The interrelationship between APC/C and Plk1
activities in centriole disengagement
Toshiyuki Hatano and Greenfield Sluder*
Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01605, USA
*Author for correspondence (firstname.lastname@example.org)
Biology Open 1, 1153–1160
Received 23rd July 2012
Accepted 6th August 2012
Mother–daughter centriole disengagement, the necessary first
step in centriole duplication, involves Plk1 activity in early
mitosis and separase activity after APC/C activity mediates
securindegradation.Plk1 activityisthought to be essential and
sufficient for centriole disengagement with separase activity
playing a supporting but non-essential role. In separase null
cells, however, centriole disengagement is substantially
delayed. The ability of APC/C activity alone to mediate
centriole disengagement has not been directly tested. We
investigate the interrelationship between Plk1 and APC/C
activities in disengaging centrioles in S or G2 HeLa and RPE1
cells,celltypesthat donotreduplicate centrioles whenarrested
in S phase. Knockdown of the interphase APC/C inhibitor
Emi1 leads to centriole disengagement and reduplication of the
mother centrioles, though this is slow. Strong inhibition of
Plk1 activity, if any, during S does not block centriole
disengagement and mother centriole reduplication in Emi1
depleted cells. Centriole disengagement depends on APC/C–
Cdh1 activity, not APC/C–Cdc20 activity. Also, Plk1andAPC/
C–Cdh1 activities can independently promote centriole
disengagement in G2 arrested cells. Thus, Plk1 and APC/C–
Cdh1 activities are independent but slow pathways for
centriole disengagement. By having two slow mechanisms for
disengagement working together, the cell ensures that
centrioles will not prematurely separate in late G2 or early
mitosis, thereby risking multipolar spindle assembly, but
rather disengage in a timely fashion only late in mitosis.
? 2012. Published by The Company of Biologists Ltd. This is
an Open Access article distributed under the terms of the
Creative Commons Attribution Non-Commercial Share Alike
Key words: APC/C, Centriole, Disengagement, Plk1
The centrosome is the primary microtubule organizing center
(MTOC) of the interphase cell and in mitosis the centrosomes act
in a dominant fashion to control spindle polarity. Since centriole
pairs collect the pericentriolar material that forms the MTOC, the
duplication of the centrosome as a whole is determined by the
duplication and separation of centriole pairs (Sluder and Rieder,
Centriole duplication starts with the functional separation, or
disengagement, of mother from daughter centrioles. This was
first reported as the disorientation of the orthogonal arrangement
of mother–daughter centrioles during late G1 (Kuriyama and
Borisy, 1981). Later live cell studies of HeLa cells revealed that
unduplicated mother and daughter centrioles inherited from
mitosis are often spatially separate and show independent
movements early in G1 (Piel et al., 2000). In a pathfinding
study Tsou and Stearns reported that the disengagement of
mother–daughter centrioles occurs in anaphase, and is dependent
upon the proteolytic activity of separase which also cleaves the
centromeric cohesin complexes that hold sister chromatids
together in metaphase (Tsou and Stearns, 2006). Reports of
cohesins at centrosomes (Wong et al., 2006) and functional
evidence that their cleavage leads to centriole disengagement
(Scho ¨ckel et al., 2011) reinforce the notion of a common
mechanism for centriole and chromosome disengagement.
Separase becomes active when its inhibitor securin is targeted
for degradation by anaphase-promoting complex/cyclosome
(APC/C) activityatthe metaphase–anaphase
Centriole disengagement is a necessary prerequisite for later
centriole duplication when the daughter cells enter S phase (Tsou
and Stearns, 2006). This model received strong support from the
finding that laser ablation of daughter centrioles in S phase cells
allows mother centrioles to assemble new daughter centrioles
(Loncarek et al., 2008). Thus, S phase is constitutively
permissive for centriole duplication and a later study showed
that prolonged G2 also is permissive for repeated centriole
reduplication (Lonc ˇarek et al., 2010). Thus, the centrosome
intrinsic block to centrosome duplication that prevents centriole
re-duplication during S and G2 (Wong and Stearns, 2003) is
based in the engagement of daughter centrioles to their mothers
thereby preventing them from duplicating again.
Separase is not the only player in centriole disengagement;
Plk1 activity also promotes disengagement. Centriole duplication
occurs in separase null HCT116 cells and is dependent upon Plk1
activity during late G2 or early mitosis. However, without
separase activity centriole disengagement is delayed into late G1
or S phases (Tsou et al., 2009). Without Plk1 activity, separase
null cells driven out of mitosis did not show centriole
disengagement or duplication by the time they entered S phase.
Even though separase activity is not absolutely required for
centriole disengagement, a number of observations have led
to the belief that Plk1 activity is necessary for centriole
disengagement and duplication (Wang et al., 2011; reviewed by
Mardin and Schiebel, 2012). For example, siRNA depletion of
Plk1 prevented centriole reduplication in S phase arrested U2OS
cells (Liu and Erikson, 2002). Additionally, the disengagement of
isolated mammalian centrioles in Xenopus egg extracts depends
upon ongoing Plk1 mediated phosphorylation of centriolar
cohesin subunits allowing them to be cleaved by separase
(Scho ¨ckelet al., 2011). Centriole
reduplication during G2 arrest is also dependent upon Plk1
activity (Lonc ˇarek et al., 2010).
Whether or not APC/C activity alone, without Plk1 activity,
can mediate centriole disengagement in live cells is uncertain and
has not been directly tested. The possibility that APC/C activity
alone can disengage centrioles is suggested by the report that
knockdown of Evi5, which stabilizes the APC/C inhibitor Emi1
in interphase, leads to an incidence of extra centrosomes/spindle
poles in mitotic human cells (Eldridge et al., 2006). However, the
basis for this was not clear and was interpreted to possibly result
from spindle abnormalities and consequent mitotic defects. On
the other hand, after siRNA depletion of Emi1 in cycling HeLa
cells, only 10% showed more than two centrosomes as seen by
gamma tubulin foci (Lonc ˇarek et al., 2010). This was interpreted
to indicate that APC/C activity alone is not sufficient to
We have further investigated the interrelationship between
APC/Cand Plk1 activities
disengagement in live cells. In particular, we were interested in
testing whether if Plk1 activity and APC/C activity represent two
pathways that can independently cause centriole disjoining or
alternatively if Plk1 activity is required with APC/C activity
playing a supporting but not essential role, as currently thought.
To avoid investigating centriole disengagement against the
complicated regulatory landscape of cells going through
mitosis, we used S phase arrested HeLa and RPE1 cells, which
normally do not disjoin or reduplicate centrioles during
prolonged S phase. This phase of the cell cycle is constitutively
permissive for procentriole assembly (Loncarek et al., 2008).
inthe controlof centriole
We used HeLa and RPE1 cells stably expressing low levels of
GFP-centrin 1 to tag the centrioles. Centriole duplication is
normal in these cells (Piel et al., 2000; LaTerra et al., 2005).
When arrested in S phase with thymidine or aphidicolin, our
HeLa cells exhibit a less than 2.5% incidence of extra centrioles
after 72 hours.
Emi1 depletion leads to centriole disengagement and
reduplication in S phase
We first determined if APC/C activity can disengage centrioles
during S phase. Asynchronous cultures were treated with
thymidine to arrest them in S phase, and 16 hours later the
interphase APC/C inhibitor Emi1 was knocked down using
siRNA constructs previously shown to be effective (Di Fiore and
Pines, 2007). In 3 experiments, transfection with Emi1_1 siRNA
resulted in a mean 58% reduction of Emi1 protein levels and a
mean 77% reduction in securin protein levels when assayed
48 hours after transfection in whole populations of transfected
plus untransfected cells (supplementary material Fig. S1A).
Functional efficacy of our Emi1 knockdowns was confirmed by
evidence of DNA re-replication in asynchronous cultures as
previously reported (Di Fiore and Pines, 2007; Machida and
Dutta, 2007; Lonc ˇarek et al., 2010). This was seen at 72 hours after
transfection by increases in nuclear size (supplementary material
Fig. S1E) and .60 clearly separate CREST positive nuclear spots in
the enlarged nuclei (not shown).
To assay for centriole disengagement/reduplication we fixed
cultures 48 and 72 hours after transfection and immunostained
for a number of recognized centriolar proteins and modifications
to centriolar microtubules. Since daughter centriole assembly
requires disengagement, the spatial separation of centrin foci
associated with a number of centriolar proteins and the assembly
of supernumerary centrioles are evidence of mother daughter
centriole disengagement in S phase. We determined the incidence
of cells containing elevated numbers of centriolar protein
immunoreactive spots associated with bright GFP-centrin foci
relative to S phase arrested cultures transfected with control
siRNA constructs. As shown in Fig. 1A–D we found a marked
increase in the incidence of cells with elevated numbers of
glutamylated tubulin (a characteristic modification of centriolar
microtubules), and pericentriolar gamma tubulin foci in Emi1
knockdown cells relative to control cells. Furthermore, we found
a similar increase in the incidence of cells with elevated numbers
of CP110 (cap protein on both mother and daughter centrioles)
foci and a lower, but still elevated, incidence of cells with
elevated numbers of acetylated tubulin foci (modification of
centriolar microtubules) and SAS-6 (cartwheel protein found in
daughter centrioles) foci after Emi1 knockdown. The incidence
of supernumerary acetylated tubulin foci may be limited by the
time-dependent nature of tubulin acetylation and the incidence of
cells with elevated numbers of SAS-6 foci may be limited by this
protein being targeted, perhaps slowly, for degradation by APC/C
activity (Strnad et al., 2007). In addition we immunostained Emi1
knockdown cells for C-Nap1 and found an increase in the
incidence of cells with supernumerary immunoreactive spots
colocalized with centrin foci relative to cells transfected with
control siRNA. More than two C-Nap1 spots in S phase cells is
indicative of centriole disengagement. Lastly, we immunostained
Emi1 knockdown cells for Cep170, a marker for the oldest
mature mother centriole (Guarguaglini et al., 2005). Almost all
cells showed one Cep170 patch associated with a pair of centrin
foci containing one larger and one smaller centrin spots in both
control and Emi1 knockdown cells (Fig. 1D). The ranges in
number of centrin spots associated with the various centriolar
proteins for control and Emi1 knockdown cells are shown in
Similar results were obtained when Emi1 was knocked down
with an independent siRNA construct (Emi1_2) (Di Fiore and
Pines, 2007) (supplementary material Fig. S1B) though the
incidence of elevated numbers of centrioles was less than that
observed in cells transfected with the Emi1_1 construct perhaps
due to a lower reduction in securin levels (supplementary material
The colocalization of a number of centriole specific proteins or
tubulin modifications with the extra centrin spots and the
spatial separationof thecentrin/centriolar
(supplementary material Fig. S1F) indicates that knockdown of
Emi1 and consequent activation of the APC/C during interphase
leads to disengagement of mother from daughter centrioles and
the repeated assembly of daughter centrioles that then disengage
from their mothers. That extra centrioles appeared to be
daughters is consistent with the report that, with the exception
APC/C and Plk1 in centriole disengagement 1154
Fig. 1. Emi1 knockdown in S phase leads to centriole
disengagement and mother centriole reduplication.
(A) Diagram of the experimental protocol. (B) Incidence
of cells with .2 Cep135 or .2 poly-glutamylated tubulin
spots associated with centrin foci at 48 hours (blue bars)
and 72 hours (brown bars). Representative images of the
Cep135, poly-glutamylated tubulin, and GFP-centrin
signals are shown for each condition. (C) Incidence of
cells with .4 CP110, .2 SAS-6 or .2 acetylated tubulin
spots associated with centrin foci at 48 hours (blue bars)
and 72 hours (brown bars). Representative images are
shown below the histograms. (D) Incidence of cells with,
.2 gamma tubulin, .2 C-Nap1 spots, or .1 Cep170
patches associated with centrin foci at 48 hours (blue
bars). (E) Emi1 knockdown without prior S phase arrest
leads to centriole disengagement and mother centriole
reduplication. Mitotic cells were collected by shake off
were transfected Emi1_1 or control siRNA constructs
1.5 hours later according to the protocol of Lonc ˇarek
et al. (Lonc ˇarek et al., 2010). The experimental protocol
is outlined at the top of this portion of the figure.
Histograms show the incidence of cells with .2 spots of
the indicated proteins associated with centrin foci.
Representative images of the indicated protein signals are
shown below for each condition. Images are maximum
intensity point projections from Z series images. Scale
bars 5 1 mm. All histogram bars indicate the average
from 3 experiments with .200 cells counted for each
condition. Error bars are one standard deviation.
APC/C and Plk1 in centriole disengagement 1155
of certain cell types such as CHO and U2OS, daughter centrioles
when separate from their mothers do not mature and duplicate
again when cells are in S phase (Lonc ˇarek et al., 2010).
Lonc ˇarek et al. reported that for cycling HeLa cells arrested in
S phase by Emi1 knockdown (Di Fiore and Pines, 2007; Machida
and Dutta, 2007), 90% of the cells contained only two
centrosomes (with two centrioles apiece) as detected by gamma
tubulin staining (Lonc ˇarek et al., 2010). The remaining 10%
showed elevated centrosome numbers. This was interpreted to
show that Emi1 knockdown does not lead to centriole
disengagement or reduplication. To explore the basis for the
difference between our results and theirs, mitotic cells were
collected by shake off and transfected Emi1_1 or control siRNA
constructs 1.5 hours later according to the protocol of Lonc ˇarek
et al. (Lonc ˇarek et al., 2010). Cultures were fixed 72 hours after
transfection and immunostained for SAS-6, Cep135, or gamma
tubulin. We counted centrioles in cells with markedly enlarged
nuclei (indicative of arrest in S phase). We found that 25% of the
cells showed more than 2 gamma tubulin foci associated with
centrin spots, 38% contained .2 Cep135 spots, and 22% had .2
SAS-6 spots (Fig. 1E). These results reveal that our findings with
S phase arrested cells were not an artifact of the means used to
arrest the cells in S or their being in S before Emi1 was knocked
down. We propose that the difference in results of the two groups
is quantitative rather than qualitative.
To test if our results were specific to HeLa cells, we knocked
down Emi1 in S phase arrested RPE1 cells – immortalized
untransformed human cells. We observed a marked increase in
the incidence of elevated centriole number relative to cells
transfected with the control siRNA construct though the
incidence of extra centrioles in the population was lower than
that found in HeLa cells (supplementary material Fig. S2A–E).
The reason why RPE1 cells show a lower incidence of centriole
disengagement/reduplication is not fully known but may reflect
the less efficient knockdown of Emi1 in this cell type (Di Fiore
and Pines, 2007).
APC/C–Cdh1, not APC/C–cdc20, activity disengages centrioles
To determine whether APC/C–Cdh1 and/or Cdc20 were
responsible for centriole disengagement, we co-depleted Emi1
and either Cdh1 or Cdc20 in S phase cells (Fig. 2A–C). 48 hours
after transfection cell populations were fixed and immunostained
for Cep135, Cp110, or SAS-6. Co-depletion of Emi1 plus Cdh1
greatly diminished the incidence of centriole disengagement and
mother centriole reduplication relative to knockdown of Emi1
alone (Fig. 2A–C). Co-depletion of Emi1 plus Cdc20 allowed
disengagement and mother centriole reduplication at levels only
slightly less than knockdown of Emi1 alone. These observations
reveal that primarily APC/C–Cdh1 rather than APC/C–Cdc20
activity promotes centriole disengagement during S phase arrest.
Plk1 activity is not required for APC/C–Cdh1 mediated centriole
Plk1 activity during S phase is low, but perhaps not zero
(Golsteyn et al., 1995). Thus, we tested if strong inhibition of
Fig. 2. After Emi1 knockdown in S phase, APC/C–Cdh1, not APC/C–
cdc20, activity mediates centriole disengagement. The experimental
conditions are indicated in the protocol diagram at the top of the figure.
(A) Incidence of cells with .2 Cep135 spots associated with centrin foci after
transfection with the indicated constructs. Representative images of the Cep135
and GFP-centrin signals are shown for each experimental condition.
(B) Incidence of cells with .4 CP110 spots associated with centrin foci after
transfection with the indicated constructs. (C) Incidence of cells with .2 SAS-
6 spots associated with centrin foci after transfection with the indicated
constructs. Representative images for each condition are shown. Images are
maximum intensity point projections from Z series images. Scale bars 5 1 mm.
In panels A and B histogram bars indicate the average from 2 experiments with
.200 cells counted for each condition. In panel C histogram bars indicate the
average from 3 experiments. Error bars are one standard deviation.
Table 1. Range for the number of immunoreactive spots per HeLa cell for the indicated centriolar proteins associated with
centrin foci under the experimental conditions listed on the left.
tubuling-tubulin Cep170 SAS-6 CP110
Control siRNA + BI200nM
siEmi1 + BI200nM
APC/C and Plk1 in centriole disengagement 1156
disengagement during S. We knocked down Emi1 in S phase
cells and simultaneously treated with 200 nM BI2536 which
should block Plk1 activity (IC50: 0.83 nM) and largely block the
activities of Plk2 and Plk3 (IC50s: 3.5 nM and 9.0 nM
respectively) (Le ´na ´rt et al., 2007). Functional efficacy of this
drug at this concentration is supported by observations that
100 nM BI2536 completely blocked centriole reduplication in G2
arrested cells (Lonc ˇarek et al., 2010) and 200 nM blocked
centriole disengagement and duplication in separase null cells
driven out of mitosis (Tsou et al., 2009).
Forty-eight hoursafter transfection and Plkinhibitorapplication
the incidence of cells with supernumerary centrin spots associated
with Cep135, CP110, gamma tubulin, and SAS-6 was equivalent
to their incidence with Emi1 knockdown alone (Fig. 3A–D). The
ranges in number of centrin spots associated with the various
centriolar proteins for Emi1 knockdown cells with and without
BI2536 application are shown in Table 1. Similar results were
obtained for RPE1 cells (supplementary material Fig. S2E) though
the incidence of cells with extra centrioles was lower than that
observed for HeLa cells. These results indicate that centriole
disengagement and mother centriole reduplication are not
dependent upon Plk1 activity when Emi1 is knocked down in S
phase.Interestingly,inhibition ofPlk1 activitydid notdiminishthe
incidence of cells with .2 gamma tubulin clouds associated with
centrin spots relative to that found when Emi1 alone was knocked
down (Fig. 3C). This indicates that acquisition of gamma tubulin
around daughter centrioles is not entirely dependent upon Plk1
activity, contrary to what was expected based on a previous report
(Wang et al., 2011).
activity affected APC/C–Cdh1 mediatedcentriole
APC/C–Cdh1 and Plk1 activities can independently disengage
centrioles during G2 arrest
Since significant Plk1 activity is not expected in S phase and
normally rises in G2, we used HeLa cells arrested in G2 with the
Cdk1 inhibitor RO-3306. By knocking down Cdh1 or inhibiting
Plk1 activity during G2 we wanted to test whether Plk1 and APC/
C activities can independently promote centriole disengagement
in this phase of the cell cycle. Although centriole disengagement/
reduplication are reported to occur in a Plk1 activity dependent
fashion in G2 (Lonc ˇarek et al., 2010), we re-examined this issue
because that Plk1 phosphorylates Emi1 to target it for SCF-Trcp1
mediated destruction (Guardavaccaro et al., 2003; Margottin-
Goguet et al., 2003; Hansen et al., 2004; Moshe et al., 2004).
Thus, inhibition of Plk1 activity starting before cells reach G2
(Lonc ˇarek et al., 2010) should also block Emi1 destruction
thereby inhibiting APC/C activity; the resulting lack of centriole
disengagement in the absence of Plk1 and APC/C activity would
thus be expected (Tsou et al., 2009).
Fig. 3. Plk1 activity is not required for APC/C–Cdh1 mediated centriole
disengagement during S. The experimental conditions are indicated in the
protocol diagram at the top of the figure. (A) Incidence of cells with .2
Cep135 spots associated with centrin foci after transfection with and without
Plk inhibitor. Representative images of the Cep135 and the GFP-centrin signals
are shown for each condition. (B) Incidence of cells with .4 CP110 spots
associated with centrin foci under the same range of conditions. Representative
images are shown below. (C) Incidence of cells with .2 gamma tubulin spots
associated with centrin foci under the conditions indicated along with
representative images. (D) Incidence of cells with .2 SAS-6 spots associated
with centrin foci under the conditions indicated. Representative images are
maximum intensity point projections from Z series of images. Scale bars 5
1 mm. In all panels histogram bars indicate the average from 3 experiments with
.200 cells counted for each condition. Error bars are one standard deviation.
APC/C and Plk1 in centriole disengagement1157
We shook off mitotic cells and 3 hours later continuously
treated them with 5 mM RO-3306 to arrest the population in G2.
Some cultures were transfected with siRNA for Cdh1 an hour and
a half after shake off and others were treated with 200 nM
BI2536 six hours after shake off (Fig. 4A, protocol diagram).
immunostained for SAS-6 and Cep135. For cultures treated
with RO-3306 alone we observed a 55.4% incidence of cells with
.2 SAS-6 spots associated with centrin foci (Fig. 4A, first bar)
and a similar incidence of cells with .2 Cep135 spots (Fig. 4B,
first bar). Inhibition of Plk1 activity with 200 nM BI2536 almost
eliminated the incidence of .2 SAS-6 and .2 Cep135 spots
(Fig. 4A,B, second bars). These observations are in accord with
those of Lonc ˇarek et al. (Lonc ˇarek et al., 2010).
Cultures transfected with siRNA to Cdh1 showed a 54%
incidence of cells with .2 SAS-6 spots associated with centrin
foci (Fig. 4A, third bar) and a similar incidence of cells with .2
Cep135 spots (Fig. 4B, third bar). These observations indicate
that Plk1 activity in the absence of APC/C–Cdh1 activity is
sufficient to disengage centrioles during G2. Knockdown of Cdh1
plus inhibition of Plk1 activity almost completely blocked
centriole disengagement/reduplication (Fig. 4A,B, fourth bars).
To test if APC/C activity alone can disengage centrioles during
G2, we shook of mitotic cells and 1.5 hours later treated them
with RO-3306 plus 200 nM BI2536 to arrest the daughter cells in
G2 and inhibit Plk1 activity. 16 hours after shake off we
transfected to knock down Emi1 and 48 hours later fixed the
cultures. These cultures exhibited a 42% incidence of cells with
supernumerary Cep135 spots associated with centrin foci, a 27%
incidence of extra CP110 spots, and a 46% incidence of extra C-
Nap1 spots (Fig. 4C–E). When we co-knocked down Emi1 and
Cdh1, the incidences of extra Cep135, CP110, and C-Nap1 spots
were greatly reduced (Fig. 4C–E). These observations indicate
that APC/C–Cdh1 activityalone
disengagement during G2.
we fixedthe culturesand
Centriole disengagement must be tightly controlled to prevent it
from occurring during G2 or early mitosis, something that can
lead to spindle multipolarity and genomic instability through
chromosome missegregation. Perhaps more pernicious, transient
spindle multipolarity can lead to one or more merotelically
attached chromosomes that lag in anaphase/telophase (Ganem
et al., 2009). These laggards may become micronuclei which do
not show complete DNA replication in the following cell cycle.
When the daughter cell with a micronucleus enters mitosis,
chromosome condensation leads to chromosome damage (Crasta
et al., 2012).
We investigated the interrelationship between APC/C and Plk1
activities in centriole disengagement, the necessary first step in
Fig. 4. APC/C–Cdh1 and Plk1 activities can independently disengage
centrioles during G2. (A,B) Incidence of cells with .2 SAS-6 or .2 Cep135
spots associated with centrin foci under the conditions indicated in the protocol
diagram at the top of panel A. Representative images of the SAS-6, Cep135 and
the GFP-centrin signals are shown for each condition. (C–E) Incidence of cells
with .2 Cep135, .4 CP110, or .2 C-Nap1 spots associated with centrin foci
under the conditions indicated in the protocol diagram at the top of panel C.
Representative images are shown for each condition. Images are maximum
intensity point projections from Z series of images. Scale bars 5 1 mm.
Histogram bars indicate the average from 3 experiments with .200 cells
counted for each condition. Error bars are one standard deviation.
APC/C and Plk1 in centriole disengagement1158
centriole duplication that normally occurs during late mitosis.
Previous work had shown that Plk1 activity alone can disengage
centrioles during G2 and mitosis (Tsou et al., 2009; Lonc ˇarek
et al., 2010). However, the ability of APC/C activity alone to
disengage centrioles had not been tested. We found that APC/C–
Cdh1, not APC/C–Cdc20, activity alone is sufficient to disengage
centrioles during S phase and G2. In S this was seen by the
assembly of multiple new centrioles as well as their spatial
separation from each other and from the original mother
centrioles. The assembly of multiple daughter centrioles
indicates that there were repeated cycles of disengagement. Our
observations that many of the centrin foci were associated with
SAS-6 and there was only one Cep170 patch associated with a
brighter centrin focus in each cell indicates that the new
centrioles are daughters (Guarguaglini et al., 2005; Strnad et al.,
2007). This is consistent with findings that laser ablation of the
daughter centriole (a definitive form of disengagement) allows
the mother to assemble new daughter centrioles in S phase
arrested cells and that daughter centrioles are not capable of
becoming mothers during S phase (Lonc ˇarek et al., 2010).
Plk1 activity by itself is also sufficient to disengage centrioles
without APC/C activity, as first shown in separase null HCT116
cells driven out of mitosis (Tsou et al., 2009) and later supported
by the finding that centriole reduplication during G2 arrest is
Plk1 dependent (Lonc ˇarek et al., 2010). We confirmed the ability
of Plk1 activity alone to disengage G2 centrioles using our
experimental system. Knockdown of Cdh1, which blocked
disengagement/reduplication of centrioles during S phase, did
not do so when cells were arrested in G2, at a time when Plk1
becomes active. However, when Cdh1 was knocked down and
Plk1 inhibited, centrioles did not disengage.
A number of studies led to the understanding that Plk1 activity
is essential for centriole disengagement whether or not APC/C
activity was present (Liu and Erikson, 2002; Tsou et al., 2009;
Lonc ˇarek et al., 2010; Scho ¨ckel et al., 2011; Wang et al., 2011;
reviewed by Mardin and Schiebel, 2012). It was proposed that
Plk1 activity was necessary to ‘‘prime’’ centriole associated
cohesin subunits for separase mediated cleavage (Tsou et al.,
2009; Scho ¨ckel et al., 2011). Significant levels of Plk1 activity
during S phase are not expected, but nevertheless we found that
strong inhibition of Plk1 (and Plk2 and Plk3) activities, which is
sufficient to block centriole reduplication during G2, did not
inhibit APC/C–Cdh1 mediated centriole disengagement during S
phase. However, we should be clear that our results do not speak
against the importance of Plk1 ‘‘priming’’ of centriolar cohesin
subunits for prompt cleavage in anaphase; rather our findings
reveal that this is not absolutely necessary in living cells if
enough time is allowed.
Thus, Plk1 and APC/C–Cdh1 activities are redundant
pathways each of which can disengage and thereby ‘‘license’’
centrioles for duplication. One can ask why the cell uses two
pathways that work cooperatively to disjoin centrioles and what
advantage that might provide. Plk1 activity during late G2 and
mitosis is slow to disengage centrioles; in separase null cells
driven out of mitosis centrioles do not disengage until sometime
in G1 (Tsou et al., 2009). We found that APC/C activity alone is
also slow in disengaging centrioles (Fig. 5) with the incidence of
cells with disengaged/reduplicated centrioles increasing steadily
until at least 72 hours after siRNA transfection when the
experiments were terminated. Given the obvious problems with
disengagement occurring too early (spindle multipolarity) or too
late (possibly incomplete centriole duplication in the next cell
cycle), the cooperative action of Plk1 and APC/C activities
ensure the timeliness and fidelity of disengagement in the short
time-window of the cell finishing mitosis. Indeed, there may be
an advantage for the cell to use two relatively slow mechanisms
to disengage centrioles. If Plk1 or APC/C activities, working
singly, were efficient in releasing the links between centrioles the
cell could be subject to the risk that when Plk1 activity rises in
G2/early mitosis and Emi1 begins to be degraded as early as
20 minutes before nuclear envelope breakdown (Di Fiore and
Pines, 2007), centrioles could prematurely separate leading to an
incidence of spindle multipolarity. Such a phenomenon can be
dramatically seen when rapidly cycling sea urchin zygotes are
held in prometaphase by a variety of methods. During prolonged
prometaphase, the centriole pairs split leading to spindle
multipolarity before anaphase onset (Sluder and Begg, 1985;
Sluder and Rieder, 1985). Additionally, by having the completion
of disengagement be linked to APC/C–Cdh1 activity and not
sensitive to APC/C–Cdc20 activity, centriole disengagement is
pushed off to late mitosis.
Our results also revealed that many of the extra centrioles were
associated with SAS-6 even 48–72 hours after the start of Emi1
knockdown. Given reports that APC/C–Cdh1 activity targets
SAS-6 for proteasomal degradation in late anaphase (Strnad et al.,
2007; Puklowski et al., 2011), persistence of this protein was at
first glance unexpected. Nevertheless, this cartwheel protein of
daughter centrioles must be stable for some time both in daughter
centrioles and in the cellular subunit pool in the presence of APC/
C–Cdh1 activity, because SAS-6 is required for procentriole
formation (Strnad et al., 2007). We would not have seen
supernumerary daughter centrioles had it been completely
degraded. Persistence of SAS-6 after Emi1 knockdown may be
due to APC/C–Cdh1 mediated degradation of the F-box protein
FBXW5, part of an SCF complex that targets SAS-6 for
proteasomal degradation (Puklowski et al., 2011). Perhaps
APC/C–Cdh1 activity alone targets SAS-6 for degradation but
does so slowly. This is consistent with our finding that the
incidence of extra SAS-6 foci was consistently lower than the
incidence of extra Cep135 and CP110 foci.
We also found that daughter centrioles are able to acquire
small gamma tubulin clouds around them during S even when
Plk1 activity, if any, is strongly suppressed by BI2536. This
indicates that acquisition of gamma tubulin by daughter centrioles is
Fig. 5. Incidence of HeLa cells with .2 Cep135 spots associated with
centrin foci as a function of time after siRNA transfection to knock down
Emi1 during S phase. The experimental protocol is outlined in Fig. 1A with
the exception that fixation times ranged from 24 to 72 hours after transfection.
Each data-point is the mean of three independent experiments (.200 cells
counted for each time-point of each experiment) and the error bars are one
APC/C and Plk1 in centriole disengagement1159
not entirely dependent upon Plk1 activity as previously proposed
(Wang et al., 2011). That the number of gamma tubulin foci
associated with centrin spots was less than the extra Cep135 and
CP110 foci suggests, however, that gamma tubulin acquisition may
be relatively slow in the absence of Plk1 activity. Also, the fact that
daughter centrioles do not duplicate during S despite presence of
gamma tubulin on some of them suggests that reproductive
maturation does not simply result from the acquisition of
pericentriolar gamma tubulin but rather depends on some other
Plk1 dependent event (Lonc ˇarek et al., 2010; Wang et al., 2011).
Materials and Methods
Cell culture and drug treatment
HeLacells expressinglow levelsofGFP-Centrin1 (La Terraetal.,2005)were culturedin
DME medium supplemented with 10% FBS and 1% Penicillin–Streptomycin cocktail
(Life Technologies, Grand Island, NY). RPE-1 cells expressing GFP-Centrin1 were
cultured in F12/DME (1:1) medium supplemented with 10% FBS and 1% Penicillin–
Streptomycin cocktail (Life Technologies, Grand Island, NY). Cells were arrested in S
phase with 1 mM thymidine, or in G2 with 5 nM RO3306 (Calbiochem, La Jolla, CA).
for cell collection, siRNA transfection, drug treatments, and fixation times are shown
diagrammatically at the top of each figure and described in the text and figure legends.
The siRNA constructs used were: siEmi1_1, sequence GATTGTGATCTCTTATT AA
(Di Fiore and Pines, 2007); siEmi1_2, sequence ACTTGCTGCCAGTTCTTCA (Di
Fiore and Pines, 2007); siCdh1 (Brummelkamp et al., 2002); and siCdc20 (Wolthuis
et al., 2008). All were synthesized by Dharmacon (Lafayette, CO). Control siRNA
constructs used: siluciferase (GL2, Dharmacon Lafayette, CO) and FITC conjugated
scrambled oligonucleotides (Santa Cruz Biotechnology Inc., Santa Cruz, CA). siRNA
constructs were transfected at a final concentration of 10 nM according to the
manufacturer’s instructions using Oligofectamine (Life Technologies, Grand Island,
NY) for HeLa cells and RNAi MAX (Life Technologies, Grand Island, NY) for RPE-1
cells. In all experiments the transfecting agent was removed by changing to fresh
medium 16 hours after initial application.
Cells were fixed in ice-cold methanol for .5 minutes. The primary antibodies used
were: SAS-6 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at 1:100 dilution;
Cep170 (Life Technologies, Grand Island, NY) at 1:500; Cep135 (Abcam, Cambridge,
MA) at 1:500; CP110 (gift from Harold Fisk ) at 1:100; gamma tubulin (Santa Cruz
CA) at 1:200; poly-glutamylated tubulin (GT335 clone, ENZO Life Science,
Farmingdale, NY) at 1:2000; CREST serum (ImmunoVision, Springdale, AR) at
1:500. Secondary antibodies conjugated to AlexaFluor 568 or 647 (Life Technologies,
Grand Island, NY) were used at 1:1000 dilutions.
Cell preparations were observed with a Leica DMR microscope equipped for
phase contrast and epifluorescence. A 1006 NA 1.3 objective lens was used to
collect Z stacks (0.2 mm steps). Image series were deconvolved using SlideBook
5.0 software (Intelligent Imaging Innovations, Denver, CO). Distances between
mother and daughter centrioles were measured using ImageJ software (National
Institute of Health, Bethesda, MD) and plotted in a graph using GraphPad Prism
for Windows version. 5.04 (GraphPad Software Inc., La Jolla, CA).
Cells were lysed with (50 mM Tris, 150 nM NaCl, 1% NP-40, 5% Glycerol)
supplimented with protease inhibitor mix (Sigma–Aldrich, St. Louis, MO). Anti-Emi1
antibody was used at 1:100. Anti-Cdh1, Cdc20 and Securin antibodies were used at
1:1000. All antibodies above are from Santa Cruz Biotechnology Inc., Santa Cruz, CA.
Band intensities were quantified using ImageJ software. Protein levels were normalized
to the actin loading control bands in the same lanes of the same gels.
We thank Drs Yumi Uetake, Paramasivam Murugan, Elizabeth Luna,
Jadranka Lonc ˇarek and Alexey Khodjakov for useful suggestions and
discussions. We are grateful to Harold Fisk for the gift of anti-CP110
antibody. This work was supported by National Institutes of Health
grant [GM 30758 to G.S.] and partial support from The Uehara
Memorial Foundation [2008 Postdoctoral Fellowship for T.H.].
The authors have no competing interests to declare.
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