Turkish Journal of Biology Turk J Biol (2018) 42: 371-381 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1805-25

The ciliopathy gene product Cep290 is required for primary formation and microtubule network organization

Elif Nur FIRAT-KARALAR* Department of Molecular Biology and Genetics, Faculty of Science, Koç University, İstanbul, Turkey

Received: 08.05.2018 Accepted/Published Online: 04.07.2018 Final Version: 25.10.2018

Abstract: The mammalian /cilium complex is composed of the centrosome, the primary cilium, and the centriolar satellites, which together function in key cellular processes including signaling. Defective assembly, maintenance, and function of the centrosome/ cilium complex cause the human genetic diseases known as ciliopathies, which are characterized by a multitude of developmental syndromes including retinal degeneration and kidney . The molecular mechanisms underlying pathogenesis in ciliopathies remain poorly understood, which requires structural and functional characterization of the mutated ciliopathy proteins at the cellular level. To this end, we elucidated the function and regulation of Cep290, which is the most frequently mutated gene in ciliopathies and importantly its functions remain poorly understood. First, we generated Cep290-null cells using the CRISPR/Cas9 genome editing approach. Using functional assays, we showed that Cep290-null cells do not ciliate and that they have defects in centriolar satellites dynamics and interphase microtubule organization. The centriolar satellites were tightly clustered around the centrosome in Cep290-null cells, and the interphase microtubule network lost its radial organization. Our results provide phenotypic insight into the disease mechanisms of Cep290 ciliopathy mutations and also the tools for studying genotype/phenotype relationships in ciliopathies.

Key words: Cep290, primary cilium, ciliopathy, centrosome, microtubules, centriolar satellites

1. Introduction Defects in the assembly and function of cilia are associated The centrosome is the main microtubule-organizing with a number of human diseases, including ciliopathies center of most animal cells and has emerged as an and primary ciliary dyskinesia (Bettencourt-Dias et al., important regulator of many critical cellular processes 2011). including division, polarity, and signaling. In cycling The centrosome is composed of two evolutionarily cells, the centrosome, composed of two centrioles and conserved microtubule-based cylindrical structures the associated pericentriolar material, organizes the termed centrioles, which recruit a matrix of associated interphase microtubule network and the mitotic spindle pericentriolar material (PCM) that nucleates and organizes (Azimzadeh and Bornens, 2007). Importantly, in some microtubules. All centrioles share a remarkable 9-fold cycling cells and most quiescent noncycling cells, one symmetric structure, but they differ in structural details, of the centrioles forms the that nucleates the age, and function. Due to the mechanism of centriole microtubule of the cilia, including the nonmotile duplication, one of the centrioles is the mother centriole primary cilium, motile cilia, and the (Nigg and and the other is the daughter centriole. The mother Raff, 2009). The primary cilium is a nonmotile sensory centriole, but not the daughter centriole, bears distal , which serves as the nexus for growth factor appendages that interact with the apical plasma membrane and mechano-sensing signaling pathways important (Sorokin, 1968; Vorobjev and Chentsov, 1982) and these in development and tissue including the appendages are critical structures in transforming the Hedgehog, Wnt, PDGF, and cyclic nucleotide pathways centriole to a basal body for the elongation of the axoneme (Johnson and Leroux, 2010; Nachury, 2014). In addition, during cilia formation (Kobayashi and Dynlacht, 2011). specialized multiciliated epithelial cells have many motile In addition to the centrioles and pericentriolar material, cilia on their surface, required for polarized movement of animal cells have an array of 70- to 100-nm granules liquid and particles along epithelial cells, and sperm cells that localize around the called centriolar have a single motile flagellum, required for sperm motility. satellites. Centriolar satellites are scaffolded by the 228- * Correspondence: [email protected] 371

This work is licensed under a Creative Commons Attribution 4.0 International License. FIRAT KARALAR / Turk J Biol kDa coiled-coil protein pericentriolar material 1 (PCM1) the entry and exit of proteins from the primary cilium. and they localize and move around the centrosome in a Over the years, the majority of the molecular components microtubule- and molecular-motor-dependent manner of the transition zone have been identified, including (Tollenaere et al., 2015). Centriolar satellites have been CC2D2A, MKS1, MKS3, MKS5, NPHP1, NPHP4, Tctn1, implicated in regulating centrosome- and cilia-related Tctn2, and Cep290 (Goncalves and Pelletier, 2017). cellular processes by sequestering and delivering proteins Cep290 is one of the most intriguing ciliopathy disease to or away from the centrosomes and cilia including the genes as mutations of Cep290 cause a wide variety of ciliopathy-associated proteins OFD1 and Cep290 (Lopes distinct phenotypes, ranging from isolated blindness, et al., 2011). , and BBS to the lethal MKS (Valente et Ciliopathies are human genetic diseases that are al., 2006; Baala et al., 2007; Brancati et al., 2007; Leitch et characterized by a broad spectrum of developmental al., 2008). The wide spectrum of diseases linked to Cep290 anomalies affecting multiple organ systems including retinal highlights the central and important roles of Cep290 in degeneration, , , hydrocephaly, cilium-related processes. Importantly, over 100 unique polycystic , and neurocognitive defects Cep290 mutations were identified in ciliopathy patients, (Van der Heiden et al., 2011). Among the ciliopathies making this gene the most frequently mutated gene in are Joubert syndrome, , Bardet–Biedl ciliopathies (Coppieters et al., 2010). However, genotype/ syndrome (BBS), oral-facial-digital syndrome (OFD1), phenotype studies for these unique mutations are very and Meckel–Gruber syndrome (MKS), which are limited and thus the molecular mechanisms underlying characterized by a distinct but overlapping set of clinical the pathogenesis of Cep290-linked ciliopathies remain symptoms. Importantly, a majority of the ciliopathy poorly understood, limiting the predictive power of a mutations identified to date have been mapped to the Cep290-related genotype. In order to understand how the genes that encode components of the centrosome/cilium unique Cep290 mutations result in the various Cep290 complex (Reiter and Leroux, 2017). Although over 187 disease phenotypes, it is essential to elucidate the function genes were shown to be mutated in ciliopathies, the and regulation of Cep290 at the cellular and organismal molecular mechanisms underlying the pathogenesis of level. these diseases remain poorly understood, which requires Cep290 forms the Y-links of the transition zone structural and functional characterization of the affected and functions as a gate keeper to control ciliary protein proteins in cells (Reiter and Leroux, 2017). composition by restricting the entry of nonciliary proteins Over the years, a number of the affected proteins (Betleja and Cole, 2010). In the green alga Chlamydomonas, in ciliopathies have been studied for their structure Cep290 localizes to the flagellar transition zone and forms and function in order to reveal the molecular defects the Y-links of the flagellar transition zone, which associates underlying ciliopathies. Ciliopathy-linked proteins were microtubules with the flagellar membrane (Betleja shown to function in the assembly, maintenance, and and Cole, 2010). The Chlamydomonas Cep290 mutant function of the primary cilium and these processes are flagella has abnormal levels of IFT complex proteins tightly regulated by selective trafficking of macromolecules and the BBSome component BBS4 (Craige et al., 2010). in and out of the primary cilium in healthy cells (Stephen Phenotypic characterization of Cep290-depleted cells by et al., 2017; Nachury, 2018). Given their strong link to different groups together showed that Cep290 localizes to ciliopathy phenotypes, dissecting the ciliary protein the centriolar satellites and the transition zone, and that transport mechanisms has been widely studied. Trafficking Cep290 functions in regulating cilium formation and of proteins from the cytosol to the primary cilium and the ciliary recruitment of the ciliogenic small GTPase Rab8 moving of proteins along the ciliary axoneme is regulated (Frank et al., 2008; Kim et al., 2008; Tsang et al., 2008; Stowe in part by vesicular trafficking (Stephen et al., 2017), et al., 2012). Finally, characterization of Cep290-/- mice (IFT) (Taschner and Lorentzen, showed that Cep290 loss in mice recapitulates ciliopathy 2016), the BBSome complex (Mourao et al., 2016; Nachury, symptoms including early vision loss and 2018), and the transition zone (Goncalves and Pelletier, and that Cep290 is required for formation of connecting 2017). cilia in photoreceptor cells and maturation of ciliated The transition zone is a selective barrier at the part ventricular ependymal calls (Rachel et al., 2015). of the primary cilium adjacent to the basal body that is Here, we describe the phenotypic consequences of composed of the transition fibers, ciliary necklace, and Cep290 ablation on cilium formation and function, Y-links (Goncalves and Pelletier, 2017). The transition zone centriolar satellite dynamics, and microtubule regulates ciliary compartmentalization through coupling organization in hTERT-RPE1 retinal pigmental epithelial the ciliary membrane to the microtubule axoneme and cells. To elucidate Cep290 function and regulation in impeding diffusion of proteins and lipids, and it controls mammalian cells, we first generated Cep290-null cells in

372 FIRAT KARALAR / Turk J Biol the RPE1::p53-/- parental background (Cep290 KO) by the 2.3. Lentivirus production and cell infection CRISPR/Cas9 genome editing approach. Using functional HIV-derived recombinant lentivirus expressing the gRNA assays, we showed that Cep290 KO cells do not ciliate and targeting Cep290 and Cas9 nuclease was made using the that they have defects in centriolar satellite distribution pLentiCRISPRv2g1 lentiviral transfer vector. Recombinant and interphase microtubule organization. The centriolar lentivirus was produced by cotransfection of HEK293T satellites were tightly clustered around the centrosome in cells with the pCMVDR8.74 packaging vector and pMD2. Cep290 KO cells, and the interphase microtubule network VSVG envelope vector (Dull et al., 1998) using PEI. Six lost its radial organization. These findings together identify hours after transfection, media were replaced with complete functions for Cep290 in regulating specific cilium, satellite, medium containing 10% FBS, and lentiviral supernatant was harvested after 48 h and passed through a 0.45-µm and microtubule-mediated activities. The results of this 5 study will provide important insight in elucidating how filter. For infection, 1 × 10 RPE1 cells were seeded on 6-well tissue culture plates the day before infection, which the unique Cep290 mutations result in the wide spectrum were infected with 1 mL of viral supernatant the following of human diseases. day. Twenty-four hours after infection, the medium was replaced with complete medium, and 48 h after infection, 2. Materials and methods cells were split and selected in the presence of 10 µg/mL 2.1. Cell culture and transfection puromycin for 5–7 days until all the control cells had died. hTERT-immortalized retinal pigment epithelial (RPE-1, LentiCRISPRv2 empty vector-transfected and puromycin- ATCC, CRL-4000) and RPE1:p53-/- cells were grown in selected cells were used as the control. After the Cep290 DMEM/F12 50/50 (Invitrogen) supplemented with 10% knockout efficiency was determined in the heterogeneous fetal bovine serum (FBS; Atlanta Biologicals, Lawrenceville, pool, cells were then trypsinized and serial dilutions were GA, USA). RPE1::p53-/- cells were generated by CRISPR- performed in normal growth medium. Colonies formed Cas9 genome editing, kindly provided by Meng-Fu Bryan within 10–14 days were then trypsinized and expanded Tsou (Izquierdo et al., 2014). Human embryonic kidney for screening for knockouts by immunofluorescence and cells (HEK293T, ATCC, CRL-3216) were grown in DMEM western blotting. supplemented with 10% FBS. All cells were cultured at 37 °C 2.4. Cloning and genome editing and 5% CO2. All cell lines were authenticated by multiplex The guide RNAs targeting the first exon of Cep290 were cell line authentication and were tested for Mycoplasma designed using the MIT CRISPR design tool (http://crispr. contamination by the MycoAlert Mycoplasma Detection mit.edu). The highest scoring guide RNA was selected Kit (Lonza). For experiments, RPE1 cells were based on the predicted specificity to the target gene in serum-starved in DMEM/F12 50/50 supplemented with order to reduce the off-target effects. Lentiviral transfer 0.5% FBS for 24 or 48 h. vector LentiCRISPRv2Cep290g1 was generated by cloning RPE1 cells were transfected with the plasmids using the following complementary oligos containing the Lipofectamine LTX according to the manufacturer’s Cep290 guide sequence and BsmBI (New England Biolabs, instructions (Invitrogen). HEK293T cells were transfected Ipswich, MA, USA) ligation adapters into LentiCRISPRv2 with the plasmids using 1 µg/µL polyethylenimine, MW (gift of Feng Zhang, Addgene plasmid #52961). Oligos 25 kDa (PEI, Sigma-Aldrich, St. Louis, MO, USA). were purchased from Macrogen (Amsterdam, the Netherlands) and resuspended to a stock concentration of 2.2. Nocodazole washout experiments 100 µm in nuclease-free water and stored at –20 °C. The Cells were seeded on glass coverslips in 24-well plates oligo sequences were as follows: and grown to 70%–80% confluency. Nocodazole (Sigma- Oligo 1: CACCGTCTTGACGGGGCAGGTCATC, Aldrich) was diluted to 10 µg/mL in culture medium Oligo2: AAACGATGACCTGCCCCGTCAAGAC. from the 10 mM stock solution in DMSO. Cells were Briefly, oligos were phosphorylated using the T4 incubated with 10 µg/mL nocodazole for 1 h at 37 °C to polynucleotide kinase (New England Biolabs), annealed, achieve complete microtubule depolymerization. Cells and ligated into the BsmBI-digested LentiCRISPRv2 were then washed 2 times with normal growth medium plasmid. The construct was verified by DNA sequencing. and incubated at 37 °C to allow regrowth of microtubules. 2.5. Antibodies During regrowth, cells were fixed at 0, 10, and 30 min in Anti-PCM1 antibody was generated and used for methanol and stained with the anti-α-tubulin and anti- immunofluorescence as previously described (Firat- γ-tubulin antibodies, markers for the microtubules and Karalar et al., 2014). Other antibodies used for the centrosome, respectively. Images shown correspond immunofluorescence in this study were goat anti-PCM1 to a representative experiment from three independent (sc-50164; Santa Cruz Biotechnology) at 1:1000, mouse experiments. anti-γ-tubulin (GTU-88; T5326; Sigma-Aldrich) at 1:4000,

373 FIRAT KARALAR / Turk J Biol rabbit anti-Cep290 (ab84870; Abcam) at 1:1000, and were then transferred onto nitrocellulose membranes mouse anti-polyglutamylated tubulin (GT335; AG-20B- via the Mini Transblot wet transfer system (Bio-Rad 0020-C100; Adipogen) at 1:500. Secondary antibodies used Laboratories). After transfer, membranes were incubated for immunofluorescence experiments were AlexaFluor in blocking solution (PBS + 5% milk, 0.1% Tween-20) for 488-, 568- or 633-coupled (Life Technologies) and they 1 h at room temperature with gentle rocking, followed by were used at 1:2000. Antibodies used for western blotting primary antibody incubation overnight at 4 °C, 3 washes were rabbit anti-Cep290 (ab84870; Abcam) at 1:1000 and with PBS + 0.1% Tween-20 (PBST), secondary antibody rabbit anti-actin (CST 4970; Cell Signaling Technology) incubation for 1 h at room temperature, and 3 washes at 1:2000. Secondary antibodies used for western blotting with PBST. Visualization of the blots was carried out experiments were IRDye 680- and IRDye 800-coupled and with the LI-COR Odyssey scanner and software (LI-COR were used at 1:1000 (LI-COR Biosciences). Biosciences). Blots were imaged using an Odyssey Infrared 2.6. Immunofluorescence, microscopy, and quantification Imaging System with scan resolution at 169 µm. For immunofluorescence experiments, cells were 2.8. Statistical analysis grown on coverslips and fixed in either methanol or 4% Statistical significance and P-values were assessed by one- paraformaldehyde in PBS. After rehydration in PBS, way analysis of variance and Student t-tests using Prism cells were blocked in 3% BSA (Sigma-Aldrich) in PBS + software (GraphPad Software, La Jolla, CA, USA). Error 0.1% Triton X-100. Coverslips were incubated in primary bars reflect SEM or SD. The following key is followed for antibodies diluted in blocking solution, and Alexa Fluor asterisk placeholders for P-values in the figures: ***P < 488-, 594-, or 633-conjugated secondary antibodies 0.001, **P < 0.01, *P < 0.05. were diluted at 1:2000 in blocking solution (Invitrogen). Coverslips of cells were imaged using LAS X software 3. Results (Premium; Leica) on a scanning confocal microscope 3.1. Generation and validation of RPE1::p53-/- Cep290- (SP8; Leica Microsystems) with Plan Apofluar 63X 1.4 NA /- cell lines objective. To elucidate the function of Cep290 in ciliopathy- Quantitative immunofluorescence for PCM1 was associated phenotypes, we first generated Cep290 null performed on cells by acquiring a z-stack of control and hTERT-immortalized retinal pigment epithelial cells depleted cells using identical gain and exposure settings. null for p53 (hereafter RPE1::p53-/-) by employing The z-stacks were used to assemble maximum-intensity the clustered regularly interspaced short palindromic projections. The centrosome region was defined by repeats CRISPR/Cas9 technology to disrupt both copies g-tubulin staining in each cell and its fluorescence pixel of the Cep290 locus. Cells ablated for most centrosome intensity was measured by defining a circular area of 3 proteins are not viable in the presence of p53 as the lack 2 µm centered on the centrosome as the region of interest. of centrosome proteins results in cell cycle arrest (Bazzi Quantifications and image processing were performed and Anderson, 2014). Therefore, we used RPE1::p53-/- using ImageJ (National Institutes of Health, Bethesda, cells for generating Cep290 nulls, which is a widely used MD, USA). The background was quantified by measuring approach for generating cell lines null for genes encoding the fluorescence intensity of a region of equal dimensions centrosome proteins (Izquierdo et al., 2014). RPE1::p53-/- in the area neighboring the centrosome and the satellites. cells were transfected with the pLentiCRISPRv2 plasmid Primary cilium formation was assessed by counting the harboring the Cep290 guide RNA (gRNA) designed to total number of cells and the number of cells with primary target exon 2 (protein-coding exon 1) or with control cilia, as detected by glutamylated tubulin staining. All data pLentiCRISPRv2 plasmid, sorted, and plated as single acquisition was done in a blinded manner. cells. We screened 10 single cell clones, detecting Cep290 2.7. Cell lysis and immunoblotting first with immunofluorescence experiments and later For preparation of cell extracts, cells were lysed in 50 with immunoblotting experiments for the clones that we mM Tris (pH 7.6), 150 mM NaCI, 1% Triton X-100, and validated as null by microscopic analysis. The two clones protease inhibitors for 30 min at 4 °C, briefly sonicated, we picked for further phenotypic characterization of and centrifuged at 15,000 × g for 20 min. The protein RPE1::p53-/- Cep290-/- (hereafter Cep290 KO1 and KO2) concentration of the resulting supernatants was determined lacked detectable Cep290 signals while the signals for the with Bradford solution (Bio-Rad Laboratories, Hercules, centrosome protein γ-tubulin were detectable at similar CA, USA) using bovine serum albumin as a standard. levels in control and Cep290 KO cells (Figure 1A). In For immunoblotting, cell extracts were first resolved on agreement with the immunofluorescence data, as shown 10% acrylamide sodium dodecyl sulfate polyacrylamide in Figure 1B, the rabbit polyclonal Cep290 antibody gel electrophoresis (SDS-PAGE) using the Mini-Protean recognized a major band above 250 kDa in control system (Bio-Rad Laboratories). Resolved proteins cells in immunoblot experiments, which disappeared

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A Cep290 gamma-tubul n Merge+ DAPI Zoomed Area RPE1::p53-/- RPE1::p53-/- CEP290-/- KO1 RPE1::p53-/- CEP290-/- KO2

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RPE1::p53-/- RPE1::p53-/- Cep290-/- KO1

CEP290 (290 kDa)

act n (42 kDa)

Figure 1. Generation and validation of Cep290 KO cells. A) Immunofluorescence analysis of control and Cep290 KO1 and KO2 cells using the Cep290 antibody (green) and γ-tubulin (red) antibody, which marks the pericentriolar material. DNA was stained with DAPI. Scale bar = 5 µm. The yellow boxes in the merged images were selected to zoom in in order to emphasize the lack of Cep290 signal in Cep290 KO cells relative to control cells that are positive for Cep290 signal at the centrosome and centriolar satellites; scale bar for the zoomed image is 1 µm. Images represent cells from the same coverslip taken with the same camera settings. Cep290 KO cells lack detectable Cep290 signal. B) Immunoblots of cell extracts of the control and Cep290 KO1 cells with anti-Cep290 antibody raised against the C-terminus of Cep290 and anti-actin antibody, which was used as a loading control. The 290 kDa band for the full-length Cep290 is absent in the Cep290 KO1 cells. in the Cep290 KO 1 clone, validating the Cep290 null together confirmed that our strategy generated a clone state. Characterization of the Cep290 KO clones by that is null for Cep290 and we used this clone in further immunofluorescence and immunoblotting experiments experiments to elucidate the cellular functions of Cep290.

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A B glutamylated tubul n PCM1 Merge+ DAPI

50 40

RPE1::p53-/- 30 20 10 % c l um format on !! !! 0

RPE1::p53-/- CEP290-/- KO1 RPE1::p53-/-RPE1::p53-/- RPE1::p53-/- CEP290-/- KO1 CEP290-/- KO2 RPE1::p53-/- CEP290-/- KO2

Figure 2. Cep290 is essential for primary cilium formation. A) Effect of Cep290 KO on cilium formation. Representative images of RPE1 control and Cep290 KO1 and KO2 cells fixed 48 h after serum starvation and stained with anti-glutamylated antibody (red), which marks the cilium, and the anti-PCM1 antibody (green), which marks the centriolar satellites. DNA was stained with DAPI. Scale bar = 10 µm. B) Quantification of percentage of cells with cilia in control and Cep290 KO1 and KO2 cells after 48 h of serum starvation. Cells were fixed, stained for cilia, and quantified. n = 500 cells for each sample, from three independent experiments. Data represent mean ± SD from three independent experiments, t-test was used for statistical analysis, **P < 0.01.

3.2. Cep290 KO cell lines are defective in primary cilium The derivation of Cep290 KO clones allowed us to assay formation ciliogenesis in a clean background, which is very different Given that Cep290 is frequently mutated in ciliopathies and from the phenotypic analysis done using cells transiently that its RNAi depletion resulted in about twofold decrease depleted for Cep290 using RNAi experiments. Our results in the ciliogenesis efficiency of mammalian cells (Kim et are significant in identifying Cep290 as an essential and al., 2008; Stowe et al., 2012), we set out to determine the critical component of the cilium biogenesis pathway ability of Cep290 KO clones to form a primary cilium and are in agreement with the cellular and organismal using quantitative immunofluorescence experiments. To phenotypes reported in the Cep290-/- mouse (Rachel et test whether Cep290 is required or essential for efficient al., 2015). ciliogenesis, we serum-starved control and Cep290 KO1 3.3. Centriolar satellites are redistributed in Cep290 KO and KO2 clones over 48 h to induce ciliogenesis and cell lines counted the percentage of cells that formed a primary Centriolar satellites localize around the centrosome in a cilium using glutamylated-tubulin antibody as the cilium microtubule- and molecular-motor-dependent manner, marker (Figure 2A). Importantly, both Cep290 KO1 and which results in a majority of them clustering around KO2 clones did not ciliate after 48 h of serum starvation, the centrosome and the remaining being homogeneously indicating that Cep290 activity is essential for primary distributed throughout the cytoplasm. Depletion of other cilia formation (Figure 2B and 2C). Previous studies satellite proteins including CCDC11 (Silva et al., 2016) identified regulatory roles for Cep290 in cilium formation and CEP72 (Stowe et al., 2012) were shown to reduce using RNAi experiments, where Cep290-depleted cells the ciliogenesis ability of the depleted cells by dispersing still ciliated to 30%–40% relative to the 80% ciliating satellites throughout the cytoplasm or tightly clustering control population (Kim et al., 2008; Stowe et al., 2012). satellites around the centrosome, respectively. Given

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A B

PCM1 Cep290 Merge+ DAPI

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RPE1::p53-/- 300 200 100 ntens ty (A.U.)

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RPE1::p53-/- RPE1::p53-/- CEP290-/- KO1 RPE1::p53-/- CEP290-/- KO1

Figure 3. Cep290 regulates centriolar satellite distribution. A) Effect of Cep290 KO on centriolar satellite organization. Control and Cep290 KO1 cells were fixed and stained with anti-PCM1 antibody (green), which marks centriolar satellites, and anti-Cep290 (red), which marks the centrosome and the centriolar satellites. DNA was stained with DAPI. Images represent cells from the same coverslip taken with the same camera settings. Scale bar = 10 µm. B) Effect of Cep290 KO1 on the pericentrosomal level of PCM1. Pericentrosomal PCM1 fluorescence intensity was measured for control and Cep290 cells in an area of 3 µm2 around the centrosome and levels are represented as arbitrary fluorescence intensity values. n = 50 cells for each sample, from two independent experiments. t-test was used for statistical analysis. Data represent mean ± SD from two independent experiments, **P < 0.01. that centriolar satellite distribution was shown to play satellites associated with an increase in their concentration important roles during primary cilium formation and that around the centrosome upon RNAi-mediated depletion of Cep290 also localizes both to the centriolar satellites and Cep290 in mammalian cells (Kim et al., 2008) and thus the centrosome, we hypothesized that the primary cilium further corroborate the function of Cep290 in regulation formation defect in Cep290 KO1 cells can be due to changes of satellite dynamics. in the cellular distribution of centriolar satellites. To test 3.4. Microtubule network organization is disrupted in this hypothesis and to test whether Cep290 is required Cep290 KO cell lines for satellite dynamics in general, we used quantitative The pericentrosomal distribution of centriolar satellites is immunofluorescence experiments to determine the in part dependent on a centrosomally focused interphase amount of pericentrosomal PCM1 in control and Cep290 microtubule radial array. Given that Cep290 was previously KO1 cells. PCM1 is the molecular scaffold for centriolar implicated to function in the formation and maintenance satellites and quantification of its levels within an area of 3 of a radial microtubule array anchored at the centrosome µm2 centered on the centrosome is a standard assay used to assay satellite distribution phenotypes in cells (Stowe et al., in interphase (Kim et al., 2008), we hypothesized that 2012; Gupta et al., 2015). Relative to control cells, Cep290 Cep290 might be regulating satellite distribution through KO1 cells had a significant increase in the PCM1 intensity its function during microtubule network organization. To around the centrosome (P < 0.01) and a corresponding determine whether redistribution of centriolar satellites reduction in cytoplasmic centriolar satellite distribution in Cep290 KO1 cells is a direct effect of Cep290 or an (Figures 3A and 3B), identifying a tight clustering indirect effect through loss of microtubule organization, phenotype for satellites in Cep290 KO1 cells. These results we assayed the organization of microtubules in Cep290 show that Cep290 plays a role in the regulation of the KO1 and control cells using nocodazole washout regulation of dynamic satellite distribution in cells, possibly experiments. Control and Cep290 KO1 cells were treated through regulating the satellite dynamics by modulating for 1 h with 10 µg/mL nocodazole at 37 °C to depolymerize the affinity of the satellites to the molecular motors or all microtubules, which we confirmed by staining for microtubules. These results are in agreement with the microtubules in cells fixed after nocodazole treatment previous reports that showed redistribution of centriolar indicated as 0 min in Figure 4A. Following nocodazole

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A B 0 m n 10 m n 30 m n M crotubules RPE1::p53-/- RPE1::p53-/- CEP290-/- KO1

Merge: m crotubules Cep290 DAPI Figure 4. Cep290 is required for the interphase microtubule organization. A) Control and Cep290 KO1 cells were treated with 10 µg/mL nocodazole for 1 h and microtubules were visualized right after nocodazole treatment (t = 0 min) and 10 and 30 min after nocodazole washout. Representative images of RPE1 control and Cep290 KO1 cells fixed throughout the nocodazole washout experiment at the indicated time points and stained with α-tubulin antibody (green), which marks the microtubules, and the anti-Cep290 antibody (green), which marks the centrosome and the centriolar satellites. DNA was stained with DAPI. Scale bar = 10 µm. B) The microtubule staining of the same images of control and Cep290 KO1 cells 30 min after nocodazole washout in A is presented in inverted form in order to emphasize the defects in interphase microtubule organization. treatment, cells were washed to remove nocodazole and situs inversus, hydrocephaly, polycystic kidney disease, and to allow regrowth of microtubules at 37 °C. We visualized neurocognitive defects. While genetic studies identified at the microtubule network in control and Cep290 KO1 cells least 187 genes mutated in ciliopathies (Reiter and Leroux, by staining with a monoclonal antibody against α-tubulin 2017), the molecular mechanisms underlying pathogenesis (Figure 4A). Ten minutes after nocodazole washout, both in the ciliopathies remain poorly understood due to the control and Cep290 KO1 cells nucleated microtubule asters lack of information on the functional and structural of similar sizes around their centrosomes, indicating that consequences of the ciliopathy mutations in affected genes. Cep290 does not result in microtubule nucleation defects An essential first step to dissecting the molecular pathways (Figure 4A). However, 30 min after nocodazole washout, underlying ciliopathy phenotypes is the structural and control cells had significantly (P < 0.05) higher percentages functional characterization of the ciliopathy genes at the of centrosomally organized distinct radial microtubule cellular level as the results of such characterization will arrays (78.5 ± 4.9%, n = 200) than Cep290 KO1 cells (52 provide insight into which cellular processes are defective ± 4.2%, n = 200) (Figure 4B). This result indicates that in ciliopathies. To address this longstanding problem, Cep290 is required for microtubule organization and that we dissected the centrosome/cilium-complex-related might in part explain the centriolar satellite distribution functions of the Cep290 ciliopathy gene product using the and ciliogenesis phenotypes of Cep290 KO1 cells. Cep290 KO clones we generated in this study. The centrosome and centriolar satellite protein Cep290 4. Discussion is the most mutated gene in ciliopathies and we chose Ciliopathies are diseases that result from defective Cep290 for further characterization of its cellular functions centrosome/cilium-complex assembly and function for two main reasons. First, over 100 unique mutations are and are characterized by a shared set of developmental mapped to the Cep290 gene, which cause a wide spectrum symptoms including retinal degeneration, polydactyly, of disorders including Joubert syndrome, MKS, and BBS

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(Valente et al., 2006; Travaglini et al., 2009), making Cep290 of the RNAi data using genetic ablation experiments the most mutated gene in ciliopathies. Second, Cep290 and also cautions against the direct interpretation of the is the gatekeeper for regulating transport of proteins in RNAi phenotypes to explain the in vivo phenotypes in and out of the cilia by forming the Y-links of the ciliary model organisms and the disease phenotypes. Second, it transition zone (Betleja and Cole, 2010). In this study, we identifies an essential role for Cep290 in primary cilium sought to determine the molecular defects underlying assembly instead of the regulatory role identified by RNAi Cep290-linked ciliopathies by generating Cep290 null experiments. The underlying mechanism of this essential cells and phenotypically characterizing the phenotypic role must be elucidated in future experiments in order consequences of the Cep290 null state in cells. Our results to determine where Cep290 functions in the ciliogenesis suggest that Cep290 is essential for cilium formation and pathway. Once the exact molecular mechanism is known, regulates centriolar satellite dynamics and organization these results will lead to understanding the disease of intact interphase microtubule arrays. These results mechanisms of Cep290-mutations. highlight the multitude of cellular functions associated Genetic ablation of Cep290 results in centrosomal with Cep290 and provide insight in understanding the clustering of centriolar satellites, which normally have a wide range of phenotypic defects underlying ciliopathies cytoplasmically distributed pool in mammalian cells. This and in the phenotypic heterogeneity associated with result is in agreement with the reported phenotypes in Cep290-mutated ciliopathies. RNAi studies and identifies a regulatory role for Cep290 in Here, we show that CRISPR/Cas9-mediated ablation of the dynamic behavior of centriolar satellites, which localize Cep290 inhibited ciliogenesis and disrupted organization and move around the centrosome/cilium-complex in a of the centriolar satellites and the interphase microtubule microtubule- and molecular-motor-dependent manner network. Overall, these results are consistent with the (Tollenaere et al., 2015). The trafficking of the centriolar previous RNAi and short hairpin RNA (shRNA) studies satellites to the centrosome requires dynein/dynactin complex activity, which was shown by dynein inhibition that showed that transient knock-down of mammalian experiments, and this requirement was studied at the Cep290 leads to reduction in ciliogenesis, defects in radial molecular level (Kubo et al., 1999; Dammermann and microtubule organization, and clustering of centriolar Merdes, 2002). The trafficking of centriolar satellites away satellites around the centrosome (Frank et al., 2008; Kim et from the centrosome possibly involves kinesin motors, al., 2008; Tsang et al., 2008; Stowe et al., 2012). One major which has not been experimentally tested before. Given the discrepancy between these CRISPR and RNAi studies is clustering phenotype we observed in Cep290-null cells, we that Cep290 KO cells do not ciliate but Cep290-depleted suggest that Cep290 is either a positive regulator of dynein- cells still ciliate despite the significant reduction in mediated transport or a negative regulator of kinesin- efficiency. This discrepancy is likely due to the incomplete mediated transport. Proteomics studies identified putative acute/transient depletion of Cep290 in siRNA and shRNA and validated interactions of Cep290 with the dynactin experiments. Our results identify Cep290 as an essential components and the kinesin motor Kif4a, supporting component of the cilium assembly pathway. our proposed model for Cep290 function in centriolar The ciliogenesis defects we observed in human satellite trafficking through modulating molecular motor RPE1 cells are in part in agreement with the phenotypic interactions or activity. Future work is required to test the characterization of Cep290-/- mouse photoreceptor predictions of this model. and ependymal cells derived from Cep290-/- mice The results of this work will be significant in that identified severe phenotypes in formation and/ correlating the phenotypes of the Cep290 null cells with or maturation of cilia in these cells (Rachel et al., 2015). the phenotypic consequences of the Cep290 ciliopathy While photoreceptor Cep290-/- photoreceptor cells mutations that result in early stop codons in the Cep290 lacked the connecting cilium and were defective in inner gene. Such analysis will contribute to determining and outer segment formation, Cep290-/- ependymal cells genotype/phenotype relationships for Cep290 mutations still had primary cilia, but these cilia showed progressive that cause a frameshift mutation introducing an early developmental defects. The variance in the severity of premature stop codon that results in complete loss of the ciliogenesis phenotypes in different tissues in mice protein. Our results suggest that Cep290 ciliopathy and in Cep290 KO RPE1 cells suggests that Cep290 have mutations might affect regulation of cilium assembly tissue-specific functions during ciliogenesis and that these and centriolar satellite distribution. As an indication of functions might contribute to the multitude of phenotypes the potential broader involvement of Cep290 in human in Cep290-linked ciliopathies. disease, Cep290 has been reported to interact with other Our characterization of Cep290 nulls for cilium disease-associated proteins, including CCDC66, which assembly defects has two important consequences. First, it causes retinal degeneration when mutated (Dekomien et highlights the importance of functional follow-up studies al., 2010; Gerding et al., 2011; Conkar et al., 2017).

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In addition to characterization of Cep290 function leads to disease and what causes the diversity of Cep290 in a clean null genetic background, the Cep290-null cells mutations discovered in patients, our study contributes generated in this study will allow us and others to address to this longstanding problem by identifying the precise another important question: what are the phenotypic functions of Cep290 in cilium-related cellular processes. consequences of the unique Cep290 mutations identified in ciliopathies? For example, experiments where Cep290 Acknowledgments gene fragments harboring the disease mutations or Cep290 I acknowledge Kübra Hazal Zırhlıoğlu and Ezgi truncation mutants that mimic the truncated proteins Odabaşı for insightful discussions regarding this work. described in patients with Cep290 mutations could be RPE1::p53-/- cells were a kind gift from Meng-Fu Bryan expressed in the Cep290 null to determine whether Tsou (Memorial Sloan-Kettering Cancer Center, New their expression rescues the satellite and microtubule York, NY, USA). This work was supported by the ERC organization defects of these cells. These experiments StG679140 Grant, an EMBO Installation Grant, TÜBİTAK will reveal the genotype and phenotype relationships Grant 115Z521, a FABED Eser Tümen Research Award, for different Cep290 mutations. While we are still a long and Turkish Academy of Sciences Distinguished Young way away from understanding how Cep290 dysfunction Scientist Award.

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