1 Departamento de Biología, Edificio de Biológicas, Universidad
Autónoma de Madrid, E-28049 Madrid, Spain
2 Institute of Cell and Molecular Biology, University of Edinburgh, Swann
Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, Scotland,
UK
* Author for correspondence (e-mail: jose.suja{at}uam.es)
Accepted 18 December 2002
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Summary |
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Key words: INCENP, Aurora-B kinase, Phosphorylated histone H3, Meiosis, Centromere, Sister-chromatid cohesion
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Introduction |
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INCENP is a protein that is conserved from budding yeast to humans
(Ainsztein et al., 1998;
Kim et al., 1999
;
Saffery et al., 1999
;
Adams et al., 2000
;
Adams et al., 2001b
;
Kaitna et al., 2000
;
Oegema et al., 2001
;
Leverson et al., 2002
). This
protein, as a chromosomal passenger protein, distributes dynamically during
mitosis (Adams et al., 2001c
;
Terada, 2001
). INCENP
concentrates at the pairing centromere domain, also called the inner
centromere domain, during metaphase, transfers to the spindle midzone and to
the cell cortex at the cleavage furrow during anaphase, and then concentrates
at the midbody during telophase (Cooke et
al., 1987
; Earnshaw and Cooke,
1991
; Mackay and Earnshaw,
1993
). Expression of truncated INCENP proteins, or of a
CENP-BINCENP chimeric protein in transfected cells, has demonstrated
that INCENP is required for prometaphase chromosome congression, sister
chromatid disjunction at anaphase and the completion of cytokinesis
(Eckley et al., 1997
;
Mackay et al., 1998
). These
processes are severely affected in INCENP-knockout mouse embryos, thus
demonstrating that INCENP is essential for cell division
(Cutts et al., 1999
). INCENP
forms a chromosomal passenger complex and colocalizes with survivin, and with
the aurora-B serine-threonine protein kinase, in all organisms studied, from
budding yeast to humans (Kim et al.,
1999
; Adams et al.,
2000
; Adams et al.,
2001a
; Adams et al.,
2001b
; Kaitna et al.,
2000
; Speliotes et al.,
2000
; Uren et al.,
2000
; Morishita et al.,
2001
; Wheatley et al.,
2001
; Leverson et al.,
2002
). It has been proposed that INCENP targets survivin to
centromeres (Wheatley et al.,
2001
) and that INCENP and survivin are essential for the targeting
of aurora-B to chromosomes (Adams et al.,
2000
; Adams et al.,
2001b
; Rajagopalan and
Balasubramanian, 2002
). This chromosomal passenger complex has
been implicated in coordinating chromosome segregation with cytokinesis in
somatic cells.
Aurora-B kinase is overexpressed in many human cancer cell lines
(Giet and Prigent, 1999), as
is INCENP (Adams et al.,
2001a
), and has also been implicated in both chromosome
congression and segregation, and efficient completion of cytokinesis
(Terada et al., 1998
;
Schumacher et al., 1998
;
Kaitna et al., 2000
;
Leverson et al., 2002
;
Murata-Hori et al., 2002
;
Kallio et al., 2002
). Several
substrates for aurora-B have been identified. In budding yeast the single
aurora kinase Ipl1p phosphorylates the kinetochore protein Ndc10p
(Biggins et al., 1999
) and is
required to allow an accurate biorientation of mitotic chromosomes
(Tanaka et al., 2002
).
Aurora-B also phosphorylates the human centromeric protein CENP-A, a histone
H3-like kinetochore protein (Zeitlin et
al., 2001
). In this sense, it has been shown in rat cells that the
kinase activity of aurora-B is required for the localization of different
microtubule motor proteins at kinetochores and for chromosome biorientation
and then congression (Murata-Hori and
Wang, 2002
; Murata-Hori et
al., 2002
). Aurora-B kinase, and their homologues, also
phosphorylates histone H3 at serine 10 in different organisms
(Hsu et al., 2000
;
Speliotes et al., 2000
;
Adams et al., 2001b
;
Giet and Glover, 2001
;
Crosio et al., 2002
;
MacCallum et al., 2002
).
Recently, different observations have led us to propose that INCENP and
aurora-B are implicated in sister-chromatid centromere cohesion. In stable
dicentric human chromosomes, INCENP is present at the inactive centromere when
sister chromatids appear joined, whereas it is no longer detectable when
centromere cohesion is lost (Vagnarelli
and Earnshaw, 2001). Additionally, the abnormalities observed in
Drosophila cells undergoing anaphase after abrogation of INCENP and
aurora-B function by RNA interference suggest that these proteins are
implicated in centromere cohesion (Adams et
al., 2001b
). The abnormal distribution of INCENP at centromeres in
chicken cells with the mutated cohesin subunit Rad21/Scc1 further support a
relationship between INCENP and centromere cohesion
(Sonoda et al., 2001
).
Although numerous studies have analysed the subcellular distribution and
pattern of expression of the chromosomal passenger complex in somatic cells
from different systems, information concerning their distribution in meiotic
cells is scarce. Recently, the subcellular localization of aurora-B has been
studied during meiosis in C. elegans. Depletion of aurora-B prevents
the release of the meiosis-specific cohesin subunit Rec8 from chromosomes. It
has been proposed that aurora-B phosphorylates Rec8, and therefore promotes
the release of sister-chromatid cohesion during both meiotic divisions
(Rogers et al., 2002
).
Owing to the absence of studies on INCENP and aurora-B kinase expression during mammalian meiosis, we analysed their subcellular distribution throughout meiosis in squashed mouse spermatocytes. For comparative purposes, we additionally studied INCENP distribution in mitotic spermatogonial cells and 3T3 cultured mouse cells. We surprisingly found that in prophase I INCENP appears at the central element of the synaptonemal complex and redistributes to the heterochromatic chromocentres during late pachytene. Then, INCENP and aurora-B concentrate at centromeres showing peculiar T-shaped signals at their inner domain in metaphase I. Both proteins are also found at a connecting strand traversing the centromere region, and joining sister kinetochores, at metaphase II centromeres. We discuss the complex dynamic relocalization of the chromosomal passenger complex during prophase I. Additionally, we suggest that this complex regulates sister-chromatid centromere cohesion during both meiotic divisions.
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Materials and Methods |
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Electrophoresis and immunoblotting
HeLa cells were harvested and washed twice with PBS. Then, they were lysed
in boiling SDS sample buffer (50 mM Tris-HCl pH 6.8, 3% SDS, 2 mM EDTA, 15%
sucrose, 9% ß-mercaptoethanol, 0.005% bromophenol blue).
Testes from adult male C57BL/6 mice were removed and placed in 2 ml of SDS solubilization solution (50 mM Tris-HCl pH 6.8, 5 mM EDTA, 3% SDS, 1% protease inhibitor cocktail). Then, testes were homogenized on ice in a Potter homogenizer. The extract was placed in a boiling water bath for 5 minutes. The appropriate quantity of extract was diluted with 5x SDS-lysis buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 5% ß-mercaptoethanol, 10% glycerol, 0.005% bromophenol blue) and boiled for 5 minutes.
SDS-PAGE was carried out in 10% polyacrylamide gels. Gels were electrically transferred to Trans-Blot sheets (Bio-Rad) for 1.5 hours at 4°C and 310 mA. Sheets were blocked for 1 hour with 4% non-fat dry milk in PBS, followed by an overnight incubation at 4°C with pAb1186 serum against INCENP at 1:500 dilution in 4% non-fat dry milk in PBS. Immunoreactive bands were visualized by incubation for 1 hour at room temperature with horseradish-peroxidase-conjugated donkey anti-rabbit Ig (Amersham Life Science) at a 1:5000 dilution in PBS and subsequently developed using an ECL (Enhanced Chemiluminescence) detection system (Amersham) according to the manufacturer instructions.
Squashing and spreading of spermatocytes
Adult male C57BL/6 mice were killed by cervical dislocation. The testes
were then removed and detunicated, and seminiferous tubules processed for
either squashing or spreading. For squashing, we followed the technique
previously described (Page et al.,
1998; Parra et al.,
2002
). Briefly, seminiferous tubules were fixed in freshly
prepared 2% formaldehyde in PBS containing 0.1% Triton X-100. After 5 minutes,
several seminiferous tubules fragments were placed on a slide coated with 1
mg/ml poly-L-lysine (Sigma) with a small drop of fixative, and the tubules
were gently minced with tweezers. The tubules were then squashed and the
coverslip removed after freezing in liquid nitrogen. For spreading of
spermatocytes, we followed the drying-down technique of Peters et al.
(Peters et al., 1997
).
Immunofluorescence microscopy
After fixation, the slides and coverslips were rinsed three times for 5
minutes in PBS and incubated for 45 minutes at room temperature with primary
antibodies. To detect INCENP we used a polyclonal rabbit serum (pAb1186)
raised against chicken INCENP, but also recognising human INCENP
(Eckley et al., 1997), at a
1:100 dilution in PBS. Centromeres were detected with the GS human
anti-centromere autoantibody (ACA) that recognises CENP-A, -B and -C
(Earnshaw and Cooke, 1989
) at
a 1:10,000 dilution in PBS. To detect SCP3 we used a polyclonal guinea pig
serum [kindly provided by Ricardo Benavente
(Alsheimer and Benavente,
1996
)], at a 1:100 dilution in PBS. To detect SCP1 we used a
polyclonal rabbit serum (A2) that recognises SCP1 [kindly provided by Christa
Heyting (Meuwissen et al.,
1992
)] at a 1:200 dilution in PBS. To detect aurora-B kinase, we
employed the mouse monoclonal AIM-1 antibody (Transduction Labs) at a 1:30
dilution in PBS. To detect survivin, we employed three polyclonal rabbit sera
against human survivin (Novus Biologicals, Calbiochem, and Autogenbioclear) at
a 1:30 dilution. Phosphorylated histone H3 (pH3) was detected with a
polyclonal rabbit serum (Upstate Biotechnology) at a 1:1000 dilution.
Following three washes in PBS, the slides were incubated for 30 minutes at
room temperature with secondary antibodies. A combination of fluorescein
isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (Jackson) at a 1:150
dilution in PBS with Texas-Red-conjugated goat anti-human IgG (Jackson) at a
1:150 dilution in PBS, a Texas-Red-conjugated goat anti-guinea pig IgG
(Jackson) at a 1:150 dilution in PBS or a Texas-Red-conjugated goat anti-mouse
IgG (Jackson) at a 1:150 dilution in PBS was used for simultaneous double
immunolabelling. The slides were subsequently rinsed in PBS and counterstained
for 3 minutes with 2 µg/ml DAPI (4',6-diamidino-2-phenylindole).
After a final rinse in PBS, the slides were mounted in Vectashield (Vector
Laboratories) and sealed with nail varnish. In double immunolabelling
experiments, primary antibodies were incubated simultaneously except for the
double localization of INCENP and SCP1. In this case slides were first
incubated with the rabbit serum against INCENP for 1 hour at room temperature,
rinsed in PBS and incubated overnight at 4°C with an FITC-conjugated goat
Fab' fragment anti-rabbit IgG (Jackson) at a 1:100 dilution in PBS.
Afterwards, slides were rinsed six times for 5 minutes in PBS, incubated with
the rabbit serum against SCP1 for 1 hour, rinsed three times for 5 minutes in
PBS and incubated with a Texas-Red-conjugated goat anti-rabbit IgG (Jackson)
at a 1:150 dilution. A control with the preimmune serum from rabbit 1186 was
made and resulted in no staining.
Observations were performed using an Olympus BH-2 microscope equipped with epifluorescence optics, and the images were recorded with an Olympus DP50 digital camera. Digital images were then treated using the Adobe PhotoShop 6.0 software.
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Results |
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The distribution of INCENP in spermatogonial mitosis is similar to
that found in cultured somatic cells
The squashing technique we employed allowed us to observe in the same
preparations mitotic spermatogonial cells and spermatocytes at different
developmental stages. To compare INCENP expression during mouse
spermatogenesis with that found during mouse mitosis, we first analysed its
distribution in spermatogonial mitosis
(Fig. 2). In interphase
spermatogonia, INCENP appeared as large bright nuclear signals corresponding
to the heterochromatic chromocentres observed after DAPI staining
(Fig. 2A,B). Some faint
nucleoplasmic staining was also observed. After a double immunolabelling of
INCENP and centromeres with the ACA serum, it became evident that centromeres
appeared as discrete foci immersed in the INCENP-labelled chromocentres
(Fig. 2A,B). In prophase
spermatogonia a bright INCENP labelling was observed at centromeric regions,
and a diffuse staining on condensing chromosomes was also seen
(Fig. 2C-E). The centromeric
INCENP signals were bright and fuzzy, and colocalized with the brightly
labelled centromeric heterochromatin detected with DAPI
(Fig. 2C-E). By contrast, the
ACA serum recognised pairs of dots, representing sister kinetochores, that
appeared at opposing surfaces of the INCENP signals (insert in
Fig. 2C,D). During metaphase,
the INCENP labelling was similar to that found during prophase. Thus, bright
and fuzzy centromeric signals were observed, and chromosome arms showed a
faint staining along their lengths (Fig.
2F,G). The INCENP labelling changed dramatically during anaphase.
In early anaphase, INCENP completely disappeared not only from centromeres but
also from chromatid arms and was only detected as thin threads at the spindle
midzone (Fig. 2H,I). During
telophase, two brightly labelled regions flanking the midbody were observed
(Fig. 2J,K). Interestingly, the
same pattern of expression was observed for the aurora-B kinase (data not
shown). We also studied the INCENP expression in somatic 3T3 cultured mouse
cells (data not shown) in order to compare it with its distribution in
spermatogonial mitosis. Our results showed that INCENP was similarly
distributed in both kinds of mouse mitotic cells, and its expression pattern
is likewise similar to that described previously in chicken
(Cooke et al., 1987),
Xenopus (Adams et al.,
2000
) and human somatic cells
(Eckley et al., 1997
).
|
INCENP distributes as nuclear threads in early prophase I
spermatocytes
In squashed seminiferous tubules, and after DAPI staining and centromere
labelling with the ACA serum, leptotene and zygotene spermatocytes showed
about 40 signals corresponding to the unpaired autosomal and sex centromeres
(Fig. 3A-D). In some of these
early prophase I spermatocytes no INCENP staining was detected
(Fig. 3A,B). However, in other
ones, INCENP was detectable as short and thin discontinuous nuclear threads
(Fig. 3C,D). Pachytene
spermatocytes were easily recognized because 21 centromere signals, nineteen
corresponding to paired autosomal centromeres and two additional ones
corresponding to unpaired sex centromeres, were detected throughout their
nuclear volume, and the sex body was discerned at the nuclear periphery
(Fig. 3E,F). In these pachytene
spermatocytes, INCENP-labelled nuclear threads appeared longer and brighter
than in early prophase I spermatocytes, and their ends reached the nuclear
periphery (Fig. 3E,F). One of
the ends of these threads colocalized with centromeres
(Fig. 3E,F). Additionally, the
entire sex body showed a faint INCENP staining that was brighter than the
nucleoplasmic background (Fig.
3E,F,K). In some favourable focal planes full-length threads were
observed (Fig. 3G). It was
apparent that these threads did not show a homogeneous thickness along their
length but that they presented some narrower regions (arrowheads in
Fig. 3G). When side views of
the insertion of the threads to the nuclear envelope were observed, it became
evident that one of their ends was always surrounded by ACA labelling
(Fig. 3H). When these proximal
ends were viewed end-on, INCENP appeared as a dot located either inside or
lateral to the ACA labelling (Fig.
3I,J). Interestingly, in some focal planes throughout the
pachytene sex body a small thread or a dot was detected. This INCENP signal
never colocalized with the two ACA signals corresponding to the unpaired sex
centromeres (Fig. 3K). The
extent and location of this INCENP labelling inside the sex body was
reminiscent of the pseudoautosomal region, the site of pairing and
recombination between sex chromosomes.
|
INCENP is found at the central element of the synaptonemal
complex
The results described above suggest that INCENP labels a synaptonemal
complex (SC) component. To test whether the INCENP labelling at putative SCs
was specific we made a control with the preimmune serum from rabbit 1186 that
resulted in no staining (data not shown). To precisely determine the early
prophase I stage when INCENP became first detectable, and to ascertain whether
INCENP really labelled SCs, we made a double immunolabelling of INCENP and
SCP3, a structural component of the SC lateral elements (LEs)
(Moens and Spyropoulos, 1995).
In leptotene spermatocytes, no INCENP labelling was detected, but during
zygotene the short INCENP threads colocalized with some SCP3-labelled
fragments that appeared thicker than those where colocalization was not found
(Fig. 5A-C). Thus, INCENP
colocalized with regions where homologous LEs were synapsed. During pachytene
the INCENP threads colocalized with SCs
(Fig. 5D-F). In squashed
spermatocytes, the guinea pig anti-SCP-3 antibody allowed us to resolve both
LEs along the entire SCs length (white arrowheads in
Fig. 5H,K,N). As a rule, SCs
appeared twisted along their length, and consequently the two LEs could only
be accurately discerned in short regions. When such regions were observed at
higher magnification it became evident that the INCENP threads were located in
between the LEs (Fig. 5G-L).
This pattern of localization was also observed at the SC ends
(Fig. 5M-O). To corroborate
these results we double immunolabelled INCENP and SCP3 on spread
spermatocytes. Results confirmed that INCENP threads colocalized with SCs.
Interestingly, in sex bivalents the unsynapsed axial elements did not show
INCENP labelling, which was only observed in the pseudoautosomal region
(Fig. 5P,Q).
|
The findings described above suggest that INCENP labels the SC central element (CE). To verify this proposal, we double immunolabelled INCENP and SCP1, a component of the CE, on squashed and spread spermatocytes. In zygotene (Fig. 6A-D) and pachytene (Fig. 6E-H) squashed spermatocytes INCENP and SCP1 signals perfectly matched, although the INCENP threads appeared more discontinuous than the SCP1 threads. As expected, the pseudoautosomal region of sex chromosomes appeared to be labelled with both antibodies during pachytene (arrow in Fig. 6E,F). Altogether, these results demonstrate that INCENP labels the synaptonemal complex CE, but that its behaviour during prophase I is different from that shown by SCP1.
|
INCENP relocalizes from synaptonemal complex CEs to heterochromatic
chromocentres from late pachytene up to late diplotene
In late pachytene spermatocytes the INCENP threads appeared to be slightly
fainter than those found in earlier pachytene nuclei. This loss of
fluorescence intensity at the threads was simultaneous with a faint INCENP
staining that began to be recognised at the heterochromatic chromocentres
found at the nuclear periphery (Fig.
3L-N). These chromocentres, which represent clustered centromere
heterochromatic regions of autosomes, appeared brighter than the labelling at
the sex body (Fig. 3L-N).
Interestingly, the centromeric regions of the sex chromosomes did not appear
to be labelled (Fig. 3M).
During early diplotene, the INCENP-labelled chromocentres appeared brighter than during late pachytene and matched with the heterochromatic chromocentres revealed with DAPI (Fig. 4A-C). Inside some chromocentres a single centromere signal was observed, whereas several centromere signals were observed in other chromocentres (Fig. 4A-G). The sex body appeared fainter than chromocentres (Fig. 4A-C). Additionally, a few short and faintly labelled INCENP threads were present either in the nuclear interior (arrows in Fig. 4A) or associated with the chromocentres (Fig. 4D,E). When early diplotene spermatocytes where observed after a double immunolabelling of INCENP and SCP3, the short INCENP threads colocalized with the still synapsed regions in desynapsing autosomal bivalents (Fig. 5R,S). Moreover, these INCENP threads colocalized with SCP1 ones, but whereas SCP1 threads were bright, INCENP ones were fainter (Fig. 6I-L). In these early diplotene spermatocytes, heterochromatic chromocentres were brightly labelled with INCENP but not with either SCP3 (Fig. 5R,S) or SCP1 (Fig. 6I-L).
|
During late diplotene, INCENP threads became progressively fainter and shorter until they finally disappeared whereas bright chromocentres and the fainter sex body appeared to be labelled (Fig. 4H,I). It is worth noting that although the fluorescence intensity of INCENP threads gradually decreased, short segments of SCP1 were still brightly labelled (Fig. 6M-P). Thus, INCENP is lost from SC fragments before SCP1. The pattern of distribution of INCENP from late pachytene up to late diplotene suggests that there is dynamic relocalization of INCENP from synaptonemal complex CEs to the heterochromatic chromocentres.
During diakinesis, the INCENP labelling at the sex body disappeared, chromocentres were no longer identified and a faint labelling was observed over the condensing chromosomes (Fig. 4J,K). A bright INCENP staining was observed at small regions partially colocalizing with the centromere signals, although INCENP domains were slightly larger (Fig. 4J,K). This result suggests that the ongoing chromosome condensation promotes the loss of INCENP from the centromeric heterochromatin.
INCENP shows an unexpected labelling at metaphase I centromeres
During metaphase I a diffuse INCENP labelling was observed on the condensed
chromatin both in autosomal and sex bivalents
(Fig. 7A-F). Interestingly, the
sex bivalent showed a bright staining at the interchromatid domain, the
contact surface between sister chromatids
(Suja et al., 1999), that was
not discerned in the autosomal bivalents
(Fig. 7A-C). However, brighter
INCENP signals were detected at centromeres
(Fig. 7). These signals showed
an unexpected T-shape when viewed from the side
(Fig. 7G,J). These signals were
smaller than the region occupied by the centromeric heterochromatin revealed
with DAPI (Fig. 7G-L). Thus,
although from late pachytene up to late diplotene the INCENP signals at
chromocentres colocalized with the centromeric heterochromatin detected with
DAPI, during diakinesis and metaphase I INCENP signals only occupy a small
region at the centromeres. This result indicates that INCENP progressively
concentrates at the centromere region during late prophase I stages. To
precisely determine the location of these signals we double immunolabelled
centromeres with INCENP and the ACA serum GS, which mainly recognises proteins
found at the inner kinetochore plate
(Earnshaw and Cooke, 1989
). We
observed that most of the T-shaped INCENP labelling was found beneath
kinetochore signals, although some degree of colocalization was detected
(Fig. 7G-L). Thus, INCENP is
mostly located beneath homologous kinetochores.
|
Similar T-shaped signals have been previously described in metaphase I
centromeres with anti-SCP3 antibodies
(Prieto et al., 2001). In
order to verify whether INCENP colocalizes with SCP3 we used double
immunolabelling. Results showed that both proteins appeared as T-shaped
structures at centromeres and that they colocalized
(Fig. 7M-O).
INCENP is retained at centromeres throughout anaphase I and
relocalizes to the spindle midzone during late anaphase I
During early anaphase I INCENP signals were only found at centromeres
(Fig. 8A-C). These centromere
signals were fuzzy and fainter than those previously found during metaphase I
(compare Fig. 7A,D with
Fig. 8A,B). At late anaphase I
a bright INCENP labelling was detected at the spindle midzone, although a
fainter and more diffuse labelling at centromeres was still visible
(Fig. 8D,E). However, at
telophase I this faint centromere labelling disappeared and only two brightly
labelled INCENP signals were observed at the midbody
(Fig. 8F,G). These results
show, in contrast to what was found during mitosis, that INCENP remains at
centromeres throughout anaphase I and that the labelling at the spindle
midzone only appears at late anaphase I.
|
INCENP appears at a connecting strand between sister kinetochores in
metaphase II centromeres
In spermatocytes undergoing interkinesis, INCENP was detected at
chromocentres that colocalized with those observed after DAPI staining
(Fig. 8H-J). All centromere
signals were observed inside those chromocentres
(Fig. 8I). This result is thus
comparable to that observed in interphase spermatogonia (compare
Fig. 2A,B with
Fig. 8H-J). In metaphase II
spermatocytes INCENP was not observed on condensed chromatin but only at
centromeres (Fig. 9A-C). Two
INCENP spots were observed on opposite faces of the centromere region. These
spots partially colocalized with sister kinetochores revealed by the ACA
serum, although most of the INCENP spots were beneath kinetochores
(Fig. 9A-C). Surprisingly,
INCENP also labelled an irregular strand between sister kinetochores that
crossed the entire centromere (arrowheads in
Fig. 9D-G), which is remarkably
similar to the CLiP staining previously described in mitotic chromosomes
(Rattner et al., 1988). At the
onset of anaphase II, just when sister chromatids began to separate, the
strand appeared to break in the middle region (arrow in
Fig. 9H,I). During early
anaphase II, however, INCENP was still detected at centromeres
(Fig. 9J,K). At late anaphase
II, as is the case during late anaphase I, INCENP labelling was detected at
the spindle midzone, finally concentrating at the midbody
(Fig. 9L,M). During telophase
II, in addition to midbody labelling, clear INCENP signals were observed to
colocalize with kinetochores (Fig.
10A-C). These INCENP signals became larger and fainter during late
telophase II (Fig. 10D-F). In
early round spermatids one or two large INCENP signals were found at the
nuclear interior colocalizing with the heterochromatic chromocentres revealed
after DAPI staining (Fig.
10G,H). In more advanced spermatids the INCENP labelling was no
longer found at chromocentres, although the centromere signals were still
observed (Fig. 10I,J).
|
|
Aurora-B kinase appears at chromocentres after INCENP association,
and both proteins colocalize in metaphase I and metaphase II centromeres
In somatic cells INCENP targets aurora-B to chromosomes. We therefore
performed a double immunolocalization of both proteins to test whether this
also occurred in mouse meiosis. Aurora-B was first detected in diplotene
spermatocytes. Surprisingly, and in contrast to INCENP, aurora-B did not label
the synaptonemal complex CE in previous prophase I stages. In diplotene
spermatocytes that still showed a faint INCENP labelling at SC remnants, and
bright staining at chromocentres, no aurora-B labelling was observed
(Fig. 11A-D). However, in late
diplotene spermatocytes, when no SC remnants were visible with INCENP,
aurora-B colocalized with INCENP at chromocentres
(Fig. 11E-H). These results
suggest that INCENP also targets aurora-B in mouse spermatocytes. From
diakinesis onwards, INCENP and aurora-B colocalized. Interestingly, the
localization of both proteins perfectly matched at the T-shaped structure at
metaphase I centromeres (Fig.
11I-L), and they also appeared to overlap at the connecting strand
between sister kinetochores at metaphase II centromeres
(Fig. 11M-P). We also tried to
localize survivin with three different antibodies; however, none of them
labelled meiotic centromeres.
|
Aurora-B appears at diplotene chromocentres before phosphorylated
histone H3
It has been demonstrated that in somatic cells the aurora-B kinase is
responsible for histone H3 phosphorylation (pH3). To test this in meiosis we
performed a double immunolocalization of aurora-B and pH3. Some diplotene
spermatocytes showed aurora-B at chromocentres but no pH3 staining
(Fig. 11Q-T). However, both
proteins colocalized at chromocentres in some other late diplotene
(Fig. 11U-X) and early
diakinesis spermatocytes. During diakinesis, aurora-B appeared to be
concentrated at individualized centromeres, as is the case with INCENP,
whereas pH3 labelled all condensing bivalents (data not shown). The first
appearance of phosphorylated H3 in late diplotene spermatocytes agrees with
previous localizations (Cobb et al.,
1999). Our results thus suggest that the presence of aurora-B may
be necessary for the initial phosphorylation of histone H3 at heterochromatic
chromocentres.
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Discussion |
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Aurora-B first appears during late diplotene at heterochromatic
chromocentres that are already labelled by INCENP. This result suggests that,
as occurs in somatic cells (Adams et al.,
2000; Adams et al.,
2001b
), INCENP may also target aurora-B to the chromocentres
during the first meiotic prophase. It is not obvious why, during male mouse
meiosis, INCENP targeting is first detected in zygotene spermatocytes at least
a week before aurora-B is first observed at diplotene. One possibility is that
INCENP does not target aurora-B during meiosis. Secondly, it may be that
aurora-B is not expressed in zygotene spermatocytes. Aurora-B expression might
occur during diplotene once INCENP has completely disappeared from CEs.
Thirdly, it is possible that these differences in targeting arise from
meiosis-specific differences in survivin or another as yet unknown subunit of
the chromosomal passenger complex. Histone H3 phosphorylated on
serine10 is first detected during late diplotene at chromocentres
that are already labelled by INCENP and aurora-B. This sequence of appearance
of aurora-B kinase and pH3 supports previous reports suggesting that aurora-B
phosphorylates histone H3 in somatic cells
(Hsu et al., 2000
;
Speliotes et al., 2000
;
Giet and Glover, 2001
;
Zeitlin et al., 2001
;
Crosio et al., 2002
;
MacCallum et al., 2002
).
INCENP and aurora-B colocalize throughout heterochromatic chromocentres
during diplotene and concentrate at individual centromeres during diakinesis
in parallel with ongoing chromosome condensation and separation. However,
during metaphase I both proteins appear as T-shaped signals that do not occupy
all the heterochromatic region, as detected by DAPI, and that are located
beneath associated sister kinetochores as detected with the ACA serum. Thus,
INCENP and aurora-B are progressively concentrated from centromeric
heterochromatin to the T-shaped centromere domain between diakinesis and
metaphase I. As these proteins are located at the inner centromere domain in
mitotic chromosomes, we propose that this T-shaped domain corresponds to the
inner centromere domain of metaphase I chromosomes. A similar T-shaped
distribution was not described when mammalian metaphase I centromeres were
stained for other centromeric components. Interestingly, we previously
reported that SCP3 appears as T-shaped signals at metaphase I centromeres
(Prieto et al., 2001). Here we
demonstrate by a double immunolabelling that INCENP colocalizes with SCP3 at
the inner centromere domain. Moreover, the cohesin subunit Rad21/Scc1 also
colocalizes with INCENP and SCP3 in this domain (J.A.S., unpublished). Thus,
the inner centromere domain in mouse metaphase I centromeres contains not only
the chromosomal passenger complex, with at least INCENP and aurora-B, but also
some subunits of the cohesin complex. Taking into account the presence of
INCENP and aurora-B at the inner centromere domain during metaphase I, their
colocalization with the cohesin subunit Rad21, and their release from
centromeres during anaphase I, it is tempting to speculate that INCENP and
aurora-B are present at the right place and time for a function in regulating
sister-chromatid centromere cohesion. This is supported by the observation
that inactivation of INCENP and aurora-B by RNAi interferes with the
disjunction of sister kinetochores in Drosophila cells undergoing
anaphase (Adams et al., 2001b
).
Additionally, it has been recently proposed that the cohesin subunit Rad21 is
necessary for the accurate targeting of INCENP to the inner centromere domain
(Sonoda et al., 2001
).
Moreover, aurora-B phosphorylates the cohesin subunit Rec8, a meiosis-specific
variant of Rad21, and therefore promotes the release of sister-chromatid
cohesion during both meiotic divisions in C. elegans
(Rogers et al., 2002
).
Another interesting result is the appearance of INCENP and aurora-B in a
connecting strand traversing the centromere region, and joining sister
kinetochores, in metaphase II centromeres. This strand disappears at the
metaphase II/anaphase II transition, and these proteins then relocalize to the
spindle midzone. A similar labelling has not been previously observed in
mammalian metaphase II centromeres. However, a connecting strand has been
reported after silver staining in grasshopper metaphase II centromeres
(Rufas et al., 1989;
Suja et al., 1992
). In somatic
cells, CLiPs (Rattner et al.,
1988
), DNA topoisomerase II
(Rattner et al., 1996
), the
mitotic centromere-associated kinesin (MCAK)
(Maney et al., 1998
) and
Mei-S332 in Drosophila (Blower and
Karpen, 2001
) have been detected at a similar connecting strand
and proposed to be somehow involved in centromere cohesion. Similarly, we
propose that INCENP and aurora-B could regulate centromere cohesion during
mammalian meiosis II, possibly through action of aurora-B on one of the other
components. Interestingly, we have recently shown that aurora-B can
phosphorylate DNA topoisomerase II
in vitro (C. Morrison and W.C.E.,
unpublished). We have observed that neither the cohesin subunits Rad21
(J.A.S., unpublished) nor STAG3 (Prieto et
al., 2001
) is present at the connecting strand during metaphase
II. However, it has been reported that the cohesin subunits Smc1ß and
Smc3 appear at metaphase II centromeres
(Revenkova et al., 2001
).
Taking into account these results, it is possible that INCENP and aurora-B
regulate centromere cohesion by acting on different cohesin complexes during
meiosis I and meiosis II. It remains to be tested whether INCENP colocalizes
with Smc1ß, Smc3 and Rec8, the meiosis-specific variant of Rad21, in
metaphase II centromeres, and whether any of these cohesin subunits is a
target of aurora-B phosphorylation. Obviously more studies are needed to
ascertain whether during meiosis the chromosomal passenger complex plays a
critical role in coordinating chromosome separation by regulating centromere
cohesion and cytokinesis.
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Acknowledgments |
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References |
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Adams, R. R., Wheatley, S. P., Gouldsworthy, A. M., Kandels-Lewis, S. E., Carmena, M., Smythe, C., Gerloff, D. L. and Earnshaw, W. C. (2000). INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow. Curr. Biol. 10,1075 -1078.[CrossRef][Medline]
Adams, R. R., Eckley, D. M., Vagnarelli, P., Wheatley, S. P., Gerloff, D. L., Mackay, A. M., Svingen, P. A., Kaufmann, S. H. and Earnshaw, W. C. (2001a). Human INCENP colocalizes with the Aurora-B/AIRK2 kinase on chromosomes and is overexpressed in tumour cells. Chromosoma 110,65 -74.[Medline]
Adams, R. R., Maiato, H., Earnshaw, W. C. and Carmena, M.
(2001b). Essential roles of Drosophila inner centromere
protein (INCENP) and Aurora B in histone H3 phosphorylation, metaphase
chromosome alignment, kinetochore disjunction, and chromosome segregation.
J. Cell Biol. 153,865
-879.
Adams, R. R., Carmena, M. and Earnshaw, W. C. (2001c). Chromosomal passengers and the (aurora) ABCs of mitosis. Trends Cell Biol. 11,49 -54.[CrossRef][Medline]
Ainsztein, A. M., Kandels-Lewis, S. E., Mackay, A. M. and
Earnshaw, W. C. (1998). INCENP centromere and spindle
targeting: identification of essential conserved motifs and involvement of
heterochromatin protein HP1. J. Cell Biol.
143,1763
-1774.
Alsheimer, M. and Benavente, R. (1996). Change of karyoskeleton during mammalian spermatogenesis: expression pattern of nuclear lamin C2 and its regulation. Exp. Cell Res. 228,181 -188.[CrossRef][Medline]
Biggins, S., Severin, F. F., Bhalla, N., Sassoon, I., Hyman, A.
A. and Murray, A. W. (1999). The conserved protein kinase
Ipl1 regulates microtubule binding to kinetochores in budding yeast.
Genes Dev. 13,532
-544.
Blower, M. D. and Karpen, G. H. (2001). The role of Drosophila CID in kinetochore formation, cell-cycle progression and heterochromatin interactions. Nat. Cell Biol. 3,730 -739.[CrossRef][Medline]
Choo, K. H. (2001). Domain organization at the centromere and neocentromere. Dev. Cell 1, 165-177.[Medline]
Cobb, J., Miyaike, M., Kikuchi, A. and Handel, M. A.
(1999). Meiotic events at the heterochromatin: histone H3
phosphorylation, topoisomerase II localization and chromosome
condensation. Chromosoma
108,412
-425.[CrossRef][Medline]
Cooke, C. A., Heck, M. M. S. and Earnshaw, W. C. (1987). The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J. Cell Biol. 105,2053 -2067.[Abstract]
Cooke, C. A., Bernat, R. L. and Earnshaw, W. C. (1990). CENP-B: A major human centromere protein located beneath the kinetochore. J. Cell Biol. 110,1475 -1488.[Abstract]
Craig, J. M., Earnshaw, W. C. and Vagnarelli, P. (1999). Mammalian centromeres: DNA sequence, protein composition, and role in cell cycle progression. Exp. Cell Res. 246,249 -262.[CrossRef][Medline]
Crosio, C., Fimia, G. M., Loury, R., Kimura, M., Okano, Y.,
Zhou, H., Sen, S., Allis, C. D. and Sassone-Corsi, P. (2002).
Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian
aurora kinases. Mol. Cell. Biol.
22,874
-885.
Cutts, S. M., Fowler, K. J., Kile, B. T., Hii, L. L. P., O'Dowd,
R. A., Hudson, D. F., Saffery, R., Kalitsis, P., Earle, E. and Choo, K. H.
A. (1999). Defective chromosome segregation, microtubule
bundling and nuclear bridging in inner centromere protein gene
(Incenp)-disrupted mice. Hum. Mol. Genet.
8,1145
-1155.
Earnshaw, W. C. and Cooke, C. A. (1989). Proteins of the inner and outer centromere of mitotic chromosomes. Genome 31,541 -552.[Medline]
Earnshaw, W. C. and Cooke, C. A. (1991). Analysis of the distribution of the INCENPs throughout mitosis reveals the existence of a pathway of structural changes in the chromosomes during metaphase and early events in cleavage furrow formation. J. Cell Sci. 98,443 -461.[Abstract]
Earnshaw, W. C. and Rattner, J. B. (1989). A map of the centromere (primary constriction) in vertebrate chromosomes at metaphase. In Aneuploidy: Mechanisms of origin (eds B. Vig and M. Resnick), pp. 33-42. New York: A. R. Liss.
Eckley, D. M., Ainsztein, A. M., Mackay, A. M., Goldberg, I. G.
and Earnshaw, W. C. (1997). Chromosomal proteins and
cytokinesis: patterns of cleavage furrow formation and inner centromere
protein positioning in mitotic heterokaryons and mid-anaphase cells.
J. Cell Biol. 136,1169
-1183.
Giet, R. and Prigent, C. (1999).
Aurora/Ipl1p-related kinases, a new oncogenic family of mitotic
serine-threonine kinases. J. Cell Sci.
112,3591
-3601.
Giet, R. and Glover, D. M. (2001).
Drosophila aurora B kinase is required for histone H3 phosphorylation
and condensin recruitment during chromosome condensation and to organize the
central spindle during cytokinesis. J. Cell Biol.
152,669
-681.
Hsu, J. Y., Sun, Z. W., Li, X., Reuben, M., Tatchell, K., Bishop, D. K., Grushcow, J. M., Brame, C. J., Caldwell, J. A., Hunt, D. F. et al. (2000). Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes. Cell 102,279 -291.[Medline]
Kaitna, S., Mendoza, M., Jantsch-Plunger, V. and Glotzer, M. (2000). INCENP and an Aurora-like kinase form a complex essential for chromosome segregation and efficient completion of cytokinesis. Curr. Biol. 10,1172 -1181.[CrossRef][Medline]
Kallio, M. J., McCleland, M. L., Stukenberg, P. T. and Gorbsky, G. J. (2002). Inhibition of aurora B kinase blocks chromosome segregation, overrides the spindle checkpoint, and perturbs microtubule dynamics in mitosis. Curr. Biol. 12,900 -905.[CrossRef][Medline]
Kim, J. H., Kang, J. S. and Chan, C. S. (1999).
Sli15 associates with the Ipl1 protein kinase to promote proper chromosome
segregation in Saccharomyces cerevisiae. J. Cell Biol.
145,1381
-1394.
Leverson, J. D., Huang, H.-K., Forsburg, S. L. and Hunter,
T. (2002). The Schizosaccharomyces pombe
aurora-related kinase Ark1 interacts with the inner centromere protein Pic1
and mediates chromosome segregation and cytokinesis. Mol. Biol.
Cell 13,1132
-1143.
MacCallum, D. E., Losada, A., Kobayashi, R. and Hirano, T.
(2002). ISWI remodeling complexes in Xenopus egg
extracts: identification as major chromosomal components that are regulated by
INCENP-aurora B. Mol. Biol. Cell
13, 25-39.
Mackay, A. M. and Earnshaw, W. C. (1993). The INCENPs: structural and functional analysis of a family of chromosome passenger proteins. Cold Spring Harb. Symp. Quant. Biol. 58,697 -706.[Medline]
Mackay, A. M., Ainsztein, A. M., Eckley, D. M. and Earnshaw, W.
C. (1998). A dominant mutant of inner centromere protein
(INCENP), a chromosomal protein, disrupts prometaphase congression and
cytokinesis. J. Cell Biol.
140,991
-1002.
Maney, T., Hunter, A. W., Wagenbach, M. and Wordeman, L.
(1998). Mitotic centromere-associated kinesin is important for
anaphase chromosome segregation. J. Cell Biol.
142,787
-801.
Meuwissen, R. L. J., Offenberg, H. H., Dietrich, A. J. J., Riesewijk, A., van Iersen, M. and Heyting, C. (1992). A coiled-coil related protein specific of the synapsed regions of the meiotic prophase chromosomes. EMBO J. 11,5091 -5100.[Abstract]
Moens, P. B. and Spyropoulos, B. (1995). Immunocytology of chiasmata and chromosomal disjunction at mouse meiosis. Chromosoma 104,175 -182.[CrossRef][Medline]
Morishita, J., Matsusaka, T., Goshima, G., Nakamura, T., Tatebe,
H. and Yanagida, M. (2001). Bir1/Cut17 moving from chromosome
to spindle upon the loss of cohesion is required for condensation, spindle
elongation and repair. Genes Cells
6, 743-763.
Murata-Hori, M. and Wang, Y.-L. (2002). The kinase activity of aurora B is required for kinetochore-microtubule interactions during mitosis. Curr. Biol. 12,894 -899.[CrossRef][Medline]
Murata-Hori, M., Tatsuka, M. and Wang, Y.-L.
(2002). Probing the dynamics and functions of aurora B kinase in
living cells during mitosis and cytokinesis. Mol. Biol.
Cell 13,1099
-1108.
Oegema, K., Desai, A., Rybina, S., Kirkham, M. and Hyman, A.
A. (2001). Functional analysis of kinetochore assembly in
Caenorhabditis elegans. J. Cell Biol.
153,1209
-1225.
Page, J., Suja, J. A., Santos, J. L. and Rufas, J. S. (1998). Squash procedure for protein immunolocalization in meiotic cells. Chromosome Res. 6, 639-642.[CrossRef][Medline]
Parra, M. T., Page, J., Yen, T. J., He, D., Valdeolmillos, A., Rufas, J. S. and Suja, J. A. (2002). Expression and behaviour of CENP-E at kinetochores during mouse spermatogenesis. Chromosoma 111,53 -61.[CrossRef][Medline]
Peters, A. H. F. M., Plug, A. W., van Vugt, M. J. and de Boer, P. (1997). A drying-down technique for the spreading of mammalian meiocytes from the male and female germ line. Chromosome Res. 5,66 -71.[CrossRef][Medline]
Pluta, A. F., Cooke, C. A. and Earnshaw, W. C. (1990). Structure of the human centromere at metaphase. Trends Biochem. Sci. 15,181 -185.[Medline]
Prieto, I., Suja, J. A., Pezzi, N., Kremer, L., Martínez-A, C., Rufas, J. S. and Barbero, J. L. (2001). Mammalian STAG3 is a cohesin specific to sister chromatid arms during meiosis I. Nat. Cell Biol. 3, 761-766.[CrossRef][Medline]
Rajagopalan, S. and Balasubramanian, M. K.
(2002). Schizosaccharomyces pombe Bir1p, a nuclear
protein that localizes to kinetochores and the spindle midzone, is essential
for chromosome condensation and spindle elongation during mitosis.
Genetics 160,445
-456.
Rattner, J. B., Kingwell, B. G. and Fritzler, M. J. (1988). Detection of distinct structural domains within the primary constriction using autoantibodies. Chromosoma 96,360 -367.[Medline]
Rattner, J. B., Hendzel, M. J., Sommer Furbee, C., Muller, M. T.
and Bazett-Jones, D. P. (1996). Topoisomerase II is
associated with the mammalian centromere in a cell cycle- and species-specific
manner and is required for proper centromere/kinetochore structure.
J. Cell Biol. 134,1097
-1107.[Abstract]
Revenkova, E., Eijpe, M., Heyting, C., Gross, B. and
Jesseberger, R. (2001). Novel meiosis-specific isoform of
mammalian SMC1. Mol. Cell. Biol.
21,6984
-6998.
Rogers, E., Bishop, J. D., Waddle, J. A., Schumacher, J. M. and
Lin, R. (2002). The aurora kinase AIR-2 functions in the
release of chromosome cohesion in Caenorhabditis elegans meiosis.
J. Cell Biol. 157,219
-229.
Rufas, J. S., Mazzella, C., Suja, J. A. and García de la Vega, C. (1989). Kinetochore structures are duplicated prior to the first meiotic metaphase. A model of meiotic behaviour of kinetochores in grasshoppers. Eur. J. Cell Biol. 48,220 -226.
Saffery, R., Irvine, D. V., Kile, B. T., Hudson, D. F., Cutts, S. M. and Choo, K. H. A. (1999). Cloning expression, and promoter structure of a mammalian inner centromere protein (INCENP). Mamm. Genome 10,415 -418.[CrossRef][Medline]
Schumacher, J. M., Golden, A. and Donovan, P. J.
(1998). AIR-2: an Aurora/Ipl1-related protein kinase associated
with chromosomes and midbody microtubules is required for polar body extrusion
and cytokinesis in Caenorhabditis elegans embryos. J. Cell
Biol. 143,1635
-1646.
Sonoda, E., Matsusaka, T., Morrison, C., Vagnarelli, P., Hoshi, O., Ushiki, T., Nojima, K., Fukagawa, T., Waizenegger, I. C., Peters, J. M. et al. (2001). Scc1/Rad21/Mcd1 is required for sister chromatid cohesion and kinetochore function in vertebrate cells. Dev. Cell 1,759 -770.[Medline]
Speliotes, E. K., Uren, A., Vaux, D. and Horvits, H. R. (2000). The survivin-like C. elegans BIR-1 protein acts with the Aurora-like kinase AIR-2 to affect chromosomes and the spindle midzone. Mol. Cell 6,211 -223.[Medline]
Suja, J. A., Antonio, C. and Rufas, J. S. (1992). Involvement of chromatid cohesiveness at the centromere and chromosome arms in meiotic chromosome segregation: A cytological approach. Chromosoma 101,493 -501.[Medline]
Suja, J. A., Antonio, C., Debec, A. and Rufas, J. S.
(1999). Phosphorylated proteins are involved in sister chromatid
arm cohesion during meiosis I. J. Cell. Sci.
112,2957
-2969.
Tanaka, T. U., Rachidi, N., Janke, C., Pereira, G., Galova, M., Schiebel, E., Stark, M. J. and Nasmyth, K. (2002). Evidence that the Ipl1-Sli15 (aurora kinase-INCENP) complex promotes chromosome bi-orientation by altering kinetochore-spindle pole connections. Cell 108,317 -329.[Medline]
Terada, Y. (2001). Role of chromosomal passenger complex in chromosome segregation and cytokinesis. Cell Struc. Func. 26,653 -657.[CrossRef][Medline]
Terada, Y., Tatsuka, M., Suzuki, F., Yasuda, Y., Fujita, S. and
Otsu, M. (1998). AIM-1: a mammalian midbody-associated
protein required for cytokinesis. EMBO J.
17,667
-676.
Uren, A. G., Wong, L., Pakusch, M., Fowler, K. J., Burrows, F. J., Vaux, D. L. and Choo, K. H. A. (2000). Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene-knockout phenotype. Curr. Biol. 10,1319 -1328.[CrossRef][Medline]
Vagnarelli, P. and Earnshaw, W. C. (2001). INCENP loss from an inactive centromere correlates with the loss of sister chromatid cohesion. Chromosoma 110,393 -401.[CrossRef][Medline]
Wheatley, S. P., Carvalho, A., Vagnarelli, P. and Earnshaw, W. C. (2001). INCENP is required for proper targeting of Survivin to the centromeres and the anaphase spindle during mitosis. Curr. Biol. 11,886 -890.[CrossRef][Medline]
Zeitlin, S. G., Shelby, R. D. and Sullivan, K. F.
(2001). CENP-A is phophorylated by aurora B kinase and plays an
unexpected role in completion of cytokinesis. J. Cell
Biol. 155,1147
-1157.
Zickler, D. and Kleckner, M. (1999). Meiotic chromosomes: Integrating structure and function. Annu. Rev. Genet. 33,603 -754.[CrossRef][Medline]