4168
Research Article
Studies of haspin-depleted cells reveal that spindlepole integrity in mitosis requires chromosome
cohesion
Jun Dai*,‡, Anna V. Kateneva and Jonathan M. G. Higgins*
Division of Rheumatology, Immunology and Allergy, Brigham & Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
*Authors for correspondence ([email protected]; [email protected])
‡
Current address: Massachusetts General Hospital, Cutaneous Biology Research Center, Charlestown, MA 02129, USA
Journal of Cell Science
Accepted 10 September 2009
Journal of Cell Science 122, 4168-4176 Published by The Company of Biologists 2009
doi:10.1242/jcs.054122
Summary
Cohesins and their regulators are vital for normal chromosome
cohesion and segregation. A number of cohesion proteins have
also been localized to centrosomes and proposed to function
there. We show that RNAi-mediated depletion of factors
required for cohesion, including haspin, Sgo1 and Scc1, leads
to the generation of multiple acentriolar centrosome-like foci
and disruption of spindle structure in mitosis. Live-cell imaging
reveals that, in haspin-depleted cells, these effects occur only
as defects in chromosome cohesion become manifest, and they
require ongoing microtubule dynamics and kinesin-5 (also
known as Eg5) activity. Inhibition of topoisomerase II in
mitosis, which prevents decatenation and separation of
chromatids, circumvents the loss of cohesion and restores
Introduction
Proteins of the cohesin system provide cohesion between replicated
chromosomes, regulate cohesion establishment during S phase and
after DNA damage, and dissolve cohesion in a regulated manner
during mitosis (Peters et al., 2008). The finding that Separase, a
protease that cleaves cohesin Scc1 (also known as Rad21), is also
required for centriole disengagement upon mitotic exit (Tsou and
Stearns, 2006) suggests that other elements of the cohesin system
might operate at centrosomes. In fact, a number of proteins that
influence chromosome cohesion, including Separase, Scc1, SMC1,
SA1 and haspin, have been reported to localize to centrosomes
(Chestukhin et al., 2003; Dai et al., 2005; Gregson et al., 2001;
Guan et al., 2008; Wang et al., 2008; Warren et al., 2000; Wong
and Blobel, 2008) and it has been suggested that at least some of
these proteins play a direct role in centrosome function. For
example, human SMC1 has been reported to associate with NuMA
at centrosomes, and immunodepletion of human SMC1 reduced the
efficiency of mitotic aster formation in a HeLa cell extract system
(Gregson et al., 2001), whereas its overexpression induced
multipolar spindles in HeLa cells (Wong and Blobel, 2008).
Furthermore, a splice variant of the cohesin protector Sgo1 is able
to reduce the number of extra centrosome-like foci induced by Sgo1
RNAi in human cells (Wang et al., 2008). These observations have
stimulated interest in the function of proteins of the cohesin system
at centrosomes.
Haspin (also known as Gsg2) is a chromosomal kinase that
phosphorylates histone H3 at threonine 3 (H3T3ph) and is required
for normal chromosome cohesion during mitosis (Dai et al., 2006;
Dai et al., 2005). Enhanced green fluorescent protein (EGFP)-haspin
integrity of the spindle poles. Although these results do not rule
out roles for cohesin proteins at centrosomes, they suggest that
when cohesion is compromised, spindle-pole integrity can be
disrupted as an indirect consequence of the failure to properly
integrate chromosome- and centrosome-initiated pathways for
spindle formation.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/122/22/4168/DC1
Key words: Chromosome cohesion, Centrosome, Mitosis, Gsg2
Spindle assembly, Kinesin-5
also localizes to spindle poles in mitosis (Dai et al., 2005). This
prompted us to examine the effect of haspin RNAi on spindle
formation. Although we observed spindle defects, our results argue
that they are largely an indirect consequence of loss of chromosome
cohesion, rather than being caused by a local failure of centrosome
function.
Results
Haspin depletion increases the number of centrosome-like foci
in mitosis
First, we confirmed by immunoblot analysis that haspin siRNA
decreased haspin protein levels in U2OS cells (supplementary
material Fig. S1A). After transfection with haspin siRNA, 76% of
mitotic U2OS cells had low levels of H3T3ph and extensive
chromosome misalignment (Fig. 1A,B), consistent with our previous
findings (Dai et al., 2006; Dai et al., 2005). In cells with misaligned
chromosomes, the majority of microtubules were incorporated into
bipolar spindles but additional small clusters of short microtubules
were noted adjacent to the fusiform spindles. These clusters were
always associated with foci of g-tubulin (Fig. 1A,D). Sixty-seven
percent of mitotic haspin-depleted cells contained three or more
foci of g-tubulin, whereas 92% of control siRNA-transfected cells
contained only two foci of g-tubulin (Fig. 1C). Similar increases in
numbers of g-tubulin foci were noted in HeLa cells transfected with
haspin siRNA (see later). Therefore, depletion of haspin caused
centrosomal defects in mitosis that correlated with the presence of
chromosome misalignment.
During mitosis, g-tubulin is a major microtubule-nucleating
component of the pericentriolar material (PCM) that surrounds the
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Fig. 1. Haspin depletion disrupts chromosome alignment and formation of normal spindles. (A)U2OS cells transfected with control or haspin siRNAs were stained
with antibodies to g-tubulin, -tubulin and H3T3ph, and with the DNA dye Hoechst 33342, and subjected to immunofluorescence microscopy. (B)Mitotic cells
treated as in A were classified into mitotic stages (prometaphase/metaphase or anaphase/telophase), or as chromosomes misaligned (a dominant bipolar spindle and
partial metaphase plate present, but other chromosomes found near the poles). (C-D)U2OS cells transfected with control siRNA (Da) or haspin siRNA (Db-e) were
stained with antibodies to g-tubulin and -tubulin, and with the DNA dye DRAQ5 and subjected to immunofluorescence microscopy to determine the number of
g-tubulin foci present per mitotic cell. (C)Over 100 cells were counted for each sample. Means ± s.d. are shown, n3; **P<0.01, ***P<0.001 by Student’s t-test.
Scale bars: 10m.
paired centrioles of each centrosome. g-tubulin dynamically
exchanges with a non-centrosomal pool (Khodjakov and Rieder,
1999) and it contributes to nucleation of microtubules initiated from
chromosomes and other microtubules during spindle formation
(Luders et al., 2006; Mahoney et al., 2006; Wilde and Zheng, 1999).
One possible cause of the multiple g-tubulin foci in haspin-depleted
cells is the presence of additional centrosomes due to overamplification in interphase or a failure of cytokinesis in a previous
cell cycle. However, there was no change in the number of g-tubulin
foci observed at interphase or mitotic prophase (data not shown),
and there was no significant increase in the number of mitotic cells
with more than four centrioles following haspin depletion (Fig.
2A,B), suggesting that neither increased centrosome numbers nor
centriole fragmentation were responsible. Furthermore, the average
fluorescence intensity of individual g-tubulin foci in haspin-depleted
mitotic cells was only one third that of bona fide centrosomes in
control cells (supplementary material Fig. S1B). Together, these
findings suggested that, following haspin depletion, g-tubulin is
redistributed to multiple foci that arise during mitosis.
To further characterize the extra foci, we examined the
distribution of additional proteins known to be components of the
PCM. In haspin-depleted cells, all g-tubulin foci contained
pericentrin (Fig. 2C) and Aurora A (see below), suggesting that
they are PCM-like structures. We also examined the distribution of
centrioles in haspin-depleted cells using the marker centrin (Paoletti
et al., 1996). Depending on centrosome orientation, a normal mitotic
cell has up to four visible distinct centrin dots (corresponding to
centrioles) that are tightly paired at the two centrosomes. Haspin
depletion caused a modest degree of centriole disengagement (Fig.
2Aa,2B), as evidenced by an increase in the percentage of mitotic
cells possessing three or four separate centrin-containing foci (24%
in haspin-depleted cells versus 7% in controls; P0.01, Student’s
t-test). However, this centriole disengagement was insufficient to
account for the presence of three or more g-tubulin foci in nearly
70% of haspin-depleted mitotic cells (Fig. 2A,B; note the existence
of g-tubulin foci that do not contain centrin).
Ectopic centrosome-like foci can nucleate microtubule asters
but do not prevent formation of a dominant bipolar spindle
A notable feature of haspin-depleted cells was the essentially
universal presence of an identifiable bipolar spindle, even when the
intensity of g-tubulin foci at the bipolar spindle poles was weaker
than at the other ectopic foci (Figs 1 and 2). In fact, in some cells,
neither g-tubulin nor centrin staining was evident at some bipolar
spindle poles (Fig. 1De, Fig. 2Ac,d,2B). This raised the issue of
what distinguished the dominant spindle poles in haspin-depleted
cells containing multiple centrosome-like foci.
The formation of focused bipolar spindles relies on microtubuleassociated motor and non-motor proteins (Manning and Compton,
2007). We asked whether three such proteins, TPX2, NuMA and
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Fig. 2. Haspin depletion causes formation of extra centrosome-like foci. (A)U2OS cells transfected with control (Aa) or haspin siRNAs (Ab-d) were stained with
antibodies to g-tubulin, -tubulin and centrin, and with the DNA dye Hoechst 33342, and subjected to immunofluorescence microscopy. (B)Mitotic cells treated as
in A were classified according to the number and location of g-tubulin- and centrin-containing foci. Means ± s.d. are shown, n3. Over 100 cells were counted for
each sample. (C)U2OS cells transfected with control or haspin siRNAs were stained with antibodies to pericentrin and g-tubulin, and with the DNA dye DRAQ5,
and subjected to immunofluorescence microscopy. Scale bars: 5m.
Aurora A, were recruited equally to ectopic g-tubulin foci and the
major bipolar spindle poles after haspin depletion. TPX2 is a key
mediator of the Ran-signaling pathway that localizes to spindle
microtubules and is required for microtubule nucleation near
kinetochores and for normal spindle formation (Bird and Hyman,
2008; Gruss et al., 2002; Tulu et al., 2006). In cells depleted of
haspin, microtubules at ectopic g-tubulin foci were able to recruit
small amounts of TPX2, but most TPX2 was associated with the
dominant bipolar spindle (Fig. 3A). Similarly, haspin depletion
showed little effect on localization of the microtubule minus-end
crosslinker NuMA to the dominant spindle pole microtubules,
although relatively low levels of NuMA were also apparent at
ectopic g-tubulin foci (Fig. 3B). The kinase Aurora A has been
implicated in both centrosome and chromosome-initiated
microtubule nucleation (Sardon et al., 2008; Tsai and Zheng, 2005).
In contrast to TPX2 and NuMA, Aurora A is localized in the PCM
of centrosomes as well as at the minus ends of spindle microtubules.
In haspin-depleted cells we found that, like TPX2 and NuMA,
Aurora A was localized on microtubules at the two poles of the
dominant bipolar spindle, even when no g-tubulin foci were
detectable at these poles. At the same time, the association of Aurora
A with multiple g-tubulin foci was more apparent than that of TPX2
and NuMA, confirming that the ectopic poles contain PCM
components, including Aurora A (Fig. 3C). Therefore, the primary
localization of TPX2 and NuMA and of microtubule-associated
Aurora A to the dominant bipolar spindle was unaffected in cells
depleted of haspin, despite the presence of ectopic centrosome-like
foci.
The above findings raised the question of whether the ectopic gtubulin foci had intrinsically low capacity to focus large microtubule
asters. To address this question, we performed microtubule regrowth
assays in which spindle disassembly by cold treatment for 1 hour
was followed by regrowth at 37°C for 2 minutes. In haspin-siRNAtransfected cells, each of the multiple g-tubulin foci was able to
nucleate microtubule asters with similar efficiency, providing no
evidence that ectopic foci were functionally compromised (Fig. 3D).
In addition, multiple g-tubulin foci were able to support the
formation of cold-stable kinetochore microtubules in haspindepleted cells (supplementary material Fig. S2). These findings led
us to ask why the presence of multiple functional centrosome-like
foci did not prevent the formation of a dominant bipolar spindle.
In particular, we wished to determine at what point during mitosis
the multiple g-tubulin foci became apparent.
Centrosome defects occur as chromosome cohesion is
compromised
To monitor the dynamics of spindle poles and chromosome
misalignment in mitosis, we generated a stable U2OS cell line
expressing g-tubulin-EGFP and histone H2B-mRFP (monomeric red
fluorescent protein) and performed live-cell imaging. All observed
control siRNA-treated cells underwent normal mitosis (Fig. 4A,
supplementary material Movie 1), whereas cells that had been
exposed to haspin siRNA fell into two groups: Group I cells
completed a near-normal mitosis (supplementary material Movie
2), whereas group II cells that were presumably more fully depleted
of haspin failed to undergo normal anaphase (Fig. 4B,
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Fig. 3. Cells depleted of haspin possess a dominant bipolar spindle and extra centrosome-like foci that can nucleate microtubules. (A-C)U2OS cells transfected
with control or haspin siRNAs were stained with the DNA dye DRAQ5 and antibodies to (A) g-tubulin and TPX2, (B) g-tubulin and NuMA, (C) g-tubulin and
Aurora A, and subjected to immunofluorescence microscopy. (D)Microtubule regrowth assays, in which spindle disassembly by cold treatment for 1 hour was
followed by regrowth at 37°C for 2 minutes, were carried out on U2OS cells transfected with control or haspin siRNAs. The cells were then stained with antibodies
to g-tubulin, -tubulin and H3T3ph, and with the DNA dye Hoechst 33342, and subjected to immunofluorescence microscopy. Scale bars: 5m.
supplementary material Movie 3 and Fig. S3). In both haspindepleted groups, prophase cells contained two centrosomes that
accumulated g-tubulin and moved to opposite poles, as in controls.
Chromosomes also condensed and congregated at a metaphase plate,
although congression took somewhat longer than normal in haspindepleted cells (28±12 and 46±23 minutes for group I and II cells,
respectively, compared to 19±3 minutes for controls; Fig. 4C).
Control and group I cells then underwent apparently normal
anaphase after 19±3 minutes and 23±9 minutes in metaphase,
respectively. By contrast, group II haspin-depleted cells remained
at a metaphase-like stage for 1-4 hours (Fig. 4D). Following this,
small numbers of chromosomes began to move away from the plate.
At this time, extra g-tubulin foci started to appear. Increasing
numbers of chromosomes then became misaligned until no clear
metaphase plate existed, and the g-tubulin foci appeared to increase
in number and mobility and to decrease in intensity. At times, there
was no obvious g-tubulin focus at at least one of the presumptive
poles. Cells remained in this state for 6-12 hours. Finally, there was
an anaphase-like movement of unequal clusters of chromosomes
to opposite poles of the cell that was quickly followed by an abortive
attempt at cytokinesis as the cells presumably exited mitosis and
the chromosomes decondensed to form micronuclei (see Fig. 4B,
535-550 minutes; supplementary material Movie 3).
As described for cells defective in other cohesion factors (see
Discussion), it appears that haspin-depleted cells enter mitosis with
cohered sister chromatids that can congress to form a metaphaselike plate, but that premature loss of cohesion leads to sister
chromatid separation, overt loss of the metaphase-like alignment,
and a prolonged mitotic arrest. Clearly, in haspin-depleted cells,
chromosome misalignment and the increase in centrosome-like foci
were preceded by a stage in which near-normal chromosome
congression and formation of bipolar spindles could occur. The
presence of a dominant bipolar spindle even in haspin-depleted cells
with severe chromosome misalignment therefore appears to be the
result of its formation earlier in mitosis, before frank cohesion
defects become evident.
Depletion of other cohesion factors causes similar centrosome
defects
The known role of haspin in maintaining centromeric cohesion
between sister chromatids during mitosis (Dai et al., 2006), coupled
with the coincidence in appearance of the chromosome alignment
and spindle defects, suggested that spindle defects following haspin
depletion might be an indirect consequence of chromosome
cohesion loss rather than an intrinsic centrosomal defect. If loss of
integrity of the spindle pole is a result of cohesion loss, depletion
of other cohesion factors should cause similar increases in the
number of g-tubulin foci. Indeed, we found that the percentage of
mitotic cells with more than four g-tubulin foci was increased from
6% to 44%, 41% and 28% following depletion of haspin, Scc1 and
Sgo1, respectively (Fig. 4E). Therefore, depletion of Scc1 and Sgo1
resulted in the appearance of spindle defects similar to those in
haspin-depleted cells. Consistent with this, live-cell imaging studies
confirmed that chromosome dynamics and the appearance of extra
g-tubulin foci in cells depleted of Scc1 were similar to those in cells
depleted of haspin (supplementary material Fig. S4 and Movie 4).
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Fig. 4. Live imaging of U2OS cells transfected with haspin, Scc1
and control siRNAs. (A-B)U2OS cells stably expressing H2BmRFP (red) and g-tubulin–EGFP (green) were transfected with
control (A) or haspin (B) siRNAs, partially synchronized by
thymidine treatment, and imaged by confocal fluorescence
microscopy every 5 minutes. The frame immediately preceding
nuclear envelope breakdown (NEB) is defined as t0. Selected
maximum intensity projections of Z-stacks are shown. For
complete movies see supplementary material Movies 1 and 3.
Scale bars: 10m. (C-D)The time from NEB to metaphase-like
chromosome alignment (C) and the duration of metaphase-like
alignment (D) are shown for siRNA-transfected mitotic cells
visualized by live-cell imaging. Haspin I cells are those that
completed mitosis (65%), and haspin II cells are those that failed
to carry out normal anaphase (35%). The results of a Bonferroni
multiple comparisons test are indicated. (E)The numbers of
g-tubulin foci in U2OS cells transfected with control, haspin, Scc1
or Sgo1 siRNAs were quantified as in Fig. 1C. Means ± s.d. are
shown, n5; *P<0.05, **P<0.01, ***P<0.001 by Bonferroni
multiple comparisons test compared with the corresponding
control.
The results suggest that the formation of multiple g-tubulin foci is
associated with precocious loss of cohesion between sister
chromatids, irrespective of the particular cohesion factor that is
depleted.
Topoisomerase II inhibition partially rescues spindle pole
integrity
If the aberrant g-tubulin localization and spindle structure
observed following haspin depletion are indirect consequences
of loss of cohesion, then artificially maintaining association
between sisters in the absence of haspin should rescue the
defect. To test this hypothesis, we treated cells with the catalytic
topoisomerase II inhibitor ICRF-193 to prevent decatenation of
chromatids during mitosis (Toyoda and Yanagida, 2006;
Vagnarelli et al., 2004), and determined the numbers of cells
with multiple g-tubulin foci. Because such drugs can prevent
entry into mitosis (Downes et al., 1994) and induce apoptosis
(Vagnarelli et al., 2004), we treated partially synchronized HeLa
cells with ICRF-193 for short periods as they entered mitosis.
Synchronization reduced the efficiency of haspin depletion as
previously reported (Dai et al., 2006), so only those cells that
showed low or absent staining for H3T3ph were included in the
analysis. ICRF-193 treatment decreased the percentage of
haspin-depleted cells exhibiting three or more g-tubulin foci in
mitosis, but had little effect on the few control cells that had
multiple foci (Fig. 5A,B), consistent with the idea that the
formation of multiple centrosome-like foci in haspin-depleted
cells is due at least in part to the loss of cohesion.
Role of microtubules in the formation of extra centrosome-like
foci
Recently it has become clear that, in vertebrate cells, the formation
of the mitotic spindle is the result of cooperation between centrosome,
chromosome and spindle-initiated microtubule nucleation
mechanisms (Karsenti and Vernos, 2001; Mahoney et al., 2006;
O’Connell and Khodjakov, 2007; Toso et al., 2009). Early in mitosis,
chromosome- and centrosome-initiated microtubule formation
mechanisms probably act relatively independently, and might even
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Cohesion and spindle pole integrity
Fig. 5. Cohesion is required for integrity of the spindle poles in haspindepleted cells. (A)HeLa cells were transfected with haspin or control siRNAs,
exposed to thymidine for 14 hours and released into drug-free medium for 11
or 13 hours. Then, 20M ICRF-193 was added for 1 hour prior to fixation.
Cells were stained with antibodies to centromeres, g-tubulin and H3T3ph and
with the DNA dye Hoechst 33342, and subjected to immunofluorescence
microscopy. (B)The numbers of g-tubulin foci in mitotic HeLa cells with low
or absent staining for H3T3ph treated as in A were quantified using
immunofluorescence microscopy. Means ± s.d. are shown. Between 23 and
116 cells were classified on two coverslips from two experiments (i.e. four
coverslips). *P<0.05, ***P<0.001 by Bonferroni multiple comparisons test
compared with corresponding ICRF-193-untreated condition. Scale bars: 5m.
be antagonistic in the absence of motor proteins that normally act to
focus microtubule asters at the centrosomes (Manning and Compton,
2007). As mitosis progresses, microtubules initiated at chromosomes,
spindle microtubules and centrosomes are integrated into a single
bipolar spindle in a process that must balance the forces generated
between microtubules formed by these different mechanisms
(O’Connell and Khodjakov, 2007). It therefore seemed possible that
loss of cohesion disrupted spindle structure in our studies because it
prevented the integration of spindle assembly mechanisms that is
required to maintain a fully bipolar state.
To determine whether the chromosome pathway of microtubule
formation was operating in haspin-depleted cells, we conducted
experiments in which chromatin-mediated microtubule aster
formation was observed in mitotic cells released from arrest with
the spindle poison nocodazole, as previously described (Tulu et al.,
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2006). In control and haspin-depleted cells, asters formed with
similar efficiency in both centrosome and chromatin regions
(supplementary material Fig. S5). Haspin depletion, therefore, did
not obviously compromise chromosome-initiated microtubule
nucleation.
To determine whether the generation of extra centrosome-like
foci following loss of chromosome cohesion was dependent on
microtubule activity, we released synchronized control and
haspin-depleted cells into medium containing the microtubulestabilizing compound taxol. Although taxol itself caused the
formation of multiple microtubule asters in mitosis, both control
and haspin-depleted cells normally each contained only two gtubulin foci in these conditions (supplementary material Fig.
S6). Microtubule depolymerization with nocodazole also
prevented the emergence of multiple spindle poles in haspindepleted cells (supplementary material Fig. S5). Therefore,
formation of multiple g-tubulin foci was prevented if
microtubule dynamics were suppressed.
Kinesin-5 (also known as Eg5) is a microtubule motor required
for centrosome separation and bipolar spindle formation in mitosis
(Blangy et al., 1995; Heck et al., 1993). We therefore determined
the effect of kinesin-5 inhibition on spindle formation in haspindepleted cells. U2OS cells were transfected with control or haspin
siRNA and, after 48 hours, exposed to monastrol for 4 hours. Cells
were then examined at 0, 20, and 60 minutes following removal of
monastrol. As expected, monastrol treatment resulted in monopolar
spindles with two adjacent centrosomes in over 90% of control
mitotic cells (Fig. 6Aa,B). Most of the remaining mitotic cells had
bipolar spindles with a centrosome at each pole. These probably
represent cells that were in mitosis when the kinesin-5 inhibitor
was added because monastrol is reported not to cause the collapse
of pre-existing cellular bipolar spindles (Kapoor et al., 2000;
Tanenbaum et al., 2008). After removal of monastrol, 86% of control
cells adopted a normal bipolar configuration within 20 minutes as
mitosis resumed (Fig. 6Ad,B).
Among haspin-depleted mitotic cells, 81% had essentially
bipolar spindles with three or more g-tubulin foci prior to
monastrol treatment. After monastrol treatment, there were a
significant number of monopolar cells with two g-tubulin foci
among haspin-siRNA transfectants (32%), indicating that haspindepleted cells that enter mitosis in monastrol fail to form bipolar
spindles, as in controls, and do not generate extra g-tubulin foci
(Fig. 6Ab,B). The remaining 68% of haspin-depleted mitotic cells
retained a bipolar configuration in monastrol, compared with 7%
in controls. This probably reflects the extended duration of
mitosis in haspin-depleted cells and the relatively high percentage
of cells that were therefore already in mitosis when monastrol
was added. Most striking, however, was the observation that
essentially all of these bipolar cells had only two g-tubulin foci,
both at the spindle poles (Fig. 6Ac,B). Therefore, pre-existing
ectopic g-tubulin foci in haspin-depleted cells were largely
eliminated by monastrol treatment. This interpretation was
strikingly confirmed using live imaging to determine the effect
of monastrol on haspin-depleted mitotic cells containing multiple
pre-existing g-tubulin foci. By 40 minutes after addition of
monastrol to such cells, ectopic foci had approached and fused
with the primary spindle poles to form fully bipolar spindles with
only two g-tubulin foci (Fig. 6C and supplementary material Movie
5). Within 20 minutes of monastrol removal, 66% of haspindepleted cells returned to a bipolar configuration with three or
more g-tubulin foci, resembling the situation prior to monastrol
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Fig. 6. Kinesin-5 activity is required to form and
maintain ectopic g-tubulin foci in haspin-depleted
cells. (A)U2OS cells transfected with control or
haspin siRNAs were incubated in monastrol for 4
hours and then immediately fixed (Aa-c) or
incubated in monastrol and then released into
monastrol-free medium for 20 minutes prior to
fixation (Ad,e). Cells were stained with
antibodies to g-tubulin and H3T3ph (together),
TPX2, and with the DNA dye DRAQ5, and
subjected to immunofluorescence microscopy.
Scale bar: 5m. (B)Cells treated as in A were
classified according to the number of g-tubulin
foci and spindle polarity as judged by TPX2
staining. Note that cells referred to as ‘bipolar’
had clear dominant bipolar spindles, but in the
noted cases also had extra g-tubulin foci.
(C)U2OS cells stably expressing H2B-mRFP
(red) and g-tubulin–EGFP (green or gray) were
transfected with haspin siRNA. After 48 hours,
cells were imaged by confocal fluorescence
microscopy every 2 minutes for 10 minutes.
Monastrol was added at t0 minutes, and imaging
was then continued every 2 minutes for another
92 minutes. Selected maximum intensity
projections of Z-stacks are shown. For complete
data see supplementary material Movie 5. Scale
bar: 10m.
treatment (Fig. 6Ae,B). These results suggest that both the
formation and maintenance of ectopic g-tubulin foci in haspindepleted cells require the activity of the microtubule motor
kinesin-5. Consistent with this, kinesin-5 localized to spindle
microtubules, including those associated with ectopic g-tubulin
foci in haspin-depleted cells (supplementary material Fig. S7).
Together, these findings suggest that the extra centrosome-like foci
formed in haspin-depleted cells are dynamic structures whose
formation and persistence require ongoing microtubule dynamics
and microtubule motor activity.
Discussion
A number of studies have shown that manipulations leading to loss
of chromosome cohesion, including RNAi directed at Scc1, Sgo1,
EFO1/2, San, Pds5A/B, PHB2, CENP-F and now haspin, cause the
appearance of extra spindle poles in mitosis (Holt et al., 2005; Hou
et al., 2007; Hou and Zou, 2005; Losada et al., 2005; Salic et al.,
2004; Takata et al., 2007; Wang et al., 2008). We suggest that in
many cases this is due to secondary consequences of defects in
chromosome cohesion that prevent the proper integration of
chromosome-initiated pathways of microtubule nucleation with
other pathways involved in formation of a fully bipolar spindle.
Disruption of chromosome cohesion prevents stable attachment of
chromosomes to the spindle during mitosis (Deehan Kenney and
Heald, 2006; Sonoda et al., 2001). It is possible that ongoing
nucleation of microtubules by such chromosomes and subsequent
focusing of these microtubules into g-tubulin-containing foci (Luders
et al., 2006; Tulu et al., 2006) provides a persistent source of
centrosome-like structures, which prevents convergence of spindle
assembly on a truly bipolar structure.
Consistent with this proposal, the appearance of extra g-tubulin
foci in haspin-depleted cells requires ongoing microtubule activity.
In particular, inhibition of the microtubule plus-end-directed motor
kinesin-5 with monastrol prevents the emergence and maintenance
of ectopic centrosome-like foci in haspin-depleted cells. This
contrasts with the reported effects of monastrol on the structure of
centrosomes when they are compromised directly by depletion of
the centrosomal protein Kizuna, or by microinjection of antipolyglutamylated tubulin antibodies. In these cases, monastrol
treatment did not prevent the appearance of multiple g-tubulin foci
(Abal et al., 2005; Oshimori et al., 2006). The requirement for
kinesin-5 activity to produce ectopic poles in haspin-depleted cells
might be analogous to the role of the Drosophila kinesin-5, Klp61F,
in the utilization of chromosome-initiated microtubules to form
acentriolar poles within monastral bipolar spindles in S2 cells
(Goshima and Vale, 2003). The ability of kinesin-5 to slide apart
anti-parallel microtubules and to prevent the relative motion of
parallel microtubules might serve to move chromatin-nucleated
microtubules away from chromosomes and aid in their subsequent
clustering to form spindle poles (Burbank et al., 2007; Kapitein et
al., 2005; Walczak et al., 1998). Interestingly, we find that kinesin5 activity is required to maintain separation of ectopic g-tubulin
foci from the dominant spindle poles, but not the separation of the
two dominant poles from one another. In cell extracts, but not in
intact cells, preformed bipolar spindles collapse to monopolar
spindles in the presence of monastrol, suggesting that attachment
of spindle poles to the cell cortex might account for the insensitivity
of pre-existing bipolar spindles to kinesin-5 inhibition (Kapoor et
al., 2000). Therefore, more robust cortical attachment of the original
bipolar spindle formed early in mitosis compared to the attachment
Journal of Cell Science
Cohesion and spindle pole integrity
of later-appearing ectopic poles is one possible explanation for the
relative monastrol-insensitivity of the dominant spindle poles in
haspin-depleted cells.
Our results also provide new insight into the nature of mitotic
defects caused by haspin depletion. Many haspin-siRNA-treated
cells reach a metaphase-like chromosome alignment before
asynchronous loss of chromosome cohesion apparently leads to
decay of the metaphase plate. It is notable that depletions of other
cohesion factors, including Scc1 (Diaz-Martinez et al., 2007a;
Sonoda et al., 2001; Toyoda and Yanagida, 2006), Sgo1
(McGuinness et al., 2005; Salic et al., 2004; Wolf et al., 2006) and
Sororin (Diaz-Martinez et al., 2007b), have all led to similar
findings. The reason that cells with defects in cohesin-mediated
chromatid cohesion are able to align chromosomes remains
incompletely defined, but might result from cohesin-independent
cohesion, perhaps via DNA catenations (Deehan Kenney and
Heald, 2006; Toyoda and Yanagida, 2006; Vagnarelli et al., 2004),
as well as from incomplete protein depletion. In addition, when
regulators of mitotic cohesion such as Sgo1 are depleted, sister
chromatids are likely to enter mitosis held together by cohesinmediated links established in S phase, and cleavage-independent
cohesin removal during early mitosis is probably required for sisters
to fully separate (Peters et al., 2008). Tension across bi-oriented
chromosomes might also contribute to complete chromatid
disjoining when the cohesin pathway is defective. This is suggested
by observed reductions in scattering (but not separation) of sister
chromatids in the presence of the microtubule-depolymerizing
agent nocodazole following Sgo1 depletion (Dai et al., 2006;
McGuinness et al., 2005), and by the maintenance of chromosome
cohesion on monopolar spindles lacking tension in cohesindepleted Xenopus extracts treated with monastrol (Deehan Kenney
and Heald, 2006).
The results of haspin depletion are consistent with a similar
scenario in which cells enter mitosis with cohered sister chromatids
that can congress to form a metaphase-like plate, but in which biorientation and attachment is unstable and incapable of satisfying
the spindle checkpoint. Progressive loss of cohesion by cleavageindependent cohesin release, topoisomerase II resolution of DNA
catenations and tension across the centromere then leads to sister
chromatid separation, overt loss of the metaphase-like alignment
and a prolonged mitotic arrest. Consistent with this, in haspindepleted cells, equatorial but not polar chromosomes appear attached
to cold-stable kinetochore microtubules (supplementary material
Fig. S2), and nocodazole treatment reduces the scattering of
separated chromatids (Dai et al., 2006).
We note that our results do not rule out direct functions for
cohesion regulators at centrosomes. A splice variant of human Sgo1,
for example, localizes to centrosomes but not centromeres and
appears to be required for centriole cohesion (Wang et al., 2008).
It remains possible that haspin has a local centrosomal function
during mitosis because EGFP-haspin can be seen at centrosomes
during mitosis (Dai et al., 2005). Nevertheless, our results make
clear the importance of chromosome cohesion in allowing the
convergence of various spindle assembly pathways on a bipolar
structure, and invite caution when interpreting the cause of apparent
centrosome defects arising when centromere integrity is disrupted.
Finally, chromosome cohesion defects have been implicated in some
human cancers (Barber et al., 2008). The results presented here
indicate that ectopic spindle poles might arise and might contribute
to instability of chromosome number when chromosome cohesion
is compromised.
4175
Materials and Methods
Cell culture and transfection
Human U2OS and HeLa cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% FBS at 37°C and 10% CO2. Human haspin,
Sgo1 and control siRNAs were described previously (Dai et al., 2006; Dai et al.,
2005). Human Scc1 (Losada et al., 2005) siRNA was synthesized by Integrated
DNA Technologies (Coralville, IA). Transfections with 40 nM siRNA were carried
out using Oligofectamine according to the manufacturer’s recommendations
(Invitrogen).
For microtubule regrowth assays, siRNA-transfected cells were incubated in cold
medium on ice for 1 hour followed by 2 minutes at 37°C in warm medium. To examine
kinetochore-associated microtubules, cells were incubated in ice-cold medium for 10
minutes prior to fixation. In other experiments, cells were treated with 100 M
monastrol (Biomol, Plymouth Meeting, PA) for 4 hours or 1.1 M nocodazole (Sigma)
for 3 hours, then fixed immediately or at various times after drug removal by three
washes in medium, or cells were treated with 10 M taxol (Sigma) for 3 hours before
fixation. For topoisomerase II inhibition, siRNA-transfected HeLa cells were released
from a 14-hour block in 2 mM thymidine and, after approximately 12 hours, ICRF193 (Biomol) was added for 1 hour prior to fixation.
Antibodies and immunofluorescence
At 48 hours after siRNA transfection, cells were fixed with 4% paraformaldehyde in
phosphate buffered saline (PBS) at room temperature for 10 minutes followed by
ice-cold methanol for 5 minutes. Antibodies used were as follows: 1:2000 rabbit antiH3T3ph B8634 (Dai et al., 2005); 1:4000 rabbit or mouse anti-g-tubulin (Sigma);
1:10,000 mouse anti--tubulin (Sigma); 1:400 goat anti--tubulin (Abcam); 1:8000
anti-TPX2 (Abcam); 1:4000 mouse anti-kinesin-5 (Abcam); and 1:2000 human
centromere autoantibodies (Immunovision, Springdale, AR). Cells were directly fixed
with ice-cold methanol for co-staining of g-tubulin with the following antibodies:
1:5000 mouse anti-centrin (Paoletti et al., 1996); 1:8000 rabbit anti-pericentrin
(Abcam); 1:2000 mouse anti-aurora A (Abcam); and 1:3000 rabbit anti-NuMA (Gaglio
et al., 1995). Secondary antibodies were donkey anti-mouse, anti-rabbit or anti-human
IgG-Cy3 or IgG-Cy5 (Jackson ImmunoResearch), or anti-rabbit, anti-goat or antimouse IgG-Alexa488 (Invitrogen). To detect DNA, 50 g/ml propidium iodide plus
100 U/ml RNAse A (Sigma), or 0.5 g/ml Hoechst 33342 (Sigma), or 1:2000 DRAQ5
(Axxora LLC) were used. Fluorescence microscopy was carried out using a Nikon
TE2000 inverted microscope equipped with a 60⫻ Plan Apo 1.4 NA oil immersion
objective, a C1 Plus Confocal Laser Scanner and a SPOT-RT CCD camera. When
four-color imaging was required, antibody staining was visualized using confocal
microscopy, and DNA staining with Hoechst 33342 was detected by wide-field
microscopy. Stacks of confocal images with 0.3-0.8 m Z-steps were acquired and
rendered as maximum intensity projections using EZ-C1 software (Nikon), and
processed with Adobe Photoshop.
Live-cell imaging
To generate the stable cell lines, U2OS cells were first transfected with pEGFP-N1–
g-tubulin vector (a gift of Alexey Khodjakov, Wadsworth Center, NY) using Fugene
6 (Roche) and selected in 1 mg/ml G418. Expanded colonies expressing g-tubulin–
EGFP were further transfected with 9:1 pmRFP-N1–H2B (Dodson et al., 2007)
and pGK-puro plasmids. After selection in 1 g/ml puromycin, EGFP-positive cells
with moderate levels of mRFP expression were collected by Dako Moflo flow
cytometric sorting (DFCI Flow Cytometry Core Facility, Boston, MA). For
imaging, cells expressing g-tubulin–EGFP and H2B-mRFP were cultured in 35mm glass bottom dishes (World Precision Instruments) in DMEM containing 10%
FBS. In some cases, 24 hours after siRNA transfection, cells were incubated in
medium containing 2 mM thymidine for 16 hours before release into phenol-redfree DMEM (Hyclone, South Logan, UT) containing 10% FBS and 25 mM HEPES
for 10 hours. Time-lapse confocal fluorescence imaging was performed using the
microscope described above in a 37°C heated chamber with CO2 supply. Two color
Z stacks with 0.8 m steps were collected with a 100-nm pinhole every 5 minutes
using a 40⫻ Plan Fluor 1.3 NA oil immersion objective, or every 2 minutes using
a 60⫻ Plan Apo 1.4 NA oil immersion objective lens. ImageJ software (NIH,
Bethesda, MD) was used to adjust brightness and contrast and to render maximum
intensity projections. Time series were converted into movies with Adobe
ImageReady.
We thank Duane Compton (Dartmouth Medical School, Hanover,
NH), Alexey Khodjakov, Ciaran Morrison (National University of
Ireland, Galway, Ireland) and Jeffrey Salisbury (Mayo Clinic
Foundation, Rochester, MN) for gifts of antibodies and expression
vectors. We also thank Nancy Kedersha, Tyler Hickman and Fangwei
Wang for assistance with U2OS cell lines, live-cell imaging and
immunoblotting, respectively. This work was funded by grants to
J.M.G.H. from the American Cancer Society (RSG 05-13401-GMC)
and the National Institutes of Health (R01 GM074210). Deposited in
PMC for release after 12 months.
4176
Journal of Cell Science 122 (22)
Journal of Cell Science
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