Karolinska Institute, Department of Biosciences/Department of Natural Sciences, University College Sodertorn, Sweden
* Author for correspondence (e-mail: karl.ekwall{at}sh.se)
Accepted 11 June 2003
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Summary |
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Key words: Fission yeast, Centromere, Mitosis, Live analysis
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Introduction |
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Materials and Methods |
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Microscopy
We performed GFP and CFP fluorescence microscopy on live cells using
Openlab software version 3.1 and a ZEISS Axioplan 2 imaging microscope
equipped with a Hamamatsu C4792-95 camera. Cells were prepared as described
previously (Facanha et al.,
2002; Pidoux et al.,
2000
). Cells were analyzed at 30°C with the exception of
mis6-302 and nuf2-1 cells, which were analyzed at 36°C.
The temperature during cultivation of mis6-302 and nuf2-1
cells was raised from 25°C to 36°C 6 hours and 4 hours, respectively,
prior to analysis. Electron microscopy was carried out according to Kniola et
al. (Kniola et al., 2001
) and
fluorescence in situ hybridization (FISH) was performed as described by Ekwall
et al. (Ekwall et al., 1995
)
using the pRS140 probe to detect otr
(Chikashige et al., 1989
).
Movies
Frames were collected every 25 seconds and are displayed at a rate of 2
frames/second. Movies are available at
http://jcs.biologists.org/supplemental
and selected images are presented in Fig.
1B,C.
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Results and Discussion |
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Using green and cyan fluorescent proteins (GFP and CFP) we were then able
to explore the centromere/kinetochore dynamics and multilayered structure in
real time. We were able to simultaneously detect two tagged proteins by using
strains expressing chromosomal copies of a combination of both green-tagged
and cyan-tagged proteins. As a reference point for spindle movement, the
spindle pole body (SPB) marker Cut12-GFP
(Bridge et al., 1998) or
Cut12-CFP was used. We also CFP tagged the S. pombe central core
binding protein Cnp1 (Takahashi et al.,
2000
). Cnp1 is the S. pombe homologue of human CENP-A
(Warburton et al., 1997
;
Vafa and Sullivan, 1997
),
Drosophila CID (Blower and Karpen,
2001
), budding yeast Cse4
(Meluh et al., 1998
) and
C. elegans HCP3 (Buchwitz et al.,
1999
). CENP-A is a histone H3 variant that creates a specialized
chromatin structure, which determines the site of kinetochore assembly.
We began an investigation of centromere domain movements in live cells by
examining the spindle growth using the Cut12-CFP marker (see Movie 1 at
http://jcs.biologists.org/supplemental).
As seen in earlier studies (Nabeshima et
al., 1998), we found that the SPBs moved in three distinct phases.
In the first phase, the SPB was duplicated. In the second phase the spindle
length remained nearly constant (2.5-3.0 µm) while the sister kinetochores
made oscillatory movements back and forth. In the third phase, which
corresponds to anaphase B, the spindle rapidly extended (up to 10 µm). By
taking advantage of the nearly constant spindle length of the second stage of
spindle growth we were able to establish the relative position on the
metaphase spindle of our marker proteins. The central core domain of the
centromere detected with CFPCnp1, and the kinetochore detected with Ndc80-GFP
can be seen occupying slightly different positions along the spindle axis when
compared to the localization of Cut12-CFP/GFP in cells with equal axis lengths
(2.7-2.9 µm distance between SPBs) (Fig.
1C; Movies 1 and 2 at
http://jcs.biologists.org/supplemental).
The slightly different localizations of Cnp1 and Ndc80 during phase two were
directly visualized in triple-tagged live cells expressing Ndc80-GFP, CFP-Cnp1
and Cut12-CFP (Fig. 1C, top;
see Movie 3 at
http://jcs.biologists.org/supplemental).
The perfect colocalization of the dual color of the control strain expression
of Cut12 (Fig. 1C, bottom)
shows that the pixel differences between Cnp1 and Ndc80
(Fig. 1C, top) are significant.
This finding reinforced the evidence for a multilayered domain structure in
mitotic fission yeast centromeres. A schematic model illustrating our
observations on S. pombe centromere structures at metaphase is
presented in the bottom panel of Fig.
1A.
Different centromere domains and different centromere
subfunctions
Kinetochore assembly, binding of kinetochore microtubules, orientation of
sister kinetochores to opposite poles and their poleward movements are
separable centromere functions which may be organized by different structural
domains of the centromere. Evidence from Drosophila shows that
cohesion function is independent from kinetochore assembly
(Lopez et al., 2000) and the
results of RNAi inhibition of CID (Drosophila CENP-A) also argues
that the centromere and flanking heterochromatin are physically and
functionally separable protein domains that are required for different
centromere functions (Blower and Karpen,
2001
). In fission yeast, mutations in cohesin, which is normally
enriched in the flanking heterochromatin, do not seem to affect Cnp1 loading
in the central core region (Toyoda et al.,
2002
). To further test if the different domains we characterized
had different centromere functions, we analyzed trans-acting mutations that
affect each of the domains. The rik1-304 mutation specifically
disrupts centromeric heterochromatin
(Allshire et al., 1995
;
Ekwall et al., 1996
). The
mis6 mutation abolishes Cnp1 loading to the central core region
(Saitoh et al., 1997
;
Takahashi et al., 2000
). The
nuf2-1 mutation disrupts the kinetochore
(Nabetani et al., 2001
).
We began by analyzing the effect of mis6 mutation on interphase
centromere clustering and kinetochore behavior using otr FISH
combined with immunofluorescence microscopy (IF) of Ndc80-GFP. It has
previously been shown that the first mitosis is normal when synchronized
mis6 mutant cells are shifted to the restrictive temperature but the
second mitosis is severely defective and lethal
(Saitoh et al., 1997).
Furthermore it was shown that a high proportion of mis6 interphase
cells in a unsynchronized culture, 6 hours after being shifted up to the
restrictive temperature, had scattered centromere FISH signal, not clustered
at the SPB (Saitoh et al.,
1997
). These results were confirmed in our study where the control
wild-type interphase cells invariably showed one large centromere FISH signal
overlapping with the Ndc80-GFP signal (Fig.
2A,B) whereas 63% of mis6 interphase cells had multiple
dispersed after 6 hours at the restrictive temperature. One of the cen
(otr) FISH signals usually overlapped with the Ndc80 signal. At the
permissive temperature, 13% of mis6 cells gave multiple cen
(otr) spots. Our interpretation of the loss of the Ndc80 kinetochore
marker that correlates with scattered cen (otr) signals is that the
central core region affected by mis6 was required to connect the
kinetochore component Ndc80 to the centromere, and that failure in this
process led to declustering of the centromeres.
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The chromodomain proteins Swi6 (fission yeast homologue of human HP1)
localizes to centromeres mating type region and telomeres
(Ekwall et al., 1995). Swi6 is
required for proper formation of heterochromatin as well as sister chromatid
cohesion, and the recruitment of Swi6 to the otr centromeric DNA is
dependent on Clr4 and Rik1 (Ekwall et al.,
1996
; Bernard et al.,
2001
; Nonaka et al.,
2002
). To investigate if centromeric heterochromatin was affected
by a mutation in mis6, we immunostained fixed mis6 cells
with anti-Swi6 and found that Swi6 was still localized on the declustered
centromeres (Fig. 2C). While
centromeric Swi6 spots were declustered the telomere and mating type spots of
Swi6 still appeared relatively normal in mis6 cells. To verify that
heterochromatin was indeed separated from the previously identified anchor
structures and plate like structures near the SPB
(Kniola et al., 2001
) we fixed
wild-type and mis6 cells using high pressure freezing (HPF) and
examined them by Swi6 immunoelectron microscopy (EM). Centromeric
electron-dense centromeric heterochromatin was normal in wild-type controls
cells but was detected at distances far away from the SPB and anchor structure
in mis6 cells, suggesting that the loss of Cnp1 played a role in
anchoring the centromere DNA to the SPB via the kinetochores
(Fig. 3B,E). The structure of
heterochromatin in rik1 cells was also analyzed by HPF-EM. Using this
method, the heterochromatin domain was not readily detectable and appeared
less electron dense than in wild-type control cells, whereas the plate-like
`anchor' structures indeed appeared to be intact
(Fig. 3C,F). This indicated
that the kinetochore assembly mediated by the central core domain was
functionally independent of the heterochromatin domain. To test this further,
we analyzed the rik1 mutant by cen (otr) FISH, and as
previously demonstrated (Ekwall et al.,
1996
) we found that the clustering of centromeres in interphase
was intact. Furthermore, the colocalization with kinetochore component Ndc80
was normal in 100% of rik1 cells (data not shown). However, the area
of the cen (otr) FISH signal in interphase was enlarged from
0.2±0.1 in wild-type cells to 0.8±0.2 µm2 in
rik1 cells (n=50). This indicated that the centromeric
heterochromatin was decondensed in cells with compromised rik1
(Fig. 2D). In contrast, the
mis6 mutant cells showed a cen (otr) FISH area of
0.2±0.1 µm2 at 25°C and 0.4±0.2
µm2 at 36°C (n=50). The slight increase in the area
of the otr signal in mis6 cells at 36°C can be accounted
for by the scattering of centromeres rather than a decondensation effect.
These findings substantiated the notion that heterochromatin and central
core/kinetochore domains have separate functions in interphase cells.
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Microtubules are not required for centromere clustering
To investigate centromere clustering in live interphase cells we first
tested whether clustering was dependent on microtubules by treating cells
expressing Cut12-GFP and CFP-Cnp1 with 10 µg/ml the microtubule poison
thiabendazole (TBZ). The resulting live-analysis movies (see Movies 4 and 5 at
http://jcs.biologists.org/supplemental)
revealed that, although TBZ suppressed the SPB movements, there was no
detectable declustering of centromere (CFP-Cnp1) signals in the TBZ-treated
cells.
Centromere declustering effects of nuf2 and mis6
In order to visualize what happens to declustered centromeric DNA in live
cells, an integrated lacI-GFP fusion protein that binds lacO repeats
integrated in the vicinity of centromere 1
(Nabeshima et al., 1998) was
used in combination with Cut12-CFP and the mutants rik1, nuf2-1 and
mis6. 73% and 59% respectively of nuf2 and mis6
mutant cells showed an abnormal positioning of the
cen1::lacO-LacI-GFP signals i.e. cen1 far away from SPB, whereas cen1
positioning was normal, at the SPB, in all wild-type and rik1 cells
(Fig. 4A). To test if the
declustered centromeres in the mis6 mutant maintained attachments to
the nuclear periphery, we immunostained the Pom152-GFP nuclear envelope marker
(Bjerling et al., 2002
) and
FISH stained cen (otr), which detects all three centromeres. This
experiment showed that most of the declustered centromeres in mis6
cells were still preferentially found at the nuclear periphery, on average
0.30 µm from the nuclear rim (Fig.
4B,C; see also supplemental Fig. S1 at
http://jcs.biologists.org/supplemental/).
Thus, since mis6 is required for association of Ndc80 to the
centromere (Fig. 2A) we
conclude that kinetochore components are normally required for anchoring
centromeres to the SPB, but an unknown, independent mechanism may be
responsible for peripheral positioning of the declustered centromeric
heterochromatin domains, even in the absence of kinetochore components.
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Consequences in mitosis
Mis6 has previously been shown to influence spindle length, as shown by the
increase of metaphase spindle length in mis6 mutants, which may be
caused by an imbalance between the kinetochore forces that are pulling and the
kinetochore-independent forces that are pushing on the centromeres
(Goshima et al., 1999). To
visualize mitotic defects in mis6 and nuf2-1 cells we
monitored cen1::lacO-LacI-GFP and the SPB marker Cut12-CFP by
time-lapse microscopy. We found that both mis6 and nuf2-1
mutant cells displayed severe defects in movement of sister-centromeres in
relation to the SPB compared with wild-type control cells
(Fig. 5). In contrast, the
majority of rik1 mutant cells displayed similar movements of
sister-centromeres to those of wild-type cells (data not shown). To
characterize the behavior of centromeres in mis6 and nuf2-1
cells we measured the distance from one SPB to the other and the distance from
one SPB to the centromeres of the two segregating cen1::lacO-LacI-GFP
signals (cen1 and cen1*, respectively).
Table 2 shows that on similar
length, 4.0-5.0 µm, spindles the average distance between the one SPB and
the closest centromere (cen1) was relatively similar in wild-type
mis6 and nuf2-1 cells (0.5±0.31, 1.72±0.42 and
0.74±0.08 µm, respectively). However, on similar length 4.0-5.0
µm spindles the furthest-away sister centromere, cen1*, was
3.85±0.78 µm from the same SPB in wild-type cells, 1.93±0.46
µm in mis6 cells and 1.1±0.36 µm in nuf2-1
cells. Thus, whereas wild-type centromeres were segregated by at least 3.5
µm, centromeres had not significantly segregated from each other in
mis6 and nuf2-1 cells. This severe mis-segregation phenotype
is in accordance with the phenotypes reported earlier for mis6 and
nuf2-1 (Saitoh et al.,
1997
; Nabetani et al.,
2001
). The fact that the two phenotypes are so similar indicates
that loss of kinetochore components from centromeres in mis6 is
functionally equivalent to the compromised kinetochore structures in
nuf2-1. The consequence in both mutants is likely the occurrence of
monotelic attachments in which only one functional sister kinetochore binds
kinetochore microtubules or completely unattached sister kinetochores,
resulting in spindle-kinetochore force imbalance and rapid spindle elongation
as previously observed (Goshima et al.,
1999
). In contrast, the defects in the heterochromatin domain
caused by rik1 lead to decondensation of the heterochromatin. This
decondensation had no effect on clustering and caused no dramatic effect on
sister kinetochore behavior (data not shown) except for the previously
observed lagging chromosome phenotype
(Ekwall et al., 1996
). This
phenotype is likely caused by a cohesin defect, since Rik1 is required to
localize Swi6 to centromeres (Ekwall et
al., 1996
) and Swi6 is required in its turn for the recruitment of
cohesins to the centromeric heterochromatin
(Bernard et al., 2001
;
Nonaka et al., 2002
).
Interestingly, we also observed a decondensation of centromeric
heterochromatin in rik1 cells
(Fig. 2D). In budding yeast,
cohesins have been shown to regulate condensin function after condensins are
associated with chromatin (Lavoie et al.,
2002
). Therefore, we speculate that the decondensation observed in
rik1 is explained by loss of Swi6 causing a loss of cohesins and
hence misregulation of condensins. In our experiments decondensation or
cohesion defects were not observed in transacting mutations that affect
central core or kinetochore. In contrast, these mutants showed defects in
clustering of centromeres at the SPB and severe mitotic defects. Thus, the
kinetochore and the central core are functionally distinct from the flanking
heterochromatin domain, both with respect to the centromere function in
interphase and the mitotic behavior of sister centromeres.
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Acknowledgments |
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Footnotes |
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Present address: Alfred Nobel's Alle 7, S-141 89, Huddinge, Sweden
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References |
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