© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 1769-1774 doi:10.1242/jcs.186221

SHORT REPORT CEP164-null cells generated by genome editing show a ciliation defect with intact DNA repair capacity Owen M. Daly1, David Gaboriau1,*, Kadin Karakaya2, Sinéad King1, Tiago J. Dantas1,‡, Pierce Lalor3, Peter Dockery3, Alwin Krämer2 and Ciaran G. Morrison1,§

ABSTRACT induced by the removal of growth factors, facilitates ciliogenesis Primary cilia are microtubule structures that extend from the distal end (Kobayashi and Dynlacht, 2011). Current models associate primary of the mature, mother . CEP164 is a component of the distal cilia with cell cycle exit and reduced proliferation, although the appendages carried by the mother centriole that is required for underlying mechanisms of such a link are not well defined (Goto primary cilium formation. Recent data have implicated CEP164 as a et al., 2013). CEP164 ciliopathy and suggest that CEP164 plays some roles in the encodes a centriolar appendage that is required DNA damage response (DDR). We used reverse genetics to test the for ciliogenesis (Graser et al., 2007; Schmidt et al., 2012). It has also role of CEP164 in the DDR. We found that conditional depletion of been implicated in modulating the DNA damage response (DDR), CEP164 in chicken DT40 cells using an auxin-inducible degron led to particularly CHK1 (Sivasubramaniam et al., 2008). CEP164 was no increase in sensitivity to DNA damage induced by ionising or initially identified in a proteomic analysis of the and, ultraviolet irradiation. Disruption of CEP164 in human retinal later, as a component of the distal appendages whose depletion by pigmented epithelial cells blocked primary cilium formation but did small interfering RNA (siRNA) treatment caused a marked not affect cellular proliferation or cellular responses to ionising or reduction in primary cilium formation (Andersen et al., 2003; ultraviolet irradiation. Furthermore, we observed no localisation of Graser et al., 2007; Schmidt et al., 2012). Immunoelectron CEP164 to the nucleus using immunofluorescence microscopy and microscopy demonstrated the localisation of CEP164 to the distal analysis of multiple tagged forms of CEP164. Our data suggest that end of the mother centriole (Graser et al., 2007). Dual CEP164 is not required in the DDR. photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) imaging has localised KEY WORDS: Primary cilium, Centrosome amplification, CEP164 in a ring around the centriole barrel with a periodic DNA damage response, DNA repair, CEP164, Ciliopathy enrichment of the signal within the ring (Sillibourne et al., 2011), and stimulated emission depletion microscopy has found that the INTRODUCTION enriched CEP164 signal corresponds to nine symmetrically- Primary cilia are membrane-enclosed, microtubule-based arranged clusters around the centriole, indicative of its association organelles that extend like antennae from the surface of most with each of the nine distal appendages (Lau et al., 2012). mammalian cell types to sense and transduce various extracellular Recent data have indicated CEP164 mutations play a role in signals. They arise from the basal body, a template provided when nephronophthisis-related ciliopathy, a rare recessive degenerative the mature, mother centriole docks to the plasma membrane (Goetz disease of the kidney, retina and brain, suggesting a link between and Anderson, 2010). display structural polarity, with the ciliopathy and a DDR role for CEP164 (Chaki et al., 2012). We set proximal ends containing microtubule triplets that taper to doublets out to explore the mechanisms that link ciliary dysfunction with at the distal ends. The distal ends of mature centrioles carry two sets DDR defects, using gene targeting to ablate CEP164 function. of appendages, which anchor cytoplasmic microtubules and which allow the docking of the mother centriole to the cell membrane RESULTS AND DISCUSSION during the formation of the primary cilium (Goetz and Anderson, To analyse the roles of CEP164 in DNA repair, we used gene 2010). The cilium core, the axoneme, consists of nine microtubule targeting in chicken DT40 cells to insert a tag that combined GFP doublets that extend from the basal body. with an auxin-inducible degron (AID; Nishimura et al., 2009) In mammalian cells, cilium formation is closely regulated and into the CEP164 locus of cells that stably expressed the TIR1 E3 linked to the cell cycle, as cilia must be resorbed to allow the basal ligase component (Fig. S1A,B). As shown in Fig. 1A, AID-GFP- body to act as a centrosome and to organise the mitotic spindle. tagged CEP164 localised to the centrosome, although we Cellular quiescence, a temporary exit from the cell cycle that can be observed no localisation of CEP164 to the nucleus, even after UV irradiation of cells to levels that induced a substantial 1Centre for Biology, School of Natural Sciences and National formation of γ-H2AX foci (Fig. 1B). Upon addition of auxin, University of Ireland Galway, Galway, Ireland. 2Clinical Cooperation Unit Molecular Hematology/Oncology, German Cancer Research Center (DKFZ) and Department AID-GFP-tagged CEP164 was depleted within 1 h (Fig. 1C; of Internal Medicine V, University of Heidelberg, Im Neuenheimer Feld 280, Fig. S1C,D). CEP164-deficient cells showed doubling times of 3 Heidelberg 69120, Germany. Anatomy, School of Medicine, National University of 8.3 h (clone 1) and 8.3 h (clone 2), compared with control times Ireland Galway, Galway, Ireland. *Present address: Facility for Imaging by Light Microscopy, Sir Alexander of 8.4 h and 8.4 h for each clone, respectively, and 8.3 h for wild- ‡ Fleming Building, Imperial College London, UK. Present address: Department of type cells. We observed no difference in sensitivity to ionising Pathology and Cell Biology, Columbia University, New York, USA. radiation or UV treatment between CEP164-deficient and wild- §Author for correspondence ([email protected]) type cells (Fig. 1D,E). In keeping with this observation, ionising- radiation-induced centrosome amplification, a potential readout

Received 18 January 2016; Accepted 8 March 2016 for the DDR (Bourke et al., 2007), occurred to the same levels in Journal of Cell Science

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Fig. 1. Wild-type DNA damage sensitivity after AID-mediated depletion of CEP164. (A) Centrosomal localisation of AID-GFP–CEP164 in chicken DT40s (green). Co-staining was for γ-tubulin (red). DNA was labelled with DAPI (blue). Scale bar: 2 µm. (B) Absence of nuclear AID-GFP-tagged CEP164 signal. Cells were treated with 10 J/m2 UV irradiation 1 h prior to fixation and staining for γ-H2AX (red) and DNA (blue). Scale bar: 2 µm. (C) Auxin- mediated depletion of AID-GFP– CEP164. The immunoblot shows total cell extracts from cells of the indicated genotype before and 24 h after treatment with 500 µM auxin. α-Tubulin was used as a loading control. (D,E) Clonogenic survival assay of cells of the indicated genotype after (D) ionising radiation or (E) UV irradiation. Curves show mean±s.d. of three independent experiments.

both CEP164-deficient and wild-type cells (Fig. S1E). These data to examine the roles of CEP164 in a cell line with high levels of show that CEP164 plays a limited role, if any, in nuclear primary ciliation. Thus, we used CRISPR-Cas9 technology to responses to ionising radiation or UV-induced DNA damage in disrupt CEP164 in hTERT-RPE1 cells, which show high levels of DT40 cells. primary cilium formation upon serum starvation. We used a guide Next, we cloned human CEP164 and expressed N- and RNA designed to direct DNA double-strand breaks in exon 9 (the C-terminally GFP- and FLAG-tagged versions in human cell 7th coding exon) of the human CEP164 locus, and selected clones lines. As shown in Fig. 2A–D, we consistently observed a that had lost CEP164 expression by immunoblot analysis (Fig. 3A). centrosomal localisation for recombinant overexpressed CEP164, Sequence analysis demonstrated that CEP164-deficient clones had but saw no nuclear signal. Immunofluorescence microscopy with incurred mutations in the CEP164 locus that led to premature stop previously published anti-CEP164 antibodies (Graser et al., 2007) codons being transcribed in-frame with the gene (Fig. S3A). also detected centrosomal, but not nuclear signals (Fig. 2E,F). Next, Immunofluorescence microscopy confirmed that these clones no we generated a new monoclonal antibody to CEP164. As shown in longer expressed CEP164, although they still carried intact Fig. S2A, monoclonal antibody 1F3G10 generated against amino centrioles (Fig. 3B). These cells proliferated as rapidly as wild- acids 6–296 of CEP164 recognised a protein of ∼200 kDa in three type cells, with doubling times of 24.1 h (clone 1) and 23.6 h (clone human cell lines. We next confirmed 1F3G10 specificity by depleting 2), compared with 23.5 h for wild-type cells. We saw no alteration CEP164 from RPE1 cells using siRNA. After CEP164 depletion, in cell cycle distribution in the absence of CEP164 (Fig. S3B). the1F3G10signaldisappeared,withnoeffectonaGAPDHcontrol Strikingly, CEP164-deficient cells showed a complete absence of (Fig. 2B). In immunofluorescence microscopy experiments, 1F3G10 primary ciliation capacity that was rescued by transgenic expression detected a signal that partly colocalised with , a component of of CEP164 (Fig. 3C,D). Transmission electron microscopy analysis the subdistal appendages, but localised adjacent to CEP135, a of the CEP164-null cells revealed no obvious structural defects in centriole proximal end component (Fig. S2C), consistent with the centriole structures, based on the dimensions of the centriole barrels known localisation of CEP164 at the distal appendages (Graser et al., (Fig. S3C). A total of 16 vesicles were identified in proximity to the 2007). Although we conclude from these experiments that the centrioles in seven CEP164-null cells, but no docking was 1F3G10 monoclonal antibody is specific for CEP164, there was no observed, consistent with a defect at the vesicle-docking stage in signal seen in the nucleus of U2OS or RPE1 cells. cilium formation seen in siRNA knockdown experiments (Schmidt Previous data have indicated a role for CEP164 in primary et al., 2012) (Fig. 3E). Thus, our disruption of the CEP164 locus ciliogenesis (Cˇ ajanek and Nigg, 2014; Graser et al., 2007; Schmidt confirms the findings made on the roles of CEP164 in primary et al., 2012; Chaki et al., 2012). Despite the feasibility of inducing ciliogenesis with siRNA experiments (Cˇ ajanek and Nigg, 2014; ciliogenesis in DT40 cells (Prosser and Morrison, 2015), we prefer Graser et al., 2007; Schmidt et al., 2012). Journal of Cell Science

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-deficient chicken DT40 cells (Dantas et al., 2011). Furthermore, we observed no localisation of CEP164 to nuclear DNA damage foci marked by γ-H2AX after ionising radiation or UV treatment in RPE1 or HeLa cells with either of the two antibodies in immunofluorescence experiments (Fig. 4B,C). Taken together, these data indicate no defect in the response to DNA damage in CEP164-deficient hTERT-RPE1 cells. The results in the two models we have explored do not support a role for CEP164 in the DDR. We did not see a proliferative decline, such as that described in IMCD3 cells after siRNA knockdown of CEP164 (Chaki et al., 2012) or an acceleration of cell cycle progression, as has been described after siRNA knockdown of CEP164 in RPE-FUCCI cells (Slaats et al., 2014). In our null lines, we observed no elevated sensitivity to ionising radiation or UV irradiation in the absence of CEP164, which contrasts with the phenotypes of UV sensitivity and loss of the G2-to-M checkpoint reported with siRNA knockdown of CEP164 in HeLa cells (Sivasubramaniam et al., 2008; Pan and Lee, 2009). These discrepancies have potential implications for understanding how CEP164 mutations cause disease. There are clear technical differences in the approaches that we have used and those previously reported. An obvious possibility is that the gradual or partial depletion imposed by siRNA treatment might lead to cellular responses different to those seen with the loss of a protein, although our degron-mediated experiment might have been expected to address this. Another possibility is that off-target effects of the siRNA treatments resulted in more marked phenotypes. Although the proliferative decline and cell cycle defects in IMCD3 cells were rescued by transgenic expression of human CEP164 (Chaki et al., 2012; Slaats et al., 2014), it is worth noting that rescues for the UV sensitivity and checkpoint defects seen in CEP164-knockdown cells were not performed (Sivasubramaniam et al., 2008; Pan and Lee, 2009), so that the specificity of these RNAi phenotypes cannot be assessed. We have not seen any significant nuclear localisation of CEP164 during the normal cell cycle or after DNA damage in one chicken and three human cell lines, using three different antibodies and multiple differently-tagged versions of transgenically expressed CEP164. Similarly to published results (Graser et al., 2007; Schmidt et al., 2012), our experiments have detected only cytosolic or centrosomal signals, in contrast to the predominantly nuclear signals reported with those antibodies generated in the original study that implicated CEP164 in the DDR (Sivasubramaniam et al., 2008). Tagging experiments and several antibodies used in a recently published study have shown predominantly cytosolic or centrosomal CEP164 signals, Fig. 2. Centrosomal, but not nuclear, localisation of CEP164 in human although these authors also observed nuclear signals using the cells. Localisation of the indicated transiently transfected tagged CEP164 original CEP164 antibodies (Chaki et al., 2012). Controls for the isoform (green) in (A) hTERT-RPE1 cells and (B–D) U2OS cells. Co-staining was for CEP135 or γ-tubulin (red). Iso., isoform. (E) Immunofluorescence specificity of the nuclear immunofluorescence signals seen with localisation of CEP164 (green) in U2OS cells using rabbit polyclonal these reagents after CEP164 knockdown or depletion have not antibodies (Graser et al., 2007). Co-staining was with antibodies for γ-tubulin been detailed (Sivasubramaniam et al., 2008; Pan and Lee, (red). (F) Immunofluorescence localisation of CEP164 (red) in hTERT-RPE1 2009). cells using rabbit polyclonal antibodies (Sigma). Co-staining was for γ-tubulin We have performed our DNA damage sensitivity and localisation (green). DNA was labelled with DAPI (blue). Scale bars: 5 µm. analyses in cell lines from different tissues. Thus, although we cannot exclude the possibility that CEP164 contributes to the DDR We next tested whether CEP164 deficiency impacted on the in certain cell types, this does not appear to be a general activity of ability of cells to withstand UV-induced DNA damage. A the protein. Our data, which support a marked defect in primary clonogenic survival assay showed that CEP164-deficient RPE1 cilium formation, but normal levels of DNA repair capacity in the cells were no more sensitive than wild-type cells (Fig. 4A). In a absence of CEP164, suggest that the principal cellular defect positive control experiment, CETN2-null RPE1 cells (Prosser and associated with CEP164 deficiency is the inability to undertake

Morrison, 2015) showed an increased UV sensitivity, as had primary ciliogenesis. Journal of Cell Science

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Fig. 3. Absence of primary ciliation in CEP164-null hTERT-RPE1 cells. (A) Immunoblot analysis of CRISPR-disrupted CEP164-null (KO) clones. (B) Absence of CEP164 signal in CEP164-null cells. Cells were stained with mouse monoclonal 1F3G10 (m) or polyclonal rabbit (r) antibodies to CEP164 then co-stained with antibodies to CEP135 or centrin2 and for DNA (blue). Scale bar: 5 µm. (C) Immunofluorescence microscopy analysis of primary cilia in cells of the indicated genotype. After 72 h serum starvation, cells were fixed and stained for detyrosinated tubulin (green) and centrin2 (red). DNA was visualised with DAPI (blue). Scale bar: 5 µm. (D) Quantitation of primary ciliation frequency in wild-type, CEP164-null cells and CEP164-null cells that were stably transfected with CEP164. Cilia were quantified by microscopy of detyrosinated tubulin and the bar graph indicates the mean±s.d. of three independent experiments in which at least 100 cells were counted. AS, asynchronous; SS, serum-starved. (E) Transmission electron micrograph analysis of ciliogenesis in cells of the indicated genotype. Panel 1 shows an assembled primary cilium in wild-type cells; panel 2 a docked ciliary vesicle; panels 3 and 4 show mother centrioles in proximity to vesicles without any docking in serum-starved CEP164-deficient cells. Scale bars: 500 nm.

MATERIALS AND METHODS irradiation where a degron-tagged protein was to be depleted, and retained in Cell culture the methylcellulose medium used for clonogenesis. For UV clonogenic Chicken DT40 cells were cultured as previously described (Takata et al., survival assays in hTERT-RPE1, cells were counted before being serially 1998). hTERT-RPE1 cells were cultured as previously described (Prosser diluted and plated in 10-cm dishes. The cells in each dish were allowed to and Morrison, 2015). HeLa and U2OS cells were obtained from ATCC and adhere for 6 h before the medium was siphoned off and they were irradiated. cultured in Dulbecco’s modified Eagle’s medium (DMEM; Lonza or PAA/ Conditioned medium (filtered medium taken from 50% confluent cells) was GE Healthcare), supplemented with 10% fetal calf serum (FCS; Lonza or used to replenish the dishes before incubation. Biochrom). Jurkat cells were from the European Collection of Animal Cell Cultures and were grown in RPMI with 10% FCS (Lonza). Auxin (Sigma- Cloning Aldrich) was prepared at 0.5 M in ethanol. Ionising radiation treatments used For targeting the chicken CEP164 locus, 5′ and 3′ homology arms and probe a 137Cs source (Mainance Engineering). For UV-C irradiation, cells were sequence were amplified from DT40 genomic DNA with KOD polymerase irradiated using an NU-6 254-nm UV-C lamp at 23 J/m2/min (Benda). DT40 (Novagen/Merck) using the following primers: 5′ arm, 5′-gacgtcCAGAC- clonogenic survival assays were performed as previously described (Takata AACAAGCTAGGATATGTACCT-3′ and 5′-ccgcggGTACCGGTACAC- et al., 1998), with 500 µM auxin added to the medium of cells 24 h prior to TTTAATTTGTCTGT-3′;3′ arm, 5′-agatctAAGGTGGGACTTGGTGTT- Journal of Cell Science

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Mutagenesis Kit (#210518, Agilent) with the following primers: deletion of GGAG, 5′-AGCAGTCCAAAGGCCTGGAAGGTTATCTCC- TC-3′ and 5′-GAGGAGATAACCTTCCAGGCCTTTGGACTGCT-3′; deletion of GTGAGTGGTGGCGGCAGCAGAGGATCGACTCAA, 5′- CCCCGCCTCACCCCCCGAGTC-TCA-3′ and 5′-TGAGACTCGGGG- GGTGAGGCGGGG-3′; insertion of GGAGAGGTACCAT, 5′-AGCAG- TCCAAAGGCCTGGAGGAGAGGTACCATAGGTTATCTCCTC-3′ and 5′-GAGGAGATAACCTATGGTACCTCTCCTCCAGGCCTTTGGACT- GCT-3′; insertion of TCGACTCAA, 5′-CCCCGCCTCACCTCGACTCA- ACCCCGAGTCTCA-3′ and 5′-TGAGACTCGGGGTTGAGTCGAGG- TGAGGCGGGG-3′.

CRISPR/Cas9 targeting of CEP164 in hTERT-RPE1 cells Primers targeting exon 9 (Mali et al., 2013) were cloned into pX330-U6- Chimeric_BB-CBh-hSpCas9 (plasmid 43330; Addgene; Cong et al., 2013): 5′-CACCGCTGTTGGCAAAGGGCGACA-3′ and 5′-AAACTGT- CGCCCTTTGCCCACAGC-3′. Transfections used Lipofectamine 2000 (Invitrogen). Genomic PCR products obtained with the diagnostic primer pair, 5′-CTGGGTGATTGATAACCATTGGG-3′ and 5′-CGCAAATGA- AGCTCCTGACTCAGT-3′ were cloned into pGEM-T-Easy and sequenced.

Monoclonal antibody generation cDNA sequence encoding CEP164 amino acids 6–296 was cloned into pGEX-4T1 (GE Healthcare) and the bacterially expressed GST fusion product was purified over a glutathione column prior to thrombin cleavage. Purified CEP164 protein fragment was used for hybridoma generation (Dundee Cell Products). Individual supernatants were screened by immunoblotting and microscopy and then concentrated antibody was purified from the best-performing 1F3G10 supernatant (Proteogenix).

RNA-mediated interference Cells were transfected with 50 nmol custom siRNA targeting CEP164 from Qiagen (5′-CAGGUGACAUUUACUAUUUCA-3′) or Silencer Select siRNA targeting GAPDH (5′-UGGUUUACAUGUUCCAAUATT-3′) using Oligofectamine (Invitrogen).

Fig. 4. Intact DDR in CEP164-null hTERT-RPE1 cells. (A) Clonogenic Immunofluorescence microscopy survival assay of cells of the indicated genotype after UV irradiation. Curves Cells were fixed for analysis as previously described (Prosser and Morrison, show mean±s.d. of three independent experiments. (B,C) Absence of 2015). Donkey and goat secondary antibodies were labelled with Cy3, Alexa nuclear CEP164 signals after DNA damage. hTERT-RPE1 (B) or HeLa (C) Fluor 488 or Alexa Fluor 594 (Jackson ImmunoResearch or Molecular 2 cells were treated with 10 Gy ionising radiation or 20 J/m UV 1 h prior to Probes). Rabbit polyclonal antibodies were against the following : γ- γ fixation and staining for CEP164 (green), -H2AX (red) and DNA (blue). Scale tubulin (1:1000, T3559, Sigma) γ-H2AX (1:1000, Ab2893, Abcam), bars: 5 µm. CEP135 (1:5000, 1420 739, Bird and Hyman, 2008), CEP164 (1:1000, HPA037606, Sigma), CEP164 (1:1000, R171, Graser et al., 2007), detyrosinated α-tubulin (1:1000, Ab48389, Abcam) and ninein (1:200, TTCAGCC-3′ and 5′-cctaggTTTGGGTTTCAGTGCCATCCCGTG-3′; ab4447, Abcam). Mouse monoclonal antibodies used were against γ-tubulin 5′ probe, 5′-CTTCTGATTTCAGTCCTGCGTGTT-3′ and 5′-CAGACA- (1:1000, GTU88, Sigma or TU-30/11–465-C100, Exbio), γ-H2AX (1:1000, TTAAATACAAGTCCCCTCC-3′ (lowercase letters represent non- JBW301, Upstate), CEP164 (1:100,000, 1F3G10) and centrin (1:1000, genomic sequences used for cloning). 20H5, Millipore). Images of DT40 and hTERT-RPE1 cells were captured on The probe for Southern analysis was labelled with digoxigenin using the an IX71 microscope (Olympus) with a 100× oil objective, NA 1.35, using PCR DIG Probe Synthesis Kit (Roche). AID-encoding sequence Volocity software (PerkinElmer), and are presented as maximum intensity (Eykelenboom et al., 2013) was subcloned into pEGFP-N1 (BD projections of z-stacks after deconvolution. Alternatively, images of U2OS Biosciences/Clontech) and a TIR1-9myc plasmid (pJE108; Eykelenboom cells were captured and processed using an Axiovert 200 M microscope et al., 2013) was stably cloned into DT40 cells to control the degron. equipped with a Plan-Apochromat 63×, NA 1.4 objective and AxioVision For cloning human CEP164 cDNA, hTERT-RPE1 RNA was extracted software (Carl Zeiss Microscopy) and are presented as single sections. using TRI reagent (Invitrogen). Reverse transcription was performed using SuperScript First-Strand (Invitrogen) and PCR with KOD Hot Start. cDNAs were cloned into pGEM-T Easy (Promega), sequenced Electron microscopy and then subcloned into pEGFP-N1, pEGFP-C1 (BD Biosciences/ hTERT-RPE1 cells were serum-starved in 0.1% FCS for 24 h prior to Clontech) or pCMV8 Tag 4A (Agilent Technologies, Santa Clara, CA, harvest. Cell pellets were prepared for transmission electron microscopy and USA). The primers used to amplify human CEP164 cDNA (isoform 1, imaged with an H-7000 Electron Microscope (Hitachi) as described (Prosser NP_055771.4) were as follows: 5′-aagcttATGGCTGGACGACCCCTCC- and Morrison, 2015). GCA-3′ and 5′-gtcgacCAGAAGCGATACACCYYCACTC-3′ (lowercase letters represent sequence added for cloning). Isoform 2 Immunoblotting (UniProt Q9UPV0, CE164_HUMAN) was cloned by mutating CEP164 Whole-cell extracts were prepared using RIPA buffer (50 mM Tris-HCl pH cDNA isoform 1 using the QuikChange Lightning Site-Directed 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA Journal of Cell Science

1773 SHORT REPORT Journal of Cell Science (2016) 129, 1769-1774 doi:10.1242/jcs.186221 and protease inhibitor cocktail). Immunoblot analyses used primary and CEP164 mutations, linking renal ciliopathies to DNA damage response antibodies against the following proteins: α-tubulin (1:10,000, B512, signaling. Cell 150, 533-548. Sigma), CEP164 (1:10,000, IF3G10), GFP (1:1000, 11814460001, Roche) Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A. et al. (2013). Multiplex genome engineering using and GAPDH (1:1000, 14C10, Cell Signaling). CRISPR/Cas systems. Science 339, 819-823. Dantas, T. J., Wang, Y., Lalor, P., Dockery, P. and Morrison, C. G. (2011). Flow cytometry Defective nucleotide excision repair with normal centrosome structures and Cells were fixed in 70% ice-cold ethanol overnight at 4°C, washed twice in functions in the absence of all vertebrate . J. Cell Biol. 193, 307-318. PBS, and incubated in 40 µg/ml propidium iodide and 200 µg/ml RNase A Eykelenboom, J. K., Harte, E. C., Canavan, L., Pastor-Peidro, A., Calvo- Asensio, I., Llorens-Agost, M. and Lowndes, N. F. (2013). ATR activates the S- in PBS for 1 h. Cytometry was performed on a FACSCanto (BD). M checkpoint during unperturbed growth to ensure sufficient replication prior to mitotic onset. Cell Rep. 5, 1095-1107. Acknowledgements Goetz, S. C. and Anderson, K. V. (2010). The primary cilium: a signalling centre We acknowledge the National Biophotonics and Imaging Platform Ireland and the during vertebrate development. Nat. Rev. Genet. 11, 331-344. NCBES Flow Cytometry core facility, which were supported by Irish Government Goto, H., Inoko, A. and Inagaki, M. (2013). Cell cycle progression by the repression Programme for Research in Third-Level Institutions cycles 4 and 5. of primary cilia formation in proliferating cells. Cell. Mol. Life Sci. 70, 3893-3905. Graser, S., Stierhof, Y.-D., Lavoie, S. B., Gassner, O. S., Lamla, S., Le Clech, M. Competing interests and Nigg, E. A. (2007). Cep164, a novel centriole appendage protein required for primary cilium formation. J. Cell Biol. 179, 321-330. The authors declare no competing or financial interests. Kobayashi, T. and Dynlacht, B. D. (2011). Regulating the transition from centriole to basal body. J. Cell Biol. 193, 435-444. Author contributions Lau, L., Lee, Y. L., Sahl, S. J., Stearns, T. and Moerner, W. E. (2012). STED O.M.D., K.K., A.K. and C.G.M. were responsible for project conception and microscopy with optimized labeling density reveals 9-fold arrangement of a direction. O.M.D., D.G., K.K., S.K., T.J.D., P.D., A.K. and C.G.M. undertook data centriole protein. Biophys. J. 102, 2926-2935. analysis. O.M.D., T.J.D., K.K. (cell biology); D.G., S.K. (monoclonal antibody) and P.L. Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E. (electron micscopy) performed experimental work. O.M.D. and C.G.M. wrote and Church, G. M. (2013). RNA-guided engineering via Cas9. the paper. Science 339, 823-826. Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. and Kanemaki, M. Funding (2009). An auxin-based degron system for the rapid depletion of proteins in Nat. Methods This work was funded by Science Foundation Ireland [Principal Investigator award nonplant cells. 6, 917-922. 10/IN.1/B2972]; and the European Commission [grant number SEC-2009-4.3-02, Pan, Y.-R. and Lee, E. Y.-H. P. (2009). UV-dependent interaction between Cep164 and XPA mediates localization of Cep164 at sites of DNA damage and UV project 242361 ‘BOOSTER’]. sensitivity. Cell Cycle 8, 655-664. Prosser, S. L. and Morrison, C. G. (2015). Centrin2 regulates CP110 removal in Supplementary information primary cilium formation. J. Cell Biol. 208, 693-701. Supplementary information available online at Schmidt, K. 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