Protein Phosphatase 2A-SUR-6/B55 Regulates
Centriole Duplication in C. elegans by Controlling
the Levels of Centriole Assembly Factors
Mi Hye Song,1,3,* Yan Liu,1D. Eric Anderson,2Wan Jin Jahng,3and Kevin F. O’Connell1,*
1Laboratory of Biochemistry and Genetics
2Proteomics and Mass Spectrometry Facility
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
3Department of Biological Sciences, Michigan Technological University, Houghton, MI 49931, USA
*Correspondence: firstname.lastname@example.org (M.H.S.), email@example.com (K.F.O.)
Centrioles play a crucial role in mitotic spindle
In worms, flies, and humans, centriole assembly is
dependent upon a key regulatory kinase (ZYG-1/
Sak/Plk4) and its downstream effectors SAS-5 and
SAS-6. Here we report a role for protein phosphatase
2A (PP2A) in centriole duplication. We find that the
PP2A catalytic subunit LET-92, the scaffolding
subunit PAA-1, and the B55 regulatory subunit
SUR-6 function together to positively regulate
embryos, the levels of ZYG-1 and SAS-5 are reduced
and the ZYG-1- and SAS-5-dependent recruitment of
SAS-6 to the nascent centriole fails. We show that
PP2A physically associates with SAS-5 in vivo and
that inhibiting proteolysis can rescue SAS-5 levels
and the centriole duplication defect of PP2A-
depleted embryos. Together, our findings indicate
that PP2A-SUR-6 promotes centriole assembly by
protecting ZYG-1 and SAS-5 from degradation.
In animal cells, the size and number of microtubule-organizing
centers, or centrosomes, are determined by the centrioles,
barrel-shaped organelles having 9-fold rotational symmetry
(Marshall, 2009). Centrioles organize centrosomes and thus,
the number of centrosomes per cell generally correlates with
the number of centrioles. A typical centrosome possesses an
orthogonally arranged pair of centrioles surrounded by a halo
of pericentriolar material (PCM), a substance from which the
microtubules are nucleated and anchored.
Centrioles duplicate precisely once per cell cycle with a new
daughter centriole assembling next to each preexisting centriole
during S phase (Strnad and Gonczy, 2008). Defects in this
process lead to abnormal numbers of centrioles, a state that
has been linked to both chromosome missegregation and tumor
formation (Basto et al., 2008; Castellanos et al., 2008; Ganem
et al., 2009; Silkworth et al., 2009). Although the structural
complexity of centrioles varies among species, studies in worms
and humans suggest the existence of a common centriole
assembly pathway that involves the activities of a small set of
conserved duplication factors (Delattre et al., 2006; Kleylein-
Sohn et al., 2007; Pelletier et al., 2006).
The centriole assembly pathway has been described most
thoroughly in the C. elegans embryo where the kinase ZYG-1
plays a key role (O’Connell et al., 2001). Related to vertebrate
Plk4 and Drosophila Sak, ZYG-1 localizes early to the nascent
centriole in a step that requires the coiled-coil protein SPD-2
(Delattre et al., 2006; Pelletier et al., 2006). ZYG-1 then recruits
a complex of two conserved coiled-coil proteins SAS-5 and
SAS-6, that is required for formation of the central tube, the first
known structural intermediate (Dammermann et al., 2004;
Delattre et al., 2004, 2006; Leidel et al., 2005; Pelletier et al.,
2006). SAS-5 and SAS-6 arealso required for the stable incorpo-
ration of SAS-4, a coiled-coil protein that promotes the addition
of nine sets of microtubules that surround the central tube
(Dammermann et al., 2008; Delattre et al., 2006; Kirkham et al.,
2003; Leidel and Gonczy, 2003; Pelletier et al., 2006).
Recently, attention has focused on the role of phosphorylation
in centriole assembly. Kitagawa et al. (2009) have found that
ZYG-1 promotes stable association of SAS-6 with the nascent
centriole through phosphorylation. Also, human Plk4 has been
shown to prevent centriole overduplication by negatively
regulating its own levels through autophosphorylation (Guderian
et al., 2010; Holland et al., 2010; Sillibourne et al., 2010).
Although roles for kinases in centriole assembly have been
established, the possible involvement of phosphatases remains
an open question. Like kinases, phosphatases regulate a myriad
of cellular processes and do so with a high degree of specificity.
The most abundant phosphatases are members of the PP1 and
PP2A family. These multimeric enzymes contain a catalytic
subunit characteristic of a specific phosphatase family and
a regulatory subunit that determines substrate specificity
(Virshup and Shenolikar, 2009).
referred to as PP2AcLET-92), physically associates with SZY-20,
a negative regulator of ZYG-1 (Song et al., 2008). We find that
PP2AcLET-92is required for centriole duplication in C. elegans,
and that it functions in this context as a holoenzyme consisting
of the catalytic subunit PP2AcLET-92, the scaffolding subunit
PP2AaPAA-1, and the regulatory subunit SUR-6. Further, we
show that the most critical role of PP2AcLET-92-SUR6 in centriole
Developmental Cell 20, 563–571, April 19, 2011 ª2011 Elsevier Inc. 563
assembly is to protect ZYG-1 and SAS-5 from degradation. Our
work thus identifies PP2AcLET-92-SUR-6 as a new component of
the centriole assembly pathway, and suggests that centriole
duplication is controlled by the coordinated action of kinases
SZY-20 Physically Interacts with Protein
Previously, we identified the RNA-binding protein SZY-20 as
(Song et al., 2008). To understand how SZY-20 controls ZYG-1,
we sought to identify proteins that physically interact with SZY-
20. Using affinity-purified a-SZY-20 antibodies, we immunopre-
cipitated SZY-20 from whole worm extracts and analyzed the
precipitated material bymass spectrometry. Among theproteins
that specifically co-precipitated with SZY-20, we identified one,
that when depleted by RNAi, reduces centrosome number in the
early embryo.Here wefocuson thisfactor, PP2AcLET-92, thesole
C. elegans protein phosphatase 2A catalytic subunit.
To confirm the SZY-20-PP2AcLET-92interaction, we immuno-
precipitated SZY-20 from whole worm extracts and analyzed
the immunoprecipitated material by western blotting. In
agreement with our mass spectrometry results, we detected
PP2AcLET-92among the material precipitated with a-SZY-20
antibodies (Figure1A).Givenourdataindicating aphysicalasso-
ciation between SZY-20 and PP2AcLET-92it seemed plausible
that SZY-20 is a substrate of PP2A. Consistent with this idea,
our mass spectrometry data indicate that SZY-20 is indeed
a phosphoprotein (see Figures S1A–S1C available online), and
depletion of PP2AcLET-92results in a significant and reproducible
reduction in mobility of SZY-20 on SDS-PAGE gels (Figure 1B).
The change in mobility appears to be due to phosphorylation,
as phosphatase treatment of SZY-20 from let-92(RNAi)-treated
worms restored its normal mobility (Figure S1D). We conclude
that PP2AcLET-92physically associates with SZY-20 in vivo and
regulates the phosphorylation level of SZY-20.
Disruption of the PP2A Holoenzyme Blocks Centriole
In C. elegans embryos, a maternal block to centrosome duplica-
tion is manifest by a distinct phenotype: a bipolar first division
followed by formation of monopolar spindles at the two-cell
stage (O’Connell et al., 2001). This phenotype arises because
the two sperm centrioles can separate and set up the poles of
the first bipolar spindle, but can only organize monopolar
spindles when partitioned to the daughter cells. Strikingly, we
found that depletion of PP2AcLET-92, although pleiotropic in
phenotype, resulted in a nearly identical centrosome defect:
a bipolar spindle during first division followed by monopolar
spindles during second division (Figure 1E; Movie S1). In addi-
tion, PP2AcLET-92-depleted embryos fail to properly align their
chromosomes on a metaphase plate, exhibit a delay in cell cycle
progression, and display defects in mitotic exit such as a failure
to reform nuclei and to decondense chromatin. A defect in
mitotic exit results from depletion of PP2A-B55a in human cells
(Schmitz et al., 2010), indicating that this role is conserved
between nematodes and humans. To quantify the effect of
PP2AcLET-92depletion on centrosome duplication, we per-
formed live imaging of let-92(RNAi) embryos expressing
GFP-SPD-2 to mark centrosomes and mCherry-histone to
mark chromatin (Movie S2). Reducing PP2AcLET-92completely
blocks centrosome duplication (0/34 successful events, n = 18
embryos). We conclude that PP2AcLET-92is required for centro-
some duplication in C. elegans.
The B55 Regulatory Subunit SUR-6 Directs the Role
of PP2A in Centriole Assembly
lytic subunit, a scaffolding subunit, and a regulatory subunit. In
C. elegans, the scaffolding subunit is encoded by a single gene
paa-1. Depletion of PP2AaPAA-1produces defects in chromo-
some alignment, cell cycle progression, and mitotic exit that
are similar to those induced by PP2AcLET-92depletion (data not
tion of monopolar spindles during the second embryonic cell
division (Figure 1F), confirming the involvement of the PP2A
holoenzyme in centrosome duplication. The specificity of the
PP2A holoenzyme is determined by association with a specific
regulatory subunit. By homology, we identified eight genes in
C. elegans that encode PP2A regulatory subunits. We used
RNAi to knock down each of these genes and screened for
a centrosome duplication defect. Only RNAi of sur-6, encoding
a B55 family member involved in Ras/Raf/MEK/ERK signaling
(Kao et al., 2004), produced an obvious duplication defect (Fig-
ure 1G). In these embryos, we observed both monopolar spin-
dles and asymmetric spindles in which one spindle pole was
smaller than the other (Figure 1G). Asymmetric spindles arise
when centriole duplication is partially inhibited (Delattre et al.,
2004; Kirkham et al., 2003). In contrast to let-92(RNAi), the effect
of sur-6(RNAi) was relatively mild; embryos did not possess
obvious defects in cell cycle progression or mitotic exit but did
occasionally exhibit anaphase bridging of chromatin. Also unlike
let-92(RNAi), sur-6(RNAi) did not completely block centrosome
duplication: 24% of spindles were monopolar and 47.5% were
asymmetric (Table S1). The lack of a completely penetrant
centriole duplication defect could reflect incomplete depletion
of SUR-6 protein by RNAi (Figure 1J), or alternatively functional
redundancy between SUR-6 and other PP2A regulatory
subunits. To address this, we rescreened these genes using
a sensitized genetic background. At the semipermissive temper-
ature of 20?C, centrosomes in the temperature-sensitive zyg-1
(it25) mutant fail to duplicate just 2.9% of the time (Figure 1I;
Table S1). When subjected to RNAi of each regulatory gene,
only sur-6(RNAi) had a significant effect, increasing the rate of
monopolar spindle formation 22-fold to 64% (Figure 1H; Table
S1). These results suggest that the role of PP2A in centrosome
duplication is predominantly, if not entirely, directed by SUR-6,
and that the lack of a more robust centriole duplication defect
upon sur-6(RNAi) is due to incomplete depletion.
To determine where in the cell SUR-6 acts, we produced
affinity-purified anti-SUR-6 polyclonal sera. The sera identified
a band of the correct size by immunoblotting, and the intensity
of this band was significantly reduced by sur-6(RNAi) (Figure 1J).
Further, the SUR-6 antibody also coprecipitated PP2AcLET-92
from worm extracts (Figure 1C). Immunostaining of embryos
produced diffuse cytoplasmic staining that was significantly
PP2A Regulates Centriole Duplication
564 Developmental Cell 20, 563–571, April 19, 2011 ª2011 Elsevier Inc.
Figure 1. PP2AcLET-92-SUR-6 Regulates Centriole Duplication
(A) PP2AcLET-92coprecipitates with SZY-20.
(B) Depletion of PP2AcLET-92results in a decrease in SZY-20 mobility consistent with hyperphosphorylation.
(C) PP2AcLET-92coprecipitates with SUR-6.
in centriole duplication defects such as monopolar spindles (left blastomere) or asymmetric spindles (right blastomere). Insets show symmetrical staining of
wild-type spindle poles (D) and asymmetrical staining of sur-6(RNAi) spindle poles (G).
(H and I) RNAi of sur-6 enhances the centriole duplication defect of zyg-1(it25) embryos raised at permissive temperature.
(J) A SUR-6 immunoblot demonstrating the specificity of the a-SUR-6 antibody. RNAi of sur-6 results in a reduction in the intensity of the SUR-6 band.
(K and L) Immunostaining reveals a broad distribution of SUR-6 (green) in the control embryo (K) and a loss of this staining in the sur-6(RNAi) embryo (L). Lower
panels show the same embryos stained for microtubules (red) and DNA (blue). Bars represent 10 mm.
For supporting data, see Figure S1, Table S1, and Movies S1 and S2.
PP2A Regulates Centriole Duplication
Developmental Cell 20, 563–571, April 19, 2011 ª2011 Elsevier Inc. 565
reduced by sur-6(RNAi) (Figures 1K and 1L). This result suggests
that the function of PP2AcLET-92-SUR-6 is not limited to the
centrosome and may function broadly in the cell to regulate
The SZY-20-PP2AcLET-92Interaction Plays a Minor Role
in Regulating Centriole Duplication
We next wanted to determine where in the centriole duplication
pathway PP2A functions. SZY-20 negatively regulates centriole
duplication (Song et al., 2008) and appears to be a substrate of
PP2AcLET-92-SUR-6 (Figure 1B; Figures S1D and S1E). Thus,
PP2A might promote duplication by repressing SZY-20. To
address this, we depleted PP2AcLET-92in strain OC470,
a szy-20(bs52) mutantthat
mCherry-histone. Strong knockdown of PP2AcLET-92in this
strain still produced a complete block to duplication (n = 38
events), indicating that the centriole duplication defect of let-92
(RNAi) embryos is not due to an overactive SZY-20 protein.
However, the szy-20(bs52) mutation could partially suppress
a weaker knockdown of PP2AcLET-92(7 of 26 centrosomes
Figure 2. PP2AcLET-92Genetically Interacts
with Other Centriole Duplication Factors
(A) A genetic interaction network of centriole
duplication genes. Shown is the percent embry-
onic lethality of indicated double heterozygotes.
Single heterozygotes are listed in last column (+).
Some single homozygotes exhibit a sterile unco-
ordinated (Stu) phenotype and others a larval
lethal (Lva) phenotype. Between 345 and 1521
embryos were counted for each strain.
(B–E) Embryos of indicated genotype stained for
microtubules (red), centrioles (SAS-4, green), and
DNA (blue). Note that let-92 (B) and zyg-1 hetero-
zygotes (not shown) assemble bipolar spindles,
but zyg-1 homozygotes (C) and let-92/+; zyg-1/+
(D) and let-92/+; +/sas-5 double heterozygotes
assemble monopolar spindles. Bar represents
For supporting data, see Movie S3.
versus 0/18 in controls), suggesting that
one minor role for PP2A is to repress
SZY-20. Overall, failure of the szy-20
(bs52) mutation to strongly affect the let-
92(RNAi) duplication defect indicates
that the critical role of PP2A in centriole
duplication lieselsewhere in thepathway.
PP2A Functions Closely with ZYG-1
We have shown that genes encoding
core centriole assembly factors exhibit
robust genetic interactions with each
other (Kemp et al., 2004). Mutations in
two different centriole assembly genes
often fail to complement one another,
with the double heterozygotes exhibiting
This interaction, termed nonallelic noncomplementation, occurs
when two genes function closely together in a common process;
in a double heterozygote, the process is rendered inefficient
enough to yield an observable phenotype. To determine whether
let-92 also behaves in this manner, we asked whether the let-92
(s504) mutation would fail to complement the zyg-1(b1) allele.
Animals heterozygous for either zyg-1(b1) or let-92(s504) had
low levels of embryonic lethality, yet strikingly the zyg-1(b1)/+;
let-92(s504)/+ double heterozygotes exhibited 100% embryonic
lethality (Figure 2A). We also tested the zyg-1(b1) mutation
against the let-92(s677) allele and again detected a very strong
interaction: 61% (n = 1197) of the embryos produced by zyg-1
(b1)/+; let-92(s677)/+ hermaphrodites were inviable compared
to 2.8% (n = 611) for let-92(s677)/+ progeny. We then examined
embryos from zyg-1(b1)/+; let-92(s504)/+ hermaphrodites and
observed a prominent centrosome duplication defect (Figures
2B–2D). Live DIC imaging of these embryos revealed that the
block to centriole duplication was complete (0 of 48 centrioles
duplicated; Movie S3). These results show that PP2AcLET-92
behaves genetically in the same manner as other core centriole
PP2A Regulates Centriole Duplication
566 Developmental Cell 20, 563–571, April 19, 2011 ª2011 Elsevier Inc.
duplication factors and indicate that PP2AcLET-92functions
closely with ZYG-1.
We next asked whether we could use this type of genetic inter-
action to map the role of PP2A, as well as other factors, in the
centriole assembly pathway. We constructed and quantified
embryonic lethality in all possible double heterozygotes from
a set of spd-2, zyg-1, sas-5, sas-6, sas-4, let-92, and paa-1 muta-
weakornointeractionswithspd-2, sas-6, and sas-4.Again,these
double heterozygotes exhibit centriole duplication defects (Fig-
ure 2E). These results suggest that the role of PP2AcLET-92is
most closely tied to zyg-1- and sas-5-dependent steps. Also of
interest, spd-2 onlyshows a stronginteraction withzyg-1, consis-
tent with the only known role of SPD-2 in localizing ZYG-1 to sites
contrast, zyg-1 shows strong genetic interactions with multiple
that ZYG-1 functions at multiple steps in this pathway.
PP2A Is Required for Recruitment of SAS-5 and SAS-6
A molecular hierarchy describing the order and dependencies
with which duplication factors localize to assembling centrioles
has been described (Delattre et al., 2006; Pelletier et al., 2006).
To determine where in this pathway PP2AcLET-92-SUR-6 func-
tions, we analyzed the effect of PP2AcLET-92depletion on the
recruitment of duplication factors. We found that let-92(RNAi)
does not block localization of ZYG-1 to nascent centrioles
(Figures 3A and 3B). We then examined SAS-5 and SAS-6, two
downstream effectors of ZYG-1 that form a complex in vivo
2005). To examine the localization of the SAS-5/6 complex, we
followed GFP-SAS-6 utilizing a previously established recruit-
mentassay(Pelletier etal.,2006). Inthisassay, matings between
wild-type males and transgenic females carrying a germ line-ex-
pressed gfp-sas-6 gene allows one to examine the accumulation
of GFP-SAS-6 protein around the unlabeled sperm-derived
centrioles. Consistent with previous observations, we found
that in control embryos, GFP-SAS-6 was initially detected at
centrioles during the first S phase (Figure 3C). In contrast, we
often failed to detect centriole-associated GFP-SAS-6 in let-92
(RNAi) embryos (Figure 3D). Of ten one-cell let-92(RNAi)
embryos that ranged from S phase to mid prophase, only two
showed even a faint GFP-SAS-6 signal around one of the two
centrioles. We conclude that PP2AcLET-92-SUR-6 is required
for proper localization of the SAS-5/6 complex to sites of
PP2AcLET-92-SUR-6 Regulates ZYG-1 and SAS-5 Levels
We next tested for direct effects of PP2AcLET-92-SUR-6 deple-
tion on centriole assembly factors. Using quantitative immuno-
blotting, we measured the levels of ZYG-1, SAS-5, and SAS-6
proteinsin controland PP2AcLET-92-depleted
Depleting PP2AcLET-92, SUR-6, or PP2AaPAA-1led to reproduc-
ible declines in the levels of ZYG-1 and SAS-5 (Figures 4A and
4B).We also notedthat
slightly upon depletion of PP2A subunits (Figure 4B). Quantita-
tively, PP2AcLET-92-depleted embryos possess 48% ± 25
(n = 6) as much ZYG-1, and 40% ± 15 (n = 7) as much SAS-5
as control embryos. As evident in Figure 3B, the reduction in
ZYG-1 protein level does not completely block the localization
of ZYG-1 to centrosomes. However, we are currently unsure
whether the centrosome levels of ZYG-1 are slightly reduced
upon PP2AcLET-92depletion. In contrast to ZYG-1 and SAS-5,
the level of SAS-6 appears unaffected by depletion of
PP2AcLET-92or SUR-6 (Figure 4C). Thus, a striking parallel exists
between our genetic and biochemical analyses; genetically let-
92 interacts with zyg-1 and sas-5 but not with sas-6, and bio-
chemically, loss of PP2AcLET-92affects the levels of ZYG-1 and
SAS-5 but not of SAS-6.
To test whether PP2AcLET-92-SUR-6 controls the levels of
SAS-5 directly, we immunoprecipitated SAS-5 from wild-type
embryonic extracts and probed for PP2AcLET-92. Consistent
tected PP2AcLET-92in the precipitated material (Figure 4D). We
also immunoprecipitated SAS-6 from wild-type embryonic
extracts but in contrast to the SAS-5 pull-down experiment, we
did not detect PP2AcLET-92among the coprecipitating proteins
(Figure 4D). It should be noted that a significant fraction of
SAS-5 exists in a cytoplasmic complex with SAS-6 (Leidel
et al., 2005). Thus, it was unexpected that we failed to detect
an interaction between SAS-6 and PP2AcLET-92. This may indi-
cate that PP2AcLET-92interacts predominantly with the pool of
SAS-5 not bound to SAS-6.
Our findings that PP2AcLET-92physically interacts with SAS-5
and controls SAS-5 levels suggests that PP2A promotes the
stability of SAS-5, and that in the absence of PP2A, the reduced
duplication. To investigate this, we determined what effect
blocking SAS-5 degradation would have on PP2AcLET-92-
depleted embryos. We knocked down both PP2AcLET-92and
RPT-4, an essential component of the 26S proteosome, and as-
sayed centriole behavior. Remarkably, we found 58% of centri-
oles (n = 26) duplicated in such embryos, whereas centrioles
never duplicated (n = 24) in paired let-92(RNAi) controls
(Movie S4). Further, knockdown of RPT-4 could also suppress
the genetic interaction between zyg-1 and let-92. Specifically,
we found (using slightly less stringent conditions than used for
the genetic interaction experiments) that 70% of centrosomes
(n = 10) duplicated in zyg-1(b1)/+; let-92(s504)/+ double hetero-
zygotes subject to RPT-4 knockdown compared to 10%
(n = 10) in controls. Further, despite the fact that RPT-4 knock-
down reduces the viability of wild-type embryos (27% viability),
we found that it actually increases the viability of embryos
produced by zyg-1(b1)/+; let-92(s504)/+ double heterozygotes
10-fold from 0.3% (n = 292) in controls to 3.0% (n = 448) in rpt-
4-treated animals. Finally, we investigated the effect of RPT-4
depletion on SAS-5 levels and found that partial knockdown of
RPT-4 could suppress the reduction of SAS-5 observed in
PP2AcLET-92-depleted embryos, the level of SAS-5 positively
correlates with the ability of centrioles to duplicate.
(Figure 4E). Therefore in
Although the involvement of kinases in centriole duplication has
been established, the potential involvement of phosphatases
PP2A Regulates Centriole Duplication
Developmental Cell 20, 563–571, April 19, 2011 ª2011 Elsevier Inc. 567
has not been well explored. Our identification of PP2AcLET-92-
SUR-6 as a positive regulator of duplication indicates that
centriole duplication is governed by both activating and inhibi-
tory phosphorylation events. A recent proteomic study in
Recruitment of the SAS-5/6 Complex
(A and B) Control (A) and let-92(RNAi) (B) embryos
stained for ZYG-1 (red), GFP-SAS-4 (green), and
DNA (blue). ZYG-1 localizes to centrosomes in
PP2AcLET-92-depleted embryos (insets).
(C and D) Control (C) and let-92(RNAi) (D) embryos
stained for endogenous SAS-4 (red), GFP-SAS-6
(green-insets only), and DNA (blue). In control
embryos (C), GFP-SAS-6is recruitedto centrioles,
as evidenced by the overlap between the GFP-
SAS-6 and SAS-4 signals (insets).
(D) In let-92(RNAi) embryos, GFP-SAS-6 does not
accumulate around the centrioles. Note that
control and let-92(RNAi) embryos are approxi-
mately the same age based on position of DNA
and separation of centrioles. The DNA of let-92
(RNAi) embryos, however,
condensed. Scale bar represents 10 mm.
Drosophila found the fly PP2A catalytic
et al., 2010). Consistent with our results,
depletion of either PP2A subunit in fly or
human tissue culture
centrosome number suggesting a defect
in centriole duplication or segregation
(Muller et al., 2010). Thus, it seems likely
that the role of PP2A in centriole duplica-
tion is conserved.
factors that are targeted by PP2AcLET-92-
SUR-6: the levels of these proteins are
diminished upon PP2AcLET-92or SUR-6
depletion. ZYG-1 is still detectable at
embryos (Figures 3A and 3B), although it
remains unclear whether ZYG-1 levels at
the centrosome are reduced relative to
the wild-type. However, because ZYG-1
and SAS-5 are both required for SAS-6
localization (Delattre et al., 2004, 2006;
Leidel et al., 2005; Pelletier et al., 2006),
the reduced levels of ZYG-1 and espe-
cially SAS-5 likely contribute to the failure
of SAS-6 to localize to nascent centrioles
in the absence of PP2AcLET-92-SUR-6.
iting proteolysis in PP2AcLET-92-depleted
embryos restores both the level of SAS-
5 and centriole duplication. Despite this,
it still remains possible that PP2AcLET-92-
SUR-6 also functions directly (and inde-
pendent of its role in regulating ZYG-1
and SAS-5 levels) to promote the recruitment of the SAS-5/6
How might the PP2AcLET-92-SUR-6 complex regulate ZYG-1
and SAS-5 levels? Our finding that SAS-5 physically associates
PP2A Regulates Centriole Duplication
568 Developmental Cell 20, 563–571, April 19, 2011 ª2011 Elsevier Inc.
with PP2AcLET-92in vivo suggests that PP2AcLET-92-SUR-6 acts
to stabilize SAS-5 by dephosphorylation. Regulating protein
stability via phosphorylation is common; in vertebrates, Plk4
negatively regulates its own levels by targeting itself for destruc-
tion via autophosphorylation (Guderian et al., 2010; Holland
et al., 2010; Sillibourne et al., 2010). Autophosphorylation
promotes association of Plk4 with bTr-CP, an adaptor for the
SCF E3 ubiquitin ligase. In flies, inhibition of SCFbTr-CPleads to
elevated levels of Plk4 and centriole amplification (Cunha-Ferre-
ira et al., 2009; Rogers et al., 2009), suggesting that autophos-
phorylation may function to maintain proper levels of Plk4.
In summary, PP2AcLET-92-SUR-6 is a core centriole duplica-
tion factor and regulates the levels of ZYG-1 and SAS-5. Unlike
the other core duplication factors, PP2AcLET-92-SUR-6 does
not appear to function exclusively at centrosomes, as we find
that depletion of RSA-1, a regulatory chain that targets
PP2AcLET-92to centrosomes (Schlaitz et al., 2007), does not
inhibit centrosome duplication (Table S1) and SUR-6 is not en-
riched at centrosomes (Figure 1K). Thus, PP2AcLET-92-SUR-6
likely acts within the cytoplasm. Future studies aimed at identi-
fying the specific residues within SAS-5, and possibly ZYG-1,
targeted by PP2AcLET-92-SUR-6 will reveal how inhibitory phos-
phorylation events contribute to the fidelity of centriole
Immunoprecipitation and Quantitative Immunoblotting
For whole worm lysates, N2 worms were grown in liquid culture until gravid,
harvested by centrifugation, washed several times in lysis buffer (50 mM
HEPES [pH 7.4], 1 mM EDTA, 1 mM MgCl2, 200 mM KCl, 10% glycerol;
Cheeseman et al., 2004), and gently pelleted. Worm slurries (7 ml) were frozen
on dry ice and lysed using a One Shot Cell Disrupter (Constant Systems
Limited, Northants, UK) set at 40 psi. Crude lysates were centrifuged three
times at 382K rpm for 45 min; the resulting high-speed supernatants were
used for immunoprecipitation.
For pull-down assays, antibodies to SUR-6, SAS-5, SAS-6, and SZY-20
(Song et al., 2008) were bound to Dynabeads coupled to protein A (Invitrogen)
at a ratio of 1 mg antibodies to1 ml beads. Anti-rabbit-IgG (Invitrogen) served as
a negative control. Antibodies were incubated with beads in PBST (1 3 PBS,
0.1% Triton X-100), rotated overnight at 4?C, and crosslinked to beads ac-
cording to manufacturer’s instructions. The beads were then mixed with
1 ml of the whole worm extract and incubated with rotation overnight at 4?C.
The lysate was removed and the beads washed with PBST ten times at
For quantitative immunoblotting, embryonic proteins were fractionated on
a NuPAGE Bis-Tris Gel (Invitrogen) and transferred to nitrocellulose. a-SZY-
20 (Song et al., 2008), a-SPD-2 (Kemp et al., 2004), a-ZYG-1 (Kemp et al.,
2007), a-PP2Ac (Clone 1D6, Millipore), a-actin (Thermo Scientific), and
DM1A (Sigma) were used at a 1:500-1500 dilution. IRDye secondary anti-
bodies (LI-COR Biosciences) were used at 1:15,000. Blots were imaged using
the Odyssey Infrared Imaging System (LI-COR Biosciences) and analyzed
using the same software or ImageJ v1.40G. For quantifying ZYG-1 and
SAS-5 levels, values were normalized against tubulin or actin.
To feed dsRNA, we placed L4 larvae on a lawn of bacteria that carried
a plasmid for inducible expression of let-92, paa-1, sur-6, sas-5, or rpt-4
dsRNA (Source Bioscience plc, Nottingham, UK) and allowed them to feed
for 1 day at 25?C. Bacteria carrying the dsRNA-feeding vector L4440 served
as a negative control. For dual RNAi, plates were seeded with mixtures of
bacteria in specified ratios based on the optical densities of the cultures; the
total amount of bacteria added to the plate was kept constant by adding the
appropriate amount of L4440 bacteria. To test for suppression of let-92 by
rpt-4, we compared the effect of a 1:1 mixture of L4440 and let-92 bacteria
(0% duplication, n = 24 events) to a 3:4:1 ratio of L4440, let-92, and rpt-4
Figure 4. The PP2AcLET-92-SUR-6 Complex Regulates the Levels of ZYG-1 and SAS-5
(A–C) Quantitative immunoblots of embryonic extracts showing that RNAi of let-92, sur-6, or paa-1 reduces the levels of ZYG1 and SAS-5 but not the levels of
(D) PP2AcLET-92coprecipitates with SAS-5, but not with SAS-6.
(E) Weak rpt-4(RNAi) restores SAS-5 levels in PP2AcLET-92-depleted embryos. Shown is a graph depicting the average relative levels of SAS-5 with standard
deviations from three experiments and a representative blot. The reduction of SAS-5 levels upon let-92(RNAi) is less pronounced in these experiments due to
dilutionoflet-92bacteria withcontrolbacteria.Ratios ofRNAibacteria used:let-92:control, 1:1;let-92:rpt-4:control: 4:1:3;rpt-4:control, 1:7.Forsupporting data,
see Movie S4.
PP2A Regulates Centriole Duplication
Developmental Cell 20, 563–571, April 19, 2011 ª2011 Elsevier Inc. 569
bacteria (58% duplication, n = 26 events) as they both contained the same
fraction of let-92 bacteria. To test for suppression of let-92(RNAi) by szy-20
(bs52), we compared N2 and szy-20(bs52) animals grown on a 1:1 mixture of
L4440 and let-92 bacteria (strong PP2AcLET-92knockdown) or on a 3:1 mixture
of L4440 and let-92 bacteria (weak knockdown). To test zyg-1(b1)/+; let-92
(s504)/+ double heterozygotes for suppression by rpt-4, we compared double
heterozygotes grown on L4440 to those grown on a 7:1 mixture of L4440 and
The following antibodies were used at a 1:500–2000 dilution: DM1A (Sigma),
a-GFP (Roche), a-SPD-2 (Kemp et al., 2004), a-SPD-5 (Hamill et al., 2002),
a-ZYG-1 (O’Connell et al., 2001), a-SAS-4 (Song et al., 2008), and Alexa Fluor
488 and 568 secondaryantibodies(Invitrogen). Methods and imaging systems
for indirect immunofluorescence and confocal and 4D-DIC microscopy have
been described (Peters et al., 2010; Song et al., 2008). Image processing
was performed with ImageJ v1.40G and Adobe Photoshop CS5.
The SAS-6 recruitment assay was performed essentially as described
(Pelletier et al., 2006). Briefly, wild-type males were mated overnight to
OD103 females on RNAi plates at 25?C. The next day embryos were immuno-
stained using the a-SAS-4 antibody to mark centrioles and the anti-GFP anti-
body to detect GFP-SAS-6.
Supplemental Material includes one figure, one table, Supplemental Experi-
mental Procedures, and four movies and can be found with this article online
We thank members of the O’Connell and Song laboratories, David Weisblat,
Alexander Dammerman, Tony Hyman, Markus Decker, Karen Oegema, Martin
Srayko, and Orna Cohen-Fix for sharing strains and reagents and/or providing
advice. Some strains were provided by The Caenorhabditis Genetics Center
and The National Bioresource Project, Japan. This work was supported by
the Intramural Research Program of the National Institutes Health and by the
National Institute of Diabetes and Digestive and Kidney Diseases.
Received: October 22, 2010
Revised: January 30, 2011
Accepted: February 28, 2011
Published: April 18, 2011
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