Centlein mediates an interaction between C-Nap1 and Cep68 to maintain centrosome cohesion

The mammalian centrosome is the primary microtubule-organizing center (MTOC) and comprises two centrioles and the surrounding pericentriolar material (PCM) (Nigg and Raff, 2009). The two centrioles are structurally and functionally distinct. Only the mother centriole, harboring distal and subdistal appendages, can migrate to the cell cortex to become a basal body for the formation of cilia during cell cycle quiescence (Kobayashi and Dynlacht, 2011). The ultrastructure shows that the centriole is polarized along its long axis, with the base referred to as the proximal end and the tip as the distal end (Gönczy, 2012). The two centrioles within a G1 centrosome are connected through a proteinous linker, which is often referred to as centrosome cohesion (Barrera et al., 2010) or the ‘G1–G2 tether’ (Nigg and Stearns, 2011), emanating from their proximal ends. As cells progress from G1 into the G2 phase, the centrosome is duplicated, and the duplicated centrosomes remain linked in order to function as a single MTOC (Nigg and Stearns, 2011). At the onset of mitosis, centrosome cohesion is disassembled by Nek2A-kinase-mediated phosphorylation, concomitant with centrosome separation in preparation for mitotic spindle assembly (Faragher and Fry, 2003). Proteins implicated in the tethering of the parental centrioles include C-Nap1 (also known as Cep250) (Mayor et al., 2000), rootletin (Bahe et al., 2005), Cep68 (Graser et al., 2007a), LRRC45 (He et al., 2013) and CDK5RAP2 (Graser et al., 2007a; Barrera et al., 2010), of which C-Nap1, rootletin and LRRC45 are phosphorylated by Nek2A and displaced from the centrosomes at the onset of mitosis. Despite a growing number of tethering proteins that have been identified, the precise composition of centrosome cohesion remains to be elucidated. Here, we characterize the centriolar protein centlein (also known as CNTLN) and demonstrate that centlein is complexed with C-Nap1 and Cep68, and is required for centrosome cohesion.

To characterize centlein, we generated both mouse and rat monoclonal antibodies spanning residues 89–437 and 901–1191, respectively, of the recombinant centlein protein (‘antigen’, supplementary material Fig. S1A). Western blotting showed that the rat antibody clone 7H2 recognized a band of ∼170 kDa, which was greatly diminished after siRNA-mediated depletion of centlein in U2OS and RPE-1 cell lysates (supplementary material Fig. S1B). By immunofluorescence staining with the antibodies, centlein was detected as punctate signals scattered in the cytoplasm and colocalized with the centrosomal marker γ-tubulin (supplementary material Fig. S1C). Silencing centlein reduced the signals from immunofluorescence to below 20% of the negative control (supplementary material Fig. S1C), confirming the specificity of the staining patterns of the antibodies.

To localize centlein at the centrosome precisely, we chose to use RPE-1 cells. Upon serum withdrawal, RPE-1 cells become quiescent and develop primary cilia. At this stage, an RPE-1 cell possesses a basal body/mother centriole and a daughter centriole, which are readily visualized by using an antibody against acetylated tubulin (Graser et al., 2007b) – a marker of primary cilia and centrioles. Centlein was detected as two dots that colocalized with acetylated tubulin but also localized within the PCM, which was marked by staining for pericentrin and γ-tubulin, and the centriolar satellites, which were marked by PCM-1 (Lopes et al., 2011), indicating that centlein is associated with the two centrioles (Fig. 1A). Further examination revealed that centlein localized distinctly from centrin2 and CP110 (also known as CCP110), which are markers of the distal ends of centrioles (Kim et al., 2011) (Fig. 1B; supplementary material Fig. S1D); by contrast, centlein colocalized substantially with the proximal centriolar markers C-Nap1 and Cep135 (Arquint et al., 2012; Fig. 1B; supplementary material Fig. S1D). In conclusion, the monoclonal antibodies specifically recognize the ∼170 -kDa protein centlein and label the proximal ends of centrioles.

Centlein was first identified by Nakanishi and colleagues; however, its precise function was unknown (Makino et al., 2008; Sakamoto et al., 2008). In HeLa lysates, western blotting analysis, by using a polyclonal antibody against centlein, has previously detected a single band of 90 kDa (Makino et al., 2008). Because the antibody had been generated against a GST-tagged fragment of centlein (amino acid residues 803–953), it is supposed to detect a band corresponding to the predicted molecular mass of human centlein (162 kDa); however, the specificity of the antibody was not verified (Makino et al., 2008), which explains discrepancies in the observed mobility of the protein.

To explore the function of centlein, we depleted the protein by using siRNAs in RPE-1 and U2OS cells and noticed an increased distance between centrosomes in the centlein-depleted interphase cells. When the distance between the parental centrioles is >2 µm, it is termed ‘centrosome splitting’ (Graser et al., 2007a). Centrosome splitting has been observed previously in cells lacking C-Nap1, rootletin or Cep68, albeit with large variation (Bahe et al., 2005; Graser et al., 2007a). The localization of C-Nap1 to the proximal ends of parental centrioles suggests that it is a centriolar docking protein (Mayor et al., 2000), whereas rootletin and Cep68 decorate fibers emanating from the proximal ends of parental centrioles (Bahe et al., 2005; Graser et al., 2007a). We quantified centrosome splitting in cells lacking centlein, C-Nap1, rootletin or Cep68 in comparison with negative controls (Fig. 1C; supplementary material Fig. S2A). In accordance with previous studies, depletion of either C-Nap1 or rootletin produced the highest splitting ratio (Fig. 1C; Graser et al., 2007a), indicating that C-Nap1 and rootletin are particularly important for the tethering of parental centrioles.

We next sought to determine the interdependence of centlein, C-Nap1, rootletin and Cep68 by using siRNA-mediated protein depletion. Centlein localization at the centrosomes was unaltered upon depletion of rootletin or Cep68, whereas knockdown of C-Nap1 resulted in a loss of centlein from centrosomes (Fig. 1D; supplementary material Fig. S2B). Depletion of any one of centlein, C-Nap1 or rootletin either displaced or diminished Cep68 staining at the centrosomes (Fig. 1E; supplementary material Fig. S2C; Bahe et al., 2005; Graser et al., 2007a), whereas silencing of centlein, Cep68 or rootletin did not produce any visible change in the localization of C-Nap1 (Fig. 1F; supplementary material Fig. S2D; Bahe et al., 2005; Graser et al., 2007a). Rootletin was displaced from centrosomes upon depletion of C-Nap1 or Cep68 (Fig. 1G; supplementary material Fig. S2E; Bahe et al., 2005; Graser et al., 2007a) but was unaffected by centlein depletion. In summary, silencing centlein diminished Cep68 staining at centrosomes significantly, whereas centrosomal centlein was reduced greatly in the C-Nap1-depleted cells (Fig. 1H; supplementary material Fig. S2F). These results indicate that centlein physically interacts with C-Nap1 and Cep68 but not rootletin.

To confirm the interactions between the proteins, reciprocal co-immunoprecipitation assays were performed. The results revealed that endogenous centlein co-immunoprecipitated with endogenous C-Nap1 (Fig. 2A, top left), which was verified by co-immunoprecipitation of Myc-tagged centlein with endogenous C-Nap1 from transiently transfected HEK293T cells (Fig. 2A, bottom left). Conversely, endogenous C-Nap1 was detected in the Myc–centlein immunoprecipitate (Fig. 2A, right). Cep68 and centlein that were epitope tagged and expressed in HEK293T cells were able to interact with each other in reciprocal immunoprecipitation experiments (Fig. 2B). Deletion analyses of centlein showed that a region spanning amino acid residues 151–600 was sufficient to bind C-Nap1 (Fig. 2C, top left; supplementary material Fig. S3A), and that a region spanning amino acids 1–600 was required for its binding to Cep68 (Fig. 2C, bottom left; supplementary material Fig. S3C). Two regions of C-Nap1, encompassing residues 608–1221 and 1838–2442, were necessary for its binding to centlein (Fig. 2C, top right; supplementary material Fig. S3B), whereas a region spanning amino acid residues 509–757 of Cep68 was sufficient to bind to centlein (Fig. 2C, bottom right; supplementary material Fig. S3D). There was no detectable interaction between rootletin and centlein (supplementary material Fig. S3E).

To further establish the association of centlein with both C-Nap1 and Cep68, we overexpressed either centlein with C-Nap1 or centlein with Cep68 in U2OS cells. Note that high-level overexpression of centlein led to the formation of filaments throughout the cytoplasm (Fig. 2D, top left panel), whereas ectopic expression of either C-Nap1 or Cep68 did not produce such structures (Fig. 2D, top panels; Bahe et al., 2005; Graser et al., 2007a). Overexpression of either C-Nap1 or Cep68 resulted in their recruitment to centlein filaments (Fig. 2D, middle and lower panels). These data demonstrate that centlein, indeed, interacts with both C-Nap1 and Cep68.

The results described above suggest that centlein exists in a complex with both C-Nap1 and Cep68. Therefore, an in vitro direct-binding assay was performed. A GST-tagged fragment of centlein (amino acid residues 89–437), immobilized on sepharose beads, selectively bound both transiently expressed Flag–Cep68 and GFP–C-Nap1 in cell extracts (Fig. 3A). This data, along with a lack of interaction between C-Nap1 and Cep68 (data not shown; Graser et al., 2007a), suggests that C-Nap1 complexes with Cep68 through the independent direct interactions of both proteins with centlein.

To elucidate an in vivo functional role of the complex, we performed knockdown-rescue experiments. We first depleted endogenous centlein by using siRNAs and observed that the centlein-depleted cells displayed split centrosomes (Fig. 3B,C; Fig. 1C). We then transfected various deletion mutants of centlein into those cells and examined the ability of each construct to localize to the centrosomes and its effect on centrosome splitting. Only the centlein mutants comprising the regions required for interaction with C-Nap1 and Cep68 were able to localize to the centrosomes and rescued centrosome splitting induced by centlein depletion (Fig. 3D–F). Thus, these experiments not only demonstrate that centlein is recruited to the centrosome through its C-Nap1-interaction domain but also substantiate the notion that centlein acts as a molecular link within the C-Nap1–centlein–Cep68 complex to mediate centrosome cohesion.

Given that C-Nap1, centlein and Cep68 form a protein complex, we asked whether centlein, like C-Nap1 and Cep68, associated with centrosomes in a cell-cycle-dependent manner (Mayor et al., 2000; Graser et al., 2007a). U2OS cells that had been synchronized for stage-specific populations were examined by using immunofluorescence microscopy. Levels of centlein were relatively high at interphase centrosomes but were reduced on mitotic spindle poles (Fig. 4A,B), which is very similar to the pattern observed for C-Nap1 – a well-characterized substrate of Nek2A (Mayor et al., 2000).

Thus, we sought to determine whether centlein and its binding partner Cep68 are Nek2A substrates. First, we examined whether Nek2A bound to and phosphorylated centlein and Cep68 in vivo. By coexpressing GFP–Nek2A and Myc–centlein in HEK293T cells, we demonstrated that Nek2A specifically co-immunoprecipitated with Myc–centlein (Fig. 4C, left), and, in a reciprocal experiment, the endogenous centlein could be readily detected in the GFP–Nek2A immunoprecipitate (Fig. 4C, middle). Similar to this, an antibody against the Flag tag co-precipitated GFP–Nek2A from lysates prepared from HEK293T cells that had been co-transfected with Flag–Cep68 and GFP–Nek2A (Fig. 4C, right). Furthermore, antibodies against the Myc or Flag tags detected more slowly migrating forms of centlein and Cep68 from HEK293T cells expressing Myc–centlein or Flag–Cep68 together with wild-type (WT), but not catalytically inactive (K37R), GFP–Nek2A (Fig. 4D). Treatment of the HEK293T cell extracts with calf intestinal alkaline phosphatase (CIAP) resulted in loss of these slower migrating centlein and Cep68 bands, confirming that the change in the gel mobility of the proteins was due to protein phosphorylation (Fig. 4D). Overexpression of GFP-Nek2A WT, but not GFP–Nek2A K37R, caused the dissociation of centlein and Cep68 from centrosomes, in turn, leading to centrosome splitting in U2OS cells (Fig. 4E). We then determined whether Nek2A could directly phosphorylate centlein and Cep68 in vitro. Bacterially expressed GST-tagged fragments of centlein (amino acid residues 89–437) and Cep68 (amino acid residues 1–282) were purified as substrates. Nek2A protein was immunoprecipitated from HEK293T cells that had been transfected with either GFP–Nek2A WT or GFP–Nek2A K37R. The in vitro kinase assay showed that both centlein and Cep68 were phosphorylated by active but not inactive Nek2A by using the antibodies specific to the phosphorylated serine and threonine residues (Fig. 4F). We thus conclude that centlein and Cep68 are novel Nek2A substrates.

In summary, centlein, together with Cep68, is a newly identified Nek2A substrate and associated with centrosomes in a cell-cycle-dependent manner (Fig. 4G). Knockdown of centlein displaces Cep68 from centrosomes and, in turn, leads to centrosome splitting. In interphase cells, centlein interacts and forms a protein complex with C-Nap1 and Cep68 at the proximal ends of centrioles (Fig. 4G). Centrosome cohesion is, thus, maintained mainly by C-Nap1–rootletin (Bahe et al., 2005; Mardin and Schiebel, 2012) and supported by C-Nap1–centlein–Cep68 (Fig. 4G).

Full-length centlein was obtained from HeLa cDNA and cloned into the vectors pEGFP-C1, pEGFP-N1 and pCMV-Myc. The truncated mutants of centlein were cloned into the vectors pCMV-Myc and pEGFP-C1. Full-length C-Nap1 was a gift from Erich A. Nigg (Bahe et al., 2005) and was subcloned into the p3×Flag-CMV vector. The truncated mutants of C-Nap1, full-length Cep68 (obtained from HeLa cDNA) and truncated mutants of Cep68 were cloned into the p3×Flag-CMV vector. GFP-tagged rootletin was a gift from Erich A. Nigg (Graser et al., 2007a). Wild-type Nek2A was obtained from HeLa cDNA and cloned into pEGFP-C1. The catalytically inactive (K37R) Nek2A was gifted from Erich A. Nigg (Bahe et al., 2005). Sequencing was performed for all constructs. For expression of recombinant proteins, base pairs 265–1311 and 2701–3573 of centlein or base pairs 1–846 of Cep68 were amplified and inserted into the expression vector pGEX-4T-1. GST-tagged fragments were expressed in Escherichia coli strain BL21 (DE3) and purified with glutathione–Sepharose-4B (17-0757-01, GE Healthcare Life Sciences).

Mouse and rat monoclonal antibodies against the N- and C-terminal regions of centlein (amino acid residues 89–437 and 901–1191, respectively) were generated by Absea Biotechnology Ltd (Beijing, China). Mouse clone 11A4 and rat Clone 7H2 were used.

U2OS, RPE-1 and HEK293T cells were cultured in Ham's F12 (Hyclone), Dulbecco's modified Eagle's medium (DMEM) with Ham's F12 (1∶1, Hyclone) and DMEM (Hyclone), respectively, and supplemented with 10% (v/v) fetal bovine serum (Hyclone), 100 IU/ml penicillin and 100 µg/ml streptomycin. The cells were grown at 37°C under 5% CO₂. To induce primary cilium formation, the growth medium was replaced with serum-free medium for RPE-1 cells for 48 h. U2OS and HEK293T cells were transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's protocol. Cells were analyzed 12–24 h post transfection.

Cells were grown on coverslips, washed in PBS and fixed in −20°C methanol for 8 min. Then coverslips were washed and blocked in 3% bovine serum albumin (BSA) for 30 min at room temperature, then incubated with primary antibodies (diluted in 3% BSA in PBS) overnight at 4°C. After three washes in PBS for 5 min each, incubations with secondary antibodies diluted in AbDil (2% BSA, 0.1% Tween-20 in PBS) were performed for 30 min. Coverslips were rinsed again in PBS and then mounted on slides with ProLong Gold (Invitrogen, P36931) containing DAPI to stain DNA. The primary antibodies mouse anti-centlein (1∶500, 11A4), rabbit anti-γ-tubulin (1∶1000, T5192, Sigma-Aldrich), mouse anti-γ-tubulin (1∶1000, C-11, Santa Cruz Biotechnology), rabbit anti-acetylated α-tubulin (1∶1200, D20G3, Cell Signaling), rabbit anti-pericentrin (1∶1000, ab4448, Abcam), rabbit anti-PCM-1 (1∶500, AP7481c. abgent), rabbit anti-CP110 (1∶500; Kunsoo Rhee, Seoul National University, South Korea), rabbit anti-Cep135 (1∶3000, A02C0240, Blue Gene), rabbit anti-C-Nap1 (1∶800, 14498-1-AP, Proteintech), rabbit anti-GFP (1∶1000, 50430-2-AP, ProteinTech), rabbit anti-rootletin (1∶3000, Erich A. Nigg), rabbit anti-Cep68 (1∶5000, Erich A. Nigg), rabbit anti-Flag (1∶5000, 20543-1-AP, ProteinTech) and mouse anti-Myc (1∶10000, M047-3, MBL International) were used. The secondary antibodies used were Alexa Fluor 488 donkey anti-rabbit IgG (1∶1500, A21206, Invitrogen), Alexa Fluor 594 goat anti-rabbit IgG (1∶1200, A11012, Invitrogen), Alexa Fluor 488 goat anti-mouse IgG (1∶500, A11029, Invitrogen) and Alexa Fluor 555 goat anti-mouse IgG (1∶1500, A21424, Invitrogen). To simultaneously visualize centlein (11A4) and centrin, a mouse anti-centrin antibody (1∶100, 20H5, Millipore) was covalently coupled to Alexa Fluor 594, using an APEX Antibody Labeling Kit (A10474, Invitrogen). Immunofluorescence microscopy was performed using a Leica DM600B microscope (Leica, Germany) equipped with a 100× oil immersion objective, and images were acquired using a digital camera Leica DFC450 C and Leica Application Suite software (version 4.0.0).

For RNA-mediated interference, cells were transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol and harvested at 72 h post transfection. ON-TARGETplus SMARTpool siRNAs against centlein were obtained from Dharmacon (L-020720-02) targeting the following sequences: 5′-GAGCTGAAGTACACGCAA-3′, 5′-GTTGAAGTATCACAGAGTA-3′, 5′-GCTAGAGGGCATCTCGGTA-3′ and 5′-ACGGAAAATTGCAGT-3′. Several proteins were depleted using siRNA duplex oligonucleotides targeting the following sequences: C-Nap1, 5′-CTGGAAGAGCGTCTAACTGAT-3′ (Bahe et al., 2005); Cep68, 5′-ACCGAAGATGATCCATCCCTA-3′ (Graser et al., 2007a) and rootletin, 5′-AAGCCAGTCTAGACAAGGA-3′ (Bahe et al., 2005). Control siRNAs were also used (ON-TARGETplus Non-Targeting pool, D-001810-10, Dharmacon). Rescue experiments were performed by transfecting siRNA-treated cells 72 h later with the indicated DNA constructs using Lipofectamine 2000 (Invitrogen).

HEK293T cells cultured in 10-cm dishes were lysed with 1 ml of cold ELB buffer [50 mM HEPES, 250 mM NaCl, 0.1% NP-40, 1 mM PMSF (Sigma) and complete EDTA-free protease inhibitor cocktail (Roche)]. The lysates were centrifuged at 12,000 g for 10 min and the supernatant was precleared by incubation with 50 µl of protein-G–Sepharose (CW0012A, Cowin Biotech). After the mixtures had been incubated at 4°C for 3 h, the supernatant was incubated with 2 µg antibody (homogeneous normal IgG as control) at 4°C for an additional 3 h, followed by addition of 20 µl Dynabeads–protein-G (10004D, Invitrogen) and incubated at 4°C overnight. Beads were washed four times using ELB buffer, followed by heating of the precipitated material in SDS-PAGE sample buffer and immunoblotting analysis.

Proteins were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore). Then the membranes were blocked in TBS-T (10 mM Tris-HCl pH 7.4, 150 mM NaCl and 0.1% Tween-20) containing 5% non-fat milk at room temperature for 1 h. Primary antibodies were diluted in TBS-T containing 1% non-fat milk and used at the following concentrations: rat anti-centlein (1∶1000, 7H2), mouse anti-centlein (1∶1000, 11A4), rabbit anti-C-Nap1 (1∶800, 14498-1-AP, ProteinTech), mouse anti-Myc (1∶1000, M047-3, MBL International), rabbit anti-Flag (1∶1000, 20543-1-AP, ProteinTech), rabbit anti-Cep68 (1∶1000, Erich A. Nigg), rabbit anti-GFP (1∶1000, 50430-2-AP, ProteinTech) and rabbit anti-GST (1∶2000, 354206, Millipore). The membranes were incubated at 4°C overnight. After incubation, the membranes were washed three times with TBS-T and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1∶10,000 GAM0072, Liankebio), swine anti-rabbit IgG (1∶5000, P0399, DakoCytomation) or goat anti-rat IgG (1∶2000, A0192, Beyotime) diluted in TBS-T containing 1% non-fat milk at room temperature for 1 h. After final washes with TBS-T, the membranes were developed by using enhanced chemiluminescence prime western blotting detection reagent (RPN 2232, GE Healthcare Life Sciences) and exposure to X-ray film (Kodak).

GST and GST fusion proteins were expressed in E. coli strain BL21 (DE3) and purified by affinity chromatography with glutathione–Sepharose-4B and then crosslinked to Sepharose beads. GST or GST fusion proteins bound to the beads (20 µg) was incubated in ELB buffer with 1 mg of extract of HEK293T cells that had been transiently transfected with GFP–C-Nap1 and Flag–Cep68. The mixture was incubated at 4°C overnight with agitation. After four washes with ELB buffer, proteins were extracted from the Sepharose beads by boiling in SDS-PAGE sample buffer and were then analysed by western blotting (Rountree et al., 2000).

For phosphatase treatments of cellular lysates, ∼1 mg of total protein was added to reaction buffer (100 mM NaCl, 50 mM Tris HCl, 10 mM MgCl₂, 1 mM DTT). The mixture was incubated with 200 units of calf intestinal alkaline phosphatase (CIAP) (M0290S, New England Biolabs) for 2 h at 37°C. The mixture was then heated in SDS-PAGE sample buffer and subjected to immunoblotting analysis.

HEK293T cells in 10-cm dishes were transfected with GFP–Nek2A WT or GFP–Nek2A K37R. At 24 h after transfection, the total cell lysate was incubated with 2 µg of an antibody against GFP. The immunoprecipitated kinases were washed four times in ELB buffer and then incubated with the GST fusion N-terminal fragment of centlein (amino acid residues 89–437 of centlein) or GST fusion N-terminal fragment of Cep68 (amino acid residues 1–282 of Cep68) (500 µg/ml) in kinase buffer (50 mM Tris HCl pH 7.7, 100 mM NaCl, 25 mM β-glycerophosphate, 25 mM MgCl₂, 1 mM DTT, protease inhibitor cocktail and 200 µM ATP) for 30 min at 30°C (Sundivakkam et al., 2013). The reaction was terminated by adding SDS-PAGE sample buffer and boiling for 5 min. The proteins were separated by SDS-PAGE on a 10% gel and then immunoblotted using rabbit antibodies against phosphorylated serine residues (1 µg/ml, 61-8100, Invitrogen) or phosphorylated threonine residues (1 µg/ml, 71-8200, Invitrogen).

U2OS cells were synchronized with a double thymidine block and then released for 0, 6, 9, 12 and 18 h. The cells were trypsinized, suspended in 100 µl PBS and fixed by adding 3 ml of cold 70% ethanol and incubating overnight. The fixed cells were washed with PBS and incubated with 10 mg/ml propidium iodide (PI) for 1 h. The cells were then analyzed using an Accuri C6 flow cytometer (BD Biosciences).

ImageJ software was used to measure the fluorescence intensity and centrosomal distance. The detailed methods of measurements are detailed elsewhere (Mardin et al., 2010). The statistical significance of the difference between two means was determined using a two-tailed Student's t-test. Differences were considered significant when P<0.01.

We thank Erich A. Nigg and Elena Nigg (University of Basel, Switzerland) for kindly providing antibodies against rootletin and Cep68, and plasmids of GFP-C-Nap1, GFP-rootletin and GFP-Nek2A K37R. We are very grateful to Kunsoo Rhee for the CP110 antibody. We also thank the members of Zhihai Qin (Institute of Biophysics, Chinese Academy of Sciences, Beijing) for technical assistance.