Title: ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis
Abstract: Article18 March 2022free access Source DataTransparent process ENKD1 promotes CP110 removal through competing with CEP97 to initiate ciliogenesis Ting Song Ting Song orcid.org/0000-0001-5945-6676 Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Data curation, Validation, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Yunfan Yang Yunfan Yang orcid.org/0000-0002-5266-7208 Cheeloo College of Medicine, Shandong University, Jinan, China Contribution: Formal analysis, Investigation Search for more papers by this author Peng Zhou Peng Zhou Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Investigation Search for more papers by this author Jie Ran Jie Ran orcid.org/0000-0002-0744-7126 Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Investigation Search for more papers by this author Liang Zhang Liang Zhang Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Investigation, Methodology Search for more papers by this author Xiaofan Wu Xiaofan Wu State Key Laboratory of Medicinal Chemical Biology, Haihe Laboratory of Cell Ecology, Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin, China Contribution: Investigation Search for more papers by this author Wei Xie Wei Xie orcid.org/0000-0002-3844-3013 Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Investigation Search for more papers by this author Tao Zhong Tao Zhong orcid.org/0000-0003-3898-057X Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Investigation Search for more papers by this author Hongbin Liu Hongbin Liu orcid.org/0000-0003-2550-7492 Cheeloo College of Medicine, Shandong University, Jinan, China Search for more papers by this author Min Liu Min Liu Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Formal analysis Search for more papers by this author Dengwen Li Dengwen Li State Key Laboratory of Medicinal Chemical Biology, Haihe Laboratory of Cell Ecology, Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin, China Contribution: Data curation, Formal analysis Search for more papers by this author Huijie Zhao Corresponding Author Huijie Zhao [email protected] orcid.org/0000-0002-8595-8159 Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Data curation, Formal analysis, Writing - review & editing Search for more papers by this author Jun Zhou Corresponding Author Jun Zhou [email protected] orcid.org/0000-0003-3131-7804 Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China State Key Laboratory of Medicinal Chemical Biology, Haihe Laboratory of Cell Ecology, Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin, China Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Ting Song Ting Song orcid.org/0000-0001-5945-6676 Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Data curation, Validation, Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Yunfan Yang Yunfan Yang orcid.org/0000-0002-5266-7208 Cheeloo College of Medicine, Shandong University, Jinan, China Contribution: Formal analysis, Investigation Search for more papers by this author Peng Zhou Peng Zhou Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Investigation Search for more papers by this author Jie Ran Jie Ran orcid.org/0000-0002-0744-7126 Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Investigation Search for more papers by this author Liang Zhang Liang Zhang Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Investigation, Methodology Search for more papers by this author Xiaofan Wu Xiaofan Wu State Key Laboratory of Medicinal Chemical Biology, Haihe Laboratory of Cell Ecology, Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin, China Contribution: Investigation Search for more papers by this author Wei Xie Wei Xie orcid.org/0000-0002-3844-3013 Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Investigation Search for more papers by this author Tao Zhong Tao Zhong orcid.org/0000-0003-3898-057X Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Investigation Search for more papers by this author Hongbin Liu Hongbin Liu orcid.org/0000-0003-2550-7492 Cheeloo College of Medicine, Shandong University, Jinan, China Search for more papers by this author Min Liu Min Liu Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Formal analysis Search for more papers by this author Dengwen Li Dengwen Li State Key Laboratory of Medicinal Chemical Biology, Haihe Laboratory of Cell Ecology, Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin, China Contribution: Data curation, Formal analysis Search for more papers by this author Huijie Zhao Corresponding Author Huijie Zhao [email protected] orcid.org/0000-0002-8595-8159 Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China Contribution: Data curation, Formal analysis, Writing - review & editing Search for more papers by this author Jun Zhou Corresponding Author Jun Zhou [email protected] orcid.org/0000-0003-3131-7804 Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China State Key Laboratory of Medicinal Chemical Biology, Haihe Laboratory of Cell Ecology, Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin, China Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Author Information Ting Song1,†, Yunfan Yang2,†, Peng Zhou1, Jie Ran1, Liang Zhang1, Xiaofan Wu3, Wei Xie1, Tao Zhong1, Hongbin Liu2, Min Liu1, Dengwen Li3, Huijie Zhao *,1 and Jun Zhou *,1,3 1Shandong Provincial Key Laboratory of Animal Resistance Biology, Institute of Biomedical Sciences, College of Life Sciences, Collaborative Innovation Center of Cell Biology in Universities of Shandong, Shandong Normal University, Jinan, China 2Cheeloo College of Medicine, Shandong University, Jinan, China 3State Key Laboratory of Medicinal Chemical Biology, Haihe Laboratory of Cell Ecology, Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin, China † These authors contributed equally to this work *Corresponding author. Tel: +86 531 8618 2518; E-mail: [email protected] *Corresponding author. Tel: +86 531 8618 2518; E-mail: [email protected] EMBO Reports (2022)23:e54090https://doi.org/10.15252/embr.202154090 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Despite the importance of cilia in cell signaling and motility, the molecular mechanisms regulating cilium formation remain incompletely understood. Herein, we characterize enkurin domain-containing protein 1 (ENKD1) as a novel centrosomal protein that mediates the removal of centriolar coiled–coil protein 110 (CP110) from the mother centriole to promote ciliogenesis. We show that Enkd1 knockout mice possess ciliogenesis defects in multiple organs. Super-resolution microscopy reveals that ENKD1 is a stable component of the centrosome throughout the ciliogenesis process. Simultaneous knockdown of ENKD1 and CP110 significantly reverses the ciliogenesis defects induced by ENKD1 depletion. Protein interaction analysis shows that ENKD1 competes with centrosomal protein 97 (CEP97) in binding to CP110. Depletion of ENKD1 enhances the CP110–CEP97 interaction and detains CP110 at the mother centriole. These findings thus identify ENKD1 as a centrosomal protein and uncover a novel mechanism controlling CP110 removal from the mother centriole for the initiation of ciliogenesis. SYNOPSIS ENKD1 is a centrosomal protein and initiates mother centriole uncapping through competing with CEP97 in binding to CP110. Loss of ENKD1 in mice causes multi-organ developmental abnormalities due to ciliogenesis defects. Depletion of ENKD1 in mice impairs the formation of both primary and motile cilia in multiple organs. ENKD1 localizes to the centrosome and interacts with CP110 directly to license mother centriole uncapping. Depletion of ENKD1 enhances the CP110-CEP97 interaction and inhibits CP110 removal from the mother centriole. Introduction Cilia are highly conserved microtubule-based organelles that protrude from the surface of most mammalian cells. Cilia are traditionally classified into two groups, primary cilia and motile cilia. Primary cilia are typically immotile and play important roles in sensing and transducing environmental cues (Anvarian et al, 2019; Nachury & Mick, 2019), whereas motile cilia are able to generate fluid flow in tissues such as the airway and brain ventricle epithelium (Spassky & Meunier, 2017; Liu et al, 2020; Pellicciotta et al, 2020). Defects in the structures and/or functions of cilia are associated with a variety of human diseases, collectively called ciliopathies (Yang et al, 2014; Reiter & Leroux, 2017). For example, primary cilia are present in the retina of the eye, and their abnormal assembly or functional impairment can cause retinal degeneration (Buskin et al, 2018; Mookherjee et al, 2018). Another example is the presence of motile cilia in the airway epithelium, and the abnormal assembly is a major cause of recurrent respiratory infections and impaired mucociliary clearance (Chivukula et al, 2020; Wallmeier et al, 2020). Ciliogenesis is a multi-step process that typically involves the formation of the basal body, assembly of the transition zone, and extension of the axoneme (Tu et al, 2018; Walia et al, 2019). Centrosomes are major microtubule-organizing centers in mammalian cells (Paz & Lüders, 2018; Meitinger et al, 2020). At the core of the centrosome are two cylindrical structures termed as mother centriole and daughter centriole. The mother centriole can be distinguished by its distal and subdistal appendages (Mönnich et al, 2018). Certain centrosomal proteins, such as centrosomal protein 192 (CEP192), CEP135, polo-like kinase 4 (PLK4), and centrosomal P4.1-associated protein (CPAP), have been identified as indispensable components for the assembly of centrosome structures (Carvalho-Santos et al, 2010; Nigg & Holland, 2018). During ciliogenesis, the mother centriole transforms into the basal body. Ciliary membrane establishment requires components of the Rab-GTPase cascade, such as Rab11 and Rab8, for the intracellular membrane trafficking (Knödler et al, 2010; Walia et al, 2019), and CEP164 for ciliary vesicle docking at the mother centriole (Schmidt et al, 2012; Čajánek & Nigg, 2014). Transition zone proteins are recruited to the mother centriole as the ciliary vesicle develops into the ciliary sheath membrane, which also tightly coincides with the growth of the axonemal microtubule doublet (Williams et al, 2011; Vieillard et al, 2016). Negative regulators of cilium assembly, in particular, the CP110-CEP97 protein complex, need to be removed from the mother centriole to initiate ciliogenesis (Spektor et al, 2007). CP110 localizes to the distal end of centrioles (Chen et al, 2002), and regulates centriole length by preventing centriolar microtubule extension (Kleylein-Sohn et al, 2007; Schmidt et al, 2009; Shoda et al, 2021). However, CP110 does not regulate cilium length, indicating that cilium elongation is a further step (Spektor et al, 2007). CEP97 is a conserved protein that also regulates centriole elongation, and might serve as a chaperone to stabilize CP110, allowing the co-recruitment of both proteins to the centrosome (Bettencourt-Dias & Carvalho-Santos, 2008; Dobbelaere et al, 2020). Removal of CP110 is critical for the conversion of the mother centriole to the basal body, and the presence of CP110 has been found to hinder cilium formation (Cao et al, 2012). However, the precise molecular mechanisms orchestrating CP110 removal remain elusive. In this study, our data demonstrate that ENKD1 is a component of the centrosome and promotes ciliogenesis by stimulating the removal of the CP110–CEP97 protein complex from the mother centriole. Results Enkd1 knockout mice display ciliogenesis defects in multiple organs In a mass spectrometry-based effort to uncover novel regulators of ciliogenesis, we identified ENKD1 in the centrosomal fractions of RPE1 human retinal pigment epithelial cells. In order to study the function of this protein, we generated Enkd1 knockout mice by using the CRISPR/Cas9 technology. The depletion of ENKD1 in the knockout mice was confirmed at both genomic and protein levels (Fig 1A–C). We then performed immunoblot analysis to examine the expression of ENKD1 in mouse tissues, and found that ENKD1 proteins were present in multiple tissues and overexpressed in the testis and lung (Fig EV1A), suggesting a universal expression of ENKD1 in tissues. Unexpectedly, Enkd1 knockout mice were born at the expected Mendelian ratio. Young adult Enkd1 knockout mice (6–12 weeks old) showed no gross defects in growth or survival when they were housed in a pathogen-free condition. Figure 1. Enkd1 knockout mice display ciliogenesis defects in multiple organs A. Strategy for the generation of Enkd1 knockout mice. Deletion of a fragment containing the 173 bp exon 3 and adjacent flanking sequences results in the disruption of ENKD1 protein expression. B. Identification of wild-type (WT), heterozygous (HZ), and knockout (KO) mice by PCR and agarose gel electrophoresis. C. Immunoblotting (IB) of ENKD1 and α-tubulin in wild-type and Enkd1 knockout mice. D–F. Immunofluorescence images (D) and quantification of the density of cilia (E, n = 20 fields from three mice) and ciliary length (F, n = 100 cilia from three mice) in mouse retinas stained with antibodies against acetylated α-tubulin (Ace-α-tubulin) and γ-tubulin and DAPI. Scale bar, 2 µm. G, H. ERG recording images (G) and a-b wave amplitudes (H, n = 5 mice) show the difference in the ERG a-b wave amplitude between wild-type and Enkd1 knockout mice. I, J. VEP recording images (I) and N2-P2 wave amplitudes (J, n = 5 mice) show the difference in the VEP N2-P2 wave amplitude between wild-type and Enkd1 knockout mice. K–M. Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 mice) and ciliary length (M, n = 100 cilia from three mice) in mouse kidneys stained with the antibody against acetylated α-tubulin and DAPI. To quantify the percentage of ciliated cells (L), > 200 cells from 12 images were analyzed for each mouse. Scale bar, 3 µm. N–Q. Images (N) and quantification of the percentage of flagellated sperm (O, n = 9 mice), flagellar length (P, n = 100 sperm from nine mice), and the percentage of sperm with normal motility among flagellated sperm (Q, n = 9 mice) for wild-type and Enkd1 knockout mice. To quantify the percentage of flagellated sperm (O), > 200 sperm were analyzed for each mouse. To quantify the percentage of sperm with normal motility among flagellated sperm (Q), > 200 flagellated sperm were analyzed for each mouse. In panel N, the arrows indicate two spermatozoa without tails. Scale bar, 50 µm. R, S. Scanning electron microscopy images of cilia (R) and quantification of the percentage of ciliated cells (S, n = 9 mice) in the mouse tracheal epithelium. To quantify the percentage of ciliated cells (S), > 200 cells from six fields were analyzed for each mouse. Scale bar, 5 µm. Data information: Data are presented as mean ± SEM. Unpaired two-tailed t-test was performed. P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), ns, not significant. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Enkd1 knockout mice display ciliogenesis defects in multiple organs A. Immunoblotting of ENKD1 and GAPDH in various tissues of wild-type and Enkd1 knockout mice. B–G. Schematic diagrams (B), ECG recording images (C), and quantifications of R wave duration (D, n = 5 mice), S wave duration (E, n = 5 mice), QRS wave duration (F, n = 5 mice,) and R-R interval (G, n = 5 mice) show the difference in ECG between wild-type and Enkd1 knockout mice. H, I. Immunofluorescence images (H) and quantification of the percentage of ciliated cells (I, n = 3 mice) of mouse trachea stained with antibodies against acetylated α-tubulin and CEP164 and DAPI. To quantify the percentage of ciliated cells (I), > 150 cells from six images were analyzed for each mouse. Scale bar, 10 µm. Data information: Data are presented as mean ± SEM. Unpaired two-tailed t-test was performed. P < 0.05 (*), P < 0.001 (***), ns, not significant. Source data are available online for this figure. Download figure Download PowerPoint To determine the potential role of ENKD1 in ciliogenesis, we first analyzed cilia in the mouse retina by immunostaining of acetylated α-tubulin, a well-known ciliary marker (L'Hernault & Rosenbaum, 1985). We found that the number of retinal photoreceptor cilia was greatly decreased in Enkd1 knockout mice, but the length of existing cilia was not significantly affected, as compared to wild-type mice (Fig 1D–F). Next, we measured the electroretinogram (ERG) and the flash visual evoked potential (F-VEP) to evaluate the effects of ENKD1 depletion on functional integrity of the retina (Ridder & Nusinowitz, 2006). In vivo ERG analysis of light response was recorded from dark-adapted mice. Strikingly, significant reductions in the amplitude of the ERG a- and b-wave were observed in Enkd1 knockout mice (Fig 1G and H). The F-VEP components of the Enkd1 knockout mice also displayed a significantly reduced voltage amplitude compared to those of wild-type mice (Fig 1I and J). These data indicate a defective vision of Enkd1 knockout mice. Moreover, we examined primary cilia in kidneys from wild-type mice and Enkd1 knockout mice. A decrease in the number of renal cilia, but not the average ciliary length, was also observed in Enkd1 knockout mice, as compared to wild-type mice (Fig 1K–M). Since primary cilia play essential roles in heart development (Willaredt et al, 2012; Klena et al, 2017; Pala et al, 2018), surface electrocardiography (ECG) was utilized to evaluate the cardiac function. We observed that the average R-wave duration of Enkd1 knockout mice was significantly reduced compared with that of wild-type mice, and the QRS duration displayed a modest reduction as well (Fig EV1B–F). Furthermore, the RR interval (the time elapsed between two successive R waves) was prolonged, although the difference did not reach statistical significance (Fig EV1C and G). Thus, these data indicate that the conduction system of the heart is moderately altered due to the loss of ENKD1. Although the Enkd1 knockout males were normally fertile, a significant increase in the number of aberrant spermatozoa with no tails was observed (Fig 1N and O). However, the flagellated sperm in Enkd1 knockout mice maintained normal flagellar length and displayed normal motility (Fig 1N, P and Q), consistent with the unaffected male fertility of Enkd1 knockout mice. We then analyzed cilia in the trachea by scanning electron microscopy and found that loss of ENKD1 significantly reduced the percentage of ciliated cells in the tracheal epithelium (Fig 1R and S). Further immunostaining revealed that quite a few CEP164-positive cells were non-multiciliated in Enkd1 knockout mice (Fig EV1H and I), suggesting that ENKD1 depletion affects axoneme formation but not centriole amplification. Taken together, these results suggest that ENKD1 is required for the assembly of both primary and motile cilia. ENKD1 is indispensable for the formation of primary cilia To confirm the requirement of ENKD1 for ciliogenesis, we examined primary cilia in mouse embryonic fibroblasts (MEFs) isolated from Enkd1 knockout mice. We found that Enkd1 knockout MEFs had ciliogenesis defects similar to that observed in the tissues of Enkd1 knockout mice (Fig 2A–D). Next, we knocked down ENKD1 in serum-starved human RPE1 cells by using two different siRNAs (Fig 2E), and found that downregulation of ENKD1 resulted in fewer cilia that were labeled with anti-acetylated α-tubulin and anti-Arl13b antibodies (Fig 2F–J). Similarly, siRNA-mediated ENKD1 depletion did not affect the average length of existing cilia in RPE1 cells (Fig 2F and H), indicating that ENKD1 is largely dispensable for the elongation or disassembly of cilia. We further knocked down ENKD1 in serum-starved mouse NIH3T3 cells and observed similar results (Fig EV2A–D). Figure 2. ENKD1 promotes the initiation of ciliogenesis in cultured cells A. Immunoblot analysis of ENKD1 and GAPDH in MEFs cultured in serum-free medium for 48 h. B–D. Immunofluorescence images (B) and quantification of the percentage of ciliated cells (C, n = 3 independent experiments) and ciliary length (D, n = 70 cilia from three independent experiments) for MEFs cultured in serum-free medium and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (C), > 100 cells were analyzed for each experiment. Scale bar, 5 µm. E. Immunoblot analysis of ENKD1 and β-actin in RPE1 cells transfected with control or ENKD1 siRNAs and cultured in serum-free medium for 48 h. F–H. Immunofluorescence images (F) and quantification of the percentage of ciliated cells (G, n = 3 independent experiments) and ciliary length (H, n = 100 cilia from three independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (G), > 150 cells were analyzed for each experiment. Scale bar, 5 µm. I, J. Immunofluorescence images (I) and quantification of the percentage of ciliated cells (J, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium, and stained with antibodies against Arl13b and Centrin and DAPI. To quantify the percentage of ciliated cells (J), > 80 cells were analyzed for each experiment. Scale bar, 2 µm. K, L. Immunofluorescence images (K) and quantification of the percentage of ciliated cells (L, n = 3 independent experiments) for RPE1 cells transfected with control or ENKD1 siRNAs and plasmids expressing GFP, GFP-ENKD1, GFP-ENKD1-N, GFP-ENKD1-M, or GFP-ENKD1-C, cultured in serum-free medium, and stained with the antibody against acetylated α-tubulin and DAPI. The siRNA-resistant forms of ENKD1 were used for these rescue experiments. To quantify the percentage of ciliated cells (L), > 120 cells were analyzed for each experiment. Scale bar, 3 µm. Data information: Data are from three independent biological repeats and presented as mean ± SEM. Unpaired two-tailed t-test was performed. P < 0.01 (**), P < 0.001 (***), ns, not significant. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. ENKD1 promotes the initiation of ciliogenesis in cultured cells A. Immunoblot analysis of ENKD1 and β-actin in NIH3T3 cells transfected with control or ENKD1 siRNAs and cultured in serum-free medium for 48 h. B–D. Immunofluorescence images (B) and quantification of the percentage of ciliated cells (C, n = 3 independent experiments) and ciliary length (D, n = 65 cilia from three independent experiments) for NIH3T3 cells transfected with control or ENKD1 siRNAs, cultured in serum-free medium and stained with antibodies against acetylated α-tubulin and γ-tubulin and DAPI. To quantify the percentage of ciliated cells (C), > 90 cells were analyzed for each experiment. Scale bar, 10 µm. Data information: Data are from three independent biological repeats and presented as mean ± SEM. Unpaired two-tailed t-test was performed. P < 0.05 (*), P < 0.01 (**), ns, not significant. Source data are available online for this figure. Download figure Download PowerPoint To explore the potential molecular mechanism underlying ENKD1-mediated regulation of ciliogenesis, we constructed the ENKD1 expression plasmid as well as a series of truncated mutants of ENKD1, including ENKD1-N (1–91 amino acid [aa]), ENKD1-M (91–250 aa), and ENKD1-C (250–346 aa), which were based on the conserved domain analysis. The carboxyl-terminal fragment contains a conserved enkurin domain. Full-length ENKD1 or various truncated mutants of ENKD1 were re-expressed in ENKD1-knockdown RPE1 cells. We found that re-expression of full-length ENKD1 or ENKD1-M, but not ENKD1-N and ENKD1-C, significantly rescued ENKD1 siRNA-induced ciliogenesis defects (Fig 2K and L). Collectively, the above results reveal a critical role for the middle domain of ENKD1 in the initiation of ciliogenesis. ENKD1 is a component of the centrosome To further investigate the association of ENKD1 with ciliogenesis, we examined its subcellular localization. The comparison of antibody reactivity in MEFs isolated from wild-type or Enkd1 knockout mice was performed to validate the antibody specificity. Immunostaining results revealed that ENKD1 displayed centrosomal localization in wild-type MEFs, which disappeared in Enkd1 knockout MEFs (Fig EV3A). Similarly, ENKD1 knockdown dramatically decreased the centrosomal localization of ENKD1 but not the centrosomal marker Centrin (Fig EV3B–D). These results confirm the antibody specificity and demonstrate that ENKD1 is a component of the centrosome. Click here to expand this figure. Figure EV3. ENKD1 is localized to the centrosome A. Immunostaining of ENKD1 and Centrin in MEF cells cultured in serum-free medium. Scale bar, 1 µm. B–D. Immunofluorescence images (B) and quantifications of fluorescence intensity of ENKD1 (C, n = 50 cells from th