CP110 Suppresses Primary Cilia Formation
through Its Interaction with CEP290, a Protein
Deficient in Human Ciliary Disease
William Y. Tsang,1Carine Bossard,2Hemant Khanna,3Johan Pera ¨nen,4Anand Swaroop,3,5Vivek Malhotra,2
and Brian David Dynlacht1,*
1Department of Pathology and Cancer Institute, New York University School of Medicine, 522 1stAvenue, New York, NY 10016, USA
2Centre for Genomic Regulation, C/ Dr. Aiguader, 88, Barcelona 08003, Spain
3Department of Ophthalmology and Visual Sciences, University of Michigan, Ann Arbor, MI 48105, USA
4Institute of Biotechnology, Program in Cellular Biotechnology, University of Helsinki, Helsinki 00014, Finland
5National Eye Institute, Neurobiology Neurodegeneration & Repair Laboratory, Bethesda, MD 20892, USA
Primary cilia are nonmotile organelles implicated in
signaling and sensory functions. Understanding
how primary cilia assemble could shed light on the
many human diseases caused by mutations in ciliary
proteins. The centrosomal proteinCP110 is known to
suppress ciliogenesis through an unknown mecha-
nism. Here, we report that CP110 interacts with
CEP290— a protein whose deficiency is implicated
in human ciliary disease—in a discrete complex sep-
arable from other CP110 complexes involved in reg-
ulating the centrosome cycle. Ablation of CEP290
prevents ciliogenesis without affecting centrosome
function or cell-cycle progression. Interaction with
CEP290 is absolutely required for the ability of
CP110 to suppress primary cilia formation. Further-
more, CEP290 and CP110 interact with Rab8a,
of CEP290 interferes with localization of Rab8a to
centrosomes and cilia. Our results suggest that
CEP290 cooperates with Rab8a to promote ciliogen-
esis and that this function is antagonized by CP110.
The centrosome is the major microtubule-organizing center in
animal cells (Bettencourt-Dias and Glover, 2007). It consists of
twocentriolessurrounded byproteinaceous pericentriolarmate-
rial from which microtubules emanate and elongate. Centro-
some duplication takes place during S phase, a period during
which two new centrioles (procentrioles) are formed adjacent
to the pre-existing mother and daughter centrioles. Procentriole
maturation takes place thereafter, such that by the end of G2
phase, a cell possesses two centrosomes containing a total of
four centrioles. In early mitosis, the two centrosomes migrate
to opposite poles to set up the spindle poles, and each of the
two incipient daughter cells receives a single centrosome upon
completion of cytokinesis. Defects in any aspect of centrosome
functioncan bedeleterious andleadtocell-cycle arrest,genome
instability, aneuploidy, cancer, and tumor formation (Badano
et al., 2005; Nigg, 2002; Sluder and Nordberg, 2004).
In addition to the role of centrosomes in anchoring and orga-
nizing microtubules, recent advances in the field have revealed
a critical connection between centrosome function and primary
cilia formation. Primary cilia are nonmotile projections found at
the surface of most quiescent vertebrate cells and are involved
in differentiation, sensory functions, and signal transduction
(Satir and Christensen, 2007; Singla and Reiter, 2006). Upon
cell-cycle exit, the mother centriole migrates toward the cell
cortex and nucleates the formation of a finger-like structure con-
sisting of a nine-fold array of doublet microtubules, termed the
axoneme, enveloped by the plasma membrane. Receptors and
signaling molecules are delivered to the primary cilium by means
of polarized trafficking via intraflagellar transport (IFT) proteins
and motor proteins such as kinesin-2 and dynein (Rosenbaum,
2002; Scholey, 2003).Deficiencies in genesimplicated in centro-
some and ciliary function give rise to a spectrum of human phe-
notypes, including cystic kidney disease, diabetes, neurological
disorders, retinal degeneration, and polydactyly. Mechanistic
details concerning the role of these proteins are not fully under-
stood (Scholey and Anderson, 2006; Singla and Reiter, 2006).
Furthermore, our understanding of the regulatory controls gov-
erning the conversion of centrosomes to basal bodies, or vice
versa, is rudimentary at best.
We have recently identified a centrosomal protein, CP110,
required for centrosome and primary cilia function (Chen et al.,
2002; Spektor et al., 2007; Tsang et al., 2006). Depletion of
CP110 by RNA interference (RNAi) results in premature centro-
some separation, abrogates centrosome reproduction in
S-phase-arrested cells, and inhibits Polo-like kinase-4-induced
centrosome amplification (Chen et al., 2002; Kleylein-Sohn
et al., 2007). In addition, CP110 functionally interacts with two
calcium-binding proteins, centrinand calmodulin(CaM),toregu-
phosphorylation results in cytokinesis failure (Chen et al., 2002;
Tsang et al., 2006). Moreover, loss of CP110 or CEP97, another
cilia in growing cells, and conversely, enforced expression of
CP110 suppresses cilium assembly in quiescent cells (Spektor
et al., 2007). Together, these data suggest that CEP97 and
Developmental Cell 15, 187–197, August 12, 2008 ª2008 Elsevier Inc. 187
CP110 collaborate to inhibit a ciliogenesis program and indicate
that cilia formation may be a default pathway engaged in the
absence of CP110 function. However, it is currently unknown
how CP110 mediates suppression of primary cilia assembly in
we initiated studies to identify additional CP110-interacting
proteins. We identified CEP290 as a CP110-interacting partner
in vivo. CEP290 is a centrosomal protein (Chang et al., 2006;
Sayer et al., 2006; Valente et al., 2006), and mutations in
CEP290 have been implicated in autosomal recessive disorders,
including nephronophthisis (Sayer et al., 2006), Senior-Loken
syndrome (Helou et al., 2007), Joubert syndrome (Sayer et al.,
eration and olfactory dysfunction (Chang et al., 2006; McEwen
et al., 2007). We have examined the function of CEP290 and
the biological significance of the CEP290-CP110 interaction.
We show that CEP290 participates strictly in primary cilia forma-
essential for suppressing cilia formation. Furthermore, CEP290
and CP110 interact with the small GTPase Rab8a, a protein
required for ciliary biogenesis. Depletion of CEP290 diminishes
the localization of Rab8a to centrosomes and prevents its entry
into the cilium. Taken together, our data demonstrate that
CP110 suppresses the activity of CEP290, which in turn cooper-
ates with Rab8a to mediate a cilia assembly program.
Biochemical Interaction between CP110 and CEP290
In an effort to understand how CP110 functions during the cen-
two-hybrid screen using full-length CP110 as bait. Eighty-two
Figure 1. CP110 Interacts with CEP290 In Vivo
(A) The number of times that each annotated protein was identified in the yeast two-hybrid screen is shown.
(B)Theindicated fragments ofFlag-tagged CEP290 wereexpressedin293T cellsand immunoprecipitated from lysates. Flag-CEP290fusion proteinsand CP110
were detected after western blotting the resulting immunoprecipitates. Input CP110 was detected in lysates from each transfection (IN).
(C) Summary of CEP290 truncation mutants and the results of in vivo binding experiments. The orange box denotes the CP110-binding domain based on the
yeast two-hybrid screen.
(D) Western blotting of endogenous CEP290, CEP97, CP110, CaM, and centrin after immunoprecipitation with anti-Flag (control), anti-centrin, anti-CEP97,
anti-CP110, or anti-CEP290 antibodies from 293T cell extracts. IN represents input.
(E) Cell extract was chromatographed on a Superose 6 gel filtration column, and the resulting fractions were blotted with antibodies against CP110, CEP290,
kendrin, or CaM. Estimated molecular weights are indicated at the top of the panel.
CP110 Inhibits CEP290-Induced Ciliogenesis
188 Developmental Cell 15, 187–197, August 12, 2008 ª2008 Elsevier Inc.
interactors were isolated, and DNA sequencing revealed that
seventy-four clones encoded CaM, a calcium-binding, CP110-
interacting protein we had identified previously (Tsang et al.,
2006), confirming the success of the screen (Figure 1A). In addi-
tion, two clones encoded CEP290 (Figure 1A), spanning regions
corresponding to amino acid residues 237–526. To confirm that
CEP290 interacts with CP110, we constructed a series of epi-
tope-tagged CEP290 truncation mutants, transfected human
cells, and performed immunoprecipitations with anti-epitope
antibodies. CP110 was readily detected on western blots after
immunoprecipitation of full-length CEP290 (1–2479; Figures 1B
and 1C). We found that amino-terminalportions of CEP290 (con-
taining residues 1–366, 221–366, and 362–822) were necessary
and sufficient to bind CP110, while other fragments did not inter-
act (Figures 1B and 1C). Thus, mapping and yeast two-hybrid
data point to CP110 binding near the amino terminus of CEP290.
To determine whether CEP290 and CP110 interact in vivo, we
performed immunoprecipitations with affinity-purified CEP290
and CP110 antibodies. CEP290 and CP110 were coimmunopre-
cipitated by CP110 antibodies, and reciprocal immunoprecipita-
tions with CEP290 antibodies confirmed a robust physiological
interaction (Figure 1D). CP110 has been shown to exist in two
mutuallyexclusive complexes. One complex, comprising centrin
andCaM,has been implicated in cytokinesis (Tsang etal.,2006).
In addition, CP110 interacts with CEP97, and this complex is re-
quired to suppress primary cilia formation (Spektor et al., 2007).
cohort of proteins. Although we detected CP110 in both centrin
and CEP97 immunoprecipitates, we failed to observe CEP290
(Figure 1D). Moreover, antibodies recognizing CEP290 did not
coprecipitate centrin, CEP97, or CaM, although CP110 was
detected (Figure 1D), suggesting that CP110 associates with
discrete centrin, CEP97, and CEP290 complexes. In addition,
we demonstrated by size exclusion chromatography that native
CEP290 reproducibly migrated as a high-molecular-weight
complex (with a mass as large as 2–3 MDa) that coeluted with
CP110 (Figure 1E). In contrast, the pericentriolar matrix protein
kendrin peaks at a somewhat higher molecular weight (Figure 1E
and Tsang et al., 2006). These observations support a physical
association of CEP290 with CP110.
Localization of CEP290
Next, we showed that CEP290 protein levels remain relatively
constant throughout the cell cycle (Figure S1A, available online).
et al., 2003; Chang et al., 2006; Sayer et al., 2006; Valente et al.,
2006), its localization during interphase has not been fully inves-
tigated. Antibodies against CEP290 stained two or four promi-
nent spots in G1 and G2 phase (Figures S1B–S1D). CEP290
staining overlapped substantially with that of centrin and
CP110 (Figures S1C and S1D), which are located at the distal
ends of centrioles (Kleylein-Sohn et al., 2007; Paoletti et al.,
1996), and to a lesser extent with that of g-tubulin, another
centrosomal marker (Figure S1B). In G0 phase, CP110 is present
at the daughter centriole, but not at the mother centriole (Figures
S1Dand S3), consistent withour prior observation that itisextin-
guished at the mother centriole in ciliated cells (Spektor et al.,
2007). CEP290, on the other hand, is found on both the mother
centrioleand thedaughter centriole(Figures S1Dand
Figure 2F). The mother centriole nucleates the formation of a pri-
mary cilium at its distal end and is attached to the daughter
centriole at the other, proximal end. Interestingly, CEP290 stain-
ing at the mother centriole is localized to the distal end near the
base of the primary cilium (Figure 2F), suggesting that it may be
present atthe transitional zoneand that itcould function in cilium
assembly. In addition, our data suggest that a portion of CEP290
protein at the mother centriole remains unassociated with
CEP290 Depletion Prevents Primary Cilia Formation
We examined the consequences of knocking down CEP290,
CP110, or both by transfecting cells with small interfering
RNAs (siRNAs) targeting the corresponding transcripts. We
transfected two distinct CEP290 siRNAs and showed that the
levels of CEP290 protein were reduced by ?75% (Figure 2A).
Depletion of either CP110 or CEP290 does not affect the levels
or localization of the other protein (Figures 2A and Figure S3
and data not shown). Since it is known that CP110 is important
for centrosome duplication (Figure 2B; Chen et al., 2002, and
Kleylein-Sohn et al., 2007), the prevention of premature centro-
depletion had any impact on these processes using appropriate
assays. Interestingly, although it interacts with CP110 in vivo,
that suppression of CEP290 did not alter cell-cycle progression
Given its association with several ciliary diseases, we tested
a role for CEP290 in the assembly of primary cilia by staining
for ciliary markers (glutamylated tubulin or acetylated tubulin).
Interestingly, when we ablated CEP290, we noticed a dramatic
alteration in the ability of cells to assemble primary ciliain cycling
human retinal pigment epithelial cells (RPE-1) (Figure 2E). In
contrast, loss of CP110 results in the opposite effect, namely,
aberrant formation of primary cilia in growing cells (Figure 2E),
consistent with our previous observations (Spektor et al.,
2007). Ablation of both CEP290 and CP110 suppressed the phe-
notype associated with loss of CP110 and mimicked CEP290
ablation (Figure 2E), suggesting that CEP290 and CP110 could
functionally interact in antagonistic ways. We also determined
the impact of CEP290 ablation on quiescent cells, which are in
a period in which the majority (?85%) form primary cilia (Figures
2F and 2G). When we depleted CEP290 protein, we observed
a consistent and striking loss of primary cilia (Figures 2F and
2G). These results are consistent with a high-throughput screen
designed to uncover ciliary proteins (Graser et al., 2007). Abla-
tion of CEP290 abolished glutamylated tubulin staining of the
axoneme, although staining at the ciliary base was unaffected
(Figure 2F). Combined suppression of CEP290 and CP110 again
resulted in a phenotype similar to loss of CEP290 alone (Fig-
ure 2G). We could show that the failure to assemble cilia in
CEP290-depleted cells was not due to an inability of the cell to
enter a quiescent state (Figures S2B and S2C). These results
provide conclusive evidence that CEP290 is required to promote
ciliogenesis in human cells, counteracting the activity of CP110.
Furthermore, when the distance between centrosomes and
nuclei was examined by immunofluorescence microscopy, we
CP110 Inhibits CEP290-Induced Ciliogenesis
Developmental Cell 15, 187–197, August 12, 2008 ª2008 Elsevier Inc. 189
found that 45% ± 6% and 21% ± 7% of the centrosomes in con-
trol and CEP290-depleted cells, respectively, were well sepa-
rated (i.e., located at least 5 mm away) from the nucleus and
were localized close to the cell surface (Figure S2D and data
not shown). Thus, depletion of CEP290 could disrupt the migra-
tion of centrioles to the cell cortex.
Ectopic Expression of the CEP290-Binding Mutant
of CP110 Does Not Suppress Primary Cilia Formation
To gain insight into the biological significance of the CP110-
CEP290 interaction, we first performed experiments to deter-
mine the regions of CP110 involved in binding CEP290. We
transfected a set of Flag-tagged CP110 mutants and performed
anti-Flag immunoprecipitation andwesternblotting todetecten-
dogenous CEP290. We found that a region of CP110 encom-
passing residues 1–223 was required for stable binding to
ther by testing a full-length CP110 mutant lacking only residues
67–82 (1–991(Daa67–82)). Excision of these residues severely
compromised its ability to interact with CEP290 (Figures 3B
and 3C). Interestingly, 1–991(Daa67–82) can nevertheless bind
to all other known CP110-interacting proteins, including
CEP97, centrin, and CaM, both in vivo (Figure 3B) and in vitro
(Tsang et al., 2006).
These data argue that CP110 binds to CEP290 independently
of other known CP110-interacting proteins, consistent with our
biochemical observations (Figure 1D), thus allowing us to func-
tionally separate and dissect these independent activities.
Next, we tested the ability of these CP110 mutants to inhibit cilia
assembly by expressing this short internal deletion mutant and
Figure 2. RNAi-Mediated Suppression of CEP290 Inhibits Primary Cilia Formation
(A) Western blotting of CP110 and CEP290 in RPE-1 cells treated with control, CP110, CEP290, or both siRNAs. a-tubulin was used as a loading control.
of centrosome overduplication, were determined.
(C) The percentages of U2OS cells with well-separated g-tubulin dots, indicative of premature centrosome separation, were determined.
(D) The percentages of binucleate WI38 cells, indicative of cytokinesis failure, were determined.
(E) The percentages of growing RPE-1 cells with primary cilia were determined using acetylated tubulin (Ac. tub.) as a marker.
(F) RPE-1 cells transiently transfected with control or CEP290 siRNAs,induced toquiescence, stained with antibodiesto glutamylated tubulin (GT335, green) and
CEP290 (red), and with DAPI (blue). Bar: 10 mM; insets: 2 mM.
(G) The percentages of quiescent RPE-1 cells with primary cilia were determined using either Ac. tub. or glutamylated tubulin (GT335) as a marker.
In (B), (C), (D), (E) and (G), average data obtained from three independent experiments is shown. Error bars represent ± standard deviations (SD). About 200 cells
for each siRNA condition were scored each time.
CP110 Inhibits CEP290-Induced Ciliogenesis
190 Developmental Cell 15, 187–197, August 12, 2008 ª2008 Elsevier Inc.
other CP110 fragments in quiescent 3T3 cells (Figures 3D and
3E). Like RPE-1, 3T3 fibroblasts are a well-established model
for cilia formation, as they readily develop primary cilia upon
entry into quiescence. We confirmed expression and proper
localization of each of these proteins by immunofluorescence
and found that only the full-length protein and the internal dele-
tion mutant localize to the centrosome (Figure 3D and data not
shown). Cells transfected with control plasmids developed
primary cilia upon induction of quiescence, as expected (Figures
3D and 3E). On the other hand, enforced expression of full-
length CP110 suppressed primary cilia formation (Figures 3D
and 3E), in line with previous observations (Spektor et al., 2007).
In striking contrast, loss of CEP290 binding led to a 3-fold re-
duction in inhibitory activity (Figures 3D and 3E), strongly sup-
porting the notion that CP110 binding to CEP290 is necessary
to prevent a CEP290-mediated cilia assembly program. Indeed,
expression of any CP110 truncation mutant unable to bind
CEP290 (200–565 and 350–991) fails to inhibit cilia assembly
(Figures 3C and 3E). Interestingly, CP110 fragments (1–223
and 1–565) that are nevertheless able to interact with CEP290
also fail to suppress ciliogenesis, most likely because they do
not localize to centrosomes (Figure 3C and data not shown).
Similar findings were also observed in RPE-1 cells (data not
shown). Theseresults establisha clearand important
Figure 3. A CP110 Mutant Unable to Bind CEP290 Fails to Inhibit Primary Cilia Formation
(A) The indicated fragments of Flag-tagged CP110 were expressed in 293T cells and immunoprecipitated from lysates. Flag-CP110 fusion proteins and CEP290
were detected after western blotting the resulting immunoprecipitates. Input CEP290 was detected in lysates from each transfection (IN).
(B) (Left panel) Flag (vector), Flag-tagged full-length CP110 (1–991), and Flag-tagged CEP290-binding mutant of CP110 (1–991(Daa67–82)) were expressed in
293T cells and immunoprecipitated from lysates. Flag-CP110 fusion proteins, CEP290, CEP97, and CaM were detected after blotting the resulting immunopre-
cipitates. IN represents input. (Right panel) Western blot of Flag-CEP290 and centrin after immunoprecipitation with anti-centrin antibodies using extracts from
293T cells transfected with the indicated constructs. IN represents input.
(C) Summary of CP110 truncation mutants and the results of in vivo binding experiments.
tub. (red), and with DAPI (blue). Bar: 10 mM; insets: 2 mM.
(E) The percentages of transfected, quiescent 3T3 cells expressing primary cilia were determined using Ac. tub. as a marker. About 100 transfected cells were
scored for each construct, and average data obtained from three independent experiments is shown. Error bars represent ±SD.
CP110 Inhibits CEP290-Induced Ciliogenesis
Developmental Cell 15, 187–197, August 12, 2008 ª2008 Elsevier Inc. 191
mechanistic link between CEP290 binding to CP110 and primary
CEP290 Depletion Affects Localization of Rab8a
to Centrosomes and to Primary Cilia
These findings prompted us to obtain further insights into
the molecular mechanisms underlying the role of CEP290 in
ciliogenesis; we examined several different well-established
centrosome/ciliary markers in CEP290-depleted quiescent cells
byimmunofluorescence. CP110 weakly butspecifically localizes
to the daughter centriole in serum-deprived cells (Figure S3).
Centrin and pericentrin are essential for centrosome and cilia
function and exhibit differential localizations at the primary cilium
(Jurczyk et al., 2004; Laoukili et al., 2000; Taulman et al., 2001).
Centrin is detected at the daughter centriole and at the mother
centriole (Figure S3), while pericentrin, a pericentriolar matrix
protein, is found at the periphery of the two centrioles and exists
as one dot (Figure S3). We observed no differences between the
staining patterns of CP110, centrin, and pericentrin in control
and CEP290-siRNA-treated cells (Figure S3), suggesting that
these proteins are targeted to centrosomes in the absence of
CEP290. This is consistent with our results indicating that cen-
trosomal function is not compromised in growing cells depleted
of CEP290 (Figures 2B–2D). Next, we examined the localization
of an IFT protein, Polaris/IFT88, found at the distal end of the
mother centriole and along the axoneme(Figure S3 andTaulman
et al. ). While we did not observe axonemal staining of
Polaris/IFT88, owing to an inhibition of cilia formation upon sup-
pression of CEP290, Polaris/IFT88 was nevertheless targeted to
one of the two centrioles (Figure S3). Similarly, the localization of
another IFT protein, IFT20 (Jurczyk et al., 2004; Yoshimura et al.,
2007), and a calcium cation channel, polycystin-2 (Jurczyk et al.,
2004), were not affected upon CEP290 depletion (Figure S3 and
data not shown). Since IFT20 and polycystin-2 localize primarily
to the mother centriole, these data suggest that localization of at
least a subset of proteins to the mother centriole is not perturbed
in the absence of CEP290.
We next asked whether other components critical for cilia
assembly might be affected by loss of CEP290. The Rab family
ical role in vesiculartrafficking(Zerialand McBride, 2001). Rab8a
isthe soleRabfamilymember enrichedatprimaryciliaandisab-
solutely essential for their formation (Yoshimura et al., 2007). It is
believed that Rab8a regulates cilia assembly by targeting and
velops the cilium and is continuous with the plasma membrane.
A molecular function for Rab8a in ciliogenesis has recently
been demonstrated, wherein it cooperates with the GTP ex-
mote ciliary membrane biogenesis (Nachury et al., 2007). Rab8a
localizes to the centrosome in growing cells and to the ciliary
membrane in quiescent cells (Figures 4A and 4C). Remarkably,
depletion of CEP290 led to a significant reduction of Rab8a at
centrosomes in growing cells (Figure 4B). These CEP290-de-
pleted cells often exhibit one or no detectable Rab8a dots
(Figure 4A, second and third rows), in contrast with two Rab8a
foci that colocalize with polyglutamylated tubulin in control cells
(Figure 4A, first row). Similar reductions in the number of Rab8a
foci were observed in serum-deprived cells that were depleted
of CEP290 and lacked primary cilia (Figures 4C and 4D). In con-
of Rab8a (as judged by the number of Rab8a foci) was not af-
fected by overexpression or RNAi-mediated depletion of CP110
(datanot shown).Our data suggestthat Rab8alocalizationto the
centrosome is dependent on CEP290 and that ciliogenesis is
regulated by a CEP290-Rab8a-dependent mechanism.
We next addressed whether CEP290 has a role in the entry of
Rab8a into the primary cilium. In a quiescent population treated
with control siRNA, 38% ± 3% of cells have cilia that are Rab8a
positive (Figures 5A and 5B). Strikingly, of the population of
CEP290 siRNA-treated cells that have retained their cilia, only
10% ± 2% were positive for Rab8a at the cilium (Figures 5A
and 5B). Interestingly, loss of CEP290 does not affect the entry
of Polaris/IFT88 into the cilium. The percentages of ciliated cells
that stained positive for Polaris/IFT88 at the cilium in control
versus CEP290 knockdown were 76% and 75%, respectively.
Next, we asked whether Rab8a protein levels were altered
upon knockdown of CEP290. We showed that there was no
difference in the abundance of total Rab8a between control
and CEP290-depleted cells (Figure 5C). Taken together, our
data suggest that CEP290 plays a role in recruiting Rab8a to
the centrosome and in regulating the entry of Rab8a into the
cilium during assembly of this organelle.
in vivo, we performed immunoprecipitations with CEP290,
CP110, and Rab8a antibodies. CEP290, CP110, and Rab8a
were immunoprecipitated with Rab8a antibodies (Figure 5D),
and reciprocal immunoprecipitations with CEP290 antibodies
(Figure 5D). Similarly, the interaction between CEP290 and
Rab8a was also observed in quiescent cells. However, both pro-
teins associated weakly with CP110, whose levels fall precipi-
tously in this setting (Figure 5D). Considered together, our data
indicate that CEP290 and Rab8a interact with one another in
growing and quiescent cells, while their interactions with
CP110 are mostly restricted to growing cells. These interactions
could provide proper cues for cilia assembly.
Rab8a in growingcells
Ectopic Expression of Certain CEP290 Fragments
Prevents Primary Cilia Formation
We next sought to determine which regions of CEP290 are in-
volved in binding Rab8a and the effect of ectopically expressing
our CEP290 truncation mutants (Figure 1C) in quiescent 3T3
cells. We found that a fragment spanning residues 580–1695
interacted with Rab8a, and use of additional deletion mutants
suggested that residues 1208–1695 were required for stable
binding to Rab8a (Figures 6A and 6B). Thus, CP110 and Rab8a
bind to mutually distinct portions of CEP290. The truncation
mutant containing residues 580–1695 is properly targeted to
the centrosome, and it supports the ability of cells to form cilia
(Figures 6B–6D). In contrast, expression of fragments containing
residues 362–822 or 1689–2050 leads to a significant decrease
in the number of ciliated cells (Figures 6B–6D). Interestingly,
both of these mutants localize to the centrosome, but they fail
to interact with Rab8a (Figures 6A–6C), suggesting that they
could exert their dominant-negative effect by displacing the
endogenous CEP290 and Rab8a from the centrosome. Thus,
CP110 Inhibits CEP290-Induced Ciliogenesis
192 Developmental Cell 15, 187–197, August 12, 2008 ª2008 Elsevier Inc.
our data clearly demonstrate a strong correlation between
Rab8a binding to CEP290 and cilia assembly.
We and others have previously described CP110 as a centroso-
mal protein that localizes to the distal end of centrioles (Chen
et al., 2002; Kleylein-Sohn et al., 2007). Here we show that
CP110interacts withCEP290,which localizesprimarily tocentri-
oles and basal bodies. CP110 is extinguished from the mother
centriole upon entry into quiescence, while CEP290 is found
on the daughter centriole and the mother centriole of a primary
cilium. These observations point to mechanistic differences in
their regulation and function and they suggest that there is
a pool of CEP290 that might function independently of CP110.
Indeed, CP110suppressesciliagrowthandformation ingrowing
cells, while CEP290 promotes primary cilia assembly. Depletion
of CEP290 has no noticeable effect on the cell cycle or the
Figure 4. CEP290 Recruits Rab8a to Centrosomes
RPE-1 cells transfected with control or CEP290 siRNAs were (A and B) grown asynchronously in the presence of serum or (C and D) induced to quiescence, and
stained with antibodies to glutamylated tubulin (GT335, green) and Rab8a (red), and with DAPI (blue). Bar: 10 mM; insets: 2 mM. (B and D) Histograms quantifying
the fold increase in the percentage of cells with fewer than two Rab8a foci at centrosomes. About 100 cells were scored for each siRNA condition, and the histo-
grams show the averages of three independent experiments. Error bars represent ±SD. The average percentage of cells with fewer than two Rab8a foci after
transfection with control siRNA was 16.5% of quiescent cells and 9.8% of growing cells.
CP110 Inhibits CEP290-Induced Ciliogenesis
Developmental Cell 15, 187–197, August 12, 2008 ª2008 Elsevier Inc. 193
centrosome cycle in growing cells. Furthermore, ablation of
CP110 does not enhance the ciliary phenotype observed after
CEP290 knockdown. These data suggest that CEP290 functions
exclusively in ciliary biogenesis and that CEP290 and CP110
functionally interact. IFT20, a component of the IFT particle
involved in anterograde transport, is localized to the centrosome
and is required only for cilia assembly (Follit et al., 2006; Yoshi-
mura et al., 2007). CEP290 and IFT20 may therefore represent
a new class of proteins that are strictly required for primary cilia
but not centrosome cycle functions, yet are assembled and tar-
bly program in quiescent cells.
Detailed molecular and cellular descriptions of the events
leading to the conversion of centrosomes to primary cilia have
not been obtained. Here, we identified a CP110 complex com-
prising CP110 and CEP290 that is distinguishable from CP110/
centrin/CaM and CP110/CEP97 complexes. In addition, CP110
and Rab8a bind to mutually distinct portions of CEP290, and
studies involving overexpression of CP110 suggest that CP110
does not compete with Rab8a in binding to CEP290 (data not
shown). Thus, CP110, CEP290, and Rab8a likely exist as one
distinct complex. It seems plausible that the main role of
CP110 in this complex is to keep CEP290 inactive in growing
cells until cells are ready to undergo ciliogenesis as they transi-
tion into the quiescent state (Figure 7). Intriguingly, our prelimi-
nary studies have shown that depletion of CEP290 disrupts
the migration of mother centrioles to the cell cortex, although
additional, higher-resolution studies will be required. In this con-
text, CP110 could act primarily on CEP290 to prevent migration
of centrioles to the cell surface. Upon transition into the quies-
cent state, CP110 is extinguished from the mother centriole
and its levels dramatically decrease (Figure 7). This could free
up CEP290 and allow proper migration and insertion of mother
centrioles into the cell cortex and subsequent implementation
of a cilia assembly program involving Rab8a as an effector.
CEP290 is present at the mother centriole and perhaps the tran-
sitional zone, a structure believed to regulate transport of pro-
tein cargos into and out of the cilium. Depletion of CEP290 re-
sults in a significant decrease of Rab8a at the centrosome and
at the cilium, raising the possibility that CEP290 first recruits
Rab8a through direct protein-protein interactions to the centro-
some in cycling cells and later promotes ciliogenesis by allowing
the entry of Rab8a into the cilium. Entry of Rab8a into the cilium
promotes the docking and fusion of membranous vesicles
thought to be critical for cilia formation. Consistent with a role
of CEP290 in mediating protein trafficking, a hypomorphic mu-
tation in the retinal degeneration 16 mouse (rd16) mouse model
is associated with redistribution of the ciliary protein Retinitis
Pigmentosa GTPase Regulator in photoreceptors (Chang et al.,
2006) and of G proteins in olfactory sensory neurons (McEwen
et al., 2007). Experiments are currently underway to further
delineate precisely how CEP290 promotes ciliogenesis and to
Figure 5. CEP290 Regulates Entry of Rab8a into the Primary Cilium and Interacts with Rab8a In Vivo
or Polaris/IFT88 (red), and with DAPI (blue). A ciliated cell is shown in each case. Bar: 10 mM; insets: 2 mM.
(B) A histogram quantifying the percentage of ciliated cells that were positive for Rab8a at the cilium. About 100 cells from the ciliated population of cells treated
with control or CEP290 siRNA were scored, and the averages of four independent experiments are shown. Error bars represent ±SD.
(C) Western blotting of CEP290 and Rab8a in RPE-1 cells treated with control or CEP290 siRNAs. a-tubulin was used as a loading control.
(D) Western blotting of endogenous CEP290, CP110, and Rab8a after immunoprecipitation with anti-Flag (control), anti-CEP290, anti-CP110, or anti-Rab8a
antibodies from growing or quiescent 3T3 cell extracts. IN, input.
CP110 Inhibits CEP290-Induced Ciliogenesis
194 Developmental Cell 15, 187–197, August 12, 2008 ª2008 Elsevier Inc.
identify additional molecular components involved in this
Mutations in CEP290 have been implicated in a broad spec-
trum of disease phenotypes, including BBS (Leitch et al.,
2008). Like CEP290, the BBS proteins, comprising a core com-
plexwithatleastseven highlyconserved members,areessential
for primary cilia formation. The ciliogenic function of the BBS
proteins appears to be mediated in part by Rab8a (Nachury
et al., 2007). Here, we have shown that CEP290 interacts with
Rab8a and that Rab8a binding to CEP290 is required for cilio-
genesis. Thus, interference with Rab8a function at the centro-
some and/or at the cilium could represent the molecular basis
Figure 6. Ecotpic Expression of Certain CEP290 Fragments Prevents Primary Cilia Formation
(A) The indicated fragments of Flag-tagged CEP290 were expressed in293T cells and immunoprecipitated from lysates. Flag-CEP290 fusion proteins and Rab8a
were detected after western blotting the resulting immunoprecipitates. Input Rab8a was detected in lysates from each transfection (IN).
(B) Summary of CEP290 truncation mutants and the results of in vivo binding experiments. The orange box denotes the CP110-binding domain based on the
yeast two-hybrid screen. NT, not tested.
(C) 3T3 cells transiently transfected with plasmids expressing Flag-CEP290 truncation mutants, induced to quiescence, and stained with antibodies to Flag
(green) and Ac. tub. (red), and with DAPI (blue) Bar: 10 mM; insets: 2 mM.
(D) The percentagesof transfected, quiescent 3T3 cells expressing primary cilia were determined using Ac. tub. as amarker. About 100 transfectedcells for each
construct were scored, and average data obtained from three independent experiments are shown. Error bars represent ±SD.
Figure 7. A Model Depicting the Role of
CEP290 in Mediating Ciliogenesis
In growing cells, CP110 antagonizes the action of
CEP290, which in turn prevents CEP290-depen-
dent Rab8a ciliogenesis. Another complex con-
taining CP110 and CEP97 also serves to suppress
primary cilia assembly. When cells exit the cell
cycle, the levels of CP110 diminish dramatically.
This frees up CEP290, which in turn cooperates
with Rab8a to promote primary cilia formation.
CP110 Inhibits CEP290-Induced Ciliogenesis
Developmental Cell 15, 187–197, August 12, 2008 ª2008 Elsevier Inc. 195
mutations in Rab8a have been found so far, disruption of Rab8a
function recapitulates the BBS phenotype in zebrafish (Nachury
et al., 2007). Given the significant overlap of clinical manifesta-
tions in patients with BBS and CEP290 mutations and the con-
nection between BBS proteins and CEP290 with Rab8a, we hy-
pothesize that BBS proteins may associate with CEP290.
Although we and others have not been able to detect an interac-
tion between BBS4 and CEP290 (McEwen et al., 2007; and data
not shown), further studies will be needed to address whether
CP110 and CEP290 are directly linked to Rab8a via other BBS
Cell Culture and Plasmids
Human 293T, T98G, U2OS, RPE-1 hTERT, and mouse 3T3 cells were grown in
DMEM supplemented with 10% FBS at 37?C in a humidified 5% CO2atmo-
sphere. Flag-tagged CP110 fusion proteins were described previously (Spek-
tor et al., 2007). To generate Flag-tagged CEP290 fusion proteins in vivo,
CEP290 fragments encoding residues 1–2479, 1–366, 221–366, 362–822,
816–1207, 580–1695, 1689–2050, and 2037–2479 were amplified by PCR
using Pfu Turbo polymerase (Stratagene) and subcloned into the EcoRV and
HindIII sites of the mammalian vector pCBF (generous gift from M. Cole). All
constructs were verified by DNA sequencing.
Antibodies used in this study included polyclonal rabbit anti-CEP290 (Chang
et al., 2006), anti-CP110 (Chen et al., 2002), anti-CEP97 (Spektor et al.,
2007), anti-centrin mouse monoclonal 20H5 (generous gift from J. Salisbury),
anti-polycystin-2 (generous gift from Y. Cai and S. Somlo), anti-CaM (Upstate),
anti-GFP (Roche), anti-Ki-67 (Zymed), anti-pericentin (Covance), anti-a-tubu-
lin, anti-acetylated tubulin, anti-Flag and anti-g-tubulin (all from Sigma-Al-
drich), anti-glutamylated tubulin GT335 (generous gift from C. Janke), anti-Po-
laris/IFT88 (generous gift from B. Yoder), and anti-Rab8a (BD Biosciences and
Peranen et al., 1996).
Yeast Two-Hybrid Screen
The Matchmaker Two-Hybrid System 3 (BD Biosciences) was used to identify
clones that interact with CP110. The full-length cDNA of CP110 was cloned
into the NcoI and SalI sites of pGBKT7 bait vector. The resultant plasmid
was transformed into the yeast reporter strain AH109. A BD Matchmaker
pretransformed human brain cDNA library (BD Biosciences) was screened
by yeast mating following the manufacturer’s recommendations. b-galactosi-
dase activity of colonies growing on stringent selection medium (SD/-Ade/-
by the manufacturer. A total of 480 positive colonies were picked and tested.
Eighty-two clones were then selected based on the X-gal assay. Plasmids
from these positive clones were extracted and sequenced. The corresponding
genes were identified by BLASTn alignment to the NCBI database.
Cell-Cycle Synchronization and FACS Analysis
T98G cells were synchronized by serum deprivation and restimulation as
described (Tsang et al., 2007). FACS analysis was performed as reported pre-
viously (Tsang et al., 2007).
Immunoprecipitation, Immunoblotting, and Immunofluorescence
Cells were lysed with buffer containing 50 mM HEPES (pH 7), 250 mM NaCl,
5 mM EDTA (pH 8), 0.1% NP-40, 1 mM DTT, 0.5 mM PMSF, 2 mg/ml leupeptin,
2 mg aprotinin, 10 mM NaF, 50 mM b-glycerophosphate, and 10% glycerol at
4?C for 30 min. After centrifugation, 2 mg of the resulting supernatant was
incubated with an appropriate antibody at 4?C for 1 hr and collected using
protein A or G-Sepharose. The resin was washed with lysis buffer, and the
bound polypeptides were analyzed by SDS-PAGE and immunoblotting. Typi-
cally, 50–100 mg of lysate was loaded into the input (IN) lane. For mapping
studies, Flag-tagged constructs were transfected into 293T cells. Cells were
harvested 48–72 hr after transfection. Flag-beads were incubated with cell
extract at 4?C for 2 hr. After washing with lysis buffer, bound proteins were
analyzed by SDS-PAGE and immunoblotting. Indirect immunofluorescence
was performed as described (Chen et al., 2002). Briefly, cells were grown on
glass coverslips and fixed with cold methanol for 2 min. The cells were per-
meabilized with 1% Triton X-100/PBS for 5 min. Slides were blocked with
3% BSA in 0.1% Triton X-100/PBS prior to incubation with primary antibodies.
Secondary antibodies used were Cy3- or FITC- conjugated donkey anti-
mouse or anti-rabbit IgG (Jackson Immunolabs). Cells were then stained
with DAPI, and slides were mounted, observed, and photographed using
a Zeiss Axiovert 200M microscope (633, NA 1.4, 1.6 Optovar) equipped
with a cooled Retiga 2000R CCD (QImaging). For analysis of the distance be-
tween centrosomes and nuclei, z-sections spaced by 0.2–0.5 mm were taken.
Synthetic siRNA oligonucleotides were obtained from Dharmacon. Transfec-
tion of siRNAs using Oligofectamine or Lipofectamine 2000 (Invitrogen) was
performed according to the manufacturer’s instructions. The 21-nucleotide
siRNA sequence for the nonspecific control was 50-AATTCTCCGAACGTGT
CACGT-30. The 21-nucleotide siRNA sequences for CEP290 used were
30. The siRNAs for CP110 silencing have been described previously (Spektor
et al., 2007).
Induction of Primary Cilia
RPE-1 or 3T3 cells transfected with siRNA or plasmid DNA were brought to
quiescence by serum starvation for 48–72 hr. Cells were examined for well-es-
tablished primary cilium markers such as acetylated tubulin or glutamylated
Superose 6 Gel Filtration Analysis
Two milligrams of extract were chromatographed over a Superose 6 gel filtra-
tion column (Pharmacia) in lysis buffer (50 mM HEPES [pH 7], 250 mM NaCl,
5 mM EDTA [pH 8], 0.1% NP-40, 0.5 mM PMSF, and 10% glycerol). Equal
volumes of each fraction were precipitated with TCA and analyzed by
SDS-PAGE and immunoblotting.
The statistical significance of the difference between two means was deter-
mined using a two-tailed Student’s t test. Differences were considered signif-
icant when p < 0.01.
Supplemental Data include three figures and are available online at http://
We wish to thank M. Takahaski and Y. Ono for the gift of anti-kendrin antibody;
J. Salisbury for anti-centrin antibody (20H5); Y. Cai and S. Somlo for anti-poly-
cystin-2 antibody (YCC2); C. Janke for anti-polyglutamylated tubulin antibody
(GT335); B. Yoder for anti-Polaris/IFT88 antibody; M. Cole for the gift of pCBF
plasmid DNA; and A. Khodjakov for providing the RPE-1 hTERT cell line. We
thank I. Sanchez, A. Spektor, and S. Vijayakumar for critical reading of the
manuscript. Weareespeciallygrateful toP.Aspinour laboratory forinvaluable
assistance with FPLC purification. We thank all members of the Dynlacht lab-
oratory for constructive advice and encouragement. This work was supported
in part by an Irma T. Hirschl Career Scientist Award to B.D.D., and by grants
from the National Institutes of Health (EY007961) and Midwest Eye Banks
and Transplantation Center to A.S. and H.K. C.B. was supported by a Human
Frontier Science Program postdoctoral fellowship. W.Y.T. was supported by
an Alberta Heritage Foundation for Medical Research full-time postdoctoral
CP110 Inhibits CEP290-Induced Ciliogenesis
196 Developmental Cell 15, 187–197, August 12, 2008 ª2008 Elsevier Inc.
Received: March 11, 2008 Download full-text
Revised: June 3, 2008
Accepted: July 7, 2008
Published: August 11, 2008
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