The deubiquitinating enzyme CYLD controls apical docking of basal bodies in ciliated epithelial cells

Abstract

CYLD is a tumour suppressor gene mutated in familial cylindromatosis, a genetic disorder leading to the development of skin appendage tumours. It encodes a deubiquitinating enzyme that removes Lys63- or linear-linked ubiquitin chains. CYLD was shown to regulate cell proliferation, cell survival and inflammatory responses, through various signalling pathways. Here we show that CYLD localizes at centrosomes and basal bodies via interaction with the centrosomal protein CAP350 and demonstrate that CYLD must be both at the centrosome and catalytically active to promote ciliogenesis independently of NF-κB. In transgenic mice engineered to mimic the smallest truncation found in cylindromatosis patients, CYLD interaction with CAP350 is lost disrupting CYLD centrosome localization, which results in cilia formation defects due to impairment of basal body migration and docking. These results point to an undiscovered regulation of ciliogenesis by Lys63 ubiquitination and provide new perspectives regarding CYLD function that should be considered in the context of cylindromatosis.

ARTICLE

Received 7 May 2014 | Accepted 3 Jul 2014 | Published 19 Aug 2014

Thibaut Eguether1,2,w, Maria A. Ermolaeva3, Yongge Zhao4, Marion C. Bonnet3,5, Ashish Jain4, Manolis Pasparakis3, Gilles Courtois6,7 & Anne-Marie Tassin1,8

CYLD is a tumour suppressor gene mutated in familial cylindromatosis, a genetic disorder leading to the development of skin appendage tumours. It encodes a deubiquitinating enzyme that removes Lys63- or linear-linked ubiquitin chains. CYLD was shown to regulate cell proliferation, cell survival and inammatory responses, through various signalling pathways. Here we show that CYLD localizes at centrosomes and basal bodies via interaction with the centrosomal protein CAP350 and demonstrate that CYLD must be both at the centrosome and catalytically active to promote ciliogenesis independently of NF-kB. In transgenic mice engineered to mimic the smallest truncation found in cylindromatosis patients, CYLD interaction with CAP350 is lost disrupting CYLD centrosome localization, which results in cilia formation defects due to impairment of basal body migration and docking. These results point to an undiscovered regulation of ciliogenesis by Lys63 ubiquitination and provide new perspectives regarding CYLD function that should be considered in the context of cylindromatosis.

1 Institut Curie/INSERM U759, Campus Universitaire, Bat 112, 91405 Orsay Cedex, France. 2 Universit Pierre et Marie Curie, 75005 Paris, France. 3 Institute for Genetics, Center for Molecular Medicine (CMMC) and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Joseph-Stelzmann-Strasse 26, 50931 Cologne, Germany. 4 Laboratory of Host Defenses, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA. 5 Excellence Research Chair, Universit Europenne de Bretagne, IRSET/INSERM UMR1085, Facult de Pharmacie, Universit de Rennes 1, 35000 Rennes, France. 6 Universit Grenoble Alpes, 38000 Grenoble, France. 7 INSERM U1038/BGE/Institut de Recherches en Technologies et Sciences pour le Vivant, CEA, 38054 Grenoble, France. 8 CNRS, Centre de Gntique Molculaire, UPR3404, Avenue de la Terrasse, F-91198 Gif-sur-Yvette, France. w Present address: University of Massachusetts Medical School, Biotech II, 373 Plantation Street, Worcester, Massachusetts 01605,

USA. Correspondence and requests for materials should be addressed to A.-M.T. (email: mailto:[email protected]

Web End [email protected] ).

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DOI: 10.1038/ncomms5585

The deubiquitinating enzyme CYLD controls apical docking of basal bodies in ciliated epithelial cells

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5585

Centrioles are conserved microtubule-based organelles displaying structural asymmetry, with the mother centriole bearing two sets of appendages at its distal end. In

resting cells, the mother centriole can turn into a so-called basal body and dock to the plasma membrane through the distal appendages, to complete primary cilia formation1. In addition to these primary cilia, multiple motile cilia are present in some mammalian epithelia (for example, brain ventricles, respiratory tract and fallopian tubes). In contrast to cycling cells, these multiciliated cells have the ability to assemble hundreds of centrioles that anchor to the plasma membrane and grow cilia2. In all cases, ciliogenesis is a multistep process that includes basal body assembly, maturation and migration, transition zone formation, docking to the cell surface and further axonemal extension, which requires intraagellar transport3.

Primary cilia are considered as cell antennas that receive extracellular signals and transmit them to the cell bodies, thus coordinating cell growth, polarity and differentiation4,5. Among their role in sensing the environment, cilia are required to mediate several signal transduction pathways6. Therefore, any defects in this organelle lead to a wide range of developmental disorders and diseases grouped under the term ciliopathies1.

Among the numerous centrosomal proteins, CAP350 serves as a platform to anchor several proteins to the centrosome. FOP (FGFR1 oncogene partner) localization at the centrosome depends on its association with CAP350 and is required to recruit EB1 at the centrosome7. The complex CAP350/FOP was shown to be required for microtubule anchoring to the centrosome. In addition, as a microtubule binding protein CAP350 stabilizes the microtubule network8 as well as the microtubules of the centriolar barrel9, allowing the maintenance of a continuous pericentrosomal golgi ribbon8. Interestingly, removal of EB1 from the centrosome by expression of a carboxy-terminal fragment of CAP350 as well as depletion of FOP inhibits primary cilia formation10.

CYLD was originally identied as a gene mutated in familial cylindromatosis, a genetic condition that predisposes patients to the development of skin appendages tumours, referred to as cylindromas. Patients with cylindromatosis carry heterozygous germ-line mutations in the CYLD gene11. During their life, some cells undergo a loss of heterozygosity with the disappearance of the wild-type (WT) CYLD allele. The remaining mutated allele leads to the expression of a truncated protein, thus resulting in the development of cylindromas. Therefore, this mode of tumour formation denes CYLD as a tumour suppressor. The CYLD gene encodes a deubiquitinating (DUB) enzyme, which removes lysine 63-linked polyubiquitin chains (K63-linked ubiquitin) as well as linear chains from target proteins12. Interestingly, most of the mutations of CYLD found in human cylindromas are predicted to cause C-terminal truncations and catalytic inactivation of the DUB domain.

The CYLD protein was shown to regulate cell survival and inammatory responses mainly through inhibiting nuclear factor-kB (NF-kB) and mitogen-activated protein (MAP) kinase signalling13,14. In addition, CYLD was also shown to negatively regulate the Wnt/b-catenin signalling pathway by removing K63-linked ubiquitin of Dishevelled (Dvl)15, and more recently CYLD was found to interact with the centrosomal protein CEP192 (ref.16), which plays a critical role in centrosome biogenesis17.

The tumour suppressing function of CYLD has been studied,

in vivo, using Cyld knockout (KO) mice. Surprisingly, these mice do not spontaneously develop tumours but their skin is more sensitive to tumorigenic chemicals compared with WT mice18. Intriguingly, transgenic mice, which carry a complete deletion of the DUB domain (CyldD9/D9) die perinatally from respiratory dysfunction and exhibit immature lungs characterized by

hyperplasic mesenchyme19. The striking difference between those two phenotypes led us to use a knockin mouse model engineered to recapitulate more closely the smallest truncation found in patients by expressing a truncated CYLD protein lacking the last 24 C-terminal amino acids of its catalytic domain (CyldD932).

To further characterize how CYLD may contribute to cylindromatosis onset, we highlight the interaction between CYLD and the centrosomal CAP350 protein, which leads to CYLD localization to the centrosome. In addition, we demonstrate that CyldD932 mice show impaired apical migration and docking of basal bodies in multiciliated cells with a concomitant loss of CYLD centrosomal localization and CAP350 interaction. To understand the mechanism by which CYLD impairs basal body migration/docking, we expressed several constructs of the protein and show that CYLD overexpression in the cytosol prevents primary cilia formation, while its specic expression at the centrosome leads to primary cilia formation. Conversely, expression of the catalytically inactive CYLD at the centrosome abolishes ciliogenesis. This demonstrates that both centrosome localization and CYLD DUB activity are required for cilia formation. Finally, we demonstrate by inhibiting the NF-kB pathway that catalytically inactive CYLD abolishes ciliogenesis in an NF-kB-independent manner. Altogether, these results point to an unravelled role of Lysine 63 ubiquitination in ciliogenesis.

ResultsCYLD interacts with CAP350 at the centrosome/basal bodies. We identied CYLD as a potential interacting partner of CAP350, by performing immunoprecipitation directed against CAP350 followed by mass spectrometry analysis. Eleven peptides were found in the 110-kDa band that matched sequences throughout the tumour suppressor protein CYLD (Fig. 1a and Supplementary Fig. 1). We conrmed the presence of CYLD in the CAP350 immunoprecipitate using a monoclonal antibody directed against the catalytic domain of CYLD (Fig. 1b), thus establishing CAP350/CYLD interaction. To further map the interacting domains of CAP350 and CYLD, we transfected Myc-tagged DNA constructs encoding various portions of CAP350 with full-length Flag-tagged CYLD into HEK 293T cells and performed immunoprecipitation with an anti-Flag antibody. The CYLD interacting domain was mapped to the C-terminal domain of CAP350 (Fig. 1c). As full-length CAP350 is insoluble, we overexpressed the CAP350 C-terminal domain with either a CYLD DN (AA394-956) or CYLD DC (AA1-585) construct. We found that the CAP350 C-terminal domain interacts with a CYLD DN (AA394-956) protein but not with the CYLD DC (AA1-585) protein, suggesting that it interacts with the DUB domain of CYLD (Fig. 1d). In addition, cells co-expressing both proteins show a co-localization of CAP350-GFP and CYLD-Flag to the centrosome and to the microtubules (Supplementary Fig. 2a). Such localization has already been observed for CAP350 overexpression alone, as previously described8, while CYLD-Flag expressed alone was present in the cytosolic fraction (Supplementary Fig. 2b). These results suggest that CAP350 directs CYLD localization to perform specic functions in cells.

CYLD localizes to the centrosome and basal bodies. This CAP350/CYLD interaction prompted us to investigate the localization of endogenous CYLD. To that end, we generated and afnity puried an antibody directed against CYLD (CYLD 089). Staining with this antibody showed that CYLD is enriched at the centrosome throughout the cell cycle (Fig. 2a,b). In addition, a faint labelling of the nucleus was observed in interphase cells. Such a nuclear staining has previously been observed for several

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CYLD

1 127 203 232 302 472 546 593 956

CAP-Gly

CAP-Gly

CAP-Gly

DUB

CYLD932

1 127 203 232 302 472 546 593 932

CAP-Gly

CAP-Gly

CAP-Gly

DUB

CAP 350

CYLD

200 kDa

116 kDa

PI PI

CAP CAP

CYLD CYLD

Unbound fractions Immunoprecitates

1 1101 1290 1502 1556 1986 2049 2499 2565 CAP FL Myc

3117

1 127 203 232 302 472 546 593 956

CYLD FL

CYLD C

CYLD N

1 983

Myc

DUB

CAP N

CAP M

CAP C

Myc

1 585

394

Flag

Flag

984 2,589

2,590 3,117

Flag

593 956

Myc

DUB

TransfectionCYLD: --------- FL ------- --------- FL --------CAP350: N M C N M C

250

150

100

100 kDa

Myc

Myc

Flag

Flag

75

50 kDa

PI Flag PI Flag PI Flag PI Flag

I.P. Un. Fr I.P.

Un. Fr

Unbound fractions Immunoprecipitates(Flag)

TransfectionCYLD: -------- C -------- ------------ N --------------

CAP350: --------- C -------- ------------- C ---------------

150

100

Figure 1 | CYLD interaction with CAP350. (a) Schematic representation of the CYLD protein with the three CAP-Gly domains and the catalyticDUB domain (593956). The locations of the 11 peptides found by mass spectrometry are underlined. In the lower panel, representation of the truncated protein expressed in CyldD932 mice, which lacks the last 24AA of the the DUB domain. (b) IP experiments were performed on RPE1 cells using pre-immune (PI, as control), polyclonal anti-CAP350 (CAP), or monoclonal anti-CYLD (CYLD) IgGs. The immunoprecipitated proteins and non-associated components are referred to as Immunoprecipitates and unbound fraction, respectively. IP products were analysed by WB analysis using antibodies directed against CAP350 and CYLD (polyclonal) as indicated. CAP, CAP350. (c) Full-length CYLD-Flag and three CAP350 fragments as schematized above were expressed in HEK293. CYLD-Flag immunoprecipitates the C-terminal domain of CAP350. (d) CAP350 C-terminal domain and either CYLD

DC (1585) and CYLD DN (394956) were expressed in HEK293. CYLD DN immunoprecipates the C-terminal domain of CAP350. Un.Fr, unbound fractions; IP, Immunoprecipitates.

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1 2

Centrosome (-Tubulin)

CYLD 089

2

1

CYLD 089

200

116

97

66

Centrosome (-Tubulin)

CYLD 089

-Tubulin

CYLD 089

S

I CTR

Cilia (TAP 952)

CAP 350

Cilia (TAP 952)

CYLD 089

Figure 2 | CYLD localizes to the centrosome and basal bodies. (a) Double labelling of RPE1 cells with polyclonal afnity-puried CYLD antibody (CYLD 089) and the monoclonal g-tubulin antibody GTU88, which recognizes the centrosome. (b) CYLD staining in RPE1 during metaphase (upper panel)

and telophase (lower panel) with antibodies directed against CYLD and g-tubulin. During metaphase, CYLD remains localized to the centrosome butis also present along the spindle as previously described for CAP350 (ref. 8). During telophase, CYLD antibodies label the centrosome and somedots around it. Staining of the midbody and the central spindle was never observed. (c) WB analysis of CYLD in Triton X-100-soluble (S) and insoluble (I) protein fractions and a highly enriched centrosomal fraction (CTR) from KE37 cells. (d) Immunostaining of isolated centrosomes with polyclonal afnity-puried CYLD antibody (CYLD089) and CTR453. (e) Deconvoluted images of cultured ependymal cells stained with antibodies against CAP350 and TAP 952. (f) Deconvoluted images of cultured ependymal cells stained with antibodies against CYLD and TAP 952. Note that CYLD stainednot only basal bodies but also cilia tips. Scale bars, 10 mm.

ciliary or centrosomal proteins20. During metaphase, CYLD was detected along some spindle microtubules and at the spindle poles (Fig. 2b), reminiscent of CAP350 staining8. To further ascertain this centrosomal localization, we used a biochemical approach. Western blot (WB) analysis with two antibodies against CYLD identied a major band at 110 kDa, which was specically enriched in the centrosomal fraction of KE37 cells but also weakly detected in Triton X-100-soluble and -insoluble fractions

(Fig. 2c). Consistent with these observations, puried centrosomes were strongly labelled by CYLD antibodies (Fig. 2d). We conclude that CYLD is a centrosomal protein.

To conrm the centrosomal localization of CYLD, we investigated its localization in multiciliated cells that contain numerous basal bodies. Serum starvation of cultured ependymal cells leads to the development of numerous motile cilia growing from these basal bodies21. Therefore, we cultured ependymal cells

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from mouse brains and stained them with antibodies against CAP350 and CYLD. Antibodies directed against glycylated tubulin, such as TAP952 (ref. 22) were used to decorate the cilia. Numerous dots located at the cilia base were labelled by antibodies directed against CAP350 or CYLD (Fig. 2e,f). A co-staining with antibodies directed against g-tubulin suggests that both proteins localize at the basal bodies of multiciliated cells (Supplementary Fig. 3). Surprisingly, CYLD antibodies, but not CAP350 antibody, stained the tips of the cilia and weakly the cilia themselves (Fig. 2f). Altogether, these results suggest that CYLD localizes at the centrosome and basal bodies but also at the ciliary tips.

Truncated CYLD inhibits primary cilia formation. The presence of CYLD at the basal bodies and at the ciliary tips prompted us to investigate the role of this protein in ciliogenesis. Therefore, we turned to CyldD932 mice23, which express the smallest truncation of CYLD causing the human pathology (a deletion of the last 24 amino acids) leading to a catalytically inactive protein24 (Fig. 1a). As previously described in mice with complete deletion of CYLD catalytic domain (CyldD9/D9)19, CyldD932 mutants die perinatally from respiratory distress with pulmonary defects (Supplementary Fig. 4) reminiscent of the phenotype observed in several mouse models exhibiting defects in signalling as Wnt5a25, Vangl2 (ref. 26) and GMAP210 (ref. 27). As these mutants show defects in cilia or in PCP signalling, we decided to investigate primary cilia formation in primary mouse embryonic broblasts (MEFs) derived from CyldWT or CyldD932 littermate embryos. We rst veried that proliferation of cells of both genotypes was comparable as proliferation rate is likely to modify cells behaviour in respect to ciliation. Results of uorescence-activated cell sorting (FACS) analysis or KI67 staining (a marker of proliferative cells) show that both WT and mutant MEFs proliferate similarly (Supplementary Fig. 5ac). As CYLD is a tumour suppressor, and primary cilia usually form in the G0/G1 phase of the cell cycle28, we also wanted to determine whether both cell types were able to undergo cell cycle arrest on serum starvation. Again, after FACS analysis and KI67

staining we found no difference in the ratio of non-cycling cells between the two conditions (Supplementary Fig. 5ac). Although both cell types proliferate similarly and are able to stop cycling on serum starvation, primary cilium staining showed that only 32% of CyldD932 cells present a primary cilium compared with 68% of WT MEFs (Fig. 3a,b), suggesting that truncated CYLD mildly affects primary cilia formation.

Truncated CYLD inhibits multiple motile cilia formation. We next examined motile cilia formation in trachea of WT and CyldD932 E18.5 embryos from several litters by scanning electron microscopy. As ciliogenesis in the trachea occurs proximo-distally29, we imaged and counted the ciliated cells in every eld from the larynx to the beginning of the main bronchi to avoid bias due to spatial differences. For each litter, the number of multiciliated cells was at least 50% lower in the mutants than in WT embryos. In addition, the motile cilia of mutant multiciliated cells were generally shorter and scarce (Fig. 4a). As a control of the specicity of this result, we took advantage of the previously published Cyld knock-out mouse model18,30,31. As CyldKO mice live without any major health issues, we were not expecting any defects in cilia formation. Indeed, there was no difference in the morphology and number of multiciliated cells between the WT and CyldKO embryos (Fig. 4a, graph). These results show that CYLDD932, a truncated catalytically inactive protein, behaves as a dominant negative form of CYLD to inhibit formation of motile cilia in multiciliated cells, whereas complete absence of CYLD has no effect on cilia formation.

As the decreased number of multiciliated trachea cells might be due to a developmental delay in the CyldD932 embryo, we used the ependymal culture system21 and compared motile cilia formation in parallel cell cultures obtained from WT and CyldD932 littermates. There was no difference in the doubling time of cells with these different genotypes, but there was a dramatic difference in the number of multiciliated cells between WT and CyldD932 cells, independently of cell-density variations (Fig. 4b). Altogether, our results in trachea and ependymal cells culture

P<0.0001

Cyld932Cyld WT

Cilia (611B1)

Basal bodies (-Tubulin)

Nuclei (DAPI)

Basal bodies (-Tubulin) Nuclei (DAPI)

Ciliated cells (%)

80.0

70.0

60.0

50.0

40.0

30.0

20.0

10.0

0.0

Wild types CYLD DeI932

Cilia (611B1)

Figure 3 | CYLDD932 affects primary cilia formation. (a) Staining of WT or CyldD932 MEFs grown at conuence and serum depleted for 48 h with anti-acetylated tubulin (green) and anti-g-tubulin (red) antibodies. WT MEFs cells show a primary cilium, while most of the CyldD932 cells do not grow a primary cilium. Scale bars, 10 mm. (b) Quantication: a primary cilium was observed in about 68% of cells (n ca. 2,000 cells out of 4 embryos) of

the WT MEFs, while only 32% of mutant cells (n ca. 4,000 cells out of 8 embryos) showed one. P-value o0.0001 after w2-test. Error bars show s.d.

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Wild types Mutants

n=1

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Cyld932

150

n=2

n=3

n=1

n=4

n=1

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n=2

n=3

n=2

n=3

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120

90

60

30

0

CYLD

Del932

N1

CYLD

Del932

N2

CYLD

Del932

N3

CYLD

Del932

N4

CYLD

Del932

N5

CYLD

KO

Cilia (GT335) Nuclei (DAPI)

P<0.0001

CyldWT

Cyld932

Ciliated cells (%)

60

50

40

30

20

10

0

Wild types Cyld Del932

Figure 4 | CYLDD932 affects multiple motile cilia formation. (a) Left: cilia analysis by scanning electron microscopy of tracheas isolated fromWT and CyldD932 embryos at E18.5. Representative elds of trachea from WT embryos (upper) or CyldD932 mutant (lower) are shown, with a magnied image on the right. Right: quantication of the percentage of ciliated cells in the trachea from WTor mutant embryos. Analysis was performed on ve litters from CyldD932 embryos and one litter from CyldKO mice. The number of WT and mutant embryos is indicated at the top of each bar. The mean was determined when possible and the s.d. is indicated. Scale bars, 10 mm for the low magnication and 1 mm for the high magnication. (b) Left: staining of a large eld of cultured ependymal cells isolated from WT (left) or CyldD932 (right) from P0 animals 15 days after differentiation with GT335 for cilia and DAPI for nuclei. Note the decreased number of multiciliated cells in the mutant. Scale bars, 10 mm. Right: quantication of the percentage of multiciliated cells in WT or CyldD932 cell cultures. Multiciliated cells in each culture were counted and reported relative to the number of nuclei. In mutant cell cultures, the number of multiciliated cells was reduced by 50%. (n for WT B2,500 cells of 5 embryos; n for CyldD932 B2,500 cells of 5 embryos).

P-value o0.0001 after w2-test. Error bars show s.d.

show that truncation of the catalytic domain of CYLD affects formation of motile cilia of multiciliated cells.

CyldD932 basal bodies duplicate normally but fail to anchor. To determine the reason of the decrease in cilia formation in CyldD932 embryos, we studied basal body formation in ependymal cell cultures from each genotype at several differentiation time points by staining with antibodies specic for basal bodies and cilia. In WT cell cultures, multiciliated cells showed abundant basal bodies decorated with anti-centrin3 antibodies (Fig. 5a). Abundant basal bodies were also observed in CyldD932 cells irrespective of the number of cilia in these cells. Quantication of cells with numerous basal bodies revealed only a slight decrease in CyldD932 mutant cells, suggesting that basal bodies amplication proceeds similarly in WT and CyldD932 cells (Fig. 5a). In addition, the timing of formation was not different between the two cell types. To further investigate a possible basal body defect, we performed detailed electron microscopy analysis of WT and mutant tracheas. We

found that a signicant number of basal bodies failed to position apically and dock at the plasma membrane in CyldD932 tracheal ciliated cells (Fig. 5b). They appeared to be positioned in different orientation (Fig. 5b, arrows). In addition, the microvilli, which interspaced the axonema, appeared to be reduced in length as well as in number, compared with the WT samples from the same litter, reminiscent of actin organization or Rho GTPase activity defect32,33. Thus, in mutant cells basal bodies multiply normally but fail to migrate apically, suggesting a defect in transport and/or docking at the apical membrane.

Centriole maturation and satellites are not affected. The failure of basal bodies to anchor could be explained by either a defect in centriole maturation or in centriolar satellite organization. Indeed, centriolar maturation is characterized by the acquisition of subdistal and distal appendages, which form the ciliary basal feet and transition bres, respectively34. Both of them are required for cilia formation. Furthermore, centriolar satellites

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Cilia (GT335)

Basal Bodies (Cen3)

Wild types Cyld Del932 P=0.005

60

50

40

30

20

10

CyldWT

Cells (%)

P=0.05

Cyld932

0 1 CTR BB multiples

Cyld WT

Cyld932

Figure 5 | CYLDD932 affects basal body anchoring of motile cilia but not duplication. (a) Left: double-staining analysis of basal bodies and cilia in WTand mutant ependymal cell cultures using GT335 and antibodies against Centrin 3, 15 days after serum removal. WT or CyldD932 multiciliated cells showa large number of basal bodies irrespective of the presence or absence of cilia. Images have been deconvoluted. Scale bars, 10 mm. Right: quantication of the number of cells with multiple basal bodies and cells with a centrosome in WT and mutant ependymal cell cultures. Mutant cells showed a slight but signicant decrease in the number of cells with multiple basal bodies compared with the WT culture. A slight decrease was also observed in the number of cells showing a centrosome. (B1,900 cells of four WT embryos; B1,900 cells of four CyldD932 embryos.) P-values after w2-test are o0.05 and0.005, respectively. Error bars show s.d. (b) Transmission electron microscopy image of WT (left) or CyldD932 (right) trachea. In the WT trachea, numerous regularly spaced anchored basal bodies are evident at the apical membrane. All of them show an axoneme. In the mutant trachea, duplicated basal bodies are organized in several orientations; some are transversally oriented, while others are longitudinally oriented (arrows). In addition, although some microvilli are observed on the apical surface most of the cells do not show any microvilli at their surface. Scale bars, 500 mm.

are believed to drive cargo to the centrosome and basal bodies35, and are also required for ciliogenesis36. To ascertain the presence of centriolar satellites and appendages, we stained ependymal cells with antibodies directed against PCM1 and OFD1, two proteins of centriolar satellites35,36, as well as with antibodies against Cep164 and CCDC123 (Cep123), two proteins located within the distal appendages37,38, and ODF2, a protein located both at distal and subdistal appendages39. All antibodies decorated centriolar satellites or basal bodies irrespective of the genotype (Fig. 6), suggesting that basal body maturation and centriolar satellite organization are not signicantly modied in CyldD932 mice.

CyldD932 does not interact with CAP350. To gain molecular insight into the defects associated with CYLDD932 expression, we tested whether CYLDD932 protein interacted with CAP350 by immunoprecipitation of CAP350 in WT and CyldD932 MEFs. In parallel experiments, CAP350 antibody immunoprecipitated CYLD (revealed by a polyclonal antibody directed against the amino-terminal domain) in WT but not CyldD932 MEFs (Fig. 7), suggesting that the truncated version of CYLD is unable to interact with CAP350. It follows that CYLDD932 would not be localized at the centrosome and, indeed, we did not detect any CYLD at the centrosome or basal bodies in CyldD932 mutant

ependymal cells (Fig. 7b,c), while CAP350 remained at the centrosome. It is worth noting that CYLD was still localized at the ciliary tip in mutant ependymal cells. These observations indicate that the lack of interaction between CYLDD932 and CAP350 results in abnormal CYLD localization.

CYLD localization and activity are required for ciliogenesis. As the impaired ciliogenesis observed in CyldD932 mice might be driven by the absence of CYLD at the centrosome, or by its compromised catalytic activity or both, we decided to investigate the effect of CYLD activity and localization on primary cilia formation. This was performed by overexpressing CYLDWT as well as its catalytically inactive version CYLDH871/N (ref. 24) in RPE1 cells. Overexpressed CYLDWT or CYLDH871/N protein is mainly cytosolic with no obvious enrichment at the centrosome (Fig. 8a). We observed a dramatic decrease in primary cilia formation in cells expressing GFP-CYLDWT, CYLDWT-Flag or CYLDH871/N-Flag (Fig. 8a) with 23%, 17% and 24% of ciliated cells, respectively, while non-transfected cells or green uorescent protein (GFP)-transfected cells showed B90%.

As this result could be due to aberrant CYLD localization, we decided to force CYLD expression at the centrosome using the centrosomal targeting domain, PACT40. The targeting of this GFP-PACT-CYLDWT to the centrioles affected ciliogenesis only

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Cyld WT Cyld932

CyldWT Cyld932

a

a

Cilia (TAP 952) PCM1

Cilia PCM1

CAP350 CYLD

200 kDa

100

150

75 kDa

b

Cilia (TAP 952) OFD1

Cilia OFD1

PI

CAP

CYLD

Unbound fractions

PI

CAP

CYLD

PI

CAP

CYLD

PI

CAP

CYLD

I.P. I.P.

Unbound fractions

b

-tub

-tub

-tub

-tub

CYLD

CYLD

CYLD

CYLD

c

Cilia (GT335) Cep123

Cilia Cep123

CyldWT

d

Cilia (GT335) Cep164

Cilia Cep164

Cyld WT

c

Cyld932

Cyld 932

e

Cilia (GT335) ODF2

Cilia ODF2

Figure 6 | Centriolar satellite organisation and centriole maturation is not affected in CyldD932 ependymal cells. Staining for cilia (TAP 952), the centriolar satellite protein PCM1 (a) and OFD1 (b) in WT (left) or CyldD932

(right) ependymal cells. Staining for cilia (GT335) and the centriole appendages proteins Ccdc123 (Cep123) (c), Cep164 (d) and ODF2 (e). Cilia are in red and PCM1, OFD1, Cep123, Cep164 and ODF2 are in green. Scale bars, 10 mm.

Figure 7 | CYLDD932 does not interact with CAP350 and does not localize at the centrosome. (a) Immunoprecipitation of WT or CyldD932

MEFs cells using rabbit pre-immune (PI), anti-CAP350 (polyclonal) and CYLD 089 (polyclonal) IgG. The immunoprecipitated proteins and non-associated components are labelled Immunoprecipitate (I.P.) and Unbound fraction, respectively. I.P. results were analysed by WB analysis using antibodies against CAP350 and CYLD (polyclonal). In WT cells, CAP350 immunoprecipitated CYLD (revealed by the polyclonal antibody(089) conrming the results obtained on Fig. 1a). However, we did not observe any CYLD in the CAP350 immunoprecipitates of CyldD932 MEFs. CAP, CAP350. (b,c) CYLD localization in WT (b) or CyldD932 (c) MEFs

(upper panel) or ependymal cells (lower pannel). CYLD localizes at the centrosome and basal bodies in WT cells, while CYLDD932 no longer localizes at the centrosome or basal bodies. Scale bars, 10 mm.

slightly, resulting in B80% of ciliated cells (Fig. 8b). By contrast, the targeting to the centrioles of CYLD with a point mutation, which impaired the catalytic activity of CYLD (GFP-PACT-CYLDH871/N), showed only 57% of ciliated cells. A similar decrease was also observed (Fig. 8b, graph) when using the truncated and catalytically inactive version of CYLD, GFP-PACT-CYLDD932 (ref. 24). To control that these observations are not caused by differences in the expression level of the constructs, we measured GFP uorescence intensity at the centrosome for each condition in a new experiment. General observation of the slides showed similar differences in cell ciliation of transfected cells (ciliated cells: GFP-PACT-CYLDWT (82%), GFP-PACT-CYLDH871/N (60%), GFP-PACT-CYLDD932 (36%)), but no signicant difference in mean uorescence intensity was observed between ciliated and unciliated cells, irrespective of the construct, which shows that ciliation is independent of expression level (Fig. 8c).

These results demonstrate that both the localization of CYLD at the centrosome/basal body and its deubiquitinase activity are essential for its function in ciliogenesis.

Ciliogenesis defects are independent of NF-jB activation. As CYLD is a negative regulator of the NF-kB signalling pathway, we were wondering whether the defective ciliogenesis observed on the trachea and ependymal cells of mutant mice or after targeting at the centrosome catalytically inactive CYLD, might be due to a deregulation of the NF-kB pathway. Therefore, we decided to cotransfect RPE1 cells with either GFP-PACT-CYLDWT or GFP-PACT-CYLDD932 with or without a non-degradable form of IkB acting a Super-repressor of NF-kB (IkBa Super-Repressor (IkBa-SR))41 to block NF-kB activation. As shown on Fig. 8d, the addition of the tumour necrosis factor (TNF) induced activation of NF-kB either in the presence of serum or after 48 h serum depletion, while the presence of IkBa-SR completely blocked this activation. In parallel, cells were xed and analysed for the

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presence of a primary cilium. Expression of IkBa-SR did not impact the ciliation process either in GFP-PACT-CYLDWT- or in GFP-PACT- CYLDD932-expressing cells as shown in Fig. 8e, suggesting that the defects in the ciliogenesis process due to the presence of GFP-PACT-CYLDD932 at the centrosome is independent of NF-kB signalling.

DiscussionIn this study we demonstrate that part of the CYLD pool is located at the centrosome/basal bodies, through its interaction with CAP350. This interaction is attested by several observations.

First of all, we identied CYLD by mass spectrometry as a partner of CAP350 and conrmed their interaction in endogenous immunoprecipitation experiments; Furthermore, we demonstrate by co-overexpression that the C-terminal portion of CYLD, including the DUB domain, interacts with the C-terminal domain of CAP350. Second, CAP350 overexpression directs the centrosomal localization of overexpressed CYLD; nally, CYLD no longer localizes at the centrosome when its interaction with CAP350 is abolished. It is worth noting that CYLD has already been found in interaction with another centrosomal protein CEP192 in co-overexpressed conditions, reinforcing the centrosomal localisation of CYLD16. Nevertheless, the relationship

Non transfected cells

Transfected cells

Non transfected cells Transfected cells

P=0.02 P<0.0001 P<0.0001 P<0.0001

P<0.001 P<0.0001 P<0.0001

Cilia (611B1) Flag

Ciliated cells (%)

100

90 80 70 60 50 40 30 20 10

0 GFP GFP-CYLD CYLD-Flag CYLD-H/N-Flag

PACT-CYLDWT PACT-CYLDH/N

Ciliated Unciliated

High expression

Low expression

Cilia (611B1) GFPNuclei (Dapi)

Ciliated cells (%)

100

90 80 70 60 50 40 30 20 10

0 GFP-PACT-CYLD

GFP-PACT-CYLD H/N

GFP-PACT-CYLD del

NS NS NS

100

Fluorescence

intensity (a.u.)

Cilia (611B1) GFP

80

60

40

20

GFP-PACT-Cyld (Ciliated)

GFP-PACT-Cyld (Unciliated)

GFP-PACT-Cyld H/N (Ciliated)

GFP-PACT-Cyld H/N (Unciliated)

0 GFP-PACT-Cyld del (Ciliated)

GFP-PACT-Cyld del (Unciliated)

TNF

12 10

6 4 2 0

8

MockHigh Low

2.5

2

1.5

1

0.5

0

C SR C SR

Non transfected cells

Transfected cells

NS NS

Fold activation

Fold activation

Ciliated cells (%)

100

90 80 70 60 50 40 30 20 10

GFP-PACT-

CYLD

0 GFP-PACT-

CYLD + SR

GFP-PACT-

CYLD del

GFP-PACT-

CYLD del + SR

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between CAP350/CEP192 and CYLD will require further investigations.

This specic centrosomal localization combined with the staining observed at the tip of motile cilia suggests that CYLD plays a role in cilia assembly/disassembly. Interestingly, localization of CYLD at the cilia tips is reminiscent of the enrichment observed for several other proteins such as EB1/EB3 (ref. 42), some IFT proteins43 and, more recently, cep104/FAP256 (ref. 44). This localization of CYLD is probably independent of its DUB activity, as the staining at the cilia tip is still observed in CyldD932 ependymal cells. As a matter of fact, an interaction between CYLD and EB1 has recently been reported45. In any case, this dual CYLD localization at the centrosome and at the cilia tip suggests that CYLD has distinct functions depending on the partners it interacts with.

Our results show that CYLD participates in ciliogenesis by allowing basal bodies to migrate and dock to the plasma membrane. However, not all motile cilia are affected, as CYLDD932 mutants do not display any leftright patterning defects, suggesting that the function of the node is unaffected by CYLD truncation. In addition, we demonstrate that CYLD has to be both catalytically active and located at the centrosome to promote ciliogenesis. Therefore, we propose a model (Fig. 9) in which ciliogenesis would be only possible if a centrosomal protein, or CAP350 itself, is deubiquitinated. In the WT situation, CYLD would be located at the centrosome/basal body via CAP350 and be able to hydrolyse K63- or linear-linked ubiquitin chains from its substrate, enabling ciliogenesis to proceed. In the mutant situation, CYLD would not be present at the centrosome, resulting in impaired ciliogenesis. On CYLD overexpression, extra CYLD in the cytoplasm would delocalize its targets and prevent ciliogenesis. However, when CYLD is overexpressed and targeted to the basal body by the PACT domain, it can deubiquitinate its target and, consequently, ciliogenesis occurs. Conversely, cells overexpressing the catalytically inactive CYLDH871/N or CYLDD932 fused with the PACT domain do not retain the ability to develop cilia. All these results argue for the involvement of K63-ubiquitination/deubiquitination in cilio-genesis. Importantly, we demonstrate that this role is NF-kB independent. Recently, several reports have suggested an inhibition of ciliogenesis caused by activation of the NF-kB pathway.

First, Lattke et al.46 have shown that the activation of this pathway by the expression of constitutively active IKK2 inhibits cilia formation. However, it is not clear whether the cilia formation defect is a consequence of a direct cell-autonomous

NF-kB activation rather than activation of inammatory processes. Moreover, it could also be a direct effect of active IKK2 in ependymal cells, independently of NF-kB, as IKK2 has

NF-kB-independent functions47. As a matter of fact, IKK2 has been found inhibiting ciliogenesis in an RNA interference screen48. Second, the pro-inammatory cytokine TNF triggered a dose-dependent loss of primary cilia in mesenchymal stromal cells49, but no molecular explanation was provided. However, our study brings several arguments supporting the fact that this DUB acts on ciliogenesis independently of NF-kB activation as trachea from CYLD KO mice do not show any defects in motile cilia formation, whereas they are defective in NF-kB signalling14,18, and inhibition of the NF-kB pathway by the expression of a non-degradable form of IkBa acting as a Super-repressor41 do not modify the ciliogenesis defect caused by expression of the CYLDD932 protein at the centrosome.

Nevertheless, it is intriguing that CyldKO mice are perfectly viable without any problem in motile cilia formation, whereas the mice expressing a truncated version of CYLD die at birth with cilia defects. These results suggest some redundancy in the control of ciliogenesis by DUBs. In the CyldKO mice another DUB may compensate for the function of CYLD, whereas in the CyldD932 mice the mutant CYLD protein probably acts as a dominant negative by delocalizing its substrate, thus preventing the action of the substitute DUB. USP 21 is a good candidate for carrying this function, as it has recently been shown to localize at the centrosome and affect primary cilia formation50. Moreover, it shares a similar target with CYLD in the regulation NF-kB signalling pathway51.

The involvement of the deubiquitinase CYLD in basal body migration/docking has to be integrated in a more general role of ubiquitination processes in ciliogenesis or centrosomal function. At rst, ubiquitinated proteins have been reported to be present in cilia and agella52, and the K63-specic E2 ubiquitinconjugating enzyme Ubc13 is present in the ciliome of various species5255. Next, protein ubiquitination was involved in agellar disassembly52. Finally, the E3 ubiquitin ligase MIB1 (ref. 56) was identied in a centrosome proteome57 and the deubiquitinase USP21 is shown to be required for primary cilia formation50. In addition, another deubiquitinase Usp33 has recently been shown to localize at the centrosome by its binding with CP110 and its activity antagonizes SCFcyclinF ubiquitination of CP110 (ref. 58).

How may CYLD regulate ciliogenesis? Our data show that CYLD is required to allow basal body migration and docking at the plasma membrane. We also show that centriole maturation

Figure 8 | Regulation of primary cilium formation by CYLD in an NF-jB-independent manner. (a) Left, immunostaining of cells overexpressing CYLD-Flag; red, anti-Flag antibodies; green, anti-acetylated tubulin antibodies. Non-transfected cells show a primary cilium (arrow), while cells transfected with CYLD-Flag show only the centrosome (arrowhead). Scale bars, 10 mm. Right: percentage of ciliated cells. Untransfected and GFP-transfected cells show a similar percentage of ciliated cells. Fewer cells are ciliated after transfection with GFP-CYLD, CYLD-Flag or CYLD H/N-Flag. For each condition, at least 500 cells in 3 experiments were counted. P-values after w2-test are 0.02 for GFP and o0.0001 for the three other conditions. (b) Left: immunostaining of cells overexpressing GFP-PACT-CYLDWT or GFP-PACT-CYLDH/N. Red, anti-acetylated tubulin antibodies; green, GFP. Scale bars, 10 mm.

Right: percentage of ciliated cells expressing GFP-PACT-CYLDWT, GFP-PACT-CYLDH/Nand GFP-PACT-CYLDD932. The targeting of CYLDWT to the centrosome does not affect ciliogenesis unless the catalytic domain is not functionnal (GFP-PACT-CYLDH/N and GFP-PACT-CYLDD932). For each condition, at least 500 cells in 3 experiments were counted. P-values after w2-test are o0.001 for GFP-PACT-CYLD and o0.0001 for the other conditions. (c) Left: cells with high expression level or low expression level of the H/N construct can be ciliated or unciliated. Cells were transfected with GFP-PACT-CYLD H/N (green) and stained with anti-acetylated tubulin antibody (red). Right: average uorescence intensity (arbitrary units) measured at the centrosome for ciliated and unciliated cells after transfection of the three constructs (GFP-PACT-CYLDWT, GFP-PACT-CYLDH/N or GFP-PACT-CYLDD932 GFP-PACT-Cyld

del) in RPE1 cells. Mean and s.d. are shown. Differences after MannWhitneys test are not statistically signicant (n.s; P 0.3; P 0.9 and P 0.65,

respectively). (d) Ig-kB-Luciferase-derived activity measured after culture of cells in high or low serum medium. In each case both basal (Mock) and induced (TNF) levels of NF-kB activity were strongly inhibited by IkBa-SR. Errors bars in all graphs show s.d. (e) Percentage of ciliated cells in

GFP-PACT-CYLDWT, GFP-PACT-CYLDH/N and GFP-PACT-CYLDD932 after inhibition of the NF-kB signalling by expression or not of the Super-repressor IkBa. It is worth noting that after w2-test statistically signicant differences can be observed irrespective of the PACT-CYLD fusion construct after

NF-kB inhibition.

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Wild type CYLD

Ub

CYLD932

CYLD Ub

CYLD CAP350

CAP350

Ciliogenesis

Ciliogenesis defect

GFP-CYLDWT overexpression GFP-PACT-CYLDWT overexpression GFP-PACT-CYLDH/N overexpression GFP-PACT-CYLD932 overexpression

CYLD Ub

CYLD Ub

CYLD

CYLD CYLD

Ub

CYLD

CYLD

CYLD

Ub

CYLD CYLD

Ub

CAP350

Ub

CAP350 CAP350

CYLD

Ub

Ciliogenesis defect Ciliogenesis Ciliogenesis defect

Figure 9 | Model explaining CYLD function at the centrosome. (a) In WT mice, CYLD is located at the centrosome and can deubiquitinate its target to promote ciliogenesis. (b) In mutant mice (CyldD932), CYLD is no longer located at the centrosome and cannot deubiquitinate its target preventing ciliogenesis. (c) After CYLD overexpression, extra CYLD is found in the cytoplasm and will delocalize its target from the centrosome. A ciliogenesis defect is observed. (d) Targeting of CYLD to the centrosome by the PACT domain (GFP-PACT-CYLD), allow CYLD to deubiquitinate its targets and ciliogenesis to occur. (e) In contrast, targeting of catalytically inactive CYLD to the centrosome by the PACT domain (GFP-PACT-CYLDH/N or GFP-PACT-CYLDD932) prevents ciliogenesis. In these cases, targets cannot be deubiquitinated and ciliogenesis defects are observed. Overexpressed CYLD is displayed in green, CYLD fused to the PACT domain is encircled with a bolder line.

proteins as well as some centriolar satellite proteins are still present in our mutants, suggesting that the anchoring defect is not a result of basal body structural defects. However, we cannot rule out that ubiquitination/deubiquitination processes might affect centriole appendages and centriolar satellite functions59,60.

Interestingly, a target of CYLD, Dvl15, has been shown in Xenopus multiciliated cells to be essential for the positioning of the apical basal bodies and for the activation of the Rho GTPase61. In addition, the RhoGEF LARG, co-immuno-precipitates with CYLD62. Therefore, we propose that CYLD would allow basal body migration by deubiquitinating, a substrate working with Dvl, and LARG to activate RhoA GTPase and subsequently remodel actin. The recent report that phosphorylated CYLD redistributes to the dorsal rufes63 supports this hypothesis of a link between CYLD and the actin network.

In conclusion, our work identies CYLD as a new regulator of ciliogenesis. Two recent studies64,65 have recently reported that impaired ciliogenesis impacts skin homeostasis and affects the pilosebaceous unit from which cylindroma are believed to originate. This convinced us that future studies designed to precisely characterize the molecular pathways regulated by CYLD during ciliogenesis might provide novel insights into how CYLD deregulation results in cylindroma in the cylindromatosis pathology.

Methods

Mice. All animals were housed in specic pathogen-free conditions at Institut Curie according to French and European Union laws. All animal procedures were

conducted in accordance with European, national and institutional guidelines and protocols, and were approved by local government authorities (Authorization for animal testing N91-518, Direction dpartementale des services vtrinaires de lEssonne).

CyldD932 and CyldKO mice were generated in a c57/Bl6J background as described previously23,30. Male and female embryos were used. As no differences between CyldD932 heterozygous mice or embryos and their WT littermates were observed throughout the study, we pooled the results of both genotypes under the classication of WT. The embryonic stage was estimated on the basis of gestational time, with day 0.5 being dened as the morning when a vaginal plug was detected. Adult mice and embryos were genotyped by PCR.

Cell culture. Human retinal pigment epithelial (RPE1) cells were obtained from ATCC and grown in DMEM/F12 medium supplemented with 10% FCS. Human lymphoblastic KE37 cells obtained from M. Bornenss laboratory were grown in RPMI medium supplemented with 10% FCS. MEFs were obtained after trypsinization of dissected embryos at embryonic day 13.5 (E13.5) and grown in DMEM medium supplemented with 10% FCS and non-essential amino acids. Ependymal cell cultures were maintained as described previously21,66. Human embryonic kidney cells (HEK293T) from ThermoScientic were transiently transfected with plasmid DNA using the calcium phosphate precipitation method. RPE1 cells were transiently transfected with plasmid DNA using X-tremeGENE HP (Roche) or Nucleofection (Amaxa) and xed 2448 h after transfection.

Cellular fractionation. Centrosomes were isolated from KE37 cells as described previously67. Briey, cells were pretreated for 1 h with Nocodazole (2 10 7 M)

and Cytochalasin D (1 mg ml 1). Cells were washed in PBS and resuspended in 8% sucrose in ten times diluted PBS before cell lysis in lysis buffer (1 mM HEPES,0.5 mM MgCl2, 0.5%NP40, 1 mM phenylmethanesulphonyl uoride and anti-proteases). After centrifugation at 2,500 g, the supernatant is ltered through a nylon-mesh and readjusted to 10 mM HEPES and treated with DNAse. Concentrated centrosomes were overlaid on a discontinuous sucrose gradient (70%, 50% and 40%) in a SW28 tube and centrifuged for 1 h 15 min at 26,000 r.p.m. Fractions were collected and analysed for centrosomes.

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One percent Triton X-100 soluble or insoluble cell fractions were isolated from KE37 or RPE1 cells as described by Tassin et al.68

Plasmid construction. Full-length CAP350 and CYLD cDNAs were subcloned into a GFP vector (GFP-C3 from Clontech) for expression of GFP-tagged proteins. Different fragments of CAP350 cDNAmyc-tagged corresponding to AA 1-893, 984-2589 and 2590-3117 were previously described8. The CYLD-Flag construct was previously described24. The DC CYLD construct was designed by introducing a stop codon in AA 586.

The PACT domain was amplied from human AKAP450 and then subcloned into the GFP-CYLD vectors. All constructions have been veried by sequencing.

Antibodies. Three antibodies directed against CYLD were used: a polyclonal antibody obtained by immunizing rabbits against the N-terminal region (amino acids 1555) of CYLD (CYLD 089) and afnity puried (diluted 1/1,000 for WB analysis and 1/300 for immunouorescence (IF), a monoclonal antibody directed against the full-length CYLD supplied by Millipore, which does not recognize endogenous protein from RPE1 and HEK293T cells (diluted 1/500 for WB analysis) and a monoclonal antibody supplied by Santa Cruz Biotechnology (E-10, SC-74435) recognizing the catalytic domain of the protein (diluted 1/100 for WB analysis). All antibodies have been characterized by the following procedure: they are recognized by WB analysis and IF overexpressed GFP-CYLD, as well as the immunoprecipitated protein. However, the polyclonal one gave a stronger signal both in IF and WB analysis. Two antibodies directed against g-tubulin were used: a previously described polyclonal antibody68 (1/500) and a monoclonal one (GTU88 from Sigma 1/ 1,000). We also used afnity-puried rabbit polyclonal antibodies directed against Centrin 3 (ref. 69) (1/1,000), CAP350 (ref. 8) (1/1,000), Ccdc123 (ref. 38) (1/250), Cep 164 (ref. 37) (1/1,000), PCM1 (ref. 70) (1/1,000), OFD1 (residues 1451,012)36 (1/100), ODF2 (residus 250632)48 (1/1,000) and several anti-tubulin antibodies were used, such as detyrosinated tubulin (T12)(1/1,000), polyglutamylated-tubulin (GT335) (1/50,000) and monoglycylated tubulin (TAP) (1/50,000) as described in Janke and Bulinski22. Antibodies against acetylated tubulin (clone 6-11 B 1) (1/ 1,000) and tubulin (clone B5-1-2) were purchased from Sigma and rabbit anti-Ki67 (1/500) (Novocastra Laboratories). DAPI (40,6-diamidino-2-phenylindole, Sigma)

was used for DNA labelling. In all experiments using Flag antibodies, we used the anti-ag M2 from Sigma (F1804)(1/1,000). Only the WB analysis of the DN CYLD was revealed by Rabbit anti-Flag antibodies (F7425) (1/500).

Western blotting. Protein extracts were run on a PAGE gel of various acrylamide/ bisacrilamyde percentages. Gels were transferred on nitrocellulose membranes with a semi-dry transfer device (Bio-Rad). Membranes were then blotted with various antibodies then revealed by electrochemiluminescence on autoradiographs. Non-cropped scans of the autoradiograps of the main WB analysis are presented in Supplementary Fig. 6.

IF microscopy and data processing. Cells were either xed with methanol for 6 min at 20 C and permeabilized for 30 s in PHEM buffer containing 0.25%

Triton X-100 before methanol xation or xed with 4% paraformaldehyde for 15 min before permeabilization with 1% Triton X-100 and processed for IF as described previously68.

After immunostaining, three-dimensional microscopy was performed using an upright motorized microscope (DM RXA2, Leica Microsystems, Mannheim, Germany) equipped with an oil immersion 40, 63, 100/numerical aperture

1.4 Plan-Apochromat objective lens and a cooled interline charge-coupled device detector (Photometrics Coolsnap HQ). Z-positioning was accomplished using a piezoelectric driver (LVPZT, Physik Instruments, Waldbronn, Germany) mounted underneath the objective lens. The whole system operated using Metamorph software (Universal Imaging Corp., Downingtown, PA). Z-stacks were generated from optical sections taken at 0.2-mm intervals. Z-stacks were deconvoluted using

Metamorph software with the appropriate point spread function (PSF) and Meinel algorithm.

In Fig. 8c, pictures were taken with a wide-eld uorescence microscope (Zeiss Axiovert 200 M equipped with an Hamammatsu C4742 camera). For each picture in the GFP channel, all parameters were kept identical (exposure time, binning, camera gain, Offset and so on). Average pixel uorescence intensity of GFP at the centrosome was then measured within a dened and constant circular area of the size of a centriole, using the measurement tools of Image J. Quantication data were compiled from measurements of 80 cells from each of the 3 constructs. Raw measurements for each 80 cells are plotted along the y axis in the graph to make comparison easier.

Immunoprecipitation experiments. Proteins from RPE1, HEK293T or MEF cells were extracted with 1D buffer15,68. After a centrifugation to eliminate trace aggregates, primary antibodies preadsorbed to protein G-sepharose beads (Pharmacia) were added to the sample and the mixture incubated for 2 h at 4 C. Protein G-sepharose beads were then sedimented. The sedimented protein G-sepharose beads were washed (ve times) with buffer and supernatants were

precipitated with cold methanol. Proteins were then processed for SDSPAGE electrophoresis and WB analysis.

For mass spectrometry analysis, the protein spots were excised from the gel after coloration with colloidal Coomassie blue (G250; Bio-Rad), reduced using dithiothreitol and alkylated using iodoacetamide. The proteins were subjected to digestion with trypsin (Sigma). The extracted peptides were analysed by matrix-assisted laser desorption-ionizationtime of ight mass spectrometry (Voyager-DETM PRO, Applied Biosystems). Peptide masses obtained by this analysis were used to search the National Center for Biotechnology Information database to identify the full-length proteins.

Scanning electron microscopy. Tracheas were dissected from embryos at embryonic day 18.5 (E18.5), xed overnight in 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.2 and post-xed with 2% osmium tetroxide (Electron Microscopy Sciences). Samples were dehydrated in a graded ethanol series and dried with hexamethyldisilazane (HMDS) or the CO2 critical point dryer Leica CPD-030. Specimens were coated with 10 nm gold/palladium in a Gatan Ion Beam Coater 681 and observed in a eld emission scanning electron microscope JSM-6700f (JEOL, Japan). Image processing and analyses were performed with ImageJ software.

Transmission electron microscopy. Tracheas were dissected from embryos at E18.5, xed overnight in 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.2 and post-xed with 2% osmium tetroxide. Samples were dehydrated in a graded ethanol series and embedded in Epon (Electron Microscopy Science). Longitudinal 200-nm-thick sections were prepared with an ultramicrotome (Leica) and mounted on copper grids (200 mesh). The samples were observed in a 200-kV eld emission gun electron microscope with an in-column energy lter (omega lter) JEM 2200 FS (JEOL, Japan).

Cilia counting and statistics. As ciliated cells in the trachea differentiate proximo-distally, we counted the cells in a series of jointed pictures taken from the larynx to the main bronchi to avoid any bias based on differentiation time. For a better visualization, results were normalized. This was achieved by taking the number of ciliated cells of WT embryos as 100%. In MEF cell cultures, primary cilia formation is not always homogeneous. Therefore, we took the pictures for counting in a randomized manner, observing the centrosome. The same eld was then examined for the presence of cilia.

Most of the experiments needing statistical validation involved paired observations on two variables for which we wanted to assess the independence of two populations. In those cases, the events being considered were mutually exclusive and the total probability was one. All observations were independent. Therefore, we used Fishers exact test or, when the sample sizes were large enough to avoid a type II error following Cochrans rules, we performed Pearsons w2-test without Yates correction. All calculations were made with GraphPad Prism 5. Statistical signicance in the uorescence intensity experiment (Fig. 8) was assessed via a MannWhitney test. The P-value is ranked based on the exact value given by the software. All error bars show s.d.

In one experiment, it was not possible to run any statistical test because of the nature of the data. To count ciliated cells in the tracheas of embryos, we had to compare individuals within the same litter to avoid bias arising from possible developmental differences between two litters. Consequently, there were too few embryos in each litter to perform statistical testing.

FACS analysis. Cell cycle proles of cycling as well as serum-starved WT or CyldD932 MEFs were determined by analysing total DNA content using propidium iodide staining (1 h incubation of ethanol-xed cells in PBS containing 20 mg ml 1 propidium iodide, 100 mg ml 1 RNase A, and 0.1% Triton X-100). Cells were subjected to ow cytometry by using a FACSCalibur analyser (BD Biosciences) and the results were analysed with FlowJo.

NF-jB inhibition by IjBa-SR. RPE1 cells were transfected by GFP-PACT-CYLDWT, GFP-PACT-CYLDH/N or GFP-PACT-CYLDD932 plasmids in the presence of an Ig-kB-Luc reporter plasmid with or without a plasmid expressing a non-degradable form of IkBa (IkBa-SR)41. Cells were serum starved 24 h after transfection to induce cilia formation and xed 48 h after serum depletion for cilia analysis. To measure the effect of IkBa-SR, cells were stimulated with 10 ng ml 1 of TNF-a just before removing the serum (24 h after transfection) or 3 h before completing the experiment. Cell extracts were prepared and Ig-kB-Luciferase-derived activity measured using a LB 9507 Berthold Luminometer.

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5585 ARTICLE

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Acknowledgements

We thank A. Hoppeler-Lebel for performing the CAP350 immunoprecipitations. We thank W. Faigle for mass spectrometry analysis and J.L. Guerquin-Kern for performing the sectioning for transmission electron microscopy. We thank Y. Bourgeois, A. Thadal,V. Dangles-Marie, I. Grandjean for general mouse care. We also thank the Institut Pasteur PFMU for providing access to the scanning electron microscope. We thank

Charlne Lasgi for performing the FACS analysis experiment and J.L. Ferrat for his advice. We thank M. Bornens, C. Janke, A. Fry, E. Nigg and G. Pereira for providing antibodies. We also thank S. Marco and I. Urbain for their preliminary observationsof tracheas in transmission electron microscopy. Finally, we thank M. Bornens, R. Basto,C. Janke, H. Hehnly, A. Fleury-Aubusson, J. Beisson and P. Guichard for critical reading of the manuscript. This work was supported by the INSERM (U759) and Institut Curie, by grant ARC3865 to A.-M.T and T.E. was funded by the French Ministry for Research and ARC (DOC20110603070).

Author contributions

A.-M.T. conceived and directed the project. T.E. and A.-M.T. designed and performed the experiments. A.-M.T., T.E. and G.C. discussed the project and wrote the manuscript. Y.Z. and A.J. performed the experiments on the CYLD KO mice; M.E., M.B. and M.P. generated and provided the CYLDD932 mice.

Additional information

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How to cite this article: Eguether, T. et al. The deubiquitinating enzyme CYLD controls apical docking of basal bodies in ciliated epithelial cells. Nat. Commun. 5:4585doi: 10.1038/ncomms5585 (2014).

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