Short Report
3455
Aneuploidy in mitosis of PtK1 cells is generated by
random loss and nondisjunction of individual
chromosomes
Liliana Torosantucci1, Marco De Santis Puzzonia1, Chiara Cenciarelli2, Willem Rens3 and
Francesca Degrassi1,*
1
IBPM Institute of Molecular Biology and Pathology, CNR National Research Council, c/o University ‘La Sapienza’, Via degli Apuli 4, 00185 Rome,
Italy
2
Department of Biology, University Roma Tre, V.le Marconi 446, 00146, Rome, Italy
3
Centre for Veterinary Science, Department of Veterinary Medicine, University of Cambridge, Cambridge CB3 OES, UK
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 19 July 2009
Journal of Cell Science 122, 3455-3461 Published by The Company of Biologists 2009
doi:10.1242/jcs.047944
Summary
Chromosome lagging at anaphase and migration of both sister
chromatids to the same pole, i.e. nondisjunction, are two
chromosome-segregation errors producing aneuploid cell
progeny. Here, we developed an assay for the simultaneous
detection of both chromosome-segregation errors in the
marsupial PtK1 cell line by using multiplex fluorescence in situ
hybridization with specific painting probes obtained by
chromosome flow sorting. No differential susceptibility of the
six PtK1 chromosomes to undergo nondisjunction and/or
chromosome loss was observed in ana-telophase cells recovering
from a nocodazole- or a monastrol-induced mitotic arrest,
suggesting that the recurrent presence of specific chromosomes
in several cancer types reflects selection effects rather than
differential propensities of specific chromosomes to undergo
missegregation. Experiments prolonging metaphase duration
during drug recovery and inhibiting Aurora-B kinase activity
Introduction
Missegregation of duplicated chromosomes within a bipolar mitosis
produces aneuploid cells, i.e. cells showing a gain or loss of entire
chromosomes. One well-known segregation error is chromosome
lagging, occurring when a single chromatid remains at the spindle
equator after chromosome migration because its kinetochore is
connected to both spindle poles through a merotelic attachment
(Cimini et al., 2001). Another segregation error is nondisjunction,
i.e. the migration of both sister chromatids to the same pole. The
term nondisjunction, originally referring to genetically identified
meiotic missegregation, has been commonly used to imply a failed
separation of homologous chromosomes or sister chromatids at the
centromere. However, this idea has been brought into question by
fluorescence in situ hybridization (FISH) data showing that comigration of sister chromatids to the same pole occurs in the vast
majority of the cases after centromere separation in somatic cultured
cells (Cimini et al., 1999). Furthermore, precocious sister-chromatid
separation due to cohesion defects has been shown to end up as
sister-chromatid co-migration in yeast (Tanaka et al., 2000; Kitajima
et al., 2004) and premature centromere splitting generates cell
aneuploidy in the mosaic variegated aneuploid (MVA) syndrome
(Kajii et al., 2001), all implying that there are mechanisms different
from retained cohesion in the origin of nondisjunction. In relation
to genetic consequences, nondisjunction produces one trisomic
on metaphase-aligned chromosomes provided evidence that
some type of merotelic orientations was involved in the origin
of both chromosome-segregation errors. Visualization of merosyntelic kinetochore-microtubule attachments (a merotelic
kinetochore in which the thicker microtubule bundle is attached
to the same pole to which the sister kinetochore is connected)
identified a peculiar malorientation that might participate in the
generation of nondisjunction. Our findings imply random
missegregation of chromosomes as the initial event in the
generation of aneuploidy in mammalian somatic cells.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/122/19/3455/DC1
Key words: Chromosome loss, Nondisjunction, Aneuploidy,
Kinetochore-microtubule attachments, Merotelic attachments
(three copies of a specific chromosome) daughter cell and a
monosomic (only one copy of a specific chromosome) one; most
lagging chromosomes will form a micronucleus (a chromatin body
separated from the main nucleus) producing a monosomic daughter
cell with or without a micronucleus in half of the cases (Cimini
and Degrassi, 2005). Finally, micronuclei can be incorporated in
one of the daughter nuclei at the following mitosis producing further
unbalanced karyotypes (Rizzoni et al., 1989). Monosomic and
trisomic cells might constitute different threats to cell populations
and individuals. All monosomic and most trisomic gametes produce
nonviable embryos and are, therefore, an important cause of
spontaneous miscarriages and stillbirths in humans. However,
chromosome 13, 18 or 21 trisomies are compatible with life and
are the cause of severe genetic syndromes (Hassold and Hunt, 2001).
In addition, aneuploidy is a nearly ubiquitous feature of human
cancers and it has been suggested to play a crucial role both in
tumour development and progression (Rajagopalan and Lengauer,
2004; Weaver and Cleveland, 2006). Most cancer cells display a
complex pattern of chromosomal abnormalities, showing both
chromosome losses and gains, and concomitant structural
aberrations (http://cgap.nci.nih.gov/Chromosomes/Mitelman).
Interestingly, recurrent gains or losses of specific chromosomes or
chromosome regions (Holloway et al., 1999; Streblow et al., 2007;
Vasuri et al., 2008) are frequently observed in tumours, although
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the biological significance of these aneusomies in cancer is hard to
establish (Sen, 2000; Albertson et al., 2003). In fact, it is still
unknown whether specific chromosomes have different propensities
to undergo errors in chromosome segregation or whether their
presence in cancer cells is the result of cellular selection of cells
bearing random aneuploidy.
In mammalian cell cultures, chromosome loss is observed at low
frequencies and its frequency is enhanced in cells recovering from
a mitotic arrest induced by microtubule-interacting drugs (Cimini
et al., 2001). Much less is known about nondisjunction. Unequivocal
detection of nondisjunction requires a rather complicated assay, in
that segregation of individual chromosomes has to be followed by
in situ hybridization on ana-telophase cells. So, very few studies
have investigated nondisjunction in human cells; however, those
that have nevertheless suggest that nondisjunction occurs at
frequencies higher than chromosome loss (Cimini at al., 1999;
Thompson and Compton, 2008). Merotelic kinetochores have been
suggested to produce nondisjunction when more microtubules on
the merotelic kinetochore are connected to the same pole to which
its sister is attached (Salmon et al., 2005). However, due to
technical difficulties in visualizing merotelic attachments, this
hypothesis has remained unproven.
To address these issues, we developed an assay for the
simultaneous detection of chromosome loss and nondisjunction for
all chromosome pairs in the marsupial PtK1 cell line. This cell line
was chosen because PtK1 cells have been the model system for
studies on chromosome loss at mitosis and possess a very low
chromosome number (2n=12), making easier the analysis of
chromosome segregation for individual chromosomes on anaphase
cells. Segregation of individual chromosomes was followed using
‘multiplex FISH’ (M-FISH) (Speicher et al., 1996), a special kind
of chromosome painting that has been applied to unambiguously
differentiate chromosomes in several marsupial species (Rens et
al., 2003a; Rens et al., 2006).
Results and Discussion
For the simultaneous detection of chromosome loss (CL) and
nondisjunction (ND) PtK1 chromosome-specific painting probes
were developed from flow-sorted chromosomes. PtK1 chromosome
preparations produced six different peaks in the flow karyotype,
corresponding to the six chromosome pairs (Fig. 1A). PtK1
chromosome-specific paints with different colours were generated
through high purity sorting and degenerate oligonucleotide primed
(DOP)-PCR amplification incorporating fluorescence tagged
dUTPs. These paints were applied in M-FISH experiments on
metaphase cells to identify each of the six marsupial chromosome
pairs in a single metaphase spread by a different colour (Fig. 1B).
Furthermore, M-FISH analysis on metaphase cells showed that the
small chromosome 5 was present in just one copy in this PtK1 cell
line, as visible in the reconstructed karyotype (Fig. 1B).
Missegregation events do not occur at a different rate for
specific chromosomes
Chromosome segregation was then evaluated by M-FISH analysis
on ana-telophase cells. The high specificity of the chromosome
paints rendered it possible to visualize the position occupied by
each chromosome in the anaphase cell, with the largest chromosome,
chromosome 1, protruding towards the periphery of the group of
migrated chromosomes and the smallest chromosome, chromosome
5, in a central position surrounded by the other chromosomes (Fig.
2A). Chromosome misdistribution was also easily identified using
Fig. 1. Chromosome characterization of Potorous tridactylus PtK1 cells.
(A) Bivariate flow karyotype of PtK1 cells. Chromosomes are separated in
individual peaks corresponding to the six chromosome pairs; each peak is
associated with the corresponding chromosome number (1-5 and X).
(B) Example of chromosome painting on a PtK1 metaphase. Chromosome
pairs, shown in different colours, are indicated below; the unpaired
chromosome 5 is present in single copy. Scale bar: 10 μm.
M-FISH. The lagging of chromosomes 1 and 3 between the two
groups of chromosomes (Fig. 2B, arrows) as well as the presence
of three copies of chromosome 4 at one pole (Fig. 2B, arrowhead),
i.e. an event of ND, could be appreciated by analyzing the images
from the individual filters. With this approach we set out to
investigate chromosome-specific differences in missegregation
rates. ND and CL are rare events in untreated cells; therefore, no
clear differences among chromosomes were observed in untreated
cultures, although the extremely low number of events did not allow
to draw any statistically sound conclusion (data not shown). A
treatment with the anti-mitotic agents nocodazole (NOC; Fig. 2C)
or monastrol (MON; Fig. 2D) was used to increase the frequencies
of ND and CL in ana-telophases harvested after drug release. To
assess whether specific chromosomes were involved at different
rates, we first analyzed the deviation from the expected value of
ND or CL calculated by assuming that all chromosomes responded
equivalently (specific chromosome ND = total ND/6). The number
of ND and CL events observed for each chromosome pair was then
compared with the expected value in a χ2 test. This analysis showed
no significant deviations from the expected values for both drugs,
indicating that no chromosome was preferentially involved in ND
or CL. To further investigate this, we performed a different data
analysis comparing missegregation rates between each chromosome
3457
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Aneuploidy and merotelic attachments
Fig. 2. Chromosome-segregation errors occur at similar rates for the six PtK1 chromosome pairs. (A) Representative anaphase from untreated PtK1 cells showing
proper chromosome segregation. Images from individual filters show that two copies (one copy for chromosome 5) of each chromosome have migrated to each
pole. (B) Example of chromosome missegregation after NOC treatment. M-FISH shows ND for chromosome 4, i.e. three intact copies at one pole and just one at
the other pole (painted blue; arrowhead), and loss of chromosome 1 and 3 (painted red and light blue, respectively; arrows). Scale bar: 10 μm (in A for A and B).
(C,D) Frequencies of ND and CL events for individual chromosomes in ana-telophases recovering from (C) a NOC-induced mitotic block or (D) a MON-induced
mitotic block. At least 600 cells in three independent experiments were counted for each treatment. Chromosome-5 missegregation frequencies are presented
doubled because chromosome 5 is present in one copy in this PtK1 cell line.
and every other one. This analysis revealed that chromosomes 1
and 5, which represent the largest and smallest chromosome in PtK1
cells, respectively, showed lower ND frequencies compared with
those observed for all other chromosomes after NOC treatment
(chromosome 1: P<0.05; chromosome 5: P<0.01). This low
representation of the two chromosomes was not observed for CL:
in this case, chromosomes 1 and 5 did not show any significant
difference from the other chromosomes. Analysis of chromosome
susceptibility to missegregate after MON treatment showed that
chromosome missegregation rates were homogeneous for all of the
chromosomes, the only exception being that the CL frequency for
chromosome 4 was significantly lower than that observed for the
other chromosomes (P<0.01). In conclusion, statistically significant
differences between chromosomes were limited and did not permit
the identification of any chromosome-specific tendency because
they were not homogeneous neither for the chromosome involved,
nor for the type of missegregation analyzed (ND vs CL), nor were
they related to a specific inducing drug. Collectively, these data
indicate that the six PtK1 chromosomes undergo ND and/or CL to
the same extent. Furthermore, they suggest that correction of
kinetochore malorientations, which involves the Aurora-Bdependent phosphorylation of the depolymerizing kinesin MCAK
(Kline-Smith et al., 2004; Lan et al., 2004; Knowlton et al., 2006;
Andrews et al., 2004) together with the activity of the Kif2b kinesin
(Bakhoum et al., 2008), and microtubule flux (Ganem et al., 2005),
is equally efficient on the different chromosomes, producing random
chromosome missegregation at mitosis.
Among the many factors that have been proposed to influence the
susceptibility of individual chromosomes to missegregate,
chromosome size and chromosome position within the mitotic
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spindle are regarded as crucial. Our data on anaphase cells (Fig. 2A;
supplementary material Fig. S1) as well as previous live imaging
data on the same cell line (Cimini et al., 2004) provide evidence that
larger chromosomes tend to position at the metaphase-plate periphery,
possibly owing to a steric hindrance of the large chromosome arms.
In this area of the spindle, chromosome oscillations tend to be shorter
and less frequent (Khodjakov and Rieder, 1996; Cimini et al., 2004),
which might be due to reduced directional instability mediated by
accumulation of the Kif18a kinesin on peripheral kinetochores
(Stumpff et al., 2008). Reduced chromosome oscillations could
theoretically decrease the correction of erroneous kinetochoremicrotubule attachments, which requires the release of microtubules
from kinetochores and their successive interaction at a different angle
(Lampson et al., 2004). Our data indicate that chromosome oscillations
at the metaphase plate do not affect missegregation rates of individual
chromosomes but rather suggest a similar correction efficiency
among the different kinetochores.
It is well known that random chromosome missegregation occurs
in meiosis. Aneuploidies for nearly all chromosomes are represented
among spontaneous abortions at very early stages of development,
but only trisomies for a few chromosomes are vital at birth,
demonstrating that meiotic aneuploidies are subjected to prenatal
selection (Hassold and Hunt, 2001). Our data suggest a similar
scenario for mitotic cells, in which the recurrent presence of specific
chromosomes in several cancer types is not the result of a different
propensity of specific chromosomes to undergo missegregation but
is the outcome of a selection process that depends on the identity of
the extra chromosome. In line with this, a recent study has shown
that aneuploid murine embryonic fibroblasts (MEFs) containing an
extra copy of only one chromosome exhibit decreased proliferation
rates and decreased cell fitness (Williams et al., 2008). However, the
timing of cell immortalization, one of the early steps in cell
transformation in culture, depended on which additional chromosome
was present in the different cell lines (Williams et al., 2008). Thus,
it can be hypothesized that, although the presence of an extra
chromosome is generally detrimental, a limited number of specific
unbalances of gene products caused by individual chromosome
aneuploidy, possibly in conjunction with additional mutations in
oncogenes and/or tumour suppressors, might promote tumorigenesis.
Merotelic attachments and nondisjunction
Having established that no preferential involvement in chromosome
missegregation occurs for specific chromosomes, we then wished to
obtain new insights into the mechanism(s) underlying ND. For correct
segregation to occur, sister kinetochores must attach to microtubules
connected to opposite poles at metaphase. This so-called amphitelic
orientation satisfies the mitotic-checkpoint requirements and allows
the segregation of the two sisters to opposite poles. Other orientations
are also possible and are transiently present during prometaphase,
including monotelic (a chromosome with only one kinetochore
attached to microtubules), syntelic (both sister kinetochores connected
to the same pole) and merotelic orientations, the latter being the cause
for chromosome lagging when persisting at anaphase (Cimini and
Degrassi, 2005). To gain insight into the role of these different
orientations in the generation of ND, we took advantage of the
different mechanisms of action of NOC and MON. NOC is a wellknown spindle poison capable of disassembling the mitotic spindle.
Following NOC removal, the mitotic spindle reassembles through
both microtubule nucleation from centrosomes and incorporation into
the centrosomal-based microtubule arrays of microtubules nucleated
at kinetochores (Tulu et al., 2006; Torosantucci et al., 2008). During
Fig. 3. Chromosome-segregation errors are efficiently induced following both
NOC or MON treatment. (A,B) ND and CL frequencies in DMSO-treated anatelophases (DMSO) and in ana-telophase cells fixed after 45 or 60 minutes of
NOC washout (A; NOC+45 and NOC+60, respectively) or MON washout (B;
MON+45 and MON+60, respectively) both without (black bars, –MG132) and
with (grey bars, +MG132) a 2-hour incubation in MG132 after drug release.
Between 200 and 400 cells were counted for each treatment from three
independent experiments.
this process, several extra-centrosomal foci of α-tubulin are present
at very short times after NOC removal (Cimini et al., 2003; Tulu et
al., 2006) (supplementary material Fig. S2); these foci favour the
formation of high numbers of incorrect kinetochore-microtubule
interactions, in large part merotelic attachments. MON is a smallmolecule inhibitor of the mitotic kinesin Eg5 that is able to arrest
cells in mitosis with monoastral spindles; chromosomes in MONtreated cells frequently exhibit sister kinetochores attached to
microtubules in a syntelic orientation (Kapoor et al., 2000). Upon
MON removal, syntelic orientations are transformed into amphitelic
ones through shortage and severing of the syntelic K-fibres and
successive attachment of other microtubules at the kinetochores
(Lampson et al., 2004) (supplementary material Fig. S2). During this
process, several types of mis-attachment are produced.
Missegregation for all PtK1 chromosomes was analyzed in
untreated cells and in cells undergoing ana-telophase 45 or 60
minutes after NOC or MON release (Fig. 3). Approximately 6%
of untreated PtK1 ana-telophase cells underwent chromosome
missegregation (35 events/569 cells), showing 4.0% ND and 2.1%
CL when controls from MON and NOC experiments were pooled
together. This suggests that ND occurs at higher rates in comparison
with CL in somatic mammalian cells, as previously observed in
human primary fibroblasts (Cimini et al., 1999). ND and CL were
efficiently induced over control values in cells harvested 45
minutes after NOC release (Fig. 3A) (P<0.005) with an
approximate twofold induction of ND in comparison with CL
(28.1% ND vs 12.6% CL). Ana-telophases recovering from MON
treatment exhibited similar rates of missegregation (19.6% ND vs
13.1% CL), demonstrating that MON also efficiently induces
chromosome missegregation (Fig. 3B) (P<0.001). Cells that were
harvested 60 minutes after drug release displayed lower frequencies
of CL and ND, both after NOC (Fig. 3A) and after MON (Fig.
3B) treatment, suggesting that the correction of faulty attachments
during metaphase decreases the frequencies of CL and ND for both
drugs. Merotelic kinetochore attachments occur frequently in early
mitosis and are partially corrected before anaphase onset when
metaphase is prolonged using the proteasome inhibitor MG132
Aneuploidy and merotelic attachments
3459
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Fig. 4. Partial inhibition of Aurora-B activity by
hesperadin increases missegregation. (A) Inhibition
of Aurora-B activity as assayed by microscope
analysis of anti-histone-H3 (Ser10) phosphoantibody staining (Phospho-H3). (B) Phospho-H3
antibody staining in DMSO-treated prometaphase
cells (DMSO) or prometaphase cells after 1-hour
incubation in 20 nM or 100 nM hesperadin (hesp)
alone, or during the last hour of MG132 incubation
of NOC-released cells. At least 100 cells from two
independent experiments were counted for each
treatment. (C) ND or CL frequencies in control anatelophases (DMSO) or in ana-telophases recovering
from a NOC-induced mitotic block (NOC) in drugfree medium (black bars, –MG132,), after a 2-hour
incubation in MG132 (grey bars, +MG132) or in
cells incubated in 100 nM hesperadin during the
last hour of MG132 treatment (blue bars,
+MG132/hesp). Data are the sum of two-three
independent experiments. (D) Different types of
merotelic kinetochores are observed after NOC
treatment. Maximum projections from deconvolved
three-dimensional (3D) image stacks showing outer
kinetochore (Hec1, red), centromere (CREST, blue)
and spindle microtubule (α-tubulin, green)
immunofluorescence are shown. The reciprocal
arrangement of outer-kinetochore and centromere
signals on single z-slices coupled with Hec1
distortion was used to identify sister kinetochores
and kinetochore-microtubule interactions.
Kinetochore-microtubule interactions were verified
by rotating reconstructed 3D images. The following
types of merotelic orientations (a single kinetochore
attached to two microtubule bundles) were
observed: mero-syntelic (the thicker microtubule
bundle is towards the same pole to which the sister
kinetochore is connected), mero-amphitelic (the
thicker microtubule bundle is connected to the pole
opposite to that of the sister kinetochore) and
balanced-merotelic (the fluorescence intensity of
the two microtubule bundles attached at the single
kinetochore is similar). Insets show a twofold
magnification of the boxed regions. The different
orientations are diagrammed below the insets. Scale
bars: 10 μm.
(Cimini et al., 2003). We also delayed anaphase onset by MG132
treatment to determine whether this had an effect on ND. The
frequencies of ND were efficiently decreased by incubation in
MG132 medium during recovery from either drug (P<0.001 and
P<0.05 at 45 and 60 minutes, respectively), although they never
reached control levels (Fig. 3A,B). CL frequencies were also
decreased (Fig. 3A,B), although to a lesser extent (P<0.05 when
data from both times were pooled together). Altogether, these data
indicate that a fraction of the faulty attachments responsible for
ND is corrected before anaphase, as already observed for lagging
chromosomes (Cimini et al., 2003).
Correction of merotelic attachments before anaphase requires
Aurora-B kinase activity as part of an inter-kinetochore tensionsensing mechanism (Kline-Smith et al., 2004; Lan et al., 2004;
Knowlton et al., 2006; Andrews et al., 2004; Cimini et al., 2006).
We therefore investigated whether Aurora-B activity was responsible
for the observed time-dependent correction of ND by using
hesperadin, a known Aurora-B inhibitor (Hauf et al., 2003). The
inhibitor was used at a dose (100 nM) that only partially suppressed
Aurora-B activity, as determined by histone H3 (Ser10)
phosphorylation (Fig. 4A,B), because partial inhibition of the kinase
activity had already been shown to increase merotelic orientations
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and lagging chromosomes at anaphase without influencing other
aspects of mitosis such as checkpoint efficiency or cytokinesis (Cimini
et al., 2006). To investigate whether merotelic attachments are
involved in ND, cells were released from a NOC-induced mitotic
arrest in MG132 medium and hesperadin was added 1 hour later. In
such a way, Aurora-B activity was inhibited when chromosomes had
already reached the metaphase configuration in the vast majority of
the cells (supplementary material Fig. S3), a moment when syntelic
attachments, which maintain chromosomes close to the poles (Cimini
et al., 2002; Cimini and Degrassi, 2005), were already corrected. Cells
were kept in MG132 and hesperadin for a further 1 hour and then
allowed to progress to anaphase by medium change. Inhibition of
Aurora B in this time window very efficiently increased CL frequency
above the value observed with MG132 alone (Fig. 4C), as already
reported (Cimini et al., 2006). Interestingly, correction of improper
attachments generating ND was also inhibited, as shown by the
increased frequencies of ND above the MG132 value (Fig. 4C)
(P=0.126). These data indicate that improper kinetochore-microtubule
attachments generating ND are present on aligned chromosomes and
that Aurora-B activity is required for their correction.
So, which type of kinetochore-microtubule interactions produces
ND? Anaphase onset in the presence of syntelic attachments would
produce chromosome ND, because both sister kinetochores are
connected to the same pole. However, several lines of evidence
converge to demonstrate that syntelic attachments activate the
mitotic checkpoint (Pinsky and Biggins, 2005; Porter et al., 2007),
rendering it less likely that syntelic chromosomes will undergo ND
in checkpoint-proficient cells. In our data, the high ND frequencies
obtained after inducing large amounts of merotelic orientations
(NOC treatment) (Fig. 3A) as compared with preferentially inducing
syntelic orientations (MON treatment) (Fig. 3B), as well as the effect
of Aurora-B inhibition on aligned chromosomes (Fig. 4C), suggest
that kinetochore-microtubule interactions that are different from
syntelic attachments, which localize chromosomes close to the
spindle poles, also contribute to ND. Monotelic orientations are very
unlikely to generate ND. Even if monotelic chromosomes were able
to reach the metaphase plate by sliding on already existing K-fibres
(Kapoor et al., 2006), the unattached kinetochore would strongly
activate the mitotic checkpoint, blocking anaphase onset. Merotelic
chromosomes congress to the metaphase plate (Cimini et al., 2002).
They have been shown to maintain connection to the two
microtubule bundles during anaphase and lag at the cell equator
when the two bundles are of similar size (balanced merotelic) or
move to the pole connected to the thicker microtubule bundle
(Cimini et al., 2004). In principle, a merotelically oriented
kinetochore, in which the thicker microtubule bundle is connected
to the same pole to which the sister is attached, would segregate to
the same pole as the sister. These so-called ‘mero-syntelic’
attachments have been shown to be responsible for ND in live cranefly meiosis (Janicke at al., 2007), a favourable system to detect
kinetochore-microtubule interactions, owing to the large dimension
of the cells. To identify these different merotelic attachments in
PtK1 cells, we analyzed Ca2+-resistant kinetochore-microtubule
interactions after z-stack acquisition and image deconvolution of
prometa-metaphase cells, which were imaged to visualize the
centromere, the outer kinetochore region and microtubules using
CREST, Hec1 and tubulin antibodies, respectively (Fig. 4D). In
cultured mammalian cells, clear identification of kinetochoremicrotubule interactions is restricted to the peripheral area of the
spindle because of the high density of microtubules into the central
area. Taking this into account, we were nevertheless able to show
that, in addition to syntelic and amphitelic attachments (not shown),
both balanced merotelic attachments, mero-amphitelics (a merotelic
kinetochore with the thicker MT bundle connected to the pole
opposite to that of the sister kinetochore) and mero-syntelics (the
thicker microtubule bundle towards the pole to which the sister
kinetochore is connected) are present during recovery from NOC
(Fig. 4D). In our microscopic analysis, about 10% of aberrant
orientations were mero-syntelics (4/45). So, by visualizing merosyntelic attachments we identified a peculiar kinetochoremicrotubule interaction that might be involved in the origin of ND
in mammalian somatic cells. However, further high-resolution livecell studies or electron-microscopy evidence are required to
unequivocally identify mero-syntelics as being responsible for ND.
In conclusion, our M-FISH studies in conjunction with the analysis
of kinetochore-microtubule interactions provide evidence indicating
that ND and CL are generated by different types of merotelic
attachments. These might originate during the extremely dynamic
process of centrosome separation and microtubule growth that occurs
during recovery from drug treatment. The position of centromeres in
relation to centrosomes during the initial stages of this process seems
to be a stronger determinant for the formation of incorrect interactions
with respect to structural differences at kinetochores among the
different chromosome pairs, as we did not observe a preferential
involvement of specific chromosomes in segregation errors. Finally,
our findings imply random missegregation of chromosomes as the
initial event in the generation of aneuploidy and in the acquisition or
loss of specific chromosomes in cancer.
Materials and Methods
Cell culture and treatments
PtK1 cells were grown as described (Rens et al., 1999). NOC, MON and MG132
(Sigma-Aldrich) were dissolved in DMSO. To promote chromosome missegregation,
cells were incubated in 2 μM NOC for 3 hours or 100 μM MON for 4 hours.
Thereafter, cells were washed three times in pre-warmed medium to release the mitotic
block, re-incubated in fresh medium and fixed at the indicated times after drug release.
In MG132 experiments, 15-25 minutes after NOC or MON release, cells were
incubated in 7 μM MG132 for 2 hours, then washed three times for 5 minutes in
pre-warmed medium and harvested after a further 45 or 60 minutes. 100 nM of the
Aurora-B kinase inhibitor hesperadin (a generous gift of Tarun Kapoor, The
Rockefeller University, NY) was supplied to some cultures for the last hour of MG132
treatment. Medium was then exchanged to remove hesperadin and MG132, and cells
were fixed after 45 or 60 minutes.
M-FISH analysis
PtK1 chromosome preparation and flow sorting were performed as previously
described (Rens et al., 1999). Flow-sorted chromosomes were used as templates for
DNA amplification by DOP-PCR (Telenius et al., 1992) and primary DOP-PCR
products were used to incorporate biotin-16–dUTP (Roche), FITC-dUTP, Cy5-dUTP,
Cy3-dUTP (Amersham), Texas-Red–dUTP (Molecular Probes) and DEAC-dUTP
(NEN Life Science Products) by PCR. FISH was performed as previously described
(Rens et al., 2003b), except that chromosome paints were denatured at 70°C for 10
minutes and slides were denatured at 55°C for 10 seconds. The biotin-labelled probe
was visualized by Cy5.5-avidin (Amersham). After detection, slides were
counterstained in 0.08 μg/ml DAPI in 2⫻SSC for 2 minutes and mounted in ProLong
Antifade (Molecular Probes).
Immunostaining
For the analysis of Ca2+-resistant kinetochore-microtubule interactions, slides were
fixed as described (Kapoor et al., 2000) except that they were post-fixed in methanol.
Primary antibodies were: CREST serum (Antibodies Incorporated, 1:50), anti-αtubulin FITC-conjugate (Sigma-Aldrich, 1:100) and anti-Hec1 (Abcam, 1:500). For
phospho-H3 staining, cells were fixed in ice-cold methanol for 5 minutes,
permeabilized 5 minutes in PBS/0.5% Triton-X and then incubated with anti-Ser10phospho-H3 antibody (Upstate Biotechnology, 1:100). DNA was counterstained in
0.1 μg/ml DAPI.
Fluorescence microscopy and image acquisition
M-FISH images were captured using Leica QFISH software (Leica Microsystems)
and a cooled Sensys CCD camera (Photometrics) mounted on a Leica DMRXA
microscope equipped with six specific filter sets and a 63⫻ (1.3 NA) objective.
Aneuploidy and merotelic attachments
Lagging chromosomes and ND were scored on anaphases with fully migrated
chromosomes (mid-anaphase), late anaphases and telophases. Chromosomes were
considered lagging if they were located inside the central 25% of the pole distance
after all chromosomes had migrated. Between 133-413 ana-telophases were scored
for each experimental point and data were analyzed using the χ2 test.
Immunofluorescence analysis of kinetochore orientation was carried out under an
Olympus Vanox microscope equipped with a 100⫻ (1.35 NA) objective and a SPOT
CCD camera (Diagnostic Instruments). Colour-encoded images were acquired using
ISO 2000 software (Deltasistemi). Three dimensional deconvolution and
reconstruction was performed on 0.3-μm-interval z-stacks of optical sections using
AutoDeblur 9.3 (AutoQuant Imaging) software.
We thank Tarun Kapoor for generously providing hesperadin, and
Daniela Cimini for many valuable suggestions and comments on this
work. This work was partially supported by grants from CNR (RTL
2007) and from AIRC.
Journal of Cell Science
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