Chromosoma (2006) 115:481–490
DOI 10.1007/s00412-006-0077-1
RESEARCH ARTICLE
Incomplete sister chromatid separation of long chromosome
arms
W. Rens & L. Torosantucci & F. Degrassi &
M. A. Ferguson-Smith
Received: 11 April 2006 / Revised: 16 August 2006 / Accepted: 20 August 2006 / Published online: 5 October 2006
# Springer-Verlag 2006
Abstract Chromosome segregation ensures the equal
partitioning of chromosomes at mitosis. However, long
chromosome arms may pose a problem for complete sister
chromatid separation. In this paper we report on the
analysis of cell division in primary cells from field vole
Microtus agrestis, a species with 52 chromosomes including two giant sex chromosomes. Dual chromosome
painting with probes specific for the X and the Y
chromosomes showed that these long chromosomes are
prone to mis-segregate, producing DNA bridges between
daughter nuclei and micronuclei. Analysis of mitotic cells
with incomplete chromatid separation showed that reassembly of the nuclear membrane, deposition of INner
CENtromere Protein (INCENP)/Aurora B to the spindle
midzone and furrow formation occur while the two groups
of daughter chromosomes are still connected by sex
chromosome arms. Late cytokinetic processes are not
efficiently inhibited by the incomplete segregation as in a
significant number of cell divisions cytoplasmic abscission
proceeds while Aurora B is at the midbody. Live-cell
imaging during late mitotic stages also revealed abnormal
cell division with persistent sister chromatid connections.
Communicated by F. Uhlmann
Electronic supplementary material Supplementary material is
available in the online version of this article at http://dx.doi.org/
10.1007/s00412-2006-0077-1 and is accessible for authorized users.
W. Rens (*) : M. A. Ferguson-Smith
Centre for Veterinary Science, University of Cambridge,
Cambridge CB3 OES, UK
e-mail: [email protected]
L. Torosantucci : F. Degrassi
Institute for Molecular Biology and Pathology CNR,
University ‘La Sapienza’,
Rome 00185, Italy
We conclude that late mitotic regulatory events do not
monitor incomplete sister chromatid separation of the large
X and Y chromosomes of Microtus agrestis, leading to
defective segregation of these chromosomes. These findings suggest a limit in chromosome arm length for efficient
chromosome transmission through mitosis.
Introduction
Sister chromatid separation at mitosis is tightly controlled
to ensure an equal partitioning of chromosomes to each of
the daughter cells. The process is governed by the
anaphase-promoting complex (APC), the activity of which
increases abruptly when all chromosomes are bioriented on
the mitotic spindle. This results in the proteasome-mediated
destruction of cyclin B and the inhibitory protein securin
(Morgan 1999). Securin destruction is required to trigger
the activation of separase that cleaves cohesin, the “glue”
protein that binds the two sister chromatids together at the
centromere (Uhlmann 2004).When all kinetochores are
attached to spindle microtubules, cohesin removal at the
centromere is completed by separase-mediated SCC1
cleavage and so anaphase ensues. The centromeres are the
sites that move apart first and then the traction exerted by
microtubules at centromeres moves the sister chromatids
apart.
However, sister chromatid separation is not instantaneous and in plants is limited in space by the cell wall.
These constraints may influence the equal partitioning of
chromosomes to the two daughter nuclei. It takes some time
for the separated chromatids to move from the equator to
one of the poles. If late-anaphase or telophase processes are
initiated during or at the end of chromosome movement,
large acrocentric or submetacentric sister chromatids might
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not be able to separate properly. While the centromeres of
these large chromosomes have reached the poles, the distal
ends of long arms may still be at the equator. This hampers
cytokinesis, as the site of furrow ingression and cell
membrane resolution is not clear of chromatin.
Very recently, a signaling pathway linking completion of
cytokinesis to chromosome separation has been proposed to
be active in yeast cells (Norden et al. 2006). The pathway
relied on the Aurora kinase Ipl1 in that spindle midzone
defects caused Ilp1-dependent activation of abscission
inhibitory proteins. Inactivation of the pathway led to
chromosome breakage by the cytokinetic machinery. This
result prompted the authors to propose that the pathway
prevents the chromosome arms still lagging in the cleavage
plane from being broken by the abscission machinery
(Norden et al. 2006). However, it is far from clear whether
this pathway operates in higher eukaryotes.
A limit in chromosome size has been demonstrated in
field beans (Vicia faba) in which the chromosome arm
length was artificially increased. This impeded proper
chromosome segregation and impaired the viability of
carriers (Schubert and Oud 1997). The same conclusion
was drawn from similar experiments with barley chromosomes (Hudakova et al. 2002). Recently, we observed that
the dimension of chromosomes influences their position in
the potoroo (Potorous tridactylus) nucleus (Rens et al.
2003b) and speculated a maximum size for potoroo
chromosome 1. The distal end of this chromosome forms
a nuclear protrusion in interphase, as it is probably wrapped
by the nuclear membrane while it is still positioned towards
the equator at the end of mitosis. What can be expected in a
species with one or two even larger chromosomes among a
large set of small chromosomes?
In this paper we report on the analysis of cell division of
the field vole, Microtus agrestis, a species with 52
chromosomes including two giant gonosomes (sex chromosomes). Both gonosomes have a large heterochromatic
region comprising around 17% of the genome (Singh et al.
2000). This region is highly enriched with L1 elements,
non-L1 retroposons and the repeated DNA sequence
pMAHae2 (Kalscheuer et al. 1996; Neitzel et al. 1998,
2002; Singh et al. 2000).
Cell division was analyzed in different ways. Dual
chromosome painting with probes specific for the X and
the Y chromosomes was used to identify and localize the
position of these large chromosomes during cell division.
An antibody against a component of the nuclear membrane
was used to observe its reassembly after nuclear division in
both Microtus agrestis and Potorous tridactylus. The latter
was included for comparison to confirm the hypothesis
made in our earlier report (Rens et al. 2003b). An antibody
against the inner-centromere protein INCENP was used to
follow the progress of cytokinesis in relation to chromo-
Chromosoma (2006) 115:481–490
some migration. After sister chromatid separation, this
passenger protein transfers from the centromeres to the
midzone of the spindle where it then indicates the site of
furrow formation (Vagnarelli and Earnshaw 2004). An
antibody against Aurora B was used to verify its possible
role in a checkpoint for abscission. Finally, Microtus
agrestis cells were transfected with an expression vector
encoding GFP-tagged histone H2B (Kanda et al. 1998) to
visualize chromosome dynamics during the late stages of
cell division by live cell imaging.
Materials and methods
Cell culture
Primary fibroblast cell cultures were obtained from biopsies
of a male Microtus agrestis (Magr) killed in the wild by a
domestic cat. The potoroo kidney line (PtK1; (Walen and
Brown 1962)) was obtained from Dr. R. Moore, Peter
McCallum Cancer Centre, Melbourne, Australia. A fibroblast cell line from Aepyprymnus rufuscens (2n=32) was
established in the Department of Genetics, La Trobe
University, Australia. The species Potorous tridactylus
(Ptri, 2n=12, 13; female, male) has a different number for
female and male due to a fusion between an autosome and
the X chromosome (Rens et al. 1999).
For 3D experiments, Ptri epithelial cells or Magr
fibroblast cells were grown overnight on coverslips in
DMEM + 10% FCS at 37°C. The coverslips were previously
immersed in 100% ethanol, dried, quickly flamed, deposited
in a petri dish, and microwaved for 4 min.
Chromosome paints
Flow sorting, chromosome paint production, and fluorescence in situ hybridization on metaphases were performed
according to the protocol described previously (Rens et al.
1999). Chromosome paints were produced from sorted
Aepyprymnus rufuscens chromosome 10, which is homologous to the distal end of potoroo chromosome 1 q (Rens
et al. 2003a,b). Microtus chromosome X and Y paints were
kindly provided by Dr. Fengtang Yang, Sanger Centre,
Cambridge, UK. The haptens used were FITC-dUTP, and
biotin-dUTP (Boehringer), the latter detected with avidinCy3 (Amersham, Little Chafont, UK).
FISH on 3D-preserved nuclei
Fluorescence in situ hybridization on 3D-preserved interphase nuclei was performed using the procedure described
in Croft et al. 1999 and Rens et al. 2003b. Coverslips with
cells were washed in 2×SSC, fixed in 4% paraformalde-
Chromosoma (2006) 115:481–490
hyde for 15 min and washed in 2×SSC. Slides were heated
to 80°C in citrate buffer for 4 min and washed in 2×SSC.
The cells were permeabilized in 1% saponin (SIGMA), 1%
Triton X-100 in PBS for 30 min and washed in 2×SSC. The
slides were incubated in 20% glycerol for 30 min and
susbsequently freeze-thawed three times in liquid nitrogen,
and washed in 2×SSC. Slides were then treated with 0.01%
pepsin in 10 mM HCl for 6 min and washed in 2×SSC. The
slides were directly denatured in 70% formamide in 2×SSC
at 68°C for 2 min and quenched in ice-cold 50% formamide
in 2×SSC.
Denatured chromosome paints were added to the nuclei
and the coverslips were sealed with rubber cement.
Hybridization occurred overnight. Care was taken that the
coverslips did not dry during the whole process. Detection
was identical to 2D FISH and was performed as described
previously (Rens et al. 1999).
Immunofluorescence: nucleoporins, INCENP,
and Aurora B
Potoroo or Microtus cells were subcultured on coverslips
the day before the procedure. Coverslips were washed in
PBS, fixed with 4% paraformaldehyde for 15 min, and
washed with PBT (PBS with 0.025% Tween-20). Subsequently, cells were permeabilized with 0.02% Triton X-100
in KB buffer (10 mM Tris–HCl, pH 7.7, 150 mM NaCl,
0.1% bovine serum albumin). The coverslips were incubated for 7 min at room temperature in KB buffer with 10%
goat serum.
The primary antibody against nuclear pore complex
proteins (mAb414, Covance) was diluted 1/1,000 in KB
buffer with 2% goat serum. The rabbit primary antibody
against (INCENP), kindly provided by Prof. WC Earnshaw
(Eckley et al. 1997; Adams et al. 2000), was diluted 1/
2,000 in KB buffer and 2% goat serum. The mouse primary
antibody against Aurora B (AIM-1, BD Biosciences) was
diluted 1/200 in KB buffer and 2% goat serum. The
secondary Alexa-488 goat anti-mouse antibody (Molecular
Probes) was diluted 1/400 in KB buffer with 2% goat
serum. The secondary Alexa-568 goat anti-rabbit antibody
(Molecular Probes) was diluted 1/500 in KB buffer and 2%
goat serum. Slides were incubated with the primary
antibody for 30 min at room temperature, washed with
PBT, and incubated with the secondary antibody for 30 min
at room temperature. After washing in PBT (3×3 min)
slides were mounted with Vectashield-DAPI (Vector).
Image analysis
Images were captured using the Leica QFISH software
(Leica Microsystems) and a cooled CCD camera (Photometrics Sensys) mounted on a Leica DMRXA microscope
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equipped with an automated filter wheel, DAPI, FITC, and
Cy3 specific filter sets and a 63×, 1.3 NA objective (for
metaphase/2D interphase) and 100×, 1.4 NA objective (for
3D interphase). The Leica DMRXA is equipped with an
automatic Z-table and the Leica QFISH software allows the
capture of Z-stacks. Images were captured with 0.3 μm
spacing. IMARIS software (Bitplane AG) was used for 3D
reconstruction. Huygens Essential software (Scientific
Imaging Solutions) was used for deconvolution and
rendering.
Analysis of chromosome segregation in live cells
Cells were plated 1 day before transfection in a 35-mm
culture dish. FuGENE 6 transfection reagent (Roche/
Boehringer Mannheim) was diluted (1/32) in 100 μl
serum-free medium, incubated for 5 min at room temperature, added to 1 μg of pEGFP-N1 vector encoding a
histone H2B-GFP fusion protein and incubated for 15 min
at room temperature. The medium in the petri dish was
replaced by fresh medium and the FuGene6/serum-free
medium/DNA was added dropwise to the petri dish. The
day after transfection the medium was replaced by fresh
medium and the petri dish was returned to the incubator for
additional 24 h. A Nikon Eclipse TE300 inverted microscope equipped with a Nikon Mats U505R30 Thermoplate
and a Nikon digital DXM1200 Camera was used to follow
mitotic progression by time-lapse microscopy. The microscope was controlled by the Nikon ACT-1 imaging
software.
Results
Chromosome painting
Figure 1 shows the result of dual colour FISH with probes
specific for the X chromosome (red) and Y chromosome
(green) on a Microtus agrestis metaphase. The large
constitutive heterochromatin sections of both chromosomes
are labeled in yellow, as these two regions hybridized to
both probes. The X-specific section in red extends just over
the X centromere and has a brighter band on the Xp arm
(see arrow). The Y-specific regions in green are localized at
both distal regions of the Y chromosome. The Y paint also
decorated two bands on the Xp arm, one adjacent to the
centromere and one in the middle of the Xp arm just above
the bright red X-paint signal (bands in yellow, see arrow
heads).
These bands are more clearly visible in the green image
(see inset). The X painting pattern is in agreement with
recently published results (Marchal et al. 2004), while the
Y-painting pattern is additional to those results. Not only
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Fig. 1 Microtus agrestis metaphase with X chromosome painted in
red and Y chromosome painted in green. The yellow parts of each
chromosome are DNA sequences shared by both sex chromosomes.
The Y-specific sequences are located at both ends of the Y
chromosome, the X-specific sequences are located on the short arm
and a region on the long arm adjacent to the centromere of the X
chromosome. In addition to the Xq-arm, two bands on the Xp arm
share sequences with the Y chromosome (see arrowheads in figure
and inset). The arrow points to a bright red band on Xp
Fig. 2 X-Y chromosome painting on Microtus agrestis anaphase and interphase cells. a and
b show two relatively normal
divisions, but c shows two
decondensed daughter nuclei
still connected by a DNA thread.
The connection consists of the
shared X-Y DNA sequences of
the long arm of chromosome
X, as the red X-specific region
can be seen on both nuclei.
The green dots seen in b are
nuclear pore proteins; see text
Chromosoma (2006) 115:481–490
Xq shares sequences with the Y chromosome but also two
bands on Xp. The band in the middle of Xp might coincide
with the repetitive sequence pMAHae2 Xp band reported in
Singh et al. 2000.
Chromosome painting with these probes was then
performed on ana-telophase cells and nuclei to identify
the position of these large chromosomes at the end of
mitosis and in interphase (Fig. 2). Figure 2a,b shows that at
late anaphase/telophase the chromosomes are in the process
of decondensation and the majority are retracted into a
sphere (green dots in Fig. 2b are nuclear pore proteins; see
next section). However, the large gonosomes are still
projected towards the equator. Figure 2b shows a telophase
cell in which chromatid separation is complete, but Fig. 2c
shows two interphase nuclei that are still connected; sister
chromatid separation of the X chromosome was not
complete. The connection can break, leaving a nucleus
with micronuclei. In every case of improper division we
observed that one of the sex chromosomes was involved;
the micronuclei were painted yellow, e.g., a section of the
constitutive heterochromatin region.
Are processes of cell division such as furrow formation
and nuclear membrane reassembly still proceeding despite
the chromatid separation defect? This will be addressed in
the next two experiments.
Chromosoma (2006) 115:481–490
Nuclear membrane reassembly
Nuclear reassembly was analyzed first in Potoroo tridactylus cells to investigate if projections seen in potoroo nuclei
[see our earlier report, (Rens et al. 2003b)] are caused by
nuclear membrane formation around large chromosomes at
telophase. Indeed, the telophase nucleus in Fig. 3a shows the
start of nuclear reassembly around one of the projections as
observed using the nucleoporin antibody (green). Nuclear
reassembly around one projection on a daughter nucleus is
also observed in a Microtus agrestis telophase (Fig. 3b).
Figure 3 is a simulated fluorescence process (SFP)
rendering of z-stack images. Figure 4 shows a set of
examples of nuclear membrane antibody staining in Microtus
agrestis mitosis.
At prophase (Fig. 4a) nuclear pore proteins are starting
to be distributed into the cytoplasm; at anaphase (Fig. 4b)
nuclear pore proteins are completely dispersed in the
cytoplasm, the X chromatids are in contact. Figure 4c,d
shows incomplete separation. A strong nuclear pore signal
is observed around a bridge between the two decondensed
nuclei (Fig. 4c). In other cases micronuclei are formed
surrounded by a nuclear membrane (Fig. 4d). In 83% of
connected nuclei the bridge was surrounded by nuclear
pores, in 16% only the nuclei exhibited signal, and in only
1% no nuclear pore signal was observed (n=104).
To assess the frequency of micronuclei formation, three
sets (from three different days of culture) of around 2,000
nuclei were observed; 5.9±2.6% of nuclei had projections,
2.4±2.4% had a dumbbell shape, 1.7±0.6% of nuclei were
connected by a thin thread of DNA, and 1.8±0.3% of the
cells had micronuclei. The mitotic index was 1.8±1.5%.
Cell division/furrow formation
To gain insight into the presence of a surveillance mechanism monitoring incomplete chromatid separation, we
investigated furrow formation at the telophase stage of
mitosis. This can be observed either by the constriction of the
cytoplasm or, more elegantly, by the deposition of INCENP
Fig. 3 Nuclear pore localization
in Potoroo tridactylus anaphase
(a) and Microtus agrestis
telophase (b). Nuclear membrane
formation starts around the two
segregated groups of chromosomes as well as around the long
Potoroo tridactylus chromosome
1 (see arrow) and the projection
on the one of the daughter nuclei
of Microtus agrestis (see arrow).
The images are SFP (simulated
fluorescence process) rendering of
Z-stack images
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to the constriction site and its concentration at the midbody
in later stages of mitosis. Figure 5 shows nucleoporin and
INCENP immunostaining. In Fig 5a,b,d the cytoplasm is
also visualized by bright field. At the onset of cytokinesis,
INCENP starts to concentrate at the furrow, and a band of
INCENP signal is visible between the two dents (Fig. 5a).
At the final stage of cytokinesis INCENP is highly
concentrated at the midbody (Fig. 5b).
Incomplete nuclear division should interrupt cytokinesis.
However, Fig. 5c,d shows two nuclei still connected by a
thread of DNA due to incomplete chromatid separation
with a clear midbody halfway along the DNA thread as
detected by the INCENP staining. The cell membrane is
constricted at the site of the midbody, but no cell abscission
took place (Fig. 5d). INCENP signals were observed in
87% (n=109) of normal telophases (not connected), while a
signal was observed in 49% (n=159) of cells in which
daughter nuclei were connected by a thread of DNA. When
a subdivision was made between telophases with relatively
condensed groups of chromosomes showing a connecting
DNA thread and decondensed connected nuclei, 88% of the
former had a positive INCENP signal compared to 22% of
the latter. This drop in the number of INCENP positive cells
indicates a delocalization of INCENP from midbody over
time.
Cell division/abscission
To evaluate the role of Aurora B in late stages of
cytokinesis, phase contrast imaging was used to observe
abscission of cells with a DNA bridge in relation to Aurora
B presence as observed by immunostaining. Table 1 shows
three categories of cytokinesis observations. In 48.5% of
the cells the DNA bridge connected two nuclei in a single
cytoplasm. The cytoplasm displayed an elongated appearance as a result of limited furrow ingression or subsequent
regression. In a large fraction of these cells Aurora B was
not detectable, as it may have dispersed after a prolonged
cytokinesis.
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Chromosoma (2006) 115:481–490
Fig. 4 Nuclear pore proteins localization in Microtus agrestis cells. At
prophase, these proteins are starting to move away from the chromatin
towards the cytoplasm (a) At anaphase these proteins are distributed in
the cytoplasm (b). Reassembly of the nuclear membrane starts although
the daughter nuclei are not fully separated (c, d). In a few cases
micronuclei are formed surrounded by a membrane (d)
In a second group of cells (30.8%), cell division was
complete and cell abscission had taken place. In Fig. 6 an
example is reported. The cell presents a broken DNA
bridge, Aurora B signal along the midbody remnants, and
two separated areas of cytoplasm (abscission). Also in this
category, part of the cells did not show Aurora B signal,
suggesting that the protein may have dispersed after
abscission. The third category in the table accounts for
cells with constricted cytoplasm due to furrow ingression,
indicative of an initial stage of cytokinesis. Thus, both
inhibition of cell separation and completion of cytokinesis
were observed, despite the presence of Aurora B in the
proximity of the chromatin bridge.
Live cell imaging
Segregation of Microtus agrestis chromosomes from anaphase onwards was visualized by cell transfection, with a
plasmid expressing a chimeric histone H2B-GFP and live
cell imaging. Supplementary movie 1 shows a normal
chromosome segregation; two cells struggling to divide
when sister chromatid separation is incomplete are presented in Supplementary movies 2 and 3. (Figure 7 shows
selected images of movie 3). In Fig. 7d a clear chromatin
connection is visible between the two groups of chromosomes, possibly representing the incomplete separation of
the telomeric regions from sister chromatids of the long X
Chromosoma (2006) 115:481–490
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Fig. 5 INCENP (red) and nuclear pores (green) localization
in dividing microtus cells.
a shows the concentration of
INCENP at the furrow. b, c, and
d show INCENP concentrated
in the mid-body. However, the
daughter nuclei in c and d are
still connected by a thread of
DNA. a, b, and d show in the
background the cytoplasm of the
dividing cell in bright field
chromosome. During telophase (Fig 7e,f), the chromosomes are not just moving longitudinally apart but even
seem to move in an agitated manner, indicating mechanical
forces on the thread of DNA connecting the decondensing
chromosomes.
Table 1 The presence of Aurora B in cells showing a DNA bridge
between daughter nuclei (n=101)
Categorya
Observation
Percentage
Defective cytokinesis
(48.5%)
Aurora B at midbody
No Aurora B staining
No Aurora B and broken
bridge
Aurora B at midbody
Aurora B on broken bridge
No Aurora B staining
No Aurora B and broken
bridge
Aurora B at midbody
No Aurora B staining
17.8
28.7
2.0
Cell abscission (30.8%)
Furrow ingression
(20.7%)
a
See text for the description of the categories
13.9
5.0
6.9
5.0
12.8
7.9
Discussion
In this report, we investigated the influence of the length of
chromosome arms on sister chromatid separation and
cytokinesis. This study was motivated by the finding that
the distal part of long chromosome arms might still be
localized near the midplane at the start of nuclear
membrane formation (Rens et al. 2003b).
The field vole Microtus agrestis has two large sex
chromosomes and its nuclei show projections as observed
previously in potoroo cells (Rens et al. 2003b). Anaphase
bridges and micronuclei were observed during Microtus
agrestis cell division; this is similar to observations in
plants with artificially enlarged chromosomes (Schubert
and Oud 1997). The bridge and micronuclei invariably
consist of a region on either the X or Y chromosome with
DNA sequences shared by the X and Y chromosomes.
Nuclear membrane reassembly was investigated to
explain the observed nuclear projections. Nuclear pore
complex staining demonstrated that nuclear membrane
reassembly is independent on sister chromatid separation
and migration. In potoroo cells nuclear reassembly starts
not only around the decondensing chromosomes but also
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Fig. 6 Final stage of cytokinesis in microtus cells. The cytoplasm is
completely separated although the daughter nuclei (blue) are still
connected by a thin DNA bridge and Aurora B (green) is present
along the chromatin bridge
around the projected long arm of the large chromosome 1,
demonstrating that initiation of nuclear membrane formation starts before all parts of every chromosomes are
Fig. 7 Chromosome segregation during late mitotic stages in a
microtus cell expressing an H2B-GFP chimeric protein. Images
a–c show the anaphase movement of Microtus agrestis chromosomes;
in d the two groups of segregating chromosomes are connected by a
Chromosoma (2006) 115:481–490
retracted into a sphere. In Microtus agrestis cells the
membrane surrounds the chromatin bridge and the micronuclei at the end of the reassembly process, indicating that
membrane reconstruction is not interrupted by the incomplete nuclear division.
It is well-known that nuclear lamin B1 accumulates at
the surface of chromosomes at late anaphase or early
telophase and that accumulation is complete when chromosomes reach the poles. Within a short time interval
thereafter, the nuclear membrane protein forms a stable
polymer around chromosomes (Moir et al. 2000). As the
polymerized lamina is required for maintaining nuclear size
and shape, projections or bridges present at this time will be
stabilized as we observed in both Potorous tridactylus and
Microtus agrestis cells. Anaphase/telophase bridges and
nuclear projections arise due to: a) limited space (Schubert
and Oud 1997) as chromosomes reach the poles while long
arms are still near the equator, and b) limited time as the
nuclear lamina and membrane start reassembling, although
not all chromosomes are included within the telophase
nuclei.
Not only is nuclear membrane formation uninterrupted,
but also the concentration of INCENP and Aurora B at the
cleavage site proceeds normally. Both in normal cells and
in cells with DNA bridges, INCENP/Aurora B concentrates
at the cell midplane and on the midbody at the end stage of
thin thread of chromatin; chromosome movements take place and
spindle pole distance increases irrespective of the connecting
bridge (e, f). The figure presents selected images from Supplementary
movie 3
Chromosoma (2006) 115:481–490
cell division. The transfer of INCENP from the kinetochores to the spindle midzone at anaphase is activated by
separase in budding yeast (Pereira and Schiebel 2003).
However, at the stage of separase activation, the onset of
anaphase, the problem of incomplete chromosome segregation has not yet occurred. Therefore, separase activity
cannot be the checkpoint sensor for the segregation of these
large chromosomes.
In the recently proposed NoCut pathway in budding
yeast (Norden et al. 2006), Ipl1 (Aurora B in higher
eukaryotes) is suggested to monitor the presence of
chromatin at the midzone. Therefore, Aurora B activity
might be a candidate in animal cells to inhibit completion
of cytokinesis if chromatin is not cleared from the midzone
(in this case the presence of long chromosome arms at the
midplane). However, the findings presented in our report
show that cytoplasmic division occurred in 30% of cells
with incomplete chromosome segregation, despite the
presence of Aurora B near the chromatin bridge in some
cells. This relatively high percentage supports the conclusion that Aurora B is not involved in a putative mammalian
NoCut pathway.
This investigation in a mammalian species was important to verify whether the control process that inhibits
completion of cytokinesis to prevent chromosome breakage
in budding yeast (Norden et al., 2006) operate in other
eukaryotes. Several lines of evidence argue against this
possibility. Cytokinesis in the absence of chromatid
separation was observed in Cut1/Separin and Cut2/Securin
mutants in fission yeast (Funabiki et al. 1996; Yanagida
2000), and reformation of interphase nuclei without
chromosome segregation was present in Xenopus egg
extracts after addition of nondegradable securin (Zou et al.
1999). Inhibition of centromere separation by interfering
with securin or separase function in mammalian cells
resulted in the uncoupling of cytokinesis from chromatid
separation. In these cases aberrant cytokinesis produced a
polyploid daughter cell and an anuclear daughter cell
(Kumada et al. 2006; Wirth et al. 2006) or gave origin to
cells connected by thin chromatin string (Zur and Brandeis
2001). This latter phenotype was also observed when sister
chromatid separation was impaired by telomere instability
due to loss of telomeric protein hPot1 (Veldman et al. 2004)
or in pre-senescent fibroblasts (Yalon et al. 2004).
In these experimental models, improper chromatin
separation was forced upon the cells by mutation or
senescence. In this report Microtus agrestis cells of a
primary cell culture were used to investigate the induction
of a checkpoint activity by the defective segregation of
large chromosomes in a genetically wild-type background.
The findings presented in our report support the conclusion
that Aurora B is not involved in a putative mammalian
NoCut pathway. It should be noted, however, that these
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abscissions may have occurred as the amount of DNA in
the thread is too small to be sensed by Aurora B or
abscission may be due to mechanical forces, which were
indeed observed in the live cell imaging experiments. The
agitated movement of the two daughter nuclei seen in the
live cell imaging experiment (Supplementary movies 2–3,
Fig. 7) demonstrates that processes to complete cell
division continues despite the connection between the
nuclei.
Conclusion
Although it is not expected that the observed incomplete
segregation does occur in vivo with the same frequency as
it was observed in the primary cell culture, we can conclude
that late mitotic regulatory events do not prevent incomplete chromosome segregation of the large X and Y
chromosomes of Microtus agrestis. The observations of
nuclear membrane reformation, INCENP/Aurora B transfer
to the cleavage site and cell abscission show that there is no
efficient checkpoint for incomplete segregation of large
chromosomes. This mis-segregation may lead to micronuclei formation and subsequent loss of DNA sequences. In
Microtus agrestis cultured cells these cells are probably lost
at a later stage. However, the fact alone of this segregation
disorder indicates that there could be a limited size for
chromosomes. Within animal species are a large variety of
karyotypes. For instance, the chicken has a large number of
chromosomes ranging from micro- to macro-chromosomes,
while other species like the Indian muntjac and some
marsupial species only have a few large chromosomes. If
number is not a limiting factor in chromosome evolution
perhaps size is, and thus should be considered as an
important parameter in theoretical models of karyotype
evolution.
Acknowledgements This work was performed at the Cambridge
Resource Centre for Comparative Genomics and was supported by a
grant from the Wellcome Trust to MAFS.
Microtus agrestis X- and Y-chromosome-specific DNA was kindly
provided by Dr. F. Yang. The inner centromere protein (INCENP)
antibody was kindly provided by Prof. WC Earnshaw. A Microtus
agrestis specimen was supplied by the house cat Tipper.
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