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
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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