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Centrosome number is controlled by a centrosomeintrinsic block to reduplication
Connie Wong1 and Tim Stearns1,2
The centrosome is the microtubule-organizing centre of animal cells
and is important for organizing the bipolar spindle during mitosis.
Aberrant centrosome number can result in the generation of
monopolar or multipolar spindles1, potentially causing aneuploidy or
cell death. Cells from many types of cancer have multiple centrosomes
and this phenotype has been postulated to function in tumorigenesis
by promoting genomic instability2–4. Centrosome duplication and
DNA replication are initiated at the G1–S transition, and both occur
only once in a single cell cycle. Rao and Johnson used a cell fusion
assay to study the regulation of DNA replication5. They showed that in
a fusion of G1- and S-phase cells, both the G1 and S nuclei replicate;
whereas in a fusion of G2 and S cells, the S nucleus replicates, but the
G2 nucleus does not re-replicate5. This demonstrated the existence of
a block to rereplication of a previously replicated nucleus.
Furthermore, this block is maintained through several mechanisms
and is regulated by the activity of cyclin-dependent kinases (Cdks)6.
Centrosome duplication is under the positive control of Cdk2 and
other factors7,8. However, it is unclear whether duplication is limited
to one round per cell cycle simply by the decline of the positive control
a
Percentage of BrdU incorporation
Percentage with one centrosome
Percentage with two centrosomes
100
Percentage of cells
The centrosome duplicates once in S phase. To determine
whether there is a block in centrosome reduplication, we used
a cell fusion assay to compare the duplication potential of
unduplicated G1 centrosomes and recently duplicated G2
centrosomes. By fusing cells in different cell cycle stages, we
found that G2 centrosomes were unable to reduplicate in a
cellular environment that supports centrosome duplication.
Furthermore, G2 cytoplasm did not inhibit centrosome
duplication in fused cells, indicating that the block to
reduplication is intrinsic to the centrosomes rather than the
cytoplasm. To test the underlying mechanism, we created
mononucleate G1 cells with two centrosomes by fusing cells
with enucleated cytoplasts. Both centrosomes duplicated,
indicating that the block is not controlled by
centrosome:nucleus ratio. We also found that human primary
cells have tight control over centrosome number during
prolonged S-phase arrest and that this control is partially
abrogated in transformed cells. This suggests a link between
the control of centrosome duplication and maintenance of
genomic stability.
80
60
40
20
0
Unsync (n = 500)
G1 (n = 900)
S (n = 700)
G2 (n = 700)
b
Figure 1 Cell cycle synchronization of fibroblast cells. (a) Fibroblasts were
arrested in G1 phase by serum starvation, S phase by double thymidine
block, and G2 phase by timed release of the S-phase cells. The degree of
synchronization was assessed by BrdU labelling and determination of
centrosome number by centrin staining (one pair of centrioles is equivalent
to one centrosome). Unsynchronized cells were used as a control. (b)
Fluorescence microscopy images of cells with one pair (left) or two pairs
(right) of centrioles. Centrin, green; DNA, blue. Scale bar represents 1 µm.
at the end of S phase or by a specific block to reduplication of previously duplicated centrosomes. We used a cell fusion assay similar to
that of Rao and Johnson5 to address this question. We reasoned that it
should be possible to distinguish these two possibilities by comparing
1Department
of Biological Sciences, Stanford University, and 2Department of Genetics, Stanford University Medical School, Stanford, CA 94305-5020, USA.
Correspondence should be addressed to T.S. (e-mail: [email protected]).
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LETTERS
a
ConA–MB, DAPI
ConA–TR
Centrin
c
b
ConA–MB, DAPI
ConA–MB, DAPI
BrdU
G2-S
fusion
ConA–TR
G1-S
fusion
d
90
Percentage of cells
80
One BrdU-positive nucleus
Two BrdU-positive nuclei
70
60
50
40
30
20
10
0
6 h (n = 66)
12 h (n = 85)
6 h (n = 43)
G1–S
12 h (n = 56)
G2–S
Time after fusion
Figure 2 DNA replication and centrosome number in fused cells. Cells were
synchronized, surface-labelled and fused using polyethylene glycol before
plating and incubation for the indicated times. DNA replication was assayed
by BrdU labelling and centrosomes were counted by staining with an anticentrin antibody. (a) A fused binucleate cell with mixed ConA–Marina Blue
(ConA–MB) and ConA–Texas Red (ConA–TR) surface label. Nucleus is
stained with DAPI. This cell has three centrosomes, revealed as three pairs
of centrin-staining centrioles, enlarged in insets. (b) Adjacent unfused cells
maintain discrete ConA surface labelling at 24 h after fusion treatment. (c)
A G2–S-fused binucleate cell containing one BrdU-labelled nucleus (top). A
G1–S binucleate cell containing two BrdU-labelled nuclei (bottom). One of
the nuclei, presumably from the S-phase cell, shows greater BrdU
incorporation. Scale bar represents 10 µm in a–c. d, Quantification of the
number of BrdU-labelled nuclei in G1–S and G2–S binucleate cells at times
after fusion. The number of cells counted at each time is indicated.
540
the duplication potential of unduplicated G1 centrosomes and
recently duplicated G2 centrosomes when placed in S-phase cytoplasm, a cellular environment that supports duplication. If duplication
was limited only by presence of a positive signal, then both G1 and G2
centrosomes introduced into the S-phase cytoplasm would be
expected to duplicate. However, if duplication was limited by a centrosome-intrinsic block to reduplication, then only the G1 centrosomes
would be expected to duplicate. As such a mechanism might be abrogated in transformed cells, which often have centrosome abnormalities, we performed all experiments with two cell types that are expected
to have normal cell cycle control: primary human diploid fibroblasts
(HDFs) isolated from infant foreskin and a human diploid fibroblast
cell line immortalized by expression of human telomerase reverse
transcriptase (hTERT-BJ1)9,10. Both cell types gave similar results in all
experiments.
A G1 population was obtained by serum starvation of cells for
approximately 72 h. A G2 population was obtained by arresting cells in
S phase using a double-thymidine block11 followed by a 7.5-h release.
Approximately 95% of cells were undergoing DNA replication at 3 h
after release from the thymidine block, as determined by 5-bromodeoxyuridine (BrdU) incorporation (Fig. 1a). In contrast, only approximately 8% of cells were undergoing DNA replication at 7.5 h after the
release, indicating that most cells had successfully entered G2 phase.
Flow cytometry analysis of the G1 and G2 cells confirmed that approximately 90% of each of the populations had the expected DNA content
(data not shown). We also determined centrosome number in the synchronized cells. As a mature centrosome contains one pair of centrioles, we used immunofluorescence microscopy analysis of the
centriolar protein centrin12 as an indicator of centrosome number
(Fig. 1b). We found that 91% of cells in the G1 population had a single
centrosome (one pair of centrioles), whereas 93% of the cells in the G2
population had two centrosomes (two pairs of centrioles; Fig. 1a).
Cells were fused using polyethylene glycol and the desired binucleate
cells were identified by microscopy. Successful analysis of cell fusions
between cells at different cell-cycle stages required the ability to distinguish between homophasic (G1–G1, S–S and G2–G2) and heterophasic (G1–S and G2–S) fusions. For this purpose, fluorescent conjugates
of concanavalin A (ConA) — a lectin that binds to cell-surface proteins — were used to label the two cell populations. Immediately
before fusion, one cell population was labelled with ConA–Texas Red
and the other with ConA–Marina Blue. Thus, only heterophasic binucleate cells should contain both Texas Red and Marina Blue. The cellsurface dyes mixed in fused cells (Fig. 2a), but were not transferable
between adjacent unfused cells (Fig. 2b). Centriole number in the
fused cells could easily be discerned by centrin staining (Fig. 2a). Note
that in several micrographs presented, the ConA–Marina Blue fluorescence is obscured by the brightness of the DAPI nuclear stain.
To test whether the fusion process affected the kinetics of centrosome
duplication, cells from two identical G1 populations were fused, allowed
to proceed through the cell cycle for 24 h and then processed for
immunofluorescence microscopy with an anti-centrin antibody. Fusion
of two G1 cells would result in a binucleate cell with two centrosomes;
thus, duplication during the post-fusion incubation would result in four
centrosomes. We found that 42% of binucleate cells had undergone
duplication by 24 h post-fusion. This was similar to the percentage of
untreated G1 cells that underwent duplication 24 h after release (49%)
and to the percentage of treated, but unfused, G1 cells that underwent
duplication 24 h after treatment (47%). Thus, centrosome duplication
occurred with normal kinetics in fused binucleate cells.
Centrosome duplication occurs in S phase, therefore it is important
that the G1–S and G2–S cell fusions remain in S phase for a sufficient
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LETTERS
a
100
Binucleate cells with three centrosomes
Binucleate cells with four centrosomes
90
Percentage of cells
80
70
60
50
40
30
20
10
0
6 h (n = 40)
12 h (n = 60)
6 h (n = 74)
G1–S
12 h (n = 43)
G2–S
Time after fusion
b
100
90
Binucleate cells with three centrosomes
Binucleate cells with four centrosomes
80
Percentage of cells
time after fusion. To test this, we compared the pattern of BrdU incorporation in G1–S and G2–S binucleate cells after fusion (Fig. 2d). In
G1–S cells at 6 h after fusion, approximately 60% of the binucleate cells
had one BrdU-labelled nucleus and 30% had two BrdU-labelled
nuclei. At 12 h after fusion, approximately 20% had one BrdU-labelled
nucleus, and more than 70% had two BrdU-labelled nuclei. In cells
with two BrdU-labelled nuclei, one of the nuclei typically showed a
more intense BrdU labelling (Fig. 2c), presumably because the S-phase
nucleus incorporated BrdU over a longer period than the G1 nucleus.
In G2–S cells at 6 h after fusion, approximately 70% of the binucleate
cells had one BrdU-labelled nucleus, whereas only 7% had two BrdUlabelled nuclei; this distribution was similar at 12 h after fusion
(Fig. 2d). These data indicate that cells in both types of fusion remain
in S phase, that the G2 nucleus does not re-replicate, and importantly,
that the G2 cytoplasm did not inhibit S-phase events, as found by Rao
and Johnson for Hela cells5.
We next compared the behaviour of the unduplicated G1 centrosomes and newly duplicated G2 centrosomes in G1–S and G2–S
fusions. Fusion of a G1 cell to an S-phase cell results in the generation
of a cell with three centrosomes. At 6 h after fusion, approximately
65% of the G1–S-fused cells had three centrosomes and 30% had four
centrosomes (Fig. 3a). In the remainder, centrosome number could
not be clearly determined. At 12 h after fusion, approximately 25% of
the G1–S-fused cells had three centrosomes, whereas 70% had four
centrosomes, indicating that one of the three had duplicated with the
same timing as DNA replication. Fusion of a G2 cell to an S-phase cell
results in a cell with four centrosomes. At 6 h after fusion greater than
90% of the G2–S binucleate cells had four centrosomes (Fig. 3a). In
contrast to the G1–S fusions, no centrosome duplication was observed
at 12 h (Fig. 3a). In several experiments, cells were analysed for up to
24 h without evidence of centrosome duplication. Thus, previously
duplicated G2 centrosomes were not able to reduplicate in the G2–S
fusions, even though DNA replication could proceed, demonstrating
the existence of a block to centrosome reduplication.
To ensure that the G2–S fusions would have sufficient time in
S phase to carry out centrosome duplication if it were possible, the
fusion experiment was repeated under conditions of S-phase arrest.
G2- and S-phase cells were fused then maintained in S phase by thymidine treatment for 24 h, well in excess of the time needed for centrosome duplication in the G1–S samples. After 24 h of S-phase arrest,
82% of the G2–S binucleate cells still had four centrosomes, and 18%
still had fewer than four (n = 27 cells), presumably through fusion of
unsynchronized cells. None of the unfused cells had more than four
centrosomes; therefore, extending the S-phase period could not overcome the block to reduplication.
There are two possible explanations for the inability of previously
duplicated G2 centrosomes to reduplicate in the G2–S fusions. First,
centrosomes contain information about their duplication status that
prevents their reduplication; second, the G2 cytoplasm contains a factor that functions dominantly to prevent reduplication, regardless of
the duplication status of the centrosome. To rule out the latter explanation, we fused G1 cells to G2 cells and asked if centrosome duplication could occur in the presence of G2 cytoplasm. In these
experiments, the G1 cells were released from serum starvation block
for 5 h before fusion. Thus, the fused cells were advanced in their cell
cycle relative to the cells above and were assayed at earlier times. This
difference, however, had no effect on the experimental outcome.
Fusion of a G1 cell to a G2 cell results in a cell with three centrosomes.
At 2 h after fusion, 70% of the G1–G2-fused cells had three centrosomes (Fig. 3b). However, at 7 h after fusion, only 18% of the G1–G2fused cells had three centrosomes, whereas 80% had four centrosomes.
70
60
50
40
30
20
10
0
2 h (n = 38)
7 h (n = 142)
18 h (n = 144)
G1–G2
7 h (n = 34)
G2–G2 (PurA)
Time after fusion
Figure 3 Centrosome duplication in G1–S, G2–S and G1–G2 cell fusions.
Cell fusions were prepared as in Fig. 2. Quantification of the number of
centrioles in (a) G1–S- and G2–S-fused binucleate cells and (b) G1–G2fused binucleate cells at various times after fusion treatment. The Cdk
inhibitor Purvalanol A (PurA) blocks centrosome duplication in fused cells.
Even at 18 h after fusion, the number of G1–G2 binucleate cells with
four centrosomes remained approximately 85% (Fig. 3b). Thus, G2
cytoplasm does not prevent centrosome duplication in fused cells.
Next, we used the Cdk inhibitors Purvalanol A and Roscovitine to
determine whether the centrosome duplication that we observed in
G1–G2 fusions occurs by the normal Cdk2-mediated pathway13–16.
Purvalanol A17 was added to G1–G2-fused cells at 1 h after fusion. At
7 h after fusion, only 30% of the purvalanol A-treated G1–G2 binucleate cells had four centrosomes, a 2.5-fold reduction from the untreated
population (Fig. 3b). Similar results were obtained with another Cdk
inhibitor, Roscovitine18 (data not shown). These results indicate that
centrosome duplication in the binucleate cells occurs through the normal Cdk-dependent pathway.
We have shown that previously duplicated G2 centrosomes cannot
reduplicate in cell fusions with either G1- or S-phase cells and that the
block to reduplication does not occur at the level of the G2 cytoplasm.
This suggests that centrosome duplication is limited to one round per cell
cycle by a centrosome-intrinsic mechanism, just as DNA replication is
limited by a nuclear-intrinsic mechanism19. We envision two models to
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LETTERS
a
b
c
70
10 h (n = 108)
36 h (n = 122)
Percentage of cells
60
50
40
30
20
10
0
1
2
4
Other
Number of centrosomes
Figure 4 Centrosome duplication in G1–G1 cytoplast–cell fusions.
(a) Differential interference contrast (DIC; left) and fluorescence (right)
images of a cytoplast adjacent to a normal cell. The cytoplast is labelled
with ConA–Texas Red and contains one centrosome (one pair of centrioles;
inset), but no nucleus. The cell is labelled with ConA–Marina Blue and
contains one centrosome (one pair of centrioles; inset) and one nucleus.
(b) A G1–G1 cytoplast–cell fusion containing two centrosomes (two pairs of
centrioles; inset) at 10 h after fusion (left). A G1–G1 cytoplast–cell fusion
containing four centrosomes (four pairs of centrioles; inset) at 36 h after
fusion (right). Scale bar represents 10 µm. c, Quantification of centriole
numbers in G1–G1 cytoplast–cell fusions at 10 h and 36 h after fusion.
account for the block in reduplication. The first is that duplication
results in a physical alteration of the centrosome such that it cannot be
reduplicated until it passes through mitosis, similar to the licensing
model for DNA replication control19. The second model is that there is a
counting mechanism by which cells assess the ratio of centrosomes to
nuclei and only duplicate the centrosome to the level of two centrosomes
per nucleus. In principle, these two models should be distinguishable by
generating mononucleate G1 cells with two G1 centrosomes. The physical alteration model predicts that both of these centrosomes would
duplicate, yielding a total of four centrosomes, whereas the counting
model predicts that neither of the centrosomes would duplicate. To test
these models, we created mononucleate G1 cells with two G1 centrosomes by fusing normal G1 cells to cytoplasts (enucleated cells) obtained
from G1 cells. The cytoplasts were prepared by centrifugation of G1
fibroblasts through a Ficoll gradient; approximately half of the cytoplasts
retained the single G1 centrosome (Fig. 4a). The cells and cytoplasts were
surface-labelled, as described above, to allow identification of the appropriate fusions. The G1 cytoplast–cell fusions were released from G1
arrest, allowed to progress to S phase and maintained in S phase with
thymidine treatment so that centrosome number could be determined.
Both unfused cells and cytoplast–cell fusions took approximately 24 h to
542
enter S phase from G1 arrest, hence the cytoplast–cell fusions were
assayed at 10 h (pre-S phase) and 36 h (S phase) after fusion.
At 10 h after fusion, 46% of the cytoplast–cell fusions had one centrosome, as expected from the fusion of a G1 cell and a cytoplast lacking a centrosome; 44% of the cytoplast-cell fusions had two
centrosomes, as expected from fusion of a G1 cell and a cytoplast containing a centrosome (Fig. 4b, c). In addition, 10% of the cytoplast–cell
fusions contained more than two centrosomes, as expected from
fusion between G1 cells and multiple cytoplasts; note that only 2% of
fusions had four centrosomes. At 36 h after fusion, 8% of the cytoplast–cell fusions contained one centrosome, whereas 56% contained
two centrosomes and 25% contained four centrosomes (Fig. 4b, c).
The cytoplast–cell fusions that contained two centrosomes at 36 h
most probably resulted from centrosome duplication in the cells that
contained one centrosome before S phase. Similarly, the cytoplast–cell
fusions that contained four centrosomes most probably resulted from
centrosome duplication in the cells that contained two centrosomes
before S phase. Thus, both centrosomes in a G1 cell with two centrosomes are able to duplicate, ruling out a centrosome/nucleus counting
mechanism for controlling centrosome number.
We have shown that there is a centrosome-intrinsic block to reduplication in somatic mammalian cells. We wondered how these results
can be reconciled with the observation that embryos and certain
somatic cells can undergo multiple rounds of centrosome duplication
during prolonged S-phase arrest21–23. It is possible that embryos lack a
block to centrosome reduplication and rely on having a limited window of opportunity for duplication. For example, the mitotic state
inhibits centrosome duplication in both sea urchin and frog
embryos14,24, so the alternation of S phase and M phase in the embryonic cell cycle would limit duplication to S phase, which might be long
enough to only allow a single round of duplication. In somatic cells,
centrosome reduplication under conditions of S-phase arrest is only
observed after prolonged incubation, equivalent in length to several
generation times21. It is possible that under these conditions, the block
to reduplication is overcome. Interestingly, centrosome number does
not increase exponentially in S-phase arrest reduplication21, consistent
with the idea that each centrosome has an intrinsic block that has a stochastic chance of being overcome.
If the delay in centrosome reduplication during S-phase arrest was
caused by a block to reduplication, then abrogation of that block
might result in more rapid reduplication. Many types of cancer cells
have been reported to have aberrant centrosome number3,4 and the
tumour suppressor protein p53 has been implicated in the regulation
of centrosome duplication25. Therefore, we compared the kinetics of
centrosome reduplication in primary HDF cells, HCT116 p53+/+
colon cancer cells and HCT116 p53−/− cells generated by somatic
knockout of the p53 gene26 (Fig. 5). Each cell type was subjected to Sphase arrest by hydroxyurea treatment and assayed for centrosome
number at various times during the arrest; any cell with more than
two centrosomes was counted as aberrant. The anti-centrin antibody
had high background labelling in HCT116 cells; therefore, we used
antibodies against γ-tubulin and pericentrin (components of the
pericentriolar material) to label centrosomes (Fig. 5a). Note that for
these antibodies, a single dot of labelling corresponds to one centrosome. Before arrest, the normal cells and cancer cells already differed
substantially in centrosome number, with no HDF cells and approximately 8% of cancer cells having an aberrant centrosome number
(Fig. 5b). This difference increased markedly by day 4 of the S-phase
arrest, with approximately 4% of HDF cells, 24% of the HCT p53+/+
cells and 50% of HCT p53−/− cells having an aberrant centrosome
number (Fig. 5b).
NATURE CELL BIOLOGY VOLUME 5 | NUMBER 6 | JUNE 2003
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LETTERS
Alto, CA). Both cell types were cultured in DMEM (Invitrogen, Carlsbad, CA)
containing 10% foetal bovine serum. G1 cells were obtained by culturing cells
in DMEM with 0.1% newborn calf serum for approximately three days. G2 cells
were obtained by releasing cells arrested in S phase for 7.5 h. Cells were arrested
in S phase using a double-thymidine block, as previously described11. For the
fusion experiments, 1 × 106 cells of each cell population to be fused were
trypsinized, resuspended in 600 µl of ConA–Texas Red or ConA–Marina Blue
(both at 16.7 µg ml−1) in PBS and incubated at 37 °C for 10 min. Cells were
then washed separately with 10 ml of growth media before being resuspended
and mixed in 600 µl of 50 µM SDS in PBS for 3 min at 37 °C. Next, cells were
centrifuged and resuspended in 600 µl of 50% polyethylene glycol-3350 for 1
min at 37 °C. Serum-free DMEM (1 ml) was added to the cells, mixed gently
and incubated at room temperature for 1 min. This process was repeated until a
total of 5 ml serum-free DMEM had been added. The cells were then washed
once with 10 ml serum-free media before being resuspended in 20 ml of growth
media and seeded onto coverslips. Cytoplasts were prepared by density gradient
centrifugation, as previously described20, except that cytochalasin B (10 µg ml−
1) was added to the cells 20 min before centrifugation. Cytoplasts generated
from 1 × 107 G1 cells were fused with 1 × 107 G1 cells, as described above.
p53+/+ and p53−/− HCT116 cells (a gift of J. Ford, Stanford University, CA) were
cultured in McCoy’s 5A Media (Invitrogen) with 10% foetal bovine serum.
Hydroxyurea (2 mM) was used for the prolonged S-phase arrest of HCT cells.
a
DAPI
γ-tubulin
Pericentrin
Day 3
Day 5
b
Percentage of cells
with two or more centrosomes
70
60
p53+/+ HDF
p53+/+ HCT116
p53–/– HCT116
50
40
30
20
10
0
Day 0
Day 3
Day 4
Figure 5 Centrosome reduplication during S-phase arrest. (a) HCT116 p53−
cells at day 3 and day 5 of hydroxyurea arrest stained for DAPI, γ-tubulin
and pericentrin to identify centrosome number. Note that these antibodies
stain the pericentriolar material rather than the centrioles, so that a single
dot of staining corresponds to one centrosome. Scale bar represents 10 µm.
(b) Percentage of HDF, HCT116 p53+/+ and HCT116 p53−/− cells with an
aberrant number of centrosomes (greater than two) before S-phase arrest
(day 0) and after three and four days of S-phase arrest.
/−
These results are consistent with the possibility that cancer cells in
general, and p53−/− cancer cells in particular, have defects in the block to
centrosome reduplication. In addition, they are consistent with previous results showing that loss of p53 results in deregulation of centrosome duplication27. However, we note that even in p53−/− cancer cells,
there is a delay in gross reduplication of the centrosomes until four days
of arrest, suggesting that regulation of centrosome number probably
occurs through several overlapping mechanisms and that the effect of
p53 might be indirect. It is also probable that failure of the block to centrosome reduplication is not the only centrosome amplification mechanism; another mechanism is the failure to undergo cytokinesis after
mitosis, resulting in a tetraploid cell with two centrosomes at G1 (refs
28, 29). The control of centrosome duplication is probably critical to
the maintenance of genome integrity and it will be of great interest to
determine the molecular basis for the block to centrosome reduplication that we have identified.
METHODS
Tissue culture, cell fusion and cytoplast preparation. HDFs were harvested
from infant foreskin and hTERT-BJ1 cells were purchased from Clontech (Palo
Cytochemistry. Cells were fixed with methanol at −20 °C then blocked with 3%
bovine serum albumin (w/v) and 0.1% Triton X-100 in PBS for 30 min. For
visualization of centrin, cells were incubated with a monoclonal mouse anticentrin antibody (20H5; a gift from J. Salisbury, Mayo Clinic, Rochester, MN
(ref. 30)), followed by Alexa 488-conjugated goat anti-mouse antibody
(Molecular Probes, Eugene, OR). DNA was visualized using 4′,6-diamidino-2phenylindole (DAPI; Molecular Probes). Cells were then observed under a Zeiss
Axioskop microscope (Zeiss, Thornwood, NJ) using a 100× oil-immersion
objective. For visualizing replicated DNA, BrdU was added to the cells to a final
concentration of 20 µM and incubated for 30 min at 37 °C. Cells were treated
with 2 M HCl for 30 min at room temperature or with 1 U ml−1 DNase I for 1 h
at 37 °C before treatment with 1000 U ml−1 exonuclease III for 1 h at 37 °C.
Staining was then performed as described above.
ACKNOWLEDGEMENTS
We thank J. Ford for cell lines, J. Salisbury for the anti-centrin antibody, and G.-W.
Fang and P. Jackson for comments on the manuscript. This work was supported by
a grant from the Human Frontier Science Program. C.W. was supported by a
Stanford Graduate Fellowship.
COMPETING FINANCIAL INTERESTS
The authors declare that they have no competing financial interests.
Received 3 January 2003; Accepted 25 March 2003;
Published online: 27 May 2003; DOI: 10.1038/ncb993
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NATURE CELL BIOLOGY VOLUME 5 | NUMBER 6 | JUNE 2003
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