1 PRESTO, The Japan Science and Technology Corporation, National Institute of
Genetics and The Graduate University for Advanced Studies, Mishima, Shizuoka
411-8540, Japan
2 CREST Research Project, The Japan Science and Technology Corporation, Kansai
Advanced Research Center, Communications Research Laboratory, 588-2 Iwaoka,
Nishi-ku, Kobe 651-2492, Japan
3 Department of Functional Genomics, Medical Research Institute, Tokyo Medical
and Dental University, Tokyo 113-8510, Japan
* Author for correspondence (e-mail: tfukagaw{at}lab.nig.ac.jp)
Accepted 1 May 2003
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Summary |
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We also created conditional loss-of-function mutants of Nuf2 and Hec1. In both mutants, the cell cycle arrested at prometaphase, suggesting that the Nuf2-Hec1 complex is essential for mitotic progression. The inner centromere proteins CENP-A, -C, and -H and checkpoint protein BubR1 were localized to chromosomes in the mutant cells arrested at prometaphase, but Mad2 localization was abolished. Furthermore, photobleaching experiments revealed that the Nuf2-Hec1 complex is stably associated with the centromere and that interaction of this complex with the centrosome is dynamic.
Key words: Nuf2, Hec1, Centrosome, Centromere, FRAP
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Introduction |
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The vertebrate centromere is a trilaminar plate structure
(Pluta et al., 1995;
Craig et al., 1999
). A number
of centromere proteins have been identified in higher vertebrates. Proteins
located in the inner plate, including CENP-A, -C, -H and -I, are required for
formation of the kinetochore structure
(Saitoh et al., 1992
;
Tomkiel et al., 1994
;
Warburton et al., 1997
;
Fukagawa et al., 1999a
;
Fukagawa et al., 2001
;
Howman et al., 2000
;
Sugata et al., 2000
;
Nishihashi et al., 2002
).
Motor proteins and spindle checkpoint proteins such as CENP-E and Mad/Bub
family proteins are associated with the outer plate
(Cooke et al., 1997
;
Jablonski et al., 1998
).
Genetic approaches have been taken to understand the molecular mechanism
underlying centromere formation in vertebrates, and several molecular
mechanisms for centromere formation in cells of higher vertebrates been
reported (Howman et al., 2000
;
Fukagawa et al., 2001
;
Nishihashi et al., 2002
). To
further understand the mechanisms underlying centromere formation and
function, it is necessary to characterize centromere components.
The Ndc80p complex in Saccharomyces cerevisiae has been identified
as a spindle pole component (Wigge et al.,
1998). Further analysis has shown that the Ndc80p complex, which
contains Ndc80p, Spc24p, Spc25p and Nuf2p, is associated with the yeast
centromere (Janke et al.,
2001
; Wigge and Kilmartin,
2001
; He et al.,
2001
). Two of the components, Ndc80p and Nuf2p, have homologues in
S. pombe, C. elegans and vertebrates
(Nabetani et al., 2001
;
Howe et al., 2001
;
McCleland et al., 2003
). The
human homologue of Ndc80p, Hec1, was originally identified as a retinoblastoma
protein (Rb)-associated protein. Microinjection of Hec1 antibodies into
cultured cells disrupts mitotic progression
(Chen et al., 1997
). Recently,
Martin-Lluesma et al. (Martin-Lluesma et
al., 2002
) reported that human Hec1 is required for recruitment of
spindle checkpoint components, including Mps1 and Mad1/Mad2 complexes, to
centromeres. It has also been reported that the human homologue of Nuf2p
localizes to centromeres during M phase and is related to mitotic function
(Nabetani et al., 2001
;
Wigge and Kilmartin, 2001
;
DeLuca et al., 2002
).
We hypothesized that both Hec1 and Nuf2 play important roles in mitotic
progression in vertebrate cells. However, it is unclear whether these proteins
form a complex in vertebrate cells or how such a complex functions in cell
cycle regulation. How this complex is involved in assembly of the vertebrate
centromere is also of interest. Although much of the detailed analysis of the
Ndc80p complex has been in yeast, we used chicken DT40 cells because they have
a high level of homologous recombination and condensed mitotic chromosomes
that are easily seen under light microscopy
(Fukagawa et al., 1999a;
Fukagawa et al., 2001
;
Nishihashi et al., 2002
). In
addition, structures such as the mitotic spindle, centrosome, and centromere
in DT40 cells are much larger and more elaborate than the analogous structures
in yeast. Furthermore, the stages of mitosis (prometaphase chromosome
congression, metaphase, and transition to anaphase) are easily distinguished
under light microscopy in DT40 cells.
To complement previous studies in yeasts, we isolated the chicken homologues of Nuf2 and Ndc80 (Hec1) for further analysis. We found that Nuf2 binds tightly to Hec1 throughout the cell cycle and that the complex localizes to the centrosome in G1 and S phases, moves to the centromere during early G2 phase, and remains at the centromere until completion of mitosis. Furthermore, we describe the generation of conditional loss-of-function mutants of both Nuf2 and Hec1 in DT40 cells to evaluate the in vivo functions of these proteins in higher vertebrates. Phenotypic analysis of these mutants revealed that both Nuf2 and Hec1 play essential roles in the formation of functional centromeres and in the progression of mitosis. Photobleaching experiments showed that interaction of the complex with the centromere is relatively stable, whereas interaction with centrosomes is dynamic.
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Materials and Methods |
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The knockout constructs were created by a standard molecular biology technique. To generate the expression construct for the Nuf2-GFP and Hec1-GFP fusion genes, the full-length cDNAs were inserted into pEGFP-N1 plasmid (Clontech). To make the expression construct for the Hec1-HcRed, a full-length Hec1 cDNA was inserted into pHcRed-N1 plasmid (Clontech). An expression construct for GFP-tubulin (pEGFP-Tub) was purchased from Clontech.
Cell culture and cell cycle analysis
DT40 cells were cultured and transfected as described previously
(Fukagawa and Brown, 1997).
Cell cycle analysis was carried out as described previously
(Fukagawa et al., 2001
).
Flow-cytometry was performed with an Epics Altra cytometer
(Beckman-Coulter).
Antibody production
A chicken Nuf2 expression construct (amino acids 91-293) and a
chicken Hec1 expression construct (amino acids 465-640) were created
in vector pET28a (Novagen). The histidine-tagged recombinant protein was
expressed in E. coli BL21 (DE3) cells. Purified recombinant proteins
were used to immunize a rabbit. Serums were affinity purified against
recombinant proteins on a CNBr-activated Sepharose 4B column.
Immunoprecipitation was performed by a standard method.
Immunocytochemistry and FISH analysis
Immunofluorescent staining of whole cells was performed as described
previously (Fukagawa et al.,
1999a). Anti-
-tubulin and
-tubulin monoclonal
antibody (Sigma), rabbit anti-chicken CENP-A antibody
(Fukagawa et al., 2001
)
(Regnier et al., 2003
), rabbit
anti-chicken CENP-C antibody (Fukagawa et
al., 1999a
), rabbit anti-chicken CENP-H antibody
(Fukagawa et al., 2001
),
rabbit anti-chicken Nuf2 antibody or rabbit anti-chicken Hec1 antibody were
used.
Metaphase chromosome spreads for immunofluorescence staining were prepared
by a method modified from Earnshaw et al.
(Earnshaw et al., 1989).
Chromosome spreads were stained with rabbit anti-chicken CENP-C antibody
(Fukagawa et al., 1999a
) or
rabbit anti-chicken BubR1 (Nishihashi et
al., 2002
).
FISH was performed as described previously
(Fukagawa et al., 1999b). All
immunofluorescence and fluorescence in situ hybridization (FISH) images were
collected with a cooled CCD camera (Photometrics Image Point) mounted on a
Zeiss Axioscope microscope with a 63x objective (Zeiss) together with a
filter wheel. Images were manipulated with Iplab software (Signal
Analytics).
Fluorescence microscopy in living cells
Fluorescently stained living cells were observed with an Olympus inverted
microscope IX70 with an oil immersion objective lens (PlanApo 60x,
NA=1.40). The DeltaVision microscope system used in this study was purchased
from Applied Precision, Inc. For temperature control during microscopic
observations, the system was assembled in a custom-made,
temperature-controlled room (Haraguchi et
al., 1997; Haraguchi et al.,
1999
). Time-lapse images were recorded at 2 minutes intervals with
an exposure time of 0.2-0.3 sesonds.
Fluorescence recovery after photobleaching (FRAP) assay
FRAP was performed using an Olympus Fluoview confocal microscope. Cells on
glass-bottomed dishes were grown at 37°C on a heated stage. One sectioned
area was photo-bleached four times. After 1 second, time-lapse images were
collected. The average signal intensity relative to the pre-bleached image was
analyzed from 20-30 live-cell sequences by measuring the net intensity of the
bleached area minus background relative to that of the whole cells in each
frame by using ImageJ version 1.28u (provided by W. Rasband, National
Institute of Health;
http://rsb.info.nih.gov./ij/).
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Results |
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The yeast homologues of Hec1, Ndc80p and Nuf2p form a complex with Spc24p
and Spc25p (Janke et al.,
2001; Wigge and Kilmartin,
2001
; He et al.,
2001
). Therefore, we examined the interaction between chicken Hec1
and Nuf2 in chicken DT40 cells. We generated a DT40 cell line in which Nuf2
was replaced with Nuf2-GFP (called Nuf39-15 cells) and a DT40 cell line in
which Hec1 was replaced with Hec1-GFP (called Hec25-1/5 cells).
Immunoprecipitation experiments with pre-immune IgG or anti-GFP antibody were
performed on extracts from Nuf39-15 cells and Hec25-1/5 cells. Immune
complexes were then analyzed by immunoblotting with anti-Hec1 and anti-Nuf2
antibodies. We found that these proteins coprecipitate
(Fig. 1A).
|
To determine the subcellular localization of these proteins, GFP and HcRed were tagged on the C termini of Nuf2 and Hec1, respectively, and a DT40 cell line was generated in which the fused genes were expressed simultaneously (called NufG/HecR cells). The fusion proteins were functional and fully suppressed the phenotypes observed in knockout cells (data not shown). Nuf2-GFP and Hec1-HcRed signals throughout the cell cycle are shown in Fig. 1B. During interphase, Nuf2-GFP was colocalized with Hec1-HcRed at or near centrosomes (Fig. 1B, column 1). Colocalization of Nuf2-GFP with Hec1-HcRed was also observed as discrete signals during prometaphase (Fig. 1B, column 2), metaphase (Fig. 1B, column 3) and anaphase (Fig. 1B, column 4). Non-overlapping GFP and HcRed signals were not observed, indicating that Nuf2 is tightly associated with Hec1 throughout the cell cycle.
To examine localization of the Nuf2-Hec1 complex in detail in DT40 cells,
cells expressing Nuf2-GFP (Nuf39-15 cells) were immunostained with
anti-CENP-C or anti -tubulin antibody. We used CENP-C as a centromere
marker (Saitoh et al., 1992
)
and
-tubulin as a centrosome marker
(Wiese and Zheng, 1999
).
During G1, Nuf2-GFP was colocalized with
-tubulin and not CENP-C
(Fig. 2A,B). We observed
Nuf2-GFP colocalized with CENP-C and
-tubulin during G2 after Nuf2-GFP
signals were trapped at the nuclear membrane. Nuf2-GFP signals localized to
the centromere during mitosis (Fig.
2A,B). Centrosomic Nuf2-GFP signals were strong and centromeric
signals were relatively weak in early G2 when the centrosome begins to split
(Fig. 2B). In contrast, the
intensity of centromeric Nuf2-GFP signals increased in late G2 when the
centrosome split was proceeding; and strong Nuf2-GFP signals were observed at
centromeres during late G2 (Fig.
2A,B).
|
To confirm that localization of Nuf2-GFP changes, we examined the behavior of Nuf2-GFP signals in individual living cells (Fig. 2C; Movie 1, http://jcs.biologists.org/supplemental). We stained the chromosomes of living cells expressing Nuf2-GFP with Hoechst 33342 and observed the cells microscopically at 37°C. An example of the time-lapse data is shown in Fig. 2C. The dynamic changes in Nuf2 localization observed during the cell cycle in living cells were similar to those identified in fixed cells.
Generation of conditional loss-of-function mutants of Nuf2 and Hec1
in DT40 cells
To investigate the roles of Nuf2 and Hec1 in higher vertebrate cells, we
generated conditional loss-of-function mutants of Nuf2 and Hec1. In our
system, expression of both Nuf2 and Hec1 was under the
control of a tetracycline (tet)-repressible promoter. A Nuf2 deletion
construct was generated such that the 8.0 kb genomic fragment encoding amino
acids 1-223 was replaced with one of several selection cassettes
(Fig. 3A). We initially
transfected a Nuf2 disruption construct containing the
histidinol-resistance cassette into DT40 cells
(Fig. 3A) and isolated
Nuf2+/ clones
(Fig. 3B). One
Nuf2+/ clone was cotransfected with a chicken
Nuf2 transgene under the control of a tet-repressible promoter and a
tet-repressible transactivator containing a zeocin-resistance cassette. We
selected zeocin-resistant colonies and identified several clones carrying
these constructs integrated at random sites in the genome
(Nuf2+//Nuf2
transgene). Six clones with the
Nuf2+//Nuf2
transgene genotype were transfected with the
puromycin-Nuf2 disruption construct to disrupt the remaining
Nuf2 allele (Fig. 3B).
We obtained two clones with the
Nuf2//Nuf2
transgene genotype, and one clone, Nuf23-63, was chosen for further
analysis.
|
We used the same strategy to generate Hec1 mutants. Three Hec1 alleles are present in the chicken DT40 genome. A Hec1 deletion construct was generated such that the 4.0 kb genomic fragment encoding amino acids 1-75 was replaced with one of several selection cassettes (Fig. 3A). We sequentially transfected Hec1 disruption constructs containing either the histidinol- or puromycin-resistance cassette into DT40 cells (Fig. 3A). After generating clones with the Hec1+///Hec1 transgene genotype, we transfected the neomycin-Hec1 disruption construct to delete the remaining Hec1 allele and obtained six clones with the Hec1///Hec1 transgene genotype (Fig. 3B). One clone, Hec25-1, was chosen for further analysis. Western blot analysis did not detect Nuf2 protein in Nuf23-63 cells 24 hours after tet addition, or Hec1 protein in Hec25-1 cells 12 hours after tet addition (Fig. 3C).
Deletion of Nuf2 and Hec1 results in accumulation of cells in
prometaphase and subsequent cell death
The growth curves of Nuf23-63 (Nuf2+, tet) and Hec25-1 (Hec1+,
tet) cells were indistinguishable from that of wild-type DT40 cells
(Fig. 4A). We then examined
cell proliferation and viability following the addition of tet to the medium.
Nuf23-63 cells (Nuf2, tet+) stopped proliferating approximately 1.5
cell cycles after addition of tet, and most cells died by 48 hours
(Fig. 4A). Hec25-1 cells
(Hec1, tet+) stopped proliferating approximately 10 hours after
addition of tet, and most cells died by 36 hours
(Fig. 4A). These findings
indicate that depletion of Nuf2 and Hec1 causes growth arrest and subsequent
cell death.
|
For cell cycle analysis following gene depletion in both knockout cells, we measured both cellular DNA content and DNA synthesis by fluorescence-activated cell sorting (FACS) after pulse-labeling with BrdU (Fig. 4B). Nuf23-63 cells (tet+) started to accumulate in the G2/M phase 18 hours after addition of tet and 48% of cells were in the G2/M phase by 24 hours. Hec25-1 cells (tet+) also started to accumulate in the G2/M phase 12 hours after addition of tet and 54% of cells were in the G2/M phase by 18 hours (Fig. 4B). After a prolonged delay in the G2/M phase, cells of both clones died suddenly. Degradation of chromosomal DNA due to massive cell death was observed (Fig. 4B and Fig. 5A).
|
To determine the exact nature of the mitotic delay, we used DNA staining
and immunocytochemical staining of microtubules to examine the time course of
events following the depletion of Nuf2 and Hec1
(Fig. 5A,B). In Nuf2-deficient
cells, the mitotic index began to increase at 12 hours and reached 44% at 24
hours after addition of tet (Fig.
5A,B). In Hec1-deficient cells, the mitotic index began to
increase at 12 hours and reached 52% at 18 hours after addition of tet
(Fig. 5A,B). We did not observe
any anaphase cells after 24 hours in either mutant cell line
(Fig. 5B). These data suggest
that both Nuf2- and Hec1-deficient cells accumulate in the M phase rather than
in the G2 phase. Although we observed the movement of these proteins to the
centromere from the centrosome, both mutant cell types displayed a terminal
phenotype during mitosis. We did not detect further uptake of BrdU after
cell-cycle arrest. We did not observe many polyploid cells, in contrast to
CENP-H- or CENP-I-deficient cells, which proceed to the next interphase after
a long delay in mitosis (Fukagawa et al.,
2001; Nishihashi et al.,
2002
). Considering the FACS data together with cytological data,
we suggest that Nuf2- and Hec1-deficient cells died after cell-cycle arrest
during mitosis.
Deletion of Nuf2 or Hec1 causes chromosome aberrations and leads to
chromosome missegregation.
During the course of cytological analyses of Nuf2- and Hec1-deficient
cells, we observed many abnormal mitotic cells that contained hypercondensed
chromosomes that failed to congress normally to the metaphase plate
(Fig. 5A). Eventually, the
cells underwent apoptosis (Fig.
5A). In control cells, the chromosomes appeared ordered and
aligned properly on the metaphase plate
(Fig. 5A).
Control cells cultured in the absence of tet showed well-ordered bipolar spindles during both metaphase and anaphase (Fig. 5A, 0 hours). In the mutant cell lines, most cells cultured in the presence of tet had normal bipolar spindles, but some contained multi- or monopolar spindles. We found, by observing living cells, that normal bipolar spindles formed just after entrance into mitosis and the abnormal spindles appeared during mitotic arrest (Fig. 6; Movies 2 and 3, http://jcs.biologists.org/supplemental). In most mutant cells with bipolar spindles, chromosome alignment was disordered. We frequently observed arch-shaped spindles away from chromosomes (Fig. 5A). These results indicate that Nuf2 and Hec1 are required either directly or indirectly for congression and/or maintenance of stable chromosome alignment and, ultimately, for progression from metaphase to anaphase.
|
We then examined whether the mitotic defects caused by depletion of Nuf2 and Hec1 were associated with induction of aneuploidy. We performed FISH analysis of metaphase spreads with chromosome painting probes specific for chicken chromosomes 1 and 2. Because DT40 cells contain three copies of chromosome 2, five fluorescent chromosomes can be observed in each wild-type cell. As shown in Fig. 5C, we observed abnormal numbers of painted chromosomes in both Nuf2- and Hec1-deficient cells in the presence of tet. The proportion of these aneuploid cells increased gradually after addition of tet (Fig. 5C), suggesting that deficiency of Nuf2 and Hec1 cause chromosome missegregation. Although Nuf2- and Hec1-deficient cells died in M phase, the proportion of aneuploid cells increased. We explain this observation as follows. At an early stage after addition of tet, cells have a small amount of Hec1 or Nuf2 protein. In this stage, several populations of cells can exit M phase with a small amount of Hec1 or Nuf2. These cells may fail to complete normal mitosis, and cells with chromosome loss accumulate.
To determine the consequences of Nuf2 deficiency in greater detail, we
observed the dynamic behavior of individual living cells after suppression of
Nuf2 expression (Fig.
6; Movies 2 and 3). To visualize microtubules in living cells, the
human -tubulin gene fused with GFP was integrated into the genome of
Nuf23-63 cells. We then stained the live Nuf23-63 cells expressing
GFP-
-tubulin with Hoechst 33342 and observed the
cells microscopically at 37°C. Representative time-lapse data for Nuf23-63
cells in the absence of tet (control) are shown in
Fig. 6A. Control cells
(n=10) required approximately 30 minutes to progress from prophase to
telophase. After addition of tet to Nuf23-63 cells, we observed abnormal
mitotic behavior (Fig. 6B). We
observed normal progression during interphase and a normal split of
centrosomes (data not shown). Time point 0 in
Fig. 6B corresponds to 18 hours
after addition of tet to cultures of Nuf23-63 cells. The cell shown in
Fig. 6B entered mitosis (20
minutes) as condensed individual chromosomes appeared and the mitotic spindle
formed. Chromosome congression to the metaphase plate and subsequent anaphase
were not observed. Instead, the cell remained arrested in prometaphase for
approximately 400 minutes with hyper-condensed chromosomes. During this
prolonged mitotic arrest, the spindle structure underwent a series of abnormal
changes. Early after mitotic entry (Fig.
6B, 16-20 minutes) a normal-looking bipolar mitotic spindle had
formed, but subsequently an abnormal spindle formed
(Fig. 6B, 126-304 minutes).
After prolonged mitotic arrest, the cell underwent apoptosis. Similar events
were observed in Hec1 mutant cells (data not shown). The time of mitotic
arrest in Nuf2 and Hec1 mutants (approximately 400 minutes) was shorter than
that observed in CENP-I-deficient cells (approximately 800 minutes)
(Nishihashi et al., 2002
).
Localization of centromere- and checkpoint-related proteins in Nuf2-
and Hec1-deficient cells
We observed that Nuf2 and Hec1 formed a complex that was maintained
throughout the cell cycle (Fig.
1). Therefore, we investigated Hec1 localization in Nuf2-deficient
cells and vice versa. We used a Nuf2-deficient cell line that stably expresses
Hec1-GFP and a Hec1-deficient cell line that stably expresses
Nuf2-GFP. We found that in Nuf2-deficient cells the Hec1-GFP signals
were diffuse at both centrosomes and centromeres. Nuf2-GFP signals were
diffuse in Hec1-deficient cells (Fig.
7A). We also found by western blot analysis that the levels of
Hec1 in Nuf2-deficient cells and of Nuf2 in Hec1-deficient cells were reduced
(Fig. 7A). These findings
support our conclusion that the Nuf2-Hec1 complex is tight during the cell
cycle.
|
In the present study, we found that the Nuf2-Hec1 complex localizes to the
centromere during mitosis. We reported previously that centromere components
were not assembled in a simple linear fashion
(Nishihashi et al., 2002).
Therefore, we examined whether inner centromere proteins localize to the
centromere in Nuf2- and Hec1-deficient cells. Metaphase spreads of Nuf2- and
Hec1-deficient cells were analyzed with antibodies against chicken CENP-A, -C,
and -H, which are typical inner centromere proteins. Representative results
are shown in Fig. 7B. In Nuf2-
and Hec1-deficient cells, strong centromeric signals for CENP-A -C, and -H
were observed, and the signal intensities were similar to those in control
cells (Fig. 7B). These results
indicate that Nuf2 and Hec1 do not influence localization of CENP-A, -C, and
-H in mitotic chromosomes.
We observed mitotic arrest in Nuf2- and Hec1-deficient cells (Figs
4,
5 and
6). This phenotype could be due
to activation of a mitotic checkpoint
(Wassmann and Benezra, 2001).
We analyzed localization of a checkpoint protein, Mad2, in Nuf2- and
Hec1-deficient cells (Fig. 7C).
We used a Nuf2-deficient cell line that stably expressed Mad2-DsRed
(Sonoda et al., 2001
) and a
Hec1-deficient cell line that stably expressed Mad2-DsRed. In both
Nuf2- and Hec1-deficient cells we did not detect Mad2-DsRed signals at
centromeres that we can detect easily in control cells
(Fig. 7C). Because both mutants
are clearly arrested in mitosis for 400 minutes before dying, we analyzed the
localization of BubR1, which is another checkpoint protein
(Chan et al., 1999
). We found
strong BubR1 signals in mitotically arrested Nuf2- and Hec1-deficient cells
(Fig. 7C). These results
suggest that the mitotic arrest in both mutants is due to activation of the
BubR1 pathway.
We have shown that the Nuf2-Hec1 complex is localized at centrosomes during
the G1 and S phases (Fig. 2).
Thus, we investigated localization of -tubulin in Nuf2- and
Hec1-deficient cells during these phases, because the ring complex with
-tubulin is a major component of centrosomes
(Wiese and Zheng, 1999
). We
stained Nuf2- and Hec1-deficient cells with an antibody against
-tubulin. We detected typical centrosome signals for
-tubulin in
both control and mutant cells (Fig.
7D), suggesting that the Nuf2-Hec1 complex is localized in the
pericentriolar material away from the
-tubulin complex.
Nuf2 and Hec1 localization in cells with disruption of the inner
centromere structure
Immunocytochemical analysis of Nuf2- and Hec1-deficient cells with inner
centromere proteins CENP-A, -C and -H revealed that Nuf2 and Hec1 are not
necessary for localization of the inner centromere proteins to the centromere
(Fig. 7B). Therefore, we
examined whether Nuf2 and Hec1 are targeted to centromeres lacking the inner
centromere proteins. We reported previously that BubR1, a checkpoint protein
in the outer centromere, is targeted to disrupted centromeres in CENP-I
knockout cells (Nishihashi et al.,
2002). We used CENP-H-deficient cells that stably expressed
Nuf2-GFP or Hec1-GFP. We also used CENP-I-deficient cells
that stably expressed Nuf2-GFP or Hec1-GFP. We did not
detect CENP-H or CENP-I signals in CENP-H- or CENP-I-deficient cell lines,
respectively, 48 hours after addition of tet. Typical results are shown in
Fig. 8. Nuf2-GFP and Hec1-GFP
signals are significantly lower in both CENP-H- and CENP-I-deficient cells in
comparison to control cells. However, we can still observe signals for Nuf2
and Hec1. These results suggest that association of Nuf2 or Hec1 with
centromeres was weak in CENP-H- and CENP-I-deficient cells.
|
Nuf2 and Hec1 turn over rapidly at centrosomes and are stably
associated with centromeres.
In the present study, we observed the dynamic behavior of the Nuf2-Hec1
complex during the cell cycle. To gain insight into the dynamics of Nuf2 and
Hec1 in living cells, we used Nuf39-15 cells in which expression of
Nuf2 is completely replaced with Nuf2-GFP and Hec25-1/5
cells in which expression of Hec1 is completely replaced with
Hec1-GFP (described in Fig.
1A). Growth of these cell lines was identical to that of wild-type
DT40 cells, which indicates that Nuf2-GFP and Hec1-GFP were functional. We
used fluorescence recovery after photobleaching (FRAP) of these cells to assay
the turnover of these proteins at centrosomes (during G1 or S phase) and at
centromeres (during mitosis). We found that Nuf2-GFP and Hec1-GFP fluorescence
at centrosomes recovered rapidly (Fig.
9; Movies 4 and 5,
http://jcs.biologists.org/supplemental)
with half-times of 11.9 seconds (n=19) and 13.2 seconds
(n=19), respectively. In contrast, fluorescent signals for both
Nuf2-GFP and Hec1-GFP at centromeres did not recover rapidly
(Fig. 9; Movies 6 and 7); it
took more than 30 minutes to recover these signals. We did not observe
substantial recovery of either GFP signal when the centromere fractions of
both cell lines were photobleached during early mitosis. These findings
indicate that the associations of Nuf2 and Hec1 with centrosomes during G1 and
S phases are dynamic, and Nuf2 and Hec1 are stable components of centromeres
during mitosis (Fig. 10).
|
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![]() |
Discussion |
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The Nuf2-Hec1 complex is required for centromere function during
mitosis
To further our understanding of the Nuf2-Hec1 complex, we generated
conditional loss-of-function mutants of Nuf2 and Hec1 in the
hyper-recombinogenic chicken DT40 cell line. We previously created several
conditional mutants of the inner centromere components CENP-C, -H and -I
(Fukagawa and Brown, 1997;
Fukagawa et al., 1999a
;
Fukagawa et al., 2001
;
Nishihashi et al., 2002
). We
then compared the phenotypes of the Nuf2 and Hec1 mutants with those of the
CENP mutants.
Despite the dynamic localization of the Nuf2-Hec1 complex during the cell
cycle, both Nuf2- and Hec1-deficient cells arrested specifically in
prometaphase. Both mutants have hypercondensed chromosomes and abnormal
spindle structures associated with prometaphase arrest. This phenotype with
the accompanying abnormal mitosis is similar to those observed in CENP-C, -H,
and -I mutants. Like CENP-C-, -H- and -I-deficient cells, Nuf2- and
Hec1-deficient cells fail to align their chromosomes at the spindle equator.
We observed by FRAP analysis that the Nuf2-Hec1 complex associates stably with
the centromere (Fig. 9). We
also used this technique to analyze turnover of CENP-H-GFP. The kinetics of
turnover CENP-H-GFP and Hec1-GFP at the centromere were similar (T.F.,
unpublished). FRAP analysis of Mad2-GFP and Cdc20-GFP at the centromere
revealed that turnover of these transient kinetochore proteins is rapid
(Howell et al., 2000;
Kallio et al., 2002
). The
association of Nuf2 and Hec1 with the centromere is more stable than that of
checkpoint regulators such as Mad2 and Cdc20. Thus, we suggest that the
Nuf2-Hec1 complex is a structural component of functional centromeres and
essential for full centromere function.
Although we found similarities between the phenotypes of Nuf2- and
Hec1-deficent cells and inner centromere protein knockouts, we also found
differences. The living-cell analysis revealed that the length of time that
Nuf2-deficient cells remained in prometaphase arrest is different from that
observed in CENP-H- or CENP-I-deficient cells. Nuf2-deficient cells arrested
for 400 minutes in prometaphase before undergoing apoptosis. In contrast,
after an 800-minute delay in mitosis, CENP-I-deficient cells proceeded to the
next cell cycle without normal cell division and then died. We reported
previously that the lengthy delays in prometaphase observed in
CENP-I-deficient cells were caused by disruption of the inner kinetochore
structure (Nishihashi et al.,
2002). In the present study, we showed that localization of inner
centromere proteins was not altered in Nuf2- and Hec1-deficient cells
(Fig. 7), suggesting that the
Nuf2-Hec1 complex is not involved in the inner kinetochore structure.
Nevertheless, we found that Nuf2 or Hec1 depletion yielded more severe mitotic
phenotypes than did depletion of CENP-C, -H, or -I. One explanation for this
phenotype difference is that the Nuf2-Hec1 complex functions not only as a
simple structural component of the centromeres but also as a regulator for
progression of mitosis.
The mitotic delay phenotype could be due to activation of mitotic
checkpoints; however, we did not observe Mad2 localization in Nuf2- and
Hec1-deficient cells. Martin-Lluesma et al.
(Martin-Lluesma et al., 2002)
showed by yeast two-hybrid analysis that human Hec1 interacts with Mad1, and
they also showed that Mad2 and Mps1 signals were reduced by Hec1 RNAi in HeLa
cells. DeLuca et al. (DeLuca et al.,
2002
) observed mitotic arrest in human Nuf2 RNAi experiments, and
they detected Mad2 signals on mitotically arrested chromosomes. In our system,
we did not detect Mad2 signals on arrested chromosomes in either Hec1- or
Nuf2-deficient cells. However, we detected strong BubR1 signals at centromeres
in both Nuf2- and Hec1-deficient cells, which suggests that mitotic arrest in
both Nuf2- and Hec1-deficient cells is caused by activation of the BubR1
pathway. Hec1-RNAi cells still underwent a Mad2-dependent checkpoint arrest,
even though they lacked Mad2 at kinetochores
(Martin-Lluesma et al., 2002
).
Therefore, it is possible that the Mad2 pathway is still active in our Nuf2-
and Hec1-deficient cells even though we cannot detect Mad2 signals.
We reported previously that the outer centromere protein BubR1 recognizes
the centromere even if the inner centromere structure is disrupted due to
CENP-H or CENP-I depletion, and we proposed that the kinetochore is not
assembled through a simple linear pathway
(Nishihashi et al., 2002). In
CENP-H- and -I-deficient cells, Nuf2 and Hec1 signal intensities are reduced,
although signals are still visible. These results indicate that centromere
localization of Nuf2 and Hec1 is in part dependent on inner centromere
structure.
What is the role of the Nuf2-Hec1 complex at centrosomes?
Although we clarified the important role of the Nuf2-Hec1 complex during
mitosis, the role of this complex during interphase remained unclear. The Nuf2
complex is localized primarily at centrosomes during G1 and S phases, and we
found by FRAP analysis that association of the complex with centrosomes is
dynamic. This dynamic behavior suggests that this complex may not be a simple
structural component of centrosomes and may function as a signal transmitter
to components localized at centrosomes. It is also possible that the Nuf2-Hec1
complex is modified at centrosomes and that the localization changes depending
on progression of the cell cycle. One possible modification is
phosphorylation. Cheeseman et al.
(Cheeseman et al., 2002)
demonstrated that the yeast homologue of Hec1, Ndc80p, is phosphorylated by
the aurora kinase Ipl1p. There are several counterparts of aurora kinase in
vertebrate cells (Adams et al.,
2001
). Aurora A localizes to centrosomes, and aurora B is targeted
to centromeres during mitosis. It is important to determine whether the
Nuf2-Hec1 complex in vertebrate cells is phosphorylated by aurora kinases. We
do not have any direct evidence for the functions of Nuf2 and Hec1 at
centrosomes, and there is a possibility that neither protein has a significant
function at centrosomes.
Before moving to the centromere from the centrosome, the Nuf2-Hec1 complex
is trapped at the nuclear membrane (Fig.
2). It has been reported that some components of the nuclear pore
redistribute in part to the kinetochore during mitosis in mammalian cells
(Belgareh et al., 2001). The
significance of this observation is unclear; however, it is possible that the
Nuf2-Hec1 complex is involved in recruitment of nuclear pore components to the
centromere.
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Acknowledgments |
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References |
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