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Research Article
Cdc48/p97–Ufd1–Npl4 antagonizes Aurora B during
chromosome segregation in HeLa cells
Grzegorz Dobrynin1,2, Oliver Popp2, Tina Romer2, Sebastian Bremer1, Michael H. A. Schmitz2,
Daniel W. Gerlich2 and Hemmo Meyer1,2,*
1
Centre for Medical Biotechnology, University of Duisburg-Essen, 45117 Essen, Germany
Institute of Biochemistry, ETH Zurich, CH-8093 Zurich, Switzerland
2
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 6 January 2011
Journal of Cell Science 124, 1571-1580
© 2011. Published by The Company of Biologists Ltd
doi:10.1242/jcs.069500
Summary
During exit from mitosis in Xenopus laevis egg extracts, the AAA+ ATPase Cdc48/p97 (also known as VCP in vertebrates) and its
adapter Ufd1–Npl4 remove the kinase Aurora B from chromatin to allow nucleus formation. Here, we show that in HeLa cells Ufd1–
Npl4 already antagonizes Aurora B on chromosomes during earlier mitotic stages and that this is crucial for proper chromosome
segregation. Depletion of Ufd1–Npl4 by small interfering RNA (siRNA) caused chromosome alignment and anaphase defects resulting
in missegregated chromosomes and multi-lobed nuclei. Ufd1–Npl4 depletion also led to increased levels of Aurora B on prometaphase
and metaphase chromosomes. This increase was associated with higher Aurora B activity, as evidenced by the partial resistance of
CENP-A phosphorylation to the Aurora B inhibitor hesperadin. Furthermore, low concentrations of hesperadin partially rescued
chromosome alignment in Ufd1-depleted cells, whereas, conversely, Ufd1-depletion partially restored congression in the presence of
hesperadin. These data establish Cdc48/p97–Ufd1–Npl4 as a crucial negative regulator of Aurora B early in mitosis of human somatic
cells and suggest that the activity of Aurora B on chromosomes needs to be restrained to ensure faithful chromosome segregation.
Key words: Mitosis, Chromosome segregation, Ubiquitin, AAA+ protein, Cdc48/p97 (VCP), Aurora B
Introduction
The proper segregation of the replicated and condensed
chromosomes during mitosis requires correct attachment of
chromatid pairs to opposite poles of the mitotic spindle and
subsequent accurate separation in anaphase, followed by timely
decondensation and nuclear envelope formation in telophase. The
kinase Aurora B plays crucial roles in various aspects of
chromosome segregation (Ducat and Zheng, 2004; Ruchaud et al.,
2007). In complex with its partner proteins INCENP, survivin and
borealin (also known as Dasra), it associates along chromosomes
in prophase where it phosphorylates histone H3 and the centromeric
histone H3 homologue CENP-A (Adams et al., 2001; Giet and
Glover, 2001; Hsu et al., 2000; Murnion et al., 2001; Zeitlin et al.,
2001). As mitosis progresses to metaphase, Aurora B dissociates
from chromosome arms and concentrates in the centromeric region,
where it controls bipolar spindle attachment to the kinetochores of
sister chromatids and thus the formation of the metaphase plate
(Adams et al., 2001; Cimini et al., 2006; Giet and Glover, 2001;
Hauf et al., 2003; Kaitna et al., 2000; Kelly and Funabiki, 2009;
Tanaka et al., 2002). Aurora B also helps to maintain the spindle
checkpoint that blocks anaphase onset and exit from mitosis until
all chromosomes properly attach to the spindle (Ditchfield et al.,
2003; Hauf et al., 2003; Kallio et al., 2002; Musacchio and
Hardwick, 2002). Upon anaphase onset, Aurora B, along with its
partners, transfers to the spindle midzone where it regulates spindle
dynamics and function. In telophase, Aurora B persists at the
midbody where it controls cytokinesis, to ensure proper division
of the daughter cells (Steigemann et al., 2009).
As a consequence of its central role, RNA interference (RNAi)mediated depletion of Aurora B, or pharmacological inhibition of
its kinase activity, in mammalian tissue culture cells leads to a
prominent chromosome alignment defect in prometaphase due to
erroneous spindle attachment to kinetochores (Ditchfield et al.,
2003; Hauf et al., 2003; Taylor and Peters, 2008). Metaphase plate
formation is largely hampered, and cells display severe anaphase
defects with lagging chromosomes, or they exit from mitosis before
completion of anaphase with resulting micronuclei, polyploidy and
multinucleated cells (Ditchfield et al., 2003; Hauf et al., 2003;
Mora-Bermudez et al., 2007). How Aurora B controls and corrects
spindle attachment to kinetochores and thus chromosome
congression to the metaphase plate is not completely understood,
but it involves regulation of a number of substrate proteins at the
kinetochore with seemingly antagonizing activities (Kelly and
Funabiki, 2009; Ruchaud et al., 2007). Phosphorylation of the
Ndc80 (also known as Hec1) complex, the major attachment module
for microtubules, is proposed to decrease its affinity for microtubules
and therefore destabilizes spindle attachment (Cheeseman et al.,
2006; Ciferri et al., 2008; DeLuca et al., 2006). By contrast,
phosphorylation of mitotic centromere-associated kinesin (MCAK;
also known as KIF2C) suppresses its microtubule-depolymerizing
activity and thus might stabilize spindle attachments (Andrews et
al., 2004; Lan et al., 2004; Ohi et al., 2004). The role of Aurora B
in correcting spindle attachment suggests that centromeric Aurora
B needs to be tightly regulated, to maintain its activity within a
limited range, to allow fine-tuning of spindle attachment and
consequently faithful chromatid segregation.
Previously, the AAA+ ATPase Cdc48/p97 (also known as VCP
in vertebrates) has been implicated in regulation of Aurora B.
Cdc48/p97 is an abundant ubiquitin-dependent chaperone involved
in protein quality control and signalling events in interphase and
mitosis (Woodman, 2003; Ye, 2006). Cdc48/p97 associates with
alternative substrate and ubiquitin adapters, including cofactor p47
Journal of Cell Science
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(officially known as NSFL1 cofactor p47; NSF1C) or the Ufd1–
Npl4 complex, to fulfil distinct functions (Alexandru et al., 2008;
Kondo et al., 1997; Meyer et al., 2000; Schuberth and Buchberger,
2008). Ufd1 and Npl4 form a stable heterodimeric complex, with
both subunits containing interaction sites for Cdc48/p97, as well as
binding domains for ubiquitin (Alam et al., 2004; Bruderer et al.,
2004; Isaacson et al., 2007; Meyer et al., 2002). As a common
activity applied in different pathways, the Cdc48/p97–Ufd1–Npl4
complex is thought to bind and extract ubiquitylated client proteins
from cellular structures and to release them for either degradation
or recycling (Jentsch and Rumpf, 2007; Ye, 2006). In mitosis,
Cdc48/p97–Ufd1–Npl4 has been associated with different
functions, such as chromosome segregation, spindle dynamics and
organelle reformation, in different organisms and developmental
stages (Meyer and Popp, 2008). In Xenopus laevis egg extracts,
Cdc48/p97–Ufd1–Npl4 removes Aurora B from chromatin during
exit from mitosis in a process that involves modification of Aurora
B with ubiquitin chains (Ramadan et al., 2007). Removal of Aurora
B from chromatin leads to a local decrease in activity, which then
allows decondensation and nuclear envelope formation (Ramadan
et al., 2007).
Ufd1–Npl4 has also been associated with chromosome
segregation in HeLa cells in two independent studies. RNAimediated depletion of Npl4 results in anaphase defects with
entangled chromosomes (Porter et al., 2007), whereas Ufd1
depletion has been shown to cause chromosome congression defects
(Vong et al., 2005). On the basis of immunohistochemistry of
small interfering RNA (siRNA)-treated cells, Vong and colleagues
concluded that Ufd1 helps to recruit Aurora B to chromosomes.
This interpretation for a positive regulation of Aurora B is in
surprising contrast with our observation in Xenopus egg extracts
(Ramadan et al., 2007), where Cdc48/p97 removes Aurora B from
chromosomes. Two other studies using RNAi-mediated depletion
of Cdc48/p97 in Caenorhabditis elegans failed to detect any role
for Cdc48/p97 or Ufd1–Npl4 in mitosis (Heallen et al., 2008;
Mouysset et al., 2008). The cellular relevance of the Cdc48/p97–
Ufd1–Npl4 complex for regulation of mitosis and its functional
relation to Aurora B has therefore remained controversial.
To address this controversy, we have re-examined the relevance
of Cdc48/p97–Ufd1–Npl4 for mitosis, and its functional relation
to Aurora B in HeLa cells. Here, we show that Ufd1 and Npl4
cooperate in proper chromosome segregation. Moreover, we
provide evidence that Ufd1–Npl4 functions by antagonizing Aurora
B during segregation. Depletion of Ufd1–Npl4 resulted in increased
levels of Aurora B associated with chromosomes during
prometaphase and metaphase, as shown by immunofluorescence
microscopy and cell fractionation. Furthermore, Ufd1 depletion
increased phosphorylation of the centromeric Aurora B substrate
CENP-A. Finally, and importantly, Ufd1 depletion partially rescued
the rate and efficiency of chromosome congression after attenuation
of Aurora B with the inhibitor hesperadin. The data establish the
Cdc48/p97–Ufd1–Npl4 complex as a negative regulator of Aurora
B early in mitosis. Moreover, the data imply that restraining the
activity of Aurora B on chromosomes is essential for its function
in controlling proper chromosome segregation.
Results
Ufd1–Npl4 is required for normal progression through
mitosis in HeLa cells
To study the role of Cdc48/p97 in regulating progression of mitosis
in HeLa cells systematically, we used an siRNA approach to deplete
its two alternative adapters implicated in mitosis, p47 or the
heterodimeric Ufd1–Npl4 complex (Kondo et al., 1997; Meyer et
al., 2000; Schuberth and Buchberger, 2008), rather than Cdc48/p97
itself, in order to reduce pleiotropic effects. Depletion for 48 hours
was efficient and did not affect Cdc48/p97 levels (Fig. 1A).
Importantly, depletion of Npl4 also specifically destabilized Ufd1,
but not vice versa, consistent with previous biochemical data
showing that Ufd1 is unstable in the absence of Npl4 (Bruderer et
al., 2004). We first monitored progression through mitosis in
unsynchronized HeLa cells stably expressing histone-H2B–mRFP
by time-lapse video microscopy and measured the time between
nuclear envelope breakdown (scored on the basis of the loss of a
defined boundary for the chromatin regions) (Beaudouin et al.,
2002) and anaphase onset (Fig. 1B,C). Ufd1- or Npl4-siRNAtreated cells displayed a delay of anaphase onset (median of ~43
minutes), compared with that of control or p47-depleted cells
(median of ~30 minutes), suggesting that the spindle assembly
Fig. 1. The Cdc48/p97 adapter Ufd1–Npl4 is required for normal
progression through mitosis of HeLa cells. (A)Western blot analysis of
HeLa Kyoto cells treated with non-silencing control (Ctrl) siRNA or two
independent oligonucleotides (S1 and S2) each targeting Ufd1, Npl4 or the
alternative adapter p47 for 48 hours. Membranes were probed with the
indicated antibodies. Note that Npl4 depletion also specifically destabilizes its
binding partner Ufd1. (B)Images from representative confocal time-lapse
fluorescence microscopy movies of unsynchronized HeLa Kyoto cells
progressing through mitosis, after treatment with indicated siRNA
oligonucleotides for 48 hours. The cells are stably express histone-H2B–mRFP
to visualize chromatin. Time (minutes) after nuclear envelope breakdown is
indicated, closed arrowheads point to anaphase onset. Note that some Npl4siRNA-treated cells did not enter anaphase (as shown in the bottom series of
images). The open arrowhead indicates a chromosome bridge. Scale bars:
5m. (C)Quantification of movies, as in B, from 5 independent experiments.
The time between nuclear envelope breakdown (NEBD) and anaphase onset
was determined and is represented in a box-and-whisker plot [Ctrl, median
30.0 minutes, mean 31.3 minutes (n48); Ufd1, median: 43.3 minutes, mean:
44.9 minutes (n28); Npl4, median: 43.3 minutes, mean: 46.3 minutes (n12);
p47, median: 29.5 minutes; mean: 31.0 minutes (n12)]. Only Npl4-siRNAtreated cells that entered anaphase within 170 minutes were quantified.
Statistical significance was tested with the Mann–Whitney U-test (n.s., not
significant). The siRNAs Ufd1-S2, Npl4-S2 and p47-S1 were used.
Cdc48/p97 antagonizes Aurora B during chromosome segregation
checkpoint is activated owing to compromised spindle–kinetochore
attachments. Indeed, inspection of the movies revealed misaligned
chromosomes at the spindle pole, and a delay in congression and
the formation of a metaphase plate, in Ufd1- or Ufd1–Npl4depleted cells (Fig. 1B). When cells eventually proceeded into
anaphase, we observed segregation defects, with separating
chromosomes remaining entangled and delayed retraction of
chromosome arms from the spindle midzone. A fraction of the
Npl4-siRNA-treated cells did not enter anaphase during the course
of the experiments (Fig. 1B). These findings confirm that the
Cdc48/p97–Ufd1–Npl4 chaperone complex is essential for proper
mitotic progression of human somatic cells.
Journal of Cell Science
Ufd1–Npl4 depletion causes chromosome congression, as
well as anaphase defects
The congression defect was confirmed in fixed cells. On average
36.8% and 46.9% of cells with an apparent metaphase plate
displayed misaligned chromosomes, often several at a time, upon
Ufd1 or Npl4 siRNA treatment, respectively, compared with 7.5%
or 6.4% in control or p47-depleted cells, respectively (Fig. 2A,C).
In Ufd1-depleted cells, the congression defect was largely rescued
by overexpression of the mouse Ufd1 cDNA that is not targeted by
the siRNA oligonucleotides used (Fig. 2C and supplementary
material Fig. S1), showing that the effect was specific. In addition,
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we observed a significant (P<0.005) increase in chromosome
segregation defects in anaphase from 7.8% in control and 7.7% in
p47-depleted cells, to 23% and 31.4% in Ufd1- or Npl4-siRNAtreated cells, respectively (Fig. 2B,D). These defects included
lagging chromosomes that probably resulted from incorrect spindle
attachment. Moreover, we observed chromosome bridges between
the separating chromatin masses, and often several entangled
chromosomes, indicating defects in sister chromatid resolution. In
particular, Npl4-siRNA-treated cells (that are depleted of the entire
Ufd1–Npl4 heterodimer) displayed aberrations in nuclear
morphology in interphase with micronuclei that probably originated
from lagging chromosomes (Fig. 2E). Even more prominently,
nuclei were often multi-lobed, a phenotype known to result from
segregation errors and defective axial compaction of anaphase
chromosomes upon inhibition of Aurora B (Hauf et al., 2003;
Mora-Bermudez et al., 2007). These results show the shared
functions of the Ufd1 and Npl4 in regulating chromosome
alignment and chromosome segregation in human somatic cells.
To address whether depletion of Ufd1–Npl4, in the same manner
as Aurora B inhibition, causes polyploidy, we treated cells with
siRNA targeting Ufd1, Npl4 or Aurora B for 2 days and measured
the DNA content by flow cytometry (Fig. 2F). Although
inactivation of Aurora B caused an increase in the number of cells
with 4N and even 8N DNA content, as previously reported (Hauf
Fig. 2. Chromosome congression and segregation defects, but
not multinucleation, upon Ufd1–Npl4 depletion.
(A,B)Confocal fluorescence images of unsynchronized
metaphase (A) and anaphase (B) HeLa Kyoto cells that had been
treated for 48 hours with the indicated siRNAs. Cells were fixed
and stained with DAPI. Scale bar: 5m. (C)Quantification
(means+s.d.) of the experiment described in A [>200 cells from
three independent experiments except for the mouse Ufd1–Myc
(Ufd1:myc) overexpression, which assessed >50 cells in one
experiment]. Statistical significance was analysed with Student’s
t-test. (D)Quantification (means+s.d.) of the experiment
described in B (>200 cells from three independent experiments).
Statistical significance was analysed with Student’s t-test.
(E)Representative fluorescence overview images of interphase
cells treated for 48 hours with the indicated siRNA. Cells were
fixed and stained with DAPI. Note the multi-lobed nuclei and
micronuclei in cells treated with the Npl4 siRNA that destabilizes
the entire Ufd1–Npl4 heterodimer. Scale bar: 10m. (F)Analysis
of the DNA content by flow cytometry of propidium-iodidestained cells after treatment with indicated siRNAs for 48 hours.
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et al., 2003), there was no dramatic shift in the DNA content in
Ufd1- or Npl4-depleted cells. Npl4 depletion did, however, cause
a slight increase of cells with intermediate DNA content between
2N and 4N, at the expense of the 2N peak, which can either be
explained by the aneuploidy due to the observed missegregation
or by a prolonged S phase. We also counted the percentage of
multinucleated cells, but did not observe any increase in Ufd1- or
Npl4-siRNA-treated cells, in contrast with Aurora-B-depleted
cells, indicating that cytokinesis after segregation was not affected
(data not shown).
Journal of Cell Science
Ufd1–Npl4 helps to remove a fraction of Aurora B from
chromosomes as cells progress to metaphase
The chromosome congression defects and the segregation errors
upon Ufd1–Npl4 depletion resemble the phenotype upon
inactivation of the kinase Aurora B (Ditchfield et al., 2003; Hauf
et al., 2003; Mora-Bermudez et al., 2007). In apparent accordance,
Vong and colleagues reported a decrease of Aurora B and survivin
on chromosomes upon Ufd1 depletion (Vong et al., 2005), which
would suggest a positive regulation of Aurora B by Ufd1–Npl4. By
contrast, we previously showed that Cdc48/p97–Ufd1–Npl4
negatively regulates Aurora B by removing it from chromatin
during exit from mitosis in embryonic systems (Ramadan et al.,
2007). We therefore re-examined Aurora B and survivin localization
in Ufd1–Npl4-depleted cells.
To do so, we fixed unsynchronized control and siRNA-treated
cells and examined the localization of Aurora B and survivin by
immunostaining. Wide-field fluorescence microscopy, with identical
exposure settings, revealed that the levels of chromosomeassociated Aurora B increased by more than 2.5–5-fold both in
Ufd1- and in Npl4-depleted cells (Fig. 3A,B). In addition, the level
of survivin on the chromosomes was increased upon Ufd1–Npl4
depletion. This finding is in sharp contrast with the observation of
Vong and colleagues, who reported loss of chromatin-associated
Aurora B upon Ufd1 depletion (Vong et al., 2005). We therefore
examined the previously published siRNA sequences
(oligonucleotides V-S1 and V-S2) and compared them with a total
of five Ufd1 and two Npl4 of our siRNA oligonucleotides. All our
sequences upregulated the localization of Aurora B to
chromosomes, although to different degrees (supplementary
material Fig. S2). Intriguingly, only the published V-S2 caused the
reported decrease (supplementary material Fig. S2). The conflicting
results were observed despite the fact that all siRNA
oligonucleotides efficiently depleted Ufd1 (supplementary material
Fig. S2C). To solve this discrepancy, we performed restoration
experiments. We introduced four silent point mutations in the
mouse Ufd1 cDNA to render it resistant to the published V-S2, in
addition to our S2 oligonucleotide, and used it to generate a
resistant Ufd1–GFP expression construct (reUfd1-GFP).
Overexpression of reUfd1-GFP was efficient in all cell populations
without affecting depletion of Ufd1 by either V-S2 or our S2 as
confirmed by western blotting (supplementary material Fig. S3A).
Importantly, the increased levels of chromatin-associated Aurora B
in Ufd1-S2-siRNA-treated cells were reduced to almost control
levels in cells overexpressing reUfd1-GFP. By contrast, loss of
Aurora B from chromosomes in cells treated with the V-S2
oligonucleotide was not restored (supplementary material Fig.
S3B,C). Consistently, reUfd1-GFP restored proper chromosome
alignment in S2-siRNA-treated cells, whereas it failed to do so in
V-S2-treated cells (supplementary material Fig. S3B,D). This result
conclusively demonstrates that the phenotype of S2 treatment was
Fig. 3. Elevated levels of Aurora B and survivin associated with
chromosomes in Ufd1–Npl4-depleted cells. (A)Cells were treated with
control (siCtrl), Ufd1 or Npl4 siRNA for 48 hours, were pre-extracted to
remove soluble protein and fixed. Aurora B and survivin was detected by
indirect immunofluorescence using wide-field fluorescence microscopy with
identical settings. (B)The intensity of Aurora B and survivin (means+s.d.) on
metaphase chromatin was determined and background-subtracted as shown in
A (n>21 for each condition). Data are presented for siUfd1-S2 and siNpl4-S2
(see supplementary material Fig. S2 for data from with additional
oligonucleotides and supplementary material Fig. S3 for restoration
experiments). (C)Western blot analysis of chromatin fractions from cells
treated with control (Ctrl), Ufd1 or Aurora B (AurB) siRNA. After nocodazole
arrest, mitotic cells were harvested by shake-off. Mitotic indices were
comparable (see supplementary material Fig. S3 for the quantification). Equal
numbers of cells were lysed with detergent, and the chromatin (pellet)
separated from the soluble fractions (sup) by centrifugation. The membrane
was probed with antibodies against Ufd1, Aurora B, or for phosphorylated
histone H3 serine 10 (pH3). Topoisomerase II alpha (TopoIIa) and GAPDH
were probed, as chromatin and cytosol controls, respectively. Note the increase
in chromatin-associated Aurora B in Ufd1-depleted cells. (D)Quantification of
the experiment shown in C. The intensities of the Aurora B band was
normalized to the TopoII signal and presented as percentage of the control
intensity for the total and pellet fractions, respectively.
specifically caused by Ufd1 depletion, whereas V-S2 affected
mitosis indirectly through an unknown mechanism.
The enhanced Aurora B association with chromosomes in Ufd1depleted cells was observed by prophase and was even observed
on segregating chromosomes in anaphase (supplementary material
Fig. S4). To confirm this increase biochemically, we synchronized
HeLa cells in nocodazole and harvested mitotic cells by shake-off.
In such a way, we analyzed control and Ufd1-depleted cells, as
well as Aurora-B-depleted cells for comparison. The mitotic index
of the collected cells ranged between 90–95% in all preparations
(supplementary material Fig. S5). Detergent-extracted cell lysates
were separated by centrifugation into soluble and chromatin
fractions. Western blotting confirmed a consistent increase of
chromatin-associated Aurora B upon Ufd1 depletion, which was in
part reflected in the total amount of cellular Aurora B (Fig. 3C,D).
Next, we ask whether Aurora B accumulated at a specific region
on the chromosome in the absence of Cdc48/p97–Ufd1–Npl4
function. Analysis of Ufd1- and Npl4-depleted cells in
Cdc48/p97 antagonizes Aurora B during chromosome segregation
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Fig. 4. Ufd1–Npl4 depletion leads to Aurora B
spreading along chromosome arms, and its increased
association at the centromere. (A)Representative
confocal images of unsynchronized HeLa cells in
prometaphase or metaphase after 48 hours of treatment
with siRNA targeting the indicated proteins. Cells were
fixed and probed with DAPI, and antibodies against
Aurora B (Aur B) or phosphorylated Aurora B (pT232).
Images were taken with identical laser settings. Note the
Aurora B localization along chromosome arms
(arrowheads) in Ufd1- and Npl4-siRNA-treated cells.
Scale bar: 5m. (B)Representative chromosome spreads
of prometaphase cells treated with control or Ufd1 siRNA.
Spreads were stained with DAPI and anti-Aurora-B
antibodies. The right-hand panel shows a higher
magnification of a representative individual chromatid
pair. Note the spreading of Aurora B along chromosome
arms upon Ufd1 depletion. (C)Control-and siUfd1-treated
cells were fixed, immunostained with antibodies against
Aurora B and the centromeric antigen CREST, and
analysed by wide-field microscopy. Scale bar: 10m.
(D)Quantification (means±s.d.) of Aurora B intensities on
chromosome arms and centromeres visualized as indicated
in C.
prometaphase and metaphase, by confocal microscopy, revealed
that Aurora B localized along the entire chromosome arms, whereas
it was confined to centromeric regions in control cells (Fig. 4A).
Aurora B on chromosome arms was active, as it was
autophosphorylated on threonine 232 (Yasui et al., 2004), which
we detected with a phosphorylation-specific antibody (Fig. 4A).
The distribution of Aurora B along the chromosome arms was also
confirmed in chromosome spreads prepared from synchronized
control- and Ufd1-depleted cells (Fig. 4B). To measure the increase,
we analyzed prometaphase cells by wide-field microscopy.
Fluorescence intensity measurement of centromeric regions (as
defined with a CREST counterstain) and areas on distal arms
revealed that Aurora B levels were significantly increased in both
chromosomal regions in Ufd1-depleted cells compared with its
localization in control treated cells (Fig. 4C,D). From these
biochemical and morphological analyses, we conclude that
impairment of Ufd1–Npl4 leads to elevated levels of Aurora B
associating with chromosomes.
Ufd1–Npl4 reduces chromosome-associated Aurora B
activity early in mitosis
To address whether the increased levels of Aurora B on
chromosomal arms and centromeres also resulted in increased
Aurora B activity associated with chromosomes, we monitored
phosphorylation of its substrates, histone H3 and the centromeric
histone H3 homologue CENP-A, by immunofluorescence
microscopy. Both proteins are first phosphorylated by Aurora B in
late G2 phase: histone H3 on serine 10 and CENP-A on serine 7.
We did not detect differences in their steady-state phosphorylation
level in control and Ufd1-depleted cells (Fig. 5C, DMSO control;
data not shown), possibly because both substrates are already
nearly maximally phosphorylated in control cells. We therefore
asked whether a possible increase in Aurora B activity would be
reflected in a partial resistance to the chemical inhibitor of Aurora
B, hesperadin.
To address this question, we first assessed whether
phosphorylation of histone H3 or CENP-A is dynamic in
prometaphase, as a result of repeated dephosphorylation and
subsequent re-phosphorylation by Aurora B, which could then be
probed in our analysis. To test this, we treated cells with 100 nM
hesperadin for between 2 and 60 minutes, to inhibit possible rephosphorylation. We then analyzed the substrate phosphorylation
state by indirect immunofluorescence with phosphorylation-specific
antibodies in prometaphase and metaphase cells. Given that
hesperadin treatment causes morphological changes, we confirmed
the mitotic stage by assessing whether the cell had broken down the
nuclear envelope but not yet degraded cyclin B1, as determined by
indirect immunofluorescence (data not shown). Histone H3
phosphorylation was not affected when cells were treated for up to
15 minutes (Fig. 5A,B). However, after 30 minutes, we observed a
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Journal of Cell Science
Fig. 5. Ufd1–Npl4 attenuates
chromosome-associated Aurora B
activity towards a centromeric
substrate. (A,B)Phosphorylation of
CENP-A, but not of histone H3 is
dynamic during prometaphase.
Unsynchronized cells were treated for
indicated times with 100 nM of the
Aurora B inhibitor hesperadin (Hesp),
fixed and phosphorylation of histone H3
(serine 10) (pH3) and CENP-A (serine 7)
(pCENP-A) detected by indirect
immunofluorescence with
phosphorylation-specific antibodies. Cells
were only assessed if they had broken
down the nuclear envelope, but not yet
degraded cyclin B1 (as identified with an
anti-cyclin-B1 antibody). A shows
fluorescence micrographs and B a
quantification (means+s.d.; n40). Note
that CENP-A, but not H3 phosphorylation
is sensitive to 15-minute incubations with
100 nM hesperadin. (C,D)Cells were
treated with increasing concentrations of
hesperadin for 15 minutes, and
phosphorylation of CENP-A was detected
as in A. C shows fluorescence
micrographs and D a quantification
(means+s.d.; n>137 from three
independent experiments). Note that
CENP-A phosphorylation is resistant to
20–50 nM hesperadin in Ufd1-siRNAbut not control-treated cells.
mixed population of cells with either fully, partially or nonphosphorylated chromatin, the latter cases most probably already
reflecting inhibition of the initial phosphorylation in those cells that
were in prophase during application of the inhibitor. This suggests
that histone H3 phosphorylation is not dynamic and that the
phosphorylation that occurs in late G2 is stable at least until anaphase
onset. In marked contrast, CENP-A phosphorylation at the
centromere was sensitive to a 15-minute incubation with hesperadin
in almost all the sample cells, indicating that it is constantly
dephosphorylated and needs to be re-phosphorylated by Aurora B
to maintain its phosphorylation status during prometaphase.
We therefore investigated the sensitivity of CENP-A
phosphorylation to 15-minute incubations with increasing
concentrations of hesperadin (from 1 to 100 nM) in control and
Ufd1-depleted cells (Fig. 5C,D). We found that phosphorylation
was undetectable at 20 nM in the majority of control cells, whereas
phosphorylation was resistant to 50 nM hesperadin and was even
partially detectable at 100 nM hesperadin. This result demonstrates
that the impairment of Ufd1–Npl4 function leads to a shift towards
stronger phosphorylation of CENP-A, which is consistent with an
elevated Aurora B activity associated with chromosomes. Together
with the observed increase in Aurora B protein, this suggests that
Ufd1–Npl4 helps to restrain the activity of Aurora B early in
mitosis, by removing a fraction of it from chromosomes as cells
progress to metaphase.
Ufd1–Npl4 antagonizes Aurora B during chromosome
congression
Next, we asked whether regulation of Aurora B activity by
Cdc48/p97–Ufd1–Npl4 had any functional relevance. We did not
observe an apparent effect on chromosome condensation in Ufd1depleted cells, even when they were challenged with hypotonic
buffer (supplementary material Fig. S6), which leads to a
decompaction of unstable chromosomes (Gassmann et al., 2004). To
address a possible effect on chromosome congression, we analyzed
the efficiency and timing of chromosome alignment both in fixed
and live cells. As a first approach, we treated control or Ufd1depleted cells with increasing concentrations of hesperadin (from
0.7 to 100 nM), fixed the cells after 1 hour and determined the
percentage of prometaphase and metaphase cells with fully aligned
chromosomes (Fig. 6A,B). As expected, both depletion of Ufd1 and
addition of hesperadin at concentrations equal or above 5 nM caused
a marked increase in misaligned chromosomes compared with that
observed in control cells. Importantly, however, chromosome
alignment in Ufd1-depleted cells was partially rescued by moderate
inhibition of Aurora B, with 1 nM of hesperadin, suggesting that the
alignment defect in Ufd1-depleted cells was, at least in part, caused
by overactivation of Aurora B. Conversely, Ufd1-depletion restored
proper alignment in the presence of 5–15 nM hesperadin, showing
that the overactivation of Aurora B due to impaired Ufd1–Npl4
function can overcome partial inhibition of Aurora B.
Journal of Cell Science
Cdc48/p97 antagonizes Aurora B during chromosome segregation
1577
Fig. 6. Ufd1–Npl4 antagonizes Aurora B during chromosome congression. (A,B)Unsynchronized control or Ufd1-depleted cells were treated with indicated
concentrations of hesperadin (Hesp) for 1 hour, and were fixed and stained with DAPI and anti-cyclin B1 antibodies. Cells were assessed if they had broken down
the nuclear envelope, but not yet degraded cyclin B1, as in Fig. 5A, and the percentage of cells with fully aligned chromosomes was determined. Data are
means+s.d. from three independent experiments (n100 for each condition). (C,D)Hela cells expressing H2B–mRFP were transfected with control (Ctrl) or Ufd1
siRNA. After 48 hours, cells were treated with hesperadin (50 nM), or equivalent amounts of its solvent DMSO, and imaged by confocal time-lapse microscopy as
in Fig. 1B. C shows images from representative movies and D shows a quantification of the time between nuclear envelope breakdown and metaphase plate
formation. Individual cells were followed in time-lapse movies and scored when they formed a distinct metaphase plate. Scored cells were plotted cumulatively for
each time point. The data are from three independent experiments (siCtrl, n146; siCtrl + Hesp, n138; siUfd1, n114; siUfd1 + Hesp, n88).
To confirm this result, we monitored metaphase plate formation
in live cells (Fig. 6C,D). Control or Ufd1-depleted cells stably
expressing histone H2B–mRFP were imaged in the absence or
presence of 50 nM hesperadin using time-lapse video microscopy
as in Fig. 1. We determined the time from nuclear envelope
breakdown to the formation of a distinct metaphase plate, when no
misaligned chromosomes could be detected. We then plotted the
percentage of cells that had formed metaphase plates cumulatively
as a function of time (Fig. 6D). Control cells readily formed a
metaphase plate within 20 minutes. In accordance with our results
in Fig. 1, Ufd1-depleted cells required longer, but eventually nearly
all cells completed congression. By contrast, and as expected, less
than 10% of the hesperadin-treated cells managed to form a
metaphase plate. Importantly, Ufd1-depletion partially restored
metaphase plate formation in hesperadin-treated cells, as
chromosome alignment occurred in more than 50% of cells, though
at a lower rate and with metaphase plates occasionally collapsing
after formation. Taken together, these results demonstrate that
Ufd1–Npl4 antagonizes Aurora B during chromosome congression
and suggest that misregulation of Aurora B, at least in part, accounts
for the congression phenotype in Ufd1–Npl4-depleted cells.
Discussion
The data presented here establish that the Ufd1–Npl4 adapter of
the Cdc48/p97 AAA+ ATPase is required for proper chromosome
segregation in human somatic cells. Cellular depletion of Ufd1–
Npl4 in HeLa cells specifically interfered with efficient
chromosome alignment to the metaphase plate, delayed
prometaphase and caused anaphase defects, which led to
missegregation and abnormal nuclear morphology. Depletion of
Ufd1 alone had a weaker effect than destabilization of the whole
Ufd1–Npl4 complex after Npl4 depletion. This suggests that Npl4
alone can still partially fulfil the function of Ufd1–Npl4. Both
Ufd1 and Npl4 have ubiquitin-binding activity, suggesting that one
binding site might compensate for a partial depletion. Interestingly,
Npl4 contains an MPN domain (residues 226–396; Kay Hofmann,
Miltenyi Biotec, personal communication), which is found in
certain deubiquitinating enzymes (Ambroggio et al., 2004).
Although the catalytic residues are not conserved, it is possible
that this domain confers a crucial function of the Ufd1–Npl4
complex.
A role for Cdc48/p97–Ufd1–Npl4 in mitosis was recently
questioned, as a mitotic delay could not be observed in C. elegans
embryos treated with RNAi against Cdc48/p97 or Ufd1–Npl4
(Mouysset et al., 2008). However, the RNAi in that study was fed
to the animals rather than injected, which can limit efficacy.
Moreover, a major delay due to chromosome misalignment is not
expected in early embryos, because they have a very weak spindle
assembly checkpoint under normal growth conditions, which can
only delay anaphase onset moderately and only when the spindle
is disrupted severely (Nystul et al., 2003). We suggest, therefore,
that the evidence presented by Mouysset and colleagues (Mouysset
et al., 2008) does not preclude a role for Cdc48/p97–Ufd1–Npl4 in
mitosis in C. elegans.
Our new results confirm the central role of Cdc48/p97–Ufd1–
Npl4 in mitosis in HeLa cells. Along with previous results from C.
elegans and Xenopus (Ramadan et al., 2007), as well as yeast (Cao
et al., 2003; Cheng and Chen, 2010; Frohlich et al., 1991; Moir et
Journal of Cell Science
1578
Journal of Cell Science 124 (9)
al., 1982), they suggest that the Cdc48/p97–Ufd1–Npl4 chaperone
complex has a conserved role in maintaining chromosome stability
and euploidy during proliferation. Here, we also provide several
lines of evidence that Cdc48/p97–Ufd1–Npl4 antagonizes Aurora
B during chromosome segregation. This is supported by the
observation that Ufd1–Npl4 depletion caused an increase in the
levels of Aurora B associated with mitotic chromosomes, as shown
by immunohistochemistry and biochemical fractionation. This
notion is based on results from a total of seven Ufd1 and Npl4
siRNA oligonucleotides. We note, however, that the depletion
efficiencies and degree of segregation errors do not completely
correlate with the various degrees of Aurora B accumulation for
the different siRNA oligonucleotides. Nevertheless, restoration
experiments with a resistant Ufd1 cDNA demonstrated that both
the Aurora B accumulation and the segregation errors are a specific
consequence of Ufd1 depletion. Moreover, the same experiments
showed that the effect of an oligonucleotide previously reported to
cause loss of Aurora B from chromosomes (Vong et al., 2005) was
indirect, as it was not rescued by overexpression of the resistant
Ufd1 cDNA.
In Xenopus egg extract, Cdc48/p97–Ufd1–Npl4 removes Aurora
B from chromosomes at the end of mitosis (Ramadan et al., 2007).
Although not shown directly in this manuscript, this suggests that
Cdc48/p97–Ufd1–Npl4 might act in a similar manner at earlier
stages of mitosis in HeLa cells and remove some Aurora B from
chromosomes as cells progress from prophase (where Aurora B
localizes along the entire chromosomes) to metaphase, thereby
attenuating its activity. Given that Cdc48/p97–Ufd1–Npl4 function
is triggered by ubiquitylation (Ramadan et al., 2007; Ye, 2006),
this implies the requirement for a specific ubiquitin ligase activity
early in mitosis. Interestingly, a cullin-3-based ligase has recently
been shown to ubiquitylate Aurora B and to be required for Aurora
B removal from chromosomes in prometaphase (Maerki et al.,
2009; Sumara et al., 2007). Moreover, cullin-3 physically interacts
with Cdc48/p97 (Alexandru et al., 2008) raising the possibility that
the two factors cooperate to regulate chromatin-associated Aurora
B. Independent of whether this holds true, both sets of findings
concur that Aurora B might be actively removed from chromatin
in a ubiquitin-regulated manner (Sumara et al., 2008). Other than
in Xenopus egg extracts, where Aurora B remains stable, however,
the available data suggests that the mobilized fraction of Aurora B
in HeLa cells might, at least in part, be degraded. This hypothesis
is supported by the increased total Aurora B levels in both Ufd1depleted cells (Fig. 4C,D) and cullin-3-depleted cells (Sumara et
al., 2007). Furthermore, proteasome inhibition leads to increased
and persisting levels of Aurora B on chromosomes (Sumara et al.,
2007).
More direct evidence for the antagonizing role of Cdc48/p97–
Ufd1–Npl4 with respect to Aurora B is the elevated activity
towards the centromeric substrate CENP-A. Ufd1-depleted cells
are less sensitive to partial inhibition of Aurora B, by addition of
its inhibitor hesperadin, with respect to CENP-A phosphorylation.
The central question arising from these findings is, therefore,
whether the activity of Cdc48/p97–Ufd1–Npl4 in antagonizing
Aurora B in prometaphase has any functional impact on
chromosome segregation and hence whether excessive Aurora B
activity can explain the defects caused by Ufd1–Npl4 depletion.
A crucial role for Ufd1–Npl4-mediated downregulation of Aurora
B in chromosome segregation is supported by the similarity of
the phenotypes observed upon Ufd1–Npl4 depletion and
inactivation of Aurora B (severe defects in chromosome
alignment, as well as lagging and entangled chromosomes in
anaphase that lead to the prominent occurrence of micronuclei
and multi-lobed nuclei) (Ditchfield et al., 2003; Hauf et al., 2003;
Mora-Bermudez et al., 2007; Vagnarelli and Earnshaw, 2004).
Segregation defects in anaphase might also be caused indirectly
through inefficient DNA replication, as observed after depletion
of Cdc48/p97 or Ufd1–Npl4 in C. elegans embryos (Mouysset et
al., 2008). However, although we do not exclude the possibility
that problems in S phase might contribute to the observed
anaphase defects, we also do not detect any major inhibition of
DNA replication in Ufd1–Npl4-depleted HeLa cells (Fig. 2F), as
described for C. elegans embryos (Mouysset et al., 2008).
Furthermore, and more importantly, our results draw a clear link
between Ufd1–Npl4 and Aurora B during mitosis, as we show
that they functionally interact in regulating chromosome
alignment. Attenuation of Aurora B activity, by chemical
inhibition with hesperadin, improved chromosome alignment to
the metaphase plate in Ufd1-depleted cells. Moreover, Ufd1depletion partially rescued congression and metaphase plate
formation in hesperadin-treated cells. This functional interaction
again suggests that Cdc48/p97–Ufd1–Npl4 antagonizes Aurora B
during chromosome congression and that overactivation of Aurora
B accounts, at least in part, for the phenotype of Ufd1–Npl4
depletion in HeLa cells.
A recent report, published during the revision of this manuscript,
revealed that in Saccharomyces cerevisiae, Cdc48/p97 also
counteracts Aurora (Ipl1) during chromosome bi-orientation (Cheng
and Chen, 2010). Taken together with our data presented here, this
suggests that the antagonizing function of Cdc48/p97 towards
Aurora B is conserved during evolution. Interestingly, Cheng and
Chen further suggest a role for the p47 adapter homologue Shp1.
We did not find any evidence for an involvement of p47 in
chromosome alignment in HeLa cells, which might point to distinct
requirements for adapters in the two systems, possibly owing to
differences like the open compared with the closed mitosis.
The implication that excessive Aurora B activity at the
centromere, upon Cdc48/p97–Ufd1–Npl4 depletion, induces
congression defects is in line with the notion that both negative
and positive regulation of Aurora B is crucial for its role in
controlling bipolar spindle attachment (Kelly and Funabiki, 2009).
Excess Aurora B activity might either overly destabilize
microtubule attachments by phosphorylation of Aurora-Bcontrolled anchor points, such as Ndc80 (DeLuca et al., 2006).
Alternatively, it might prevent adjustment of incorrect spindle
attachment to kinetochores by phosphorylation and thus inhibition
of the microtubule-depolymerizing activity of MCAK (Andrews
et al., 2004; Lan et al., 2004; Ohi et al., 2004). The requirement
for negative regulation to limit Aurora B activity has been shown
before in the case of protein phosphatase-1, which antagonizes
Aurora B at the kinetochore to ensure proper chromosome
segregation (Emanuele et al., 2008; Francisco et al., 1994; Pinsky
et al., 2006). It will be interesting to clarify whether phosphatases
and the ubiquitin system, including Cdc48/p97, cooperate to
control Aurora B activity.
Materials and Methods
Antibodies and plasmids
Monoclonal (1:250; 5E2) and polyclonal (HME14) anti-Ufd1, anti-Npl4 (HME16)
(all 1:500), anti-p47 (1:5000; HME22) and anti-Cdc48/p97 (1:2000; HME01)
antibodies were as described previously (Meyer et al., 2000). Antibodies against the
following proteins were purchased and used at the indicated dilutions: Aurora B
(1:500; 611082, BD Bioscience), phosphorylated Aurora B (serine 10) (1:200;
Rockland), TopoIIa (1:1000; Boehringer Ingelheim), phosphorylated histone H3
Journal of Cell Science
Cdc48/p97 antagonizes Aurora B during chromosome segregation
1579
(serine 10) (1:4000; 06-570, Upstate), GAPDH (G8795, Sigma) 1:5000,
phosphorylated CENP-A (1:1000; 04-792, Millipore), cyclin B1 (1:500; sc-245,
Santa Cruz Biotechnology), anti-survivin (1:500; AF886, R&D Systems) and human
anti-centromere (kinetochore) CREST (1:400, Antibodies).
Mouse Ufd1 cDNA was cloned into pcDNA3.1 (Invitrogen) with a sequence
coding for a C-terminal Myc tag. Mouse Ufd1 cDNA was cloned into the BamHI
site of pEGFP-N3 (Clontech) and the sequence stretch 5⬘-CTGCGGGTGATGGAGACCAAA-3⬘ was mutated to 5⬘-CTGCGGGTAATGGAAACTAAG-3⬘
by the Quikchange protocol to render it resistant to V-S2 Ufd1 siRNA.
We thank Izabela Sumara, Matthias Peter, Monica Gotta and Patrick
Meraldi for help and critical discussions. We thank Catherine Brasseur
for technical help. This work was supported by a grant of the Deutscher
Akademischer Austauschdienst (to O.P.), and grants of the
Schweizerischer Nationalfond (3100A0-113749), the ETH (TH-03
07-1) and the Novartis Research Foundation (to H.M.). M.H.A.S.
generated H2B–mRFP-expressing cells and, together with D.W.G.,
designed quantitative live imaging assays.
Cell culture, establishment of stable cell lines and RNAi
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/124/9/1571/DC1
For routine cell culture, HeLa Kyoto cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum
and 1% penicillin/streptomycin (routine media). For RNAi, cells were plated 1 day
before transfection with 100–200 nM siRNA (Microsynth, Switzerland) using
Oligofectamine, or 10 nM siRNA using Lipofectamine RNAiMAX in OptiMEM
minimal medium according to the manufacturer’s instructions (Invitrogen). Cells
were analysed 48 hours after transfection. As a negative control in each experiment,
a non-silencing siRNA with the sense sequence 5⬘-UUCUCCGAACGUGUCACGUdTdT-3⬘ (adapted from Ambion, Austin, TX) was used. The following siRNAs,
targeting Cdc48/p97 cofactors Ufd1, Npl4 and p47 were used (sense strand): Ufd1S1, 5⬘-GGGCUACAAAGAACCCGAAdTdT-3⬘; Ufd1-S2, 5⬘-GUGGCCACCUACUCCAAAUdTdT-3⬘; Ufd1-S3, 5⬘-CTACAAAGAACCCGAAAGAdTdT-3⬘;
Ufd1-S4, 5⬘-ACAAAGAACCCGAAAGACAdTdT-3⬘; Ufd1-S5, 5⬘-CTGGGCTACAAAGAACCCGAAdTdT-3⬘; Npl4-S1, 5⬘-CGUGGUGGAGGAUGAGAUUdTdT-3⬘; Npl4-S2, 5⬘-CAGCCUCCUCCAACAAAUCdTdT-3⬘; p47-S1,
5⬘-AGCCAGCUCUUCCAUCUUAdTdT-3⬘;
and
p47-S2,
5⬘-UCAGAGCCUACCACAAACAdTdT-3⬘. If not otherwise stated, Ufd1-S2, Npl4-S2 and p47-S1
were used for functional assays. Aurora B siRNA, 5⬘-GGTGATGGAGAATAGCAGT-3⬘ (Hauf et al., 2003), Ufd1-V-S1 and -V-S2 were as published previously
(Vong et al., 2005).
Cell extracts
For western blot analysis, HeLa cells were lysed for 15 minutes on ice in 150 mM
KCl, 25 mM Tris-HCl pH 7.4, 5 mM MgCl2, 5% glycerol, 1% Triton X-100, 1 mM
dithiothreitol (DTT) and EDTA-free protease inhibitor cocktail (Roche), and
centrifuged at 10,000 g at 4°C for 20 minutes, with the supernatant recovered for
analysis. For fractionation, cells were synchronized in 330 nM nocodazole for 16
hours. Mitotic cells were harvested by shake-off and the mitotic index determined.
Equal numbers of cells were incubated for 15 minutes in lysis buffer (10 mM
HEPES pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 340 mM sucrose, 10% glycerol, 0.25%
Triton X-100, 1 mM DTT and protease inhibitors) and lysed by 20 strokes in a
douncer. The chromatin was sedimented by centrifugation at 1300 g for 5 minutes
and washed twice in lysis buffer; this supernatant, which contained the soluble
proteins, was centrifuged at 16,000 g for 15 minutes and the supernatant harvested
for analysis. Equal fractions were subjected to western blotting.
Confocal live cell imaging (mitotic timing)
Stable H2B–mRFP HeLa Kyoto cells growing on Ibidi chambers were transfected
with siRNAs. Cells were cultured, for data acquisition, in minimal essential medium
with Hank’s F12 (MEM/F12; Gibco) supplemented with 15 mM HEPES pH 7.4.
Confocal fluorescence time-lapse movies were taken with a Leica SP2 AOPS
confocal microscope equipped with a 20⫻ 0.7 NA Apofluor objective lens. Cells
were kept at 37°C during imaging. For acquisition, pictures from one focal plane of
living cells were taken every 1–2 minutes. Microscope settings were as follows:
picture average, 6; 400 Hz scan speed; 8-bit resolution; logical size, 1024⫻1024,
PMT 1: 0.7 Offset, 736.2 volts; PMT2: 3.5 Offset, 677.60 volts. Images were
processed using Imaris (Bidplane) and ImageJ software.
Indirect immunofluorescence
Cells were grown on glass coverslips and transfected with siRNAs as described
above. HeLa cells were either first treated with pre-extraction buffer (25 mM Hepes
pH 7.5, 50 mM NaCl, 1 mM EGTA, 3 mM MgCl2, 300 mM sucrose and 0.5%
Triton X-100) for 5 minutes at 4°C, or fixed directly with 4% paraformaldehyde in
PBS for 10–15 minutes. For hypotonic treatment, cells were incubated for 5 minutes
in RSB buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl and 5 mM MgCl2) before
fixing. Cells were permeabilized with 0.1% Triton X-100 (Sigma) in PBS for 5–10
minutes and washed as described above. Permeabilized cells were blocked with 3%
BSA in PBS for 30 minutes, followed by indirect immunostaining. Samples were
mounded with Mowiol and analyzed using a Leica SP2 AOPS or a SP1 AOPS
confocal microscope, or Zeiss Axiovert TV or Leica DM6000B epifluorescence
microscope. Images were processed and intensities quantified with the Imaris or
ImageJ software, respectively.
Statistical analysis
Datasets were quantified with Sigma Plot and Past (http://palaeoelectronica.pangaea.de/2001_1/past/issue1_01.htm) software. Normal and non-normal
distributed and datasets with equal and different sample numbers, were analyzed by
Student’s t-test, except for Fig. 1C where a Mann–Whitney U-test was used.
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