Supernumerary Centrosome

Current Biology 22, 1628–1634, September 11, 2012 ª2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.06.057

Reports

Nucleate Extra Cilia and CompromisePrimary Cilium Signaling

Moe R. Mahjoub1 and Tim Stearns1,2,*1Department of Biology, Stanford University, Stanford,CA 94305, USA2Department of Genetics, Stanford School of Medicine,Stanford, CA 94305, USA

Summary

The primary cilium is a nexus of cell signaling, and ciliarydysfunction is associated with polycystic kidney disease,

retinal degeneration, polydactyly, neural tube defects, andobesity (ciliopathies) [1]. Signaling molecules for cilium-

associated pathways are concentrated in the cilium, andthis is essential for efficient signaling. Cilia are nucleated

from centrioles, and aberrant centriole numbers are seenin many cancers and in some ciliopathies [2]. We tested

the effect of supernumerary centrioles on cilium functionand found that cells with extra centrioles often formed

more than one cilium, had reduced ciliary concentrationof Smoothened in response to Sonic hedgehog stimulation,

and reduced Shh pathway transcriptional activation. Thisciliary dilution phenotype was also observed with the

serotonin receptor Htr6, fibrocystin PKHD1, and Arl13b.The presence of extra centrioles and cilia disrupted epithe-

lial organization in 3D spheroid culture. Cells mutant forthe tuberous sclerosis gene Tsc2 also had extra cilia

and diluted ciliary protein. In most cells, extra cilia were

clustered and shared the same ciliary pocket, suggestingthat the ciliary pocket is the rate-limiting structure for

trafficking of ciliary proteins. Thus, extra centrioles andcilia disrupt signaling and may contribute to disease

phenotypes.

Results and Discussion

The primary cilium grows from the older of the two centriolesin quiescent cells. Centriole number is maintained by aduplication and segregation mechanism linked to the cellcycle [3], and centriole amplification is a common character-istic of many cancers [4]. Centriole amplification has alsobeen observed in renal tissues from mouse models andhuman patients bearing mutations in a subset of ciliopathygenes, including cells from PKD1 deletion mice and humanautosomal dominant polycystic kidney disease patients[5], fibroblasts and mesenchymal cells from PKD2 dele-tion mice [6], cells from tuberous sclerosis (TSC1 deletion)mice [7], and cells from Mks1 or Mks3 mouse and humanpatients with Meckel syndrome [8]. It is unclear what pheno-types are conferred upon cells having extra centrosomesthat might lead to cilia-related diseases. Here we considerthe possibility that extra centrosomes might result in morethan one primary cilium and thus affect normal ciliarysignaling.

*Correspondence: stearns@stanford.edu

Supernumerary Centrioles Cause Superciliated Cells

Defective in Shh SignalingTo investigate the effects of multiple cilia, we manipulated thenumber of cilia independent of genome ploidy. Overexpres-sion of the kinase Plk4 induces the formation of multiple cen-trioles in a diversity of cell types; thus, we tested whetherPlk4-induced centriole amplification would result in the forma-tion of extra cilia. Plk4 was overexpressed by two methods:transient transfection of a Plk4-expressing plasmid and tran-sient induction of a tet-inducible Plk4 stable cell line. BecausePlk4 is an unstable protein, both methods resulted in a tran-sient increase in Plk4 levels, and both gave similar results,causing the formation of extra centrioles as described [9].To test whether the extra centrioles created by Plk4 over-

expression can make cilia, we held cells overexpressing Plk4in S phase for 16 hr by thymidine treatment and then releasedfrom S phase for two subsequent rounds of cell division toallow the newly assembled centrioles tomature and potentiallybecome competent for cilium formation [10]. We found thatin many cells with extra centrioles, some of those centriolesformed a cilium, resulting in superciliated cells containingtwo to six primary cilia per cell (Figures 1A and 1B; see TableS1 available online). As expected, the fraction of ciliated cellswith more than one cilium increased over time due to matura-tion of centrioles by passage through the cell cycle (Table S1).Quantification of cilium length in cells with one, two, or threeprimary cilia showed that, on average, the length of each ciliumin these cells was similar (w4 mm, Figure 1C).We considered that an increased number of primary cilia

might cause changes in signaling pathways that rely on ciliumfunction. We focused first on the transmembrane proteinSmoothened (Smo), which translocates into the cilium inresponse to Sonic Hedgehog (Shh) ligand [11]; this transloca-tion is essential in activating downstream signaling in mam-malian cells [12]. Plk4 was expressed in mouse embryonicfibroblasts to generate superciliated cells, which were treatedwith Shh and stained for Smo (Figure 1D; [12]). We used quan-titative single-cell fluorescence to measure the total ciliaryamounts of Smo in cells with one, two, or three primary cilia(Figure 1D). Surprisingly, the total amount of ciliary Smo wassimilar in mono-, bi-, and triciliated cells (Figure 1E). Becausethe total cilium length increased linearly with cilium number(Figure 1C), this was most likely due to reduced concentrationof Smo in each cilium in superciliated cells. To confirm this, wedetermined the amount of Smo per unit length of cilium bymeasuring the ratio of Smo signal intensity with respect tociliary axoneme tubulin staining. The amount of Smo per unitlength was reduced in biciliated cells compared to monocili-ated cells and further reduced in triciliated cells (Figure 1F),indicating that the Smo protein is diluted in the cilia of super-ciliated cells.We tested whether the dilution of ciliary Smo in superciliated

cells perturbed the response of the pathway. Hedgehogresponse was assayed using an NIH3T3 reporter cell linethat expresses GFP under the control of Gli response elements[13, 14]. NIH 3T3::Gli-GFP cells were transfected with Plk4to generate superciliated cells and treated with Shh, induc-ing expression of Gli-GFP. Quantification of GFP levels in

Figure 1. Superciliated Cells Are Defective in Shh Signaling

(A) Schematic depicting generation of superciliated cells.

(B) Examples of NIH 3T3 cells with amplified centrioles and cilia, stained for glutamylated tubulin (centrioles and cilia; red) and DNA (blue). Scale bars repre-

sent 10 mm and 2 mm (right panel).

(C) Average lengths of cilia in mono- and superciliated cells.

(D) Endogenous Smo localization (green) in mono- and superciliated cells. Cilia were costained for glutamylated tubulin (G.T., red) and DNA (blue). Scale bar

represents 10 mm.

(E) Distribution of total Smo signal intensity in cilia of mono-, bi- and triciliated cells.

(F) Ratio of Smo/tubulin levels in cilia of mono-, bi-, and triciliated cells.

(G) Graph of GFP signal intensity in mono- and biciliated cells before and after Shh treatment. For all graphs, n = 50 for each sample (from two independent

experiments; *** indicates p < 0.0001).

Error bars are SD.

Extra Cilia Compromise Ciliary Signaling1629

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mono- versus biciliated cells revealed a significant reductionin the latter (Figure 1G), demonstrating that dilution of Smoin cilia of superciliated cells results in defective activation ofthe Shh pathway.

Generality of the Ciliary Dilution Phenotype

in Superciliated CellsWe next addressed the generality and nature of the dilutionphenotype in superciliated cells by examining the ciliaryconcentrations of additional proteins that localize to cilia. Wefound that the serotonin 6 (Htr6) receptor, the fibrocystinprotein, and the GTPase Arl13b all exhibited a dilution pheno-type similar to that of Smo in superciliated cells (SupplementalResults; Figures S1 and S2). The ciliary dilution phenotypewas observed even under conditions in which the total con-centration of the transported protein was not limiting (Supple-mental Results; Figure S1). Because superciliated cellsassembled cilia of similar length to those in monociliated cells,we reasoned that components of the ciliary machinery mightnot display the ciliary dilution phenotype. Consistent withthis hypothesis, the ciliary concentration of IFT88, a compo-nent of intraflagellar transport, was equal in mono- and bicili-ated cells (Figure S2C). Lastly, we found that the ciliary dilutionphenotype was dependent on number of cilia rather thannumber of centrioles (Figure S2B) and was independent ofnuclear ploidy (Figure S2D), suggesting that the mechanismdetermining ciliary protein levels assesses the number of ciliaper cell, rather than cilia per unit genome.

Disease-Related Defects Associated with Presenceof Extra Cilia

We sought to determine the functional consequences ofhaving extra cilia in a cell or tissue system that relies on ciliumfunction. Ciliary signaling is essential in organizing the archi-tecture of epithelial cells; components of both the canonicaland noncanonical (planar cell polarity) Wnt signaling path-ways localize to cilia and depend on proper cilia function toestablish cell polarity [15]. Growth of epithelial cells in a 3Dgel matrix allows cells to organize into polarized, spheroidstructures that resemble in vivo epithelium architecture [16].Spheroid systems have been used to study the activities ofoncogenes with regard to their ability to disrupt epithelialarchitecture early during tumor formation [16] and haverecently been used to assay cilia dysfunction related to muta-tions implicated in ciliopathies [17, 18]. IMCD-3 cells grownin Matrigel for 4 days formed spheroids with a prominentlumen, apically located cilia projecting into the lumen, andwell defined apical and basolateral junctions (Figure 2A). Incontrast, 84% of superciliated IMCD-3 cells made by transientexpression of Plk4 failed to develop spheroid structures,instead forming clusters of cells with irregular or nonexistentlumens, disorganized apical and basolateral junctions, andrandomly oriented cilia (Figures 2A and 2B). Note that it isnot possible to distinguish between the contribution of theextra centrioles versus the extra cilia in this assay, becauseperturbing ciliary structure and function (even in cells withthe normal complement of centrioles) leads to defectivespheroid formation [17, 18]. Thus, the presence of extracentrosomes and/or cilia disrupts epithelial organization inthis in vitro tissue model.

We next examined the effect of extra cilia caused by anaturally occurring disease mutation. Tuberous sclerosis isa multisystemic disease in which patients develop benigntumors in the brain, retina, kidney, heart, and skin [19].

Causative mutations occur in two tumor suppressor genes(TSC1 and TSC2), the gene products of which function asa complex to inhibit mTOR [20]. Interestingly, mutations inTSC1 and TSC2 have recently been linked to ciliary dysfunc-tion. MEFs isolated from Tsc12/2 and Tsc22/2 mice show en-hanced ciliary formation, and many Tsc22/2 MEFs have extracentrosomes and cilia [21]. To test whether Tsc22/2 supercili-ated MEFs showed the ciliary dilution phenotype, we treatedTsc22/2 and control Tsc2+/+ MEFs with Shh and the amountof Smo per unit cilium length was determined. Ciliary Smoconcentration was reduced in biciliated Tsc22/2 cells com-pared to monociliated cells of either Tsc22/2 or Tsc2+/+ geno-type (Figure 2C). This phenotypemight be relevant to tuberoussclerosis disease, given the similarity between the diseasephenotype at the tissue level and the epithelial disorganiza-tion we observed in spheroid cultures.Finally, to address potential mechanisms underlying the

ciliary dilution effect, we examined the ultrastructure of thecilia in superciliated cells. In the experiments above, we notedthat in most superciliated cells (>95%) the centrioles wereclustered and that the primary cilia were situated next toeach other. This raised the possibility that the multiple cilia insuperciliated cells shared the same membrane compartmentat the surface of the cell. The proximal portion of the primarycilium is part of a specialized membrane compartment, theciliary pocket [22], an invagination of the plasma membranein which the primary cilium is rooted. The ciliary, or flagellar,pocket is important for trafficking of ciliary components intrypanosomatids and is the unique site of endocytosis andexocytosis [23] and may be performing a similar role inmammalian cells [24]. We tested whether the multiple cilia inour superciliated cells might occupy the same ciliary pocket,limiting the amount of ciliary signaling proteins available toeach cilium. To test this hypothesis, we used transmissionelectron microscopy to examine the ciliary pocket in supercili-ated RPE-1 cells. Longitudinal sections through the ciliaryregion of control monociliated cells showed the typical ciliarypocket structure (Figure 3A). Remarkably, serial sections ofsuperciliated cells revealed that in each cell in which multiplecilia could be identified (n = 6 cells), those cilia occupied thesame ciliary pocket (Figure 3A). To test whether the ciliary dilu-tion phenotype might be due to this clustering of cilia, wecompared the concentration of ciliary Arl13b in cells wherethe cilia were clustered to that in the small fraction of cells(<5%) in which the cilia were unclustered and presumablyresiding in separate ciliary pockets (Figure 3B). We foundthat the concentration of Arl13b in unclustered cilia was similarto that in cells with one cilium (Figure 3C), supporting thehypothesis that the ciliary pocket is the rate-limiting structurefor trafficking of ciliary proteins.

Conclusion

We show that extra centrioles often result in extra cilia and thatpresence of extra cilia results in reduced ciliary concentrationof signaling molecules in cilia. This dilution phenotype is likelya direct effect of extra cilia because the same effect wasobserved in superciliated cells generated by transient Plk4expression or by a cytokinesis block, as well as in Tsc22/2

null MEFs. That the presence of extra cilia can lead to alteredcilium structure and function is supported by results demon-strating a reduction in canonical Wnt pathway activation inbiciliated cells [25]. How might the presence of extra ciliaresult in a signaling defect? In the case of the Shh pathway,we propose that decreased pathway activation results from

Figure 2. Disease-Related Defects Associated with Presence of Extra Cilia

(A–B) Immunofluorescence images of monociliated (Control) and superciliated (SCC) IMCD-3 cells grown in 3D culture. Most monociliated cells formed

spheroids with a prominent lumen (93%, n = 400), whereas most superciliated cells failed to develop spheroids (84%, n = 400); p < 0.0001. Arrow in (A)

denotes the region that is magnified 3-fold and displayed in (A’). Arrows in (B) mark cells in an SCC cluster with multiple cilia; arrowheads mark misoriented

cilia. Scale bars represent 10 mm.

(C) Immunofluorescence images of endogenous Smo (green) in monociliated Tsc2+/+ and biciliated Tsc22/2 MEFs, costained with glutamylated tubulin

(G.T., red) and DNA (blue). Scale bar represents 10 mm. Graph (right panel) shows ratio of Smo/glutamylated tubulin levels. n = 50 for each sample (from

two independent experiments; p < 0.0001).

Extra Cilia Compromise Ciliary Signaling1631

decreased ciliary concentration of Smo, as well as othercomponents of that pathway, several of which are known tobe localized to the cilium [12]. This would result in reducedinteraction between these components in the ciliary compart-ment and compromised signaling (Figure 3D). This contrastswith the mechanism of ciliary dysfunction caused by muta-tions in canonical ciliopathy genes, in which the moleculardefect appears to be the absence of particular ciliary proteins(Figure 3D). Although the mechanisms of ciliary dysfunctionare different in these cases, we posit that ciliary dilution due

to supernumerary centrosomes and cilia might lead to thesame phenotypic output.Centrosome aberrations are associated with renal cystic

disease [2], but a causative role for centrosome defects incystogenesis has not been established. We speculate thatthe ciliary signaling defects in superciliated cells might playa role in the initiation of cyst formation. Interestingly, cells inkidney tissue from a patient bearing a mutation in MKS3were found to have extra centrioles and cilia in vivo [8], andkidney cysts in patients with mutations in PKD1 have

Figure 3. Architecture of the Ciliary Pocket in Superciliated Cells

(A) Transmission electronmicroscopy of wild-type (Control) and superciliated (SCC) RPE-1 cells. Images show a set of four serial sections through the ciliary

pocket of a superciliated cell. In this cell, there are three centrioles with associated cilia (numbered 1–3), as well as other centrioles not associated with cilia,

below the pocket. Themembrane of the ciliary pocket is indicatedwith black stippling, and themembranes of the three cilia are colored red, blue, and green,

as indicated. Cilia 1 and 3 are orientedmostly in the plane of the sections, whereas cilium 2 is oriented obliquely to the section plane and is thus only partially

visible. Scale bars represent 0.5 mm.

(B) Immunofluorescence images of endogenous Arl13b in RPE-1 cells, containing either 1, 2-clustered, or 2-unclustered cilia. Cells were costained with glu-

tamylated tubulin. Scale bar represents 2 mm.

(C) Level of endogenous Arl13b per unit length, in monociliated, biciliated clustered, and unclustered cells. n = 40 for each sample (from two independent

experiments; p < 0.0001).

(D) Model depicting ciliary dysfunction caused by mutation of a ciliopathy protein, resulting in loss of a cilium signaling component, or by dilution of such

components in superciliated cells where the cilia are residing in a single ciliary pocket.

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superciliated cells in vivo (unpublished data). Thus, defectscaused by extra cilia might be an important aspect of a setof cilia-related diseases.

How might the concentration of ciliary proteins be affectedby extra cilia? Localization to the cilium is controlled at several

levels, including a barrier to diffusion of cytoplasmic andmembrane proteins into the cilium [26, 27], specificmembrane-targeting pathways for ciliary membrane proteins[28] and a cilium-specific transport mechanism (intraflagellartransport; [29]). We propose that these pathways act at the

Extra Cilia Compromise Ciliary Signaling1633

level of the ciliary pocket, the unique membrane structure atthe base of the cilium. We find that more than one cilium canoccupy the ciliary pocket in superciliated cells, suggestingthat the ciliary pocket is the rate-limiting structure for traf-ficking of ciliary proteins and that the ciliary dilution phenotypederives from the distribution of trafficked proteins among thecilia sharing that pocket. Further work on the factors involvedin centriole clustering in interphase and on the assembly of theciliary pocket will help to establish the molecular mechanismof the ciliary dilution phenotype.

Experimental Procedures

Generating Superciliated Cells

Extra centrioles were generated by transfecting cells with a construct ex-

pressing Plk4 using Lipofectamine 2000 (Invitrogen), according to the

manufacturer’s protocol. Cells were arrested in S phase by treatment with

2 mM thymidine for 16 hr to induce centriole amplification, then incubated

with fresh medium and allowed to grow for 2 additional days to allow

centriole maturation. Alternatively, tetracycline-inducible RPE-1::Tet-Plk4

were incubated with 1 mg/mL doxycycline and 2 mM thymidine for 16 hr to

induce expression of Plk4 while maintaining arrest in S phase. Cells were

then grown in fresh medium (without doxycycline) for 2 additional days to

allow centriole maturation. To induce ciliogenesis, we incubated cells with

low-serum (0.5%) medium for 24 hr. Activation of the Shh pathway was

achieved by treating cells (MEFs or NIH 3T3) with Shh-conditioned medium

for 16 hr, as previously described.

Immunofluorescence Microscopy

Samples were fixed in either 100%methanol at220�C for 10 min or with 4%

paraformaldehyde in PBS at room temperature for 10 min, depending on

antigen. Following fixation, cells were rinsed twice with PBS and briefly

extracted with 0.1% Triton X-100 in PBS and blocked for 1 hr at room

temperature with 3% BSA (Sigma-Aldrich) in PBS. Samples were incubated

with primary antibodies for either 1 hr at room temperature or 4�C overnight.

Primary antibodies used in this study were as follows: rabbit anti-Smo

(1:2,000; gift from Rajat Rohatgi, Stanford, CA), mouse anti-acetylated

tubulin (clone 6-11b-1; 1:5,000; Sigma), mouse anti-glutamylated tubulin

(GT335; 1:2,000; gift from C. Janke, Centre de Recherches de Biochimie

Macromoleculaire, Montpellier, France), rabbit anti-GFP (generated in our

lab), rabbit anti-IFT88 (1:1,000; gift from Bradley Yoder, University of

Alabama at Birmingham, AL), rat anti-Zo-1 (1:1,000; American Research

Products), and rabbit anti-b-catenin (1:1,000; Santa Cruz Biotechnology).

Alexa dye-conjugated secondary antibodies (Invitrogen) were used at a

dilution of 1:500 at room temperature for 1 hr. Samples were mounted using

Mowiol mounting medium containing N-propyl gallate (Sigma-Aldrich).

Images were captured using Openlab 4.0.4 (Improvision) controlling an

Axiovert 200M microscope (Carl Zeiss MicroImaging, Inc.). Images of

spheroids were captured on a Zeiss LSM 510 Confocal Laser Scanning

microscope using a 25X oil-immersion objective and a scan zoom of 4. A

series of digital optical sections consisting of 12 sections in the z axis

(total of 25 mm) was captured, and three-dimensional image reconstruc-

tions were produced.

Quantification of Signal Intensity and Statistical Analysis

The ciliary fluorescence intensity was calculated using ImageJ software

(http://rsbweb.nih.gov/ij/). Briefly, a region encompassing each cilium was

selected, and the total fluorescence intensity for a particular protein was

determined for each cilium. Subsequently, the total fluorescence intensity

of glutamylated tubulin in each cilium was obtained using the same param-

eters. In both cases, a background subtraction was performed by quanti-

fying the fluorescence intensity of a region of equal dimensions in the

area neighboring the cilium. The ratio of protein/tubulin was determined

and graphs were generated using Excel software (Microsoft). For statistical

analysis, unpaired two-tailed Student’s t test was performed using Excel. N

value for each experiment is noted in the respective figure legend.

Supplemental Information

Supplemental Information includes two figures, one table, Supplemental

Results, and Supplemental Experimental Procedures and can be found

with this article online at http://dx.doi.org/10.1016/j.cub.2012.06.057.

Acknowledgments

We thank R. Rohatgi for anti-Smo antibody, C. Janke for anti-glutamylated

tubulin, B. Yoder for anti-IFT88, M. Nachury for IMCD-3::Htr6-GFP and

IMCD-3:: CTSPKHD1-GFP cells, B. Tsou for RPE-1::Tet-Plk4 cells, E. Henske

for Tsc2+/+ and Tsc22/2 MEFs, and J. Chen for NIH3T3:Gli-GFP reporter

cells. We also thank John Perrino (Stanford Cell Sciences Imaging Facility)

for help with electron microscopy. This work was supported by National

Institutes of Health grant GM52022 to T.S.

Received: April 11, 2012

Revised: May 30, 2012

Accepted: June 18, 2012

Published online: July 26, 2012

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