proline were mutated to
alanine.) As expected, GST–sororin9Awas poorly phosphorylated
by Cdk1–cyclin B1 in vitro (Fig. 1C).
To analyze sororin phosphorylation in vivo, we added a V5 tag
to the C-terminus of the sororin mutants shown in Fig. 1A and
used recombinant adenoviruses to overexpress a nuclear targeted
cyclin B1 (NB1) and a constitutively active mutant Cdk1-AF
(Cdk1 T14A Y15F) (Jin et al., 1998). HeLa M cells were blocked
in S phase with hydroxyurea and then infected with adenovirus to
express NB1 and Cdk1-AF. Western blotting showed that
overexpression of Cdk1–cyclin B1 reduced the mobility of
sororinWT–V5but not sororin9A–V5(Fig. 2A). This result suggests
that sororin is phosphorylated in response to Cdk1–cyclin B1.
Next, we tested the effect of mutating potential phosphorylation
sites on the mobility of sororin during mitosis. HeLa M cells were
transfected with sororin cDNAs and treated with nocodazole to
block the cells in mitosis. SororinWT–V5migrated as a doublet,
whereas sororin9A–V5failed to exhibit a mobility shift (Fig. 2B).
We also mutated each of the nine Cdk1 sites to alanine,
individually as well as in various combinations (Fig. 1A). Every
single and multiple mutant (excluding sororin9A–V5) showed a
mobility shift (supplementary material Fig. S1A,B). Thus,
phosphorylation of a number of sites appears to be responsible
for reducing the electrophoretic mobility of sororin from cells in
To further investigate the notion that Cdk1 is the kinase that
phosphorylates sororin, inhibitor studies were performed. HeLa
M cells were transfected with sororinWT–V5and treated with
nocodazole in the presence or absence of purvalanol A to inhibit
Cdk1. Extracts from cells treated with nocodazole exhibited a
mobility shift compared with untreated cells (Fig. 2C). Cells
treated with both nocodazole and purvalanol A contained less of
the slower migrating species (Fig. 2C). These results produce
further evidence that Cdk1–cyclin B1 is responsible for the
phosphorylation of sororin in vivo.
Phosphorylation-deficient sororin fails to be released from
chromatin during mitosis
To better understand the importance of phosphorylation, we
fused a GFP tag to the C-terminus of wild-type and mutant
sororin. GFP-tagged sororinWTand sororin9Awere first compared
by transfecting the constructs into HeLa M cells and performing
live-cell imaging. As previously observed, sororinWT–GFPwas
displaced from the chromatin and localized to the cytoplasm in
prometaphase but relocalized to the chromatin in anaphase
(Rankin et al., 2005). By contrast, sororin9A–GFPremained
localized to the chromatin throughout mitosis (Fig. 3A).
Continued imaging revealed that the intensity of both wild-type
and 9A sororin–GFP was rapidly reduced after telophase,
consistent with recognition by the APC/C (supplementary
material Fig. S2) (Rankin et al., 2005). In order to confirm that
sororin9A–GFPcolocalizes with chromatin in mitosis, we co-
transfected HeLa M cells with sororin–GFP and histone H2A–
RFP. Sororin9A–GFP, but not sororinWT–GFP, colocalized with
phosphorylation of sororin is necessary to remove the protein
from the chromatin (Fig. 3B). To quantify staining patterns, we
determined the ratio of sororinGFPon the chromatin to that in the
cytoplasm. Sororin9A–GFPwas significantly more enriched on
chromatin than sororinWT–GFPfrom G2 through metaphase,
whereas at anaphase similar staining was observed for both
sororinWT–GFPand sororin9A–GFP(Fig. 3C). For both forms of
sororin, we observed a large drop in chromatin enrichment as
cells entered prophase, consistent with the release of sororin into
the cytoplasm at nuclear envelope breakdown (Fig. 3C). This
observation also indicates that a proportion of sororin9A–GFPis
released from chromatin at the onset of mitosis.
Overexpression of sororinWT–GFPin HeLa M cells increased the
duration of mitosis when compared with cells transfected with
H2B–GFP alone (Fig. 3D). Sororin9A–GFPalso increased the
duration of mitosis, but not quite as efficiently as sororinWT–GFP.
In addition we observed a subtle but significant increase in the
percentage of cells with lagging chromosomes after overexpression
Phosphorylation of sororin 2977
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of sororinWTcompared with sororin9A(Fig. 3E, supplementary
material Fig. S3A). The average pixel intensities of sororinWT–GFP
and sororin9A–GFPin the live-cell image series were similar
(Fig. 3F). Therefore, the increase in lagging chromosomes, and
not expression level, could be responsible for the increased mitotic
duration in cells overexpressing sororinWT. We considered the
possibility that overexpression of sororin might alter the ratio of
phosphorylated to unphosphorylated endogenous sororin, thereby
altering chromosome dynamics and lengthening mitosis. After
transfecting HeLa M cells with sororin and treating cells with
nocodazole,immunoblotting wasperformed. There wasno apparent
difference in the ratio of phosphorylated to unphosphorylated
sororin when it was overexpressed (supplementary material Fig.
Multiple sites of phosphorylation contribute to chromatin
release of sororin
In order to identify the phosphorylation sites required for the
removal of sororin from chromosomes, HeLa M cells were
transiently transfected with a number of single and multiple point
mutants. Cells were then treated with nocodazole and the
localization of the sororin–GFP fusions was determined in live
Fig. 1. Cdk1 phosphorylates sororin.
(A) Diagram of wild-type and mutant
forms of sororin used in this study. Nine
potential sites of phosphorylation by
Cdk1 are indicated above the wild-type
sororin. Each one of these sites appears
to be phosphorylated in vivo, as
determined by proteomic analysis of
phosphopeptides isolated from cells
(information obtained from phosida and
phosphosite websites) (Beausoleil et al.,
2006; Cantin et al., 2008; Chen et al.,
2009; Dephoure et al., 2008; Gnad et al.,
2007; Olsen et al., 2006; Van Hoof et al.,
2009). Each of these nine sites has a
serine or threonine followed by proline
(minimal Cdk consensus), whereas sites
marked with an asterisk conform to the
full Cdk consensus ([S/T]Px[K/R]).
RDLEM is the potential CIM. Below the
wild-type sororin are the various
truncation and multi-site mutants of
sororin that were generated. In addition, a
single mutant at each of the nine sites
was generated (not shown).
(B) Phosphorylation of sororin by Cdk1
in vitro. Recombinant Cdk1–cyclin B1
was mixed with GST–sororin and a
phosphorylation reaction was carried out
in vitro with [32P]ATP. Reactions were
separated by SDS-PAGE and analyzed
by autoradiography. (C) Effect of serine
to alanine mutations on sororin
phosphorylation. Recombinant sororinWT
or sororin9Awith an N-terminal GST tag
was phosphorylated in an in vitro
reaction with Cdk1–cyclin B1 and
[32P]ATP. Reactions were analyzed by
autoradiography with histone H1 serving
as a positive control. CBB, Coomassie-
Journal of Cell Science 124 (17)2978
Journal of Cell Science
cells. Sororin8A–GFPand sororin9A–GFPboth localized to the
chromatin (Fig. 4A; sororin8Ais identical to sororin9Aexcept that
S209 is wild type). Similar results were obtained when we
created N-terminal GFP constructs of sororinWTand sororin9A,
suggesting that this effect is not due to the C-terminal tag (Dreier
and Taylor, unpublished data). Fixed cells expressing V5- or
GFP-tagged sororin9Ashowed only minimal colocalization of
sororin with chromatin. Therefore, analysis of subcellular
localization was carried out using C-terminal GFP tags in live
cells. All the single point mutants localized to the cytoplasm in
mitotic cells (Fig. 4A). Cells transfected with additional multi-
site mutants of sororin showed intermediate patterns of
(Fig. 4B). In order to quantify these patterns we captured
digital images of live GFP-transfected cells and measured the
standard deviation of pixel intensities within the bounds of a cell
[cells with uniform sororin staining have lower standard
deviations (Huang et al., 2009)]. The standard deviations was
then corrected by mean pixel intensity of the same cell and the
corrected standard deviation derived from many cells was
presented as an average value (Fig. 4C). One of the sororin
mutants with two Cdk1 sites mutated (2Aa) showed a slightly
higher corrected standard deviations compared with wild-type
sororin. Sororin3A–GFPand sororin6A–GFPshowed intermediate
sororin9A–GFP(Fig. 4C). We also measured the fold enrichment
as described for Fig. 3C, which indicated similar trends of
staining with the multiple mutants. Overall, these results suggest
that multiple sites of phosphorylation, possibly acting in an
additive manner, are required to remove sororin from the
Phosphorylation-deficient sororin shows increased
association with the cohesin complex
To further investigate the mechanism by which phosphorylation
controlstherelease of sororin
investigated the interaction between sororin and the cohesin
complex. We compared cells progressing through S to G2 phases
to those blocked in prometaphase with nocodazole (Fig. 5C).
Using coimmunoprecipitation, we observed that in prometaphase,
more sororin9A–V5than sororinWT–V5was immunoprecipitated
with SMC3 (Fig. 5A). In S–G2, more sororinWT–V5
associated with SMC3 than in prometaphase (Fig. 5A). Sororin
did not detectably change SMC3 levels in chromatin (Fig. 5B).
These results suggest that sororinWT–V5is associated with the
cohesin complex throughout S phase, but dissociates in
suggests that dissociation from the cohesin complex might be
triggered by Cdk1-mediated phosphorylation of sororin.
In order to obtain more insight into the association of sororin
with chromatin, we tested whether the protein could be
precipitated from cell lysates with DNA–cellulose. For those
experiments, HeLa M cells were transfected with wild-type or 9A
forms of sororin bearing C-terminal V5 tags. Cell lysates were
incubated with DNA–cellulose and the bound fractions were
analyzed by western blotting. Both sororinWT-V5and sororin9A–V5
associated with DNA–cellulose (Fig. 6A). Interestingly, only the
fast migrating form of sororinWT–V5was found in the bound
fraction, suggesting that phosphorylation reduces the association
of sororin with DNA–cellulose (Fig. 6B).
Effect of phosphorylation-deficient sororin on sister
We hypothesized that constitutive binding of sororin9Ato
chromosomes increases sister chromatid cohesion. To examine
sister chromatid cohesion in prometaphase, HeLa M cells were
transfected with V5-tagged sororin. Transiently transfected cells
were blocked in mitosis with nocodazole, and examined using
chromosome spreads. Cells transfected with sororin9A, but not
sororinWTshowed a significant increase in the number of cells with
closed arms (Fig. 7A,B). Chromosome spreads were prepared after
chromosomes on glass slides before staining. To test whether
by fixationand dryingof
Fig. 2. Electrophoretic mobility of sororin. HeLa M cells were transiently
transfected with sororinWT–V5or mutant forms of sororin and analyzed by
western blot to detect a mobility shift. (A) Overexpression of Cdk1–cyclin
induces a sororin mobility shift. HeLa M cells were blocked in S phase with
hydroxyurea and infected with recombinant adenovirus that expressed Cdk1
T14A Y15F (Cdk1-AF) and cyclin B1 fused to the nuclear targeting signal of
SV40 T antigen (NB1). Cell lysates were analyzed by western blotting with
an antibody to the V5 tag on sororin. (B) Effect of mutations on the
electrophoretic migration of sororin. HeLa M cells were transiently
transfected with sororin–V5 constructs expressing the indicated proteins.
Transfected cells were either left untreated or treated with nocodazole (noc)
for 20 hours to block them in mitosis. Cell lysates were separated on a 12.6%
SDS-polyacrylamide gel and analyzed by western blotting with an antibody to
the V5 tag. (C) Effect of purvalanol A (purv) on the phosphorylation of
sororin. Purvalanol A inhibits phosphorylation of sororin. HeLa M cells were
transiently transfected with sororinWT-V5, treated with nocodazole with and
without purvalanol A for 16 hours, and analyzed by western blotting.
Phosphorylation of sororin2979
Journal of Cell Science
cohesion is affected by sororin9A–V5in intact fixed cells, we
measured interkinetochore distances using immunofluorescence
(for example see supplementary material Fig. S3B). We observed
no significant difference in interkinetochore distances in cells
(Fig. 7C). Overall, these observations suggest that overexpression
of sororin9A–V5increases sister chromatid cohesion. The fact that
sororin9A–V5did not alter interkinetochore distances might mean
that sororin9A–V5stabilizes the complex during chromosome
preparation, but does not directly block the removal of cohesion.
Alternatively, sororin might be more important in regulating arm
cohesion than pericentromeric cohesion.
Phosphorylation-deficient sororin rescues mitotic arrest
induced by sororin knockdown
To investigate the effect of phosphorylation on the function of
sororin, we generated a short hairpin (sh) RNA to the 39UTR of
sororin, allowing us to reconstitute cells with either sororinWT–V5
or sororin9A–V5. Transiently transfecting HeLa M cells with
sororin shRNA caused sororin knockdown (Fig. 8A) and resulted
in a prometaphase arrest (Fig. 8B,C). Cells blocked in mitosis as
a result of sororin knockdown were characterized by misaligned
chromosomes that did not form a metaphase plate (Fig. 8B),
similar to previous reports (Diaz-Martinez et al., 2007; Schmitz
et al., 2007). Reconstituting with either sororinWT–V5
Fig. 3. Phosphorylation of sororin releases it from
chromatin during mitosis. (A) Localization of
sororin–GFP during mitosis. H2B–GFP, sororinWT–GFP
and sororin9A–GFPwere transiently transfected into
HeLa M cells. Time-lapse images of cells progressing
through mitosis as seen by fluorescence microscopy
(image interval: 12 minutes). Entry into mitosis was
indicated by release of sororin–GFP from the nucleus
(nuclear envelope breakdown; NEBD). The end of
mitosis was indicated by anaphase separation of
chromatids. As a control, cells were transiently
transfected with H2B–GFP to visualize DNA. In this
case, entry into mitosis was taken as the first frame in
which DNA condensation was visible.
(B) Sororin9A–GFPlocalizes to DNA in mitosis.
Histone H2A–RFP and sororin9A–GFPwere transiently
transfected into HeLa M cells. An example of a cell
showing colocalization of sororin9A–GFPand H2A–
RFP is shown. (C) Enrichment of sororinGFPon
chromatin. Frames from time-lapse microscopy were
used to quantify mean pixel intensities in a defined
region of the chromatin and compared with
cytoplasmic intensity of the same-sized region.
Because cells spent different amounts of time in
metaphase, only the first two metaphase frames were
quantified. Also, the onset of anaphase occurred at a
different time for each cell; this effect is indicated by
the dashed line. Averages of at least eight cells with
standard errors are shown. (D) SororinWT–V5and
sororin9A–V5increase the length of mitosis. HeLa M
cells were transiently transfected with either
sororinWT–GFPor sororin9A–GFP. Frames from time-
lapse microscopy were used to quantify the length of
mitosis for each cell. (E) Quantification of lagging
chromosomes. HeLa M cells were transfected with
either sororinWT–V5or sororin9A–V5, fixed and
analyzed by immunofluorescence with antibodies to
the V5 tag and to H2A phosphorylated at T121 to
indicate centromeres. V5-positive cells in metaphase
were assessed for the presence of chromosomes that
had not aligned at the metaphase plate. Values are
average percentages of cells with lagging
chromosomes; bars indicate ¡ s.e.m. (F) Average
expression level of either sororinWT–GFPor
sororin9A–GFPin live cells. HeLa M cells were
transfected with GFP-tagged sororin and visualized by
time-lapse fluorescence microscopy as in A. Single
frames of GFP-positive cells in metaphase were used to
measure average pixel intensities within the whole cell
(a.u., arbitrary units). Values are average from at least
28 cells for each condition; bars indicate ¡ s.e.m.
Journal of Cell Science 124 (17) 2980
Journal of Cell Science
sororin9A–V5reduced the percentage of cells in mitosis (Fig. 8C).
Also, knocking down sororin increased the percentage of cells
with more than one nucleus, presumably as a result of progress
through a defective mitosis. SororinWT–V5and sororin9A–V5were
similarly able to suppress the formation of multinucleated cells
when combined with the shRNA against sororin (Fig. 8C). These
results suggest that phosphorylation-deficient sororin retains
those activities of wild-type sororin required for cells to progress
The role of the spindle assembly checkpoint in mitotic
arrest after sororin knockdown
The mitotic block that occurs upon sororin knockdown is most
probably triggered by sister chromatids that separate prematurely
because of a lack of proper cohesion (Diaz-Martinez et al., 2007;
Rankin et al., 2005; Schmitz et al., 2007). Consistent with this
idea, transfecting HeLa M cells with sororin shRNA increased
the number of cells with separated sister chromatids (Fig. 9A,B).
In addition, chromosomes were approximately half as long after
sororin knockdown compared
(Fig. 9C). This shortening of chromosomes upon sororin
knockdown was previously attributed to hypercondensation
during a prolonged mitosis (Rankin et al., 2005). In order to
test the role of the SAC in the arrest that occurs upon sororin
knockdown, we determined the effect of the Aurora kinase
inhibitor, ZM447439, on the mitotic block. Cells transfected with
sororin shRNA spent ,10 hours in mitosis, with many cells
dying before being able to exit the block (Fig. 9D). When
sororin-shRNA-transfected cells were exposed to ZM447439, the
length of mitosis was reduced to ,1 hour (Fig. 9D). These results
suggest that single sister chromatids activate an Aurora-kinase-
dependent mitotic block. Consistent with these findings, we were
able to detect punctate staining of histone H3-like centromeric
protein (CENP-A), phosphorylated at Ser7, in sororin-shRNA-
transfected mitotic cells (Fig. 9E). These areas of staining were
closely associated with inner centromere protein (INCENP), a
with control chromosomes
Fig. 4. Multiple sites of phosphorylation
contribute to the release of sororin from
chromatin during prometaphase. SororinWT–GFP
and mutant forms of sororin were transiently
transfected into HeLa M cells. Transfected cells
were exposed to nocodazole to block them in
prometaphase. (A) Localization of single point
mutants of sororin. Representative cells transfected
with the indicated single point mutants of sororin
are shown. SororinT159A–GFPshowed undetectable
levels of fluorescence. (B) Localization of sororin–
GFP with multiple S/T to A mutations in HeLa M
cells. Specific mutations in each of the constructs
are shown in Fig. 1. (C) Staining uniformity of
various forms of sororin. To measure the uniformity
of staining, digital images were captured of live
transfected cells. The standard deviation (SD) of
pixel intensities was then determined for each cell.
This value was then corrected for the average pixel
intensity of each corresponding cell. This corrected
standard deviation was averaged over many cells
and is shown with standard errors indicated by bars.
The various mutants were compared with either
sororinWTor sororin9Ausing a Student’s t-test.
P-values are indicated in the table.
Phosphorylation of sororin2981
Journal of Cell Science
marker for the inner centromere. In the absence of the primary
antibody there was some modest cytoplasmic staining using the
secondary antibody for CENP-A detection (M.E.B., M.R.D. and
W.R.T., unpublished data). Phosphorylation of CENP-A at Ser7
is catalyzed by Aurora kinase, suggesting that chromatids that
have prematurely separated are able to recruit this kinase to
activate the SAC.
Sister chromatid cohesion is essential for faithful chromosome
segregation, keeping sister chromatids together until the exact
time at the metaphase–anaphase transition when the duplicated
genome is equally segregated to daughter cells. Chromosomes
that fail to disjoin contribute to aneuploidy, a condition
commonly found in tumor cells. The exact role of aneuploidy
in tumor progression is under debate (Schvartzman et al., 2010).
It is clear that without sister chromatid cohesion, segregation of
complex genomes would be impossible. Sister chromatid
cohesion in animal cells is mediated by mechanisms that
include chromatin catenation as well as the proteinaceous ring
cohesin (Skibbens, 2009; Uhlmann, 2004; Wang et al., 2010).
Sororin is a substrate of the APC/C with a key role in sister
chromatid cohesion (Rankin et al., 2005). High levels of sororin
added to Xenopus laevis extracts cause an increase in cohesin
association with metaphase chromosomes, which leads to failed
segregation of the sister chromatids (Rankin et al., 2005). In
human cells, sororin is essential for cohesion at G2 (Schmitz
et al., 2007). Depletion of sororin causes mitotic arrest and failed
sister chromatid cohesion, which is comparable to the phenotypes
observed upon depletion of shugoshin (Diaz-Martinez et al.,
Sororin is phosphorylated during mitosis, resulting in a
reduced electrophoretic mobility. Here we have investigated the
role of Cdk1 in sororin phosphorylation and have uncovered a
function of this modification in regulating the subcellular
localization of the protein. We expressed a GFP-tagged version
Fig. 5. Phosphorylation of sororin reduces its association
with the cohesin complex. SororinWTor sororin9A, which
contained C-terminal V5 epitope tags, were transiently
transfected into HeLa M cells. Some samples were
untransfected (UNT). The cells were then synchronized in S
phase with 2 mM thymidine for 24 hours. Thymidine was
removed and nocodazole was added for 14 hours to
synchronize the cells in mitosis. To prepare an S–G2
population, cells were released from the thymidine block for
6 hours. Chromatin was prepared as described in the
Materials and Methods. (A) Sororin9Aassociates with
chromatin in mitosis. Cohesin was immunoprecipitated with
an antibody to SMC3. Immune complexes were loaded onto
12.6% SDS-polyacrylamide gels and analyzed by western
blotting with an anti-V5 antibody. (B) Sororin does not alter
the levels of SMC3. Samples were prepared as in A and
loaded onto 12.6% SDS-polyacrylamide gels and analyzed by
western blotting with SMC3 and anti-V5 antibodies.
(C) Verification of the cell cycle stage for each of the cell
populations. Flow cytometry was performed using propidium
iodide-stained cells. Numbers of cells in G1, S or G2–M were
determined from flow cytometry. M, the mitotic index of
parallel cultures prepared by the chromosome dropping
method; AYSN, asynchronous; UNT, untransfected.
Journal of Cell Science 124 (17)2982
Journal of Cell Science
of sororin and found that it localizes to the nucleus of HeLa M
cells, and as previously observed, in mitosis disperses from the
chromatin and localizes throughout the cytoplasm. Sororin
contains nine residues that match the minimal Cdk consensus,
and one potential CIM. GST–sororin is phosphorylated by Cdk1,
and mutating the nine serines/threonines followed by proline to
alanines severely reduces this phosphorylation. When expressed
in HeLa M cells, all of the sororin mutants tested exhibited a
reduced electrophoretic mobility after nocodazole treatment,
except for sororin9A–V5. This suggests that many phosphorylation
sites contribute to the reduced electrophoretic mobility of sororin.
chromosomes and the cohesin complex throughout prometaphase,
whereas sororoinWT–GFPwas dispersed from chromosomes during
mitosis. Also, wefound that hypophosphorylated
precipitates with DNA–cellulose. These results suggest that Cdk1
phosphorylation of sororin releases sororin from the chromosomes
by weakening its interaction with the cohesin complex. The
precipitation of sororin with DNA–cellulose might indicate an
ability to bind to DNA. Alternatively, because these experiments
were carried out with cell lysates, sororin might indirectly bind to
DNA through the cohesin complex. In either case, phosphorylation
appears to influence this association. Sororin is targeted for
destruction by the APC/C. Consistent with this, we observed a loss
of sororin–GFP intensity after anaphase. The behavior of
sororin9A–GFP suggests that loss of phosphorylation at the nine
sites does not alter the kinetics of degradation.
In order to narrow down the phosphorylation sites that are
needed to remove sororin from the chromatin, we analyzed a
number of multi-site mutants of sororin fused to GFP and analyzed
their subcellular localization. Interestingly, sororin3A–GFPand
showed intermediatephenotypes between
Fig. 6. The nonphosphorylated form of sororin binds to DNA–cellulose.
(A) Association of sororin with DNA–cellulose. HeLa M cells were
transiently transfected with either sororinWT–V5or sororin9A–V5. UNT,
untransfected. Cells were blocked in mitosis by exposure to nocodazole for
16 hours. Cell lysates were incubated with DNA–cellulose for 16 hours and
the bound fraction was washed extensively. Proteins remaining associated
with the DNA–cellulose were analyzed by western blotting. An aliquot of the
lysate used for the binding reaction (Input) was also analyzed for comparison.
(B) Desitometric scans of the lanes. The fastest migrating nonspecific band
was used to register the scans.
Fig. 7. Sororin9Aalters sister chromatid cohesion. (A) Examples of
chromosome spreads with closed or open arms. SororinWT–V5and
sororin9A–V5were transfected into HeLa M cells. The cells were then treated
for 24 hours with 2 mM thymidine, after which the thymidine was washed off
and 100 ng/ml of nocodazole was added for 24 hours. Then chromosome
drops were performed. Images of Giemsa-stained cells are shown. Arrows
indicate sister chromatids. (B) Quantification of closed sister chromatids in
prometaphase. Cells were prepared as in A, and the percentage of spreads
where sister chromatids were closed was assessed. Values are averages ¡
s.e.m. (C) Measurement of interkinetochore distance. HeLa M cells were
transfected with sororinWT–V5or sororin9A–V5and analyzed by
immunofluorescence using antibodies to Hec1 and H2A phosphorylated at
T121 [H2A T121(P)]. Antibodies to the V5 tag were also used to identify
cells expressing the transfected sororin proteins. H2A T121(P) staining
indicates which Hec1-positive dots belong to sister chromatids (see
supplementary material Fig. S3B). At least 10 Hec1 pairs per cell were
analyzed and at least 14 cells were measured for each condition. Values are
averages ¡ s.e.m.
Phosphorylation of sororin 2983
Journal of Cell Science
sororinWT–GFPand sororin9A–GFP. By increasing the number of
sites that were blocked from phosphorylation, we observed a
graded increase in the association of sororin with chromosomes.
This observation suggests that each site of phosphorylation
contributes, in an additive manner, to the release of sororin from
chromosomes. Despite the ability of Cdk1 to induce sororin
phosphorylation, mutations that are expected to disrupt the
putative CIM (R134A; L134A) had little effect on the sororin
protein. SororinR134A;L134Awas released from chromatin in
mitosis, still rescued the mitotic arrest induced by sororin
knockdown and also exhibited an electrophoretic mobility shift
(R. Coffman, M.R.D. and W.R.T., unpublished data). These
observations suggest that Cdk1 phosphorylates sororin in a CIM-
Because sororin is involved in sister chromatid cohesion, high
levels of a non-phosphorylatable protein might cause defects in
cell division, such as impairing the segregation of chromosomes.
However, we observed that although overexpressing sororin9A–V5
increased the duration of mitosis, sororinWT–V5increased the
duration of mitosis even more. This difference might be related to
the fact that a higher frequency of sororinWT-transfected cells
contain lagging chromosomes than sororin9A-expressing cells. It
is not known how sororin overexpression increases the frequency
of lagging chromosomes. One possibility is that high levels of
phosphorylated sororin bind to a cytoplasmic target to interfere
with chromosome alignment. Sororin9Amight also induce this
effect by displacing a population of endogenous sororin from
chromosomes. In either case it appears that phosphorylation-
competent sororin is linked to the persistence of lagging
chromosomes in our studies. The lagging chromosomes we
observed were mainly only one or two chromosomes that had not
yet aligned to the metaphase plate. This phenotype could be a
result of delayed attachments or failure to resolve inappropriate
attachments of chromosomes to the spindle.
Sororin appears to antagonize the cohesin destabilizer Wapl
(Nishiyama et al., 2010). Phosphorylation of sororin by Cdk1
Fig. 8. SororinWT–V5and sororin9A–V5alleviate a mitotic arrest
induced by sororin knockdown. (A) Efficiency of shRNA knockdown
of sororin. HeLa M cells were transiently transfected with either a
plasmid that produced an shRNA targeting the 39UTR of sororin
(shRNA sororin) or empty pSUPER. Cell lysates were separated on
12.6% SDS-polyacrylamide gels and analyzed by western blotting with
an antibody to endogenous sororin. Actin served as a loading control.
(B) Defective metaphase plates after sororin knockdown. pSUPER,
shRNA sororin or shRNA sororin with sororinWT–V5were transiently
transfected into HeLa M cells. Sororin cDNA clones used in this study
lacked a 39UTR and are not targeted by the shRNA construct. H2B–
GFP was also transfected to visualize DNA. Examples of live mitotic
cells are shown. (C) Rescue of mitotic arrest by reconstituted sororin.
HeLa M cells were transfected simultaneously with a mixture of three
plasmids: (1) shRNA against sororin in pSUPER; (2) either
sororinWT–V5or sororin9A–V5in a mammalian expression construct; and
(3) H2B–GFP to visualize the DNA and to mark transfected cells.
Positively transfected cells were then quantified 3 days later to
determine whether they were in mitosis or interphase. Multinucleate
cells were also counted. Living cells were quantified to avoid loss of
mitotic cells during fixation.
Journal of Cell Science 124 (17)2984
Journal of Cell Science
during prometaphase might be required to neutralize sororin,
allowing Wapl to coordinate prophase removal of cohesin from
chromosome arms. Consistent with this idea, overexpression of
sororin9Aincreased sister chromatid cohesion, as assessed in
chromosome spreads. We also expected that the sororin9Amutant
might increase the frequency of chromosome nondisjunction at
anaphase; however, this defect was very rare and did not appear
to be exacerbated by overexpression of sororin9A(M.R.D. and
W.R.T., unpublished data). The fact that cells are able to progress
through anaphase in the presence of sororin9Amight be taken as
evidence that prophase removal of cohesin is not essential for
cells to separate chromosomes at anaphase. In support of this idea
is the observation that most cells in which Wapl levels are
reduced with RNAi still progress from prometaphase to anaphase
with normal kinetics, and show no major defects in chromosome
separation (Gandhi et al., 2006). This raises the question of the
physiological significance of the prophase pathway. Importantly,
cells transfected with Wapl RNAi, show a significant increase in
the percentage of multi-lobed nuclei, suggesting that mitosis is
defective in some cells. This suggests that cleavage of Scc1 by
separase isa dominant activity,
segregation even under conditions of suboptimal prophase
chromosome resolution. Prophase removal therefore, could be
most importantin supporting
segregation needed to avoid aneuploidy.
Phosphorylation by Cdk1 does not appear to be required to
activate sororin to carry out its role in establishing cohesion. This
latter point is supported by our observation that sororin9A–V5can
rescue the mitotic block that occurs upon sororin knockdown.
This is also consistent with the fact that sororin establishes
cohesin during interphase, when Cdk1 activity is low. The
mitotic block induced by sororin knockdown is probably
triggered by an accumulation of single sister chromatids (Diaz-
Martinez et al., 2007; Schmitz et al., 2007). These single
chromatids presumably trigger the SAC. Along these lines, we
have observed that inhibiting aurora kinases with ZM447439
abolishes the mitotic block induced by sororin knockdown.
Aurora B plays an essential role in sensing tension defects at the
high fidelity chromosome
Fig. 9. Knockdown of sororin causes activation of the
spindle assembly checkpoint. (A) Giemsa-staining of
chromosomes to show connected and separated
chromosomes. The ‘connected’ spread was from mock-
transfected HeLa M cells, and the ‘separated’ spread was
from HeLa M cells transfected with sororin shRNA.
(B) Chromatid separation after sororin knockdown. HeLa M
cells transfected with pBABEpuro (mock) or sororin shRNA
were analyzed by chromosome dropping 72 hours post-
transfection. (C) Sororin shRNA decreases chromosome
length. Cells were transfected as in B and chromosome
spreads were analyzed using Slidebook software to measure
chromosome lengths. (D) Effect of ZM447439 on mitotic
arrest induced by sororin knockdown. HeLa M cells were
co-transfected with H2B–GFP, used to mark transfected cells,
and with sororin shRNA. 72 hours later, the length of mitosis
of GFP-positive cells were determined by time-lapse
microscopy. In the +ZM sample, 2.5 mM ZM447439 was
added 72 hours post-transfection and filming began at the
time of drug addition. Values are averages ¡ s.e.m.
(E) Phosphorylated CENP-A in sororin knockdown cells.
HeLa M cells were transfected with sororin shRNA, and
72 hours later analyzed by immunofluorescence microscopy.
Cells were stained with antibodies to INCENP and CENP-A
phosphorylated at Ser7. Examples of mitotic cells are shown.
Phosphorylation of sororin2985
Journal of Cell Science
inner centromere and in triggering the SAC (Nezi and
Musacchio, 2009). Aurora B might be acting at the prematurely
separated sister chromatids to invoke the SAC-dependent arrest.
Along these lines, INCENP still localizes to a region near the
centromere after suppressing Scc1 with RNAi, although the
localization pattern is altered compared with control cells
(Sonoda et al., 2001). This localization of the chromosomal
passenger complex to separated sister centromeres might allow
Aurora B to constitutively phosphorylate kinetochore targets to
destabilize monotelic attachments and activate the SAC.
Consistent with this, CENP-A was still phosphorylated at Ser7
in sororin knockdown cells. The fact that sororin9A–V5can relieve
the mitotic block induced by sororin knockdown would be
consistent with an ability to establish sister chromatid cohesion.
Altogether, our experiments uncover an important role for Cdk1
phosphorylation of sororin in inhibiting its association with the
cohesin complex and inactivating its stabilization of cohesion
Materials and Methods
Cell line and culture conditions
HeLa M cells, a subline of HeLa (Tiwari et al., 1987), were incubated in a
humidified atmosphere containing 10% CO2 in Dulbecco’s modified Eagle’s
medium (Mediatech, Inc.) supplemented with 10% fetal bovine serum (Atlanta
Biologicals). All chemicals were from Sigma unless otherwise noted. We used
100 ng/ml nocodazole, 2 mM thymidine and 2.5 mM ZM447439 (AstraZeneca).
Transfections were carried out using Expressfect (Denville Scientific, Metuchen,
NJ), Fugene (Roche) or polyethyleneimine (Polysciences). A typical experiment in
a six-well plate would include 2–5 mg DNA, which was mixed with 50–100 ml of
DMEM and 6–15 ml polyethyleneimine (1 mg/ml) followed by a 15 minute
incubation before adding to cells. This was scaled up accordingly.
Cloning of sororin
mRNA was isolated from HeLa M cells using TRIzol (Invitrogen) according to the
manufacturer’s instructions, and reverse transcription-polymerase chain reaction
(RT-PCR) was carried out using enzymes from Promega. The primers are listed in
supplementary material Table S1.
To construct a C-terminal V5-tagged sororin protein, the PCR products were
cloned into the pcDNA 3.2 expression vector using TOPO cloning (Invitrogen).
All plasmids were confirmed by sequencing.
Coimmunoprecipitation and flow cytometry
Sororin was coimmunoprecipitated with cohesin from chromatin essentially as
described previously (Schmitz et al., 2007). SororinWT–V5and sororin9A–V5were
transiently transfected into HeLa M cells that were then treated with 2 mM
thymidine for 24 hours, arresting the cells in S phase. After thymidine was washed
off, cells were left either for 6 hours so that they could progress through S phase or
blocked in mitosis with nocodazole for 14 hours. Then, the cells were harvested
and lysed on ice for 20 minutes in IP buffer (20 mM Tris pH 7.7, 100 mM NaCl,
5 mM MgCl2, 0.1% Triton X-100, 10% glycerol) containing 1 mM dithiothreitol
(DTT), 1 mM NaF, and protease inhibitors (1 mg/ml aprotinin, 2 mg/ml leupeptin,
1 mg/ml pepstatin A). To pellet the chromatin, samples were spun for 10 minutes
at 16,000 g (4˚C). Pellets were solubilized by sonication and spun at 16,000 g for
10 minutes at 4˚C. The supernatant was digested with 0.6 IU/ml DNase (Fisher
BioReagents)for 20 minutesat37˚C
immunoprecipitation was performed with an antibody to SMC3 (Millipore)
coupled to protein A beads and immunoblotted with an antibody to V5 directly
conjugated to horseradish peroxidase (Bethyl Laboratories, Montgomery, TX). In
order to confirm the position in the cell cycle, cells were analyzed by flow
cytometry for DNA content, and by chromosome dropping (described below) to
quantify mitotic cells. For flow cytometry, cells were collected by trypsinization
and fixed in 70% ethanol. Cells were resuspended in PBS and stained with a
mixture of propidium iodide (50 mg/ml) and RNase A (5 mg/ml).
(Schmitzet al.,2007). The
HeLa M cells were scraped into PBS and then centrifuged (16,000 g, 4˚C) for
5 minutes and stored at –80˚C. Pellets were lysed by adding RIPA buffer containing
10 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 1% DOC, 0.1% SDS
(supplemented with 1 mg/ml aprotinin, 2 mg/ml leupeptin, 1 mg/ml pepstatin A,
1mM DTT,0.1M phenylmethylsulfonylfluoride,1 mM sodium fluorideand1 mM
4˚C (Dreier et al., 2009). To ensure equal loading, the protein concentration of each
lysate was determined using the BSA Protein Assay Kit (Pierce). Proteins were then
separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE) and the gels were transferred to polyvinylidene difluoride membranes
(Millipore). The membranes were then blocked for 1.5 hours with blocking buffer,
which consisted of 5% (w/v) non-fat dry milk dissolved in PBST [PBS containing
0.05% (v/v) Tween 20], and then incubated with primary antibody for 16 hours
(Rabbit-V5 or SMC3; Millipore). The membranes were then washed three times for
10 minutes in PBST. Goat anti-rabbit secondary antibodies conjugated to
horseradish peroxidase (Biorad) were used at a dilution of 1:10,000 in blocking
buffer for 1 hour. Membranes were washed again three times for 10 minutes in
PBST. Antibody binding was detected using an enhanced chemiluminescence kit
Analysis of truncated forms of sororin
We identified three exact matches to the Cdk consensus phosphorylation site and
one potential cyclin interaction motif (CIM) in the sororin sequence. Sororin
contains another six serines/threonines followed by proline that might be Cdk sites.
We engineered three different GST fusion proteins, each containing the CIM but
lacking a different group of potential phosphorylation sites. PCR, using primers
shown in supplementary material Table S1, was used to engineer the GST fusions
and the fragments were cloned into pGEX-3X that was digested with EcoRI and
BamHI. All plasmids were confirmed by sequencing. In vitro kinase assays were
conducted with each GST fusion protein purified from E. coli using glutathione
beads. GST fusions were incubated with [32P]ATP and purified Cdk1–cyclin B1
(Cell Signaling). The beads were washed and loaded onto an SDS-PAGE gel,
which was dried and exposed to film.
Generation of sororin point mutants
QuikChange multi site-directed mutagenesis (Stratagene) was carried out with
the primers shown in supplementary material Table S1 to create point mutations
in sororin that we had previously inserted into pcDNA3.2. Gateway cloning
(Invitrogen) was used to make GFP constructs with all the point mutants. For
this purpose, the mutants were first transferred into the donor vector pDONR
221 from pcDNA3.2. Then, mutants were transferred into destination vector
pcDNA-DEST47 to create C-terminal GFP fusions. All constructs were
confirmed by sequencing. In some experiments sororin was knocked down
using shRNA. For this purpose, a previously defined target sequence,
(Schmitz et al., 2007).
clonedinto pSUPER (Oligoengine)
HeLa M cells were blocked in mitosis with nocodazole, exposed to 0.075 M KCl
to swell the cells, and fixed with methanol:acetic acid (3:1 v/v) (Taylor et al.,
1999). The cells were dropped onto slides, briefly exposed to steam, and stained
with Giemsa. Chromosome morphology was determined by a person with no
knowledge of the sample origin, and included data from at least two independent
experiments performed in triplicate. To distinguish spreads with no sister
separation (‘single’) from those with centromere separation, two methods were
used. If a spread contained some chromosomes with sister chromatids still attached
and others that had completely separated, it was scored as ‘centromere separated’.
However, if a spread only contained single chromosome figures, chromosomes
were counted to distinguish full cohesion (,92 chromosomes for the tetraploid
HeLa genome) or full centromere separation (,184 chromosomes).
DNA binding assay
DNA–cellulose binding assays were performed essentially as described previously
(Klein et al., 2006). HeLa M cells were transfected with sororinWT–V5,
sororin9A–V5or pBABEpuro as a negative control using Expressfect (Denville).
At 1 day post-transfection, the cells were treated with nocodazole (100 ng/ml) for
16 hours. Cells were lysed (lysis buffer: PBS, 10% glycerol, 0.5% NP-40) and
protein concentrations determined using a Bradford assay (Pierce). DNA–cellulose
(12 mg) was suspended in 400 ml of protein binding buffer [50 mM Tris-HCl,
pH 8.0, 4 mM MgCl2, 1 mM DTT, 150 mM NaCl and 0.1% (w/v) Triton X-100],
spun, and washed twice. The DNA–cellulose was resuspended in 150 ml of protein
binding buffer and distributed into three 50 ml aliquots. Equilibrated lysate was
added to each tube of DNA–cellulose and the mixture incubated for 16 hours on a
rocker at 4˚C. Then, samples were spun at 1500 g for 2 minutes, supernatant was
aspirated, and the pellet was resuspended in 0.5 ml of protein binding buffer. This
wash was repeated three times. Bound proteins were separated by SDS-PAGE and
analyzed by western blotting with an antibody to the V5 tag (Millipore).
Immunofluorescence techniques were carried out as we have described previously
(Kaur et al., 2010). Briefly, cells on coverslips were given a brief wash with PBS,
fixed with 2% formaldehyde for 15 minutes, followed by permeabilization
[150 mM NaCl, 10 mM Tris (pH 7.7), 0.1% Triton X-100 and 0.1% BSA] for
9 minutes, and blocked with PBS containing 0.1% BSA for 1 hour at room
Journal of Cell Science 124 (17)2986
Journal of Cell Science
temperature. Lagging chromosomes were quantified in cells transfected with V5- Download full-text
tagged sororinWTor sororin9A. H2A phosphorylated at T121 [H2A T121(P);
AssaybioTech] was immunofluorescently stained, and lagging chromosomes were
identified by H2A T121(P)-positive centromeres associated with Hoechst 33342-
stained chromatin. Only V5-positive cells were included in the analysis.
Interkinetochore distances were obtained for cells transfected with V5-tagged
sororin. Cells were simultaneously stained with antibodies to Hec1 (Abcam) to
mark the kinetochore, H2A T121(P), to mark the inner centromere, and V5 to
identify sororin. Hec1-positive dots on either side of a H2A T121(P)-positive dot
identified sister kinetochores (supplementary material Fig. S3B). Phosphorylated
S7 CENP-A was detected with a polyclonal antibody (Cell Signaling).
Magnification of images were kept the same within each figure panel.
This work was supported by NIH grants R15GM084410-01
and R15GM073758-01 to W.R.T. and Sullivan Fellowship and
Undergraduate Research and Creative Activity Program Fellowship
to M.R.D. Deposited in PMC for release after 12 months.
Supplementary material available online at
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