ODF2 Maintains Centrosome Cohesion by Restricting Β-Catenin Accumulation

RESEARCH ARTICLE ODF2 maintains centrosome cohesion by restricting β-catenin accumulation Kefei Yang, Marco Andreas Tylkowski*, Daniela Hüber‡, Constanza Tapia Contreras and Sigrid Hoyer-Fender§

ABSTRACT body is ODF2 (also known as cenexin), first identified as the major The centrosome, as the main microtubule-organizing center, protein component of the sperm cytoskeletal outer dense fibers safeguards chromosome segregation by supporting the bipolar (ODFs) (Brohmann et al., 1997; Shao et al., 1997; Turner et al., 1997; spindle. Centrosome aberrations are causally related to chromosome Lange and Gull, 1995; Nakagawa et al., 2001; Hoyer-Fender et al., segregation disorders, both characterizing cancer cells. Thus, a 2003; Schweizer and Hoyer-Fender, 2009). ODF2 is essential for cilia restriction to only having one centrosome per cell and cell cycle- formation and, beyond that, is critically important for embryonic dependent duplication of the centrosome is mandatory. Duplicated development, as ODF2 deficiency in mice leads to pre-implantation centrosomes remain physically connected, in order to function as a lethality (Ishikawa et al., 2005; Anderson and Stearns, 2009; Salmon Odf2 single entity, until onset of mitosis when centrosome disjunction is et al., 2006). Alternative splicing of creates functionally diverse licensed by disassembly of linker proteins and accumulation of isoforms illustrated by the N-terminal cenexin insertion present in so- β-catenin. The crucial role β-catenin plays in centrosome disjunction called cenexin isoforms but not in the abundant testicular ODF2 form inevitably demands for restricting its premature accumulation. ODF2 (Hüber and Hoyer-Fender, 2007). This insertion in conjunction with (also known as cenexin) is an essential centrosomal component, but the coiled-coil region is required for centrosomal targeting and for the its relevance for the interphase centrosome has not been elucidated. formation of subdistal appendages and basal feet (Hüber et al., 2008; ∼ We show here that ODF2 plays a central role in centrosome cohesion. Tateishi et al., 2013). Furthermore, a C-terminal extension of 150 Depletion of ODF2 induces premature centrosome splitting and amino acids, which specifies human and rat cenexin isoforms, is formation of tripolar spindles that are likely caused by the observed important for centrosomal targeting and ciliogenesis as well as for the accumulation of centrosomal β-catenin. Our data collectively indicate recruitment of Polo-like kinase 1 (Plk1) to the centrosome (Soung that ODF2 restricts β-catenin accumulation at the centrosome, thus et al., 2006, 2009; Rivkin et al., 2008; Chang et al., 2013; Tateishi preventing premature centrosome disjunction. et al., 2013). The binding site for the polo-box domain of Plk1 is generated by Cdk1-mediated phosphorylation of S796 in the C- KEY WORDS: ODF2, Cenexin, Centrosome, Cohesion, β-catenin terminal extension of human isoform denoted cenexin 1 (Lee et al., 1998; Barr et al., 2004; Elia et al., 2003; Soung et al., 2006, 2009). At INTRODUCTION the onset of mitosis, activated Plk1 promotes centrosome separation The centrosome functions as microtubule-organizing center and the formation of a bipolar spindle that is fundamental to ensure (MTOC) in interphase and constitutes the spindle poles in correct partitioning of duplicated chromosomes. mitosis. Centrosomes are unique organelles consisting of a pair of Although centrosomes are duplicated synchronously with DNA centrioles and its associated pericentriolar material (PCM), and are replication, they are tightly connected and function as a single entity duplicated once every cell cycle (Kellogg et al., 1994; Doxsey, until the onset of mitosis. Parental centrioles are tethered by a 2001; Bornens, 2002). However, as centriole duplication is semi- proteinaceous linker until the G2/M transition when the linker is conservative, centrosomes comprise structurally and functionally severed by the action of kinases. At the onset of mitosis, cyclin diverse centrioles. The older or mother centriole is characterized B2–Cdk1 activates Plk1, which is recruited to the centrosome by the presence of distal and subdistal appendages, and initiates by ODF2. Activated Plk1 in turn phosphorylates Mst2, which microtubule (MT) polymerization and anchoring. Moreover, it is the counteracts the phosphatase activity of the Nek2–PP1γ complex. mother centriole that is transformed into a basal body to initiate Subsequently, the activated Nek2 kinase phosphorylates the pivotal the formation of a primary cilium, an essential sensory organelle tethering proteins C-Nap1 (also known as Cep250) and rootletin, present on nearly every cell of the vertebrate body (Dawe et al., leading to their dissociation and severing of the proteinaceous linker 2007; Gerdes et al., 2009; Veland et al., 2009; Hoyer-Fender, 2010, to allow for centrosome separation (Fry et al., 1998; Mayor et al., 2013). A signature protein of the mature centriole and the basal 2000, 2002; Faragher and Fry, 2003; Bahe et al., 2005; Mardin et al., 2011; Nam and van Deursen, 2014; Hardy et al., 2014). Likewise important for centrosome separation and establishment of Johann-Friedrich-Blumenbach-Institute of Zoology and Anthropology– a bipolar spindle is β-catenin, which is in accordance with the Developmental Biology, GZMB, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, Georg-August-UniversitätGöttingen, 37077 Göttingen, Germany. observation that Wnt signaling can induce centrosome splitting, *Present address: Gymnasium Harksheide, Falkenbergstr. 25, 22844 Norderstedt, eventually causing the centrosome aberrations observed in cancer Germany. ‡Present address: Horizon Discovery Group Co., 8100 Cambridge cells (Kaplan et al., 2004; Hadjihannas et al., 2010). Logically, Research Park, Waterbeach, Cambridge CB25 9TL, UK. several Wnt pathway components have been localized at the §Author for correspondence ([email protected]) centrosome (Fumoto et al., 2009; Itoh et al., 2009; Kim et al., 2009; Hadjihannas et al., 2010; Mbom et al., 2013). β-catenin, K.Y., 0000-0002-8253-4203; M.A.T., 0000-0001-5504-2716; C.T., 0000-0003- 2054-0963; S.H., 0000-0002-6349-594X specifically, colocalizes with rootletin and is also phosphorylated by Nek2. However, increased Nek2 activity results in rootletin-

Received 5 June 2018; Accepted 10 September 2018 independent binding of (stabilized) β-catenin to centrosomal sites, Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs220954. doi:10.1242/jcs.220954 and this is essential for centrosome separation (Bahmanyar et al., of tripolar spindles from 15% (n=32 from a total of 215 cells) in 2008). Nek2, although phosphorylating the same regulatory sites in control cells to 23% (n=23 from a total of 100 cells) in siRNA- the N-terminus of β-catenin as GSK3β, seems to inhibit binding of treated cells. the E3 ligase β-TrCP, thus preventing β-catenin ubiquitylation and We next asked whether decreased spindle pole cohesion is degradation. Nek2-mediated phosphorylation of β-catenin therefore already reflected by a relaxation of centrosome cohesion in the G2 stabilizes β-catenin at the centrosome (Mbom et al., 2014). The phase. NIH3T3 cells were transfected either with Odf2 siRNA or a negative regulator of β-catenin, conductin (also known as Axin2), non-target control siRNA and the centrosomes immunologically binds to C-Nap1. Conductin promotes β-catenin phosphorylation detected by staining for either γ-tubulin or pericentrin. The distances to stimulate its proteasomal degradation. Consequently, knockdown of the two spots, which characterize the G2 centrosome, were of conductin reduces the phosphorylation of β-catenin at the measured using the software program Amira (Amira 5.3.2; Stalling centrosome, and knockdown of C-Nap1 or rootletin abolishes et al., 2005). We found a statistically significant increase of the β-catenin phosphorylation. However, since loss of Nek2 neither distance between the two γ-tubulin spots (Odf2 siRNA n=69, affected centrosomal linker dissolution nor cell cycle progression, control siRNA n=104) as well as between the two pericentrin spots an alternative pathway for linker dissolution must operate (Fletcher (Odf2 siRNA n=43, control siRNA n= 64) when ODF2 is depleted, et al., 2004; Mardin and Schiebel, 2012). This model proposes that indicative of a relaxation of centrosome cohesion (Fig. 1C,D). centrosome cohesion is promoted by phosphorylation-mediated These data were corroborated by experiments where ODF2 β-catenin degradation whereas unphosphorylated, and therefore depletion was mediated by the short hairpin vector sh3 (Fig. S2). To stabilized, β-catenin promotes centrosome splitting (Hadjihannas this end, NIH3T3 cells were transfected either with the control et al., 2010). Separated centrosomes afterwards form the two poles vector K07, the short hairpin vector sh3,orsh3 and human cenexin of the bipolar spindle. (hCenexin) vectors for rescue. Simultaneous transfection of a Since formation of a bipolar spindle is crucial for correct plasmid encoding human histone H2A fused to Emerald (H2A–Em) chromosome segregation, the association of centrosomal served to identify transfected cells. At 48 h post transfection, cells abnormalities with genome instability and tumorigenesis is hardly were incubated with the Eg5 inhibitor VS-83 for 2 h and finally surprising (Salisbury et al., 1999; Wang et al., 2004). ODF2 is a processed for immunodecoration of acetylated tubulin. The marker protein of the mature centrosome and essential for cell numbers of monopolar, bipolar and multipolar spindles were viability. Since aberrant expression of Odf2 is frequently observed counted in cells transfected with K07 (n=687), sh3 (n=588), and sh3 in cancer cells, it has been acknowledged as both a cancer and testis plus hCenexin (n=293) that were H2A–Em positive (Fig. 1E,F). antigen (Whitehurst, 2014). Previous data, obtained in human sh3-mediated depletion of ODF2 significantly promoted the cancer cells, have hinted that ODF2 is involved in bipolar spindle formation of bipolar and multipolar spindles, whereas hCenexin formation and normal mitotic progression (Soung et al., 2006, expression rescued the phenotype (P<0.05 and P<0.01, 2009). We used here the mouse fibroblast cell line NIH3T3 to respectively) (Fig. 1F). We additionally observed an increase in determine the involvement of ODF2 in centrosome cohesion. We the distance of centrosomes in monopolar spindle poles when ODF2 show that depletion of ODF2 induced centrosome splitting. is depleted, from 1.4290 µm in K07-transfected control cells (n=54) Depletion of ODF2 is accompanied by an increase in centrosomal to 1.623 µm in ODF2-depleted cells (n=51) that was rescued β-catenin. These data are corroborated by reporter gene assays that by hCenexin expression to 1.408 µm (n=42; P<0.05) (Fig. 1G). show that overexpression of ODF2 inhibits canonical Wnt signaling. Our data indicate that relaxation of centrosome cohesion that occurs Our results thus demonstrate that ODF2 reduces β-catenin stability, upon ODF2 depletion eventually provokes centrosome amplification, thus maintaining centrosome cohesion and preventing premature causing the formation of multipolar spindle poles. centrosome splitting. ODF2 controls centrosomal β-catenin RESULTS ODF2 is a marker protein of the mature centriole. The Wnt pathway ODF2 depletion affects centrosome and spindle pole cohesion component β-catenin localizes to the centrosome and its To determine the importance of ODF2 for centrosome cohesion, we stabilization has been reported to impair centrosome cohesion, investigated the effect of ODF2 depletion on centrosome and thus provoking premature centrosome splitting (Lim et al., 2009; spindle pole splitting. To this end, NIH3T3 cells were transfected Mbom et al., 2013). We therefore queried whether the centrosome either with Odf2 siRNA or a non-targeting control siRNA and separation as observed upon ODF2 depletion is reflected by a shift treated with the Eg5 (also known as KIF11) inhibitor VS-83 in the amount of centrosomal β-catenin. Centrosomal localization of (depletion of ODF2 by siRNA is shown in Fig. S1). The mitotic ODF2 and β-catenin is shown in Fig. 2. Furthermore, there is an kinesin Eg5 is essential for spindle pole separation leading to obvious increase of the amount of β-catenin at the centrosome when monoaster formation when inhibited (Kapitein et al., 2005). Spindle ODF2 is reduced. Depletion of ODF2 was mediated by siRNA poles were detected by γ-tubulin staining, and their appearance was transfection in NIH3T3 cells and monitored 48 h later. Expression considered either monopolar and bipolar as shown in Fig. 1A. In of Centrin2–GFP was used as a centrosomal marker. Successful control siRNA-treated cells more than 70% of cells showed depletion of ODF2 by Odf2 siRNA was verified by anti-ODF2 monopolar spindles 48 h after transfection (n=1077). However, staining (Fig. 2E–H). In control siRNA-transfected cells, ODF2 is depletion of ODF2 decreased the proportion of cells with clearly visible at the centrosome, which is highlighted by Centrin2– monopolar spindles, with a concurrent increase in the proportion GFP colocalization (Fig. 2A–D). Odf2 siRNA transfection caused of cells with separated spindle poles up to ∼35% (n=1113), an obvious decrease of ODF2. Depletion of ODF2 by means of corresponding to a 1.3-fold change, which is statistically significant Odf2 siRNA transfection, additionally, affected centrosomal (P<0.05, Student’s t-test type 2) (Fig. 1B). Decreased centrosome β-catenin (Fig. 2I–P). Compared to control siRNA-transfected cohesion might in turn provoke chromosome segregation defects as cells, the amount of centrosomal β-catenin is increased when ODF2 indicated by the formation of tripolar spindles. When ODF2 was is depleted (Fig. 2M–P compared to I–L), which was additionally depleted by siRNA transfection, we found an increase of the number confirmed by quantification of centrosomal β-catenin (Fig. 2Q). Journal of Cell Science

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Fig. 1. See next page for legend. Journal of Cell Science

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Fig. 1. ODF2 maintains centrosome cohesion. (A,B) Spindle pole cohesion OT and the control plasmid phRL. Transfection of the Odf2 in NIH3T3 cells transfected either with control siRNA or Odf2 siRNA, expression plasmid 13.8NC-GFP (OT; phRL; ODF2/13.8NC-GFP) γ respectively, and analyzed for -tubulin staining at 48 h post-transfection. did not significantly alter the transcriptional activity of OT beyond The Eg5 inhibitor VS-83 was added 2 h before fixation. Spindle poles were considered as monopolar or bipolar (split), respectively, for counting. Scale the background level (OT; phRL). However, ODF2/13.8NC bars: 5 µm (A). Fold change of split spindle poles in NIH3T3 cells depleted for significantly inhibited the β-catenin-mediated transcriptional ODF2 by siRNA transfection is compared to cells transfected with the non- upregulation of the reporter vector (P<0.001) [OT; phRL; β- target control siRNA. Result of two independent biological replicas (B). catenin; ODF2 (13.8NC-GFP) compared to OT; phRL; β-catenin]. *P<0.05. (C,D) Relaxation of centrosome cohesion upon siRNA-mediated siRNA-mediated knockdown of ODF2 did not significantly alter the ODF2 depletion. NIH3T3 cells were transfected either with a non-target control transcriptional activity of OT (OT; phRL; β-catenin; Odf2 siRNA siRNA or with Odf2 siRNA, and centrosomes stained for γ-tubulin (red) or β pericentrin (green). Nuclei are stained with DAPI. Scale bars: 10 µm (C). compared to OT; phRL; -catenin) most likely owing to the general Increase of the distance between the two centrosomal spots, which low amount of endogenous ODF2 (Fig. 4). Moreover, inhibition of characterize the G2 centrosome, when ODF2 is depleted (D). ***P<0.001. canonical Wnt reporter gene activity was also found upon (E–G) Relaxation of spindle pole cohesion upon sh3-mediated ODF2 expression of ODF2/13.8NCΔGFP, in which the GFP tag is depletion. NIH3T3 cells where transfected with the non-target control plasmid omitted, demonstrating that the GFP tag is not responsible for K07, the short hairpin plasmid targeting ODF2 sh3,orsh3 and hCenexin canonical Wnt reporter gene inhibition (data not shown). encoding human ODF2. Transfected cells were identified by expression of histone H2A fused to Emerald (H2A-Em; green). Immunostaining was also β performed for detection of acetylated α-tubulin (red). DNA stained with DAPI ODF2 promotes -catenin degradation (blue). Spindle poles were considered as monopolar, bipolar or multipolar (E). Our studies so far have demonstrated that overexpression of ODF2 Merged images. Scale bars: 2.5 µm (E). Fold change of split spindle poles. represses β-catenin-mediated reporter gene activity and inhibits the *P<0.05; **P<0.01 (F). Distance of centrosomes in monopolar spindles accumulation of β-catenin at the centrosome. To further substantiate (P<0.05) (G). Triplicate experiments. For the box plots in D and G, the box whether ODF2 affected β-catenin stability, the relative amount of – represents the 25 75th percentiles. The median is indicated by a line, and the β-catenin was quantified on western blots. HEK293 cells were mean by a cross. The whiskers show the minimum and maximum values inside β the range given by Q1–1.5×IQR and Q3+1.5×IQR, and outliers (D) and all transfected with either -catenin or ODF2 (13.8NC-GFP) expression points (G) are indicated by circles. Only the 25–75th percentiles, mean and plasmids or both, and the quantity of β-catenin (at ∼100 kDa) relative median are shown for B and F. to α-tubulin quantity was assessed (Fig. 5). Overexpression of ODF2 (13.8NC-GFP) caused a reduction of the relative amount of β-catenin siRNA-mediated depletion of ODF2 resulted in an ∼2-fold increase both of the endogenous as well as of the overexpressed β-catenin of β-catenin in interphase centrosomes (P<0.01; Student’s t-test; (Fig. 5A). Calculation of the relative amount of β-catenin in three n=14 for control siRNA-treated cells and n=27 for Odf2 siRNA- independent biological replicas revealed an overall reduction to ∼40– treated cells). To further substantiate these data, we depleted ODF2 60% compared to the control (Fig. 5B). The reduction in the relative via expression of a short hairpin construct (sh3). Cells were amount of β-catenin is statistically significant [P<0.05 for co-transfected with either the non-target control plasmid (K07) and endogenous β-catenin and P<0.01 in cells overexpressing both an expression vector encoding Centrin2 fused to Cherry (Centrin2- β-catenin and ODF2 (13.8NC-GFP)]. Cherry), or sh3 and Centrin2-Cherry (sh3/Centrin2-Cherry). To rescue sh3-mediated ODF2 knockdown, a human cenexin ODF2 is a component of the β-catenin–Axin1 multi-protein expression plasmid (hCenexin) was transfected concurrently with complex sh3/Centrin2-Cherry plasmids. At 48 h post transfection, cells were Collectively, our data support the view that ODF2 promotes β-catenin processed for immunocytology and the centrosomal area, indicated degradation. Accordingly, depletion of ODF2 caused an increase in by Centrin2–Cherry labelling, quantified (Fig. 3A–C). Expression centrosomal β-catenin. We thus asked whether both proteins are of the short hairpin sequence targeting ODF2 (sh3) resulted in a present in the same complex. Cells transfected with ODF2 constructs significant decrease of ODF2 protein at the centrosome compared to were lysed, and the endogenous β-catenin was immunoprecipitated. that seen upon the expression of the non-target control K07 Western blotting revealed that not only ODF2 (13.8NC, without GFP (Student’s t-test; P<0.001; K07, n=30; sh3, n=30) (Fig. 3D). In tag) but also its C-terminally (NC2-GFP) and N-terminally (N2C- contrast, the centrosomal amount of β-catenin is increased when GFP) truncated versions co-precipitated with β-catenin (Fig. 6A, co- ODF2 is depleted (P<0.001; K07, n=26; sh3, n=28). Concurrent IP). Neither β-catenin nor ODF2 (here fused to GFP, 13.8NC-GFP) expression of human cenexin counteracted the effects of sh3- were precipitated using an unrelated, control IgG (Fig. 6A, co-IP, mediated ODF2 depletion, resulting in a significant decrease in the +control IgG) whereas both proteins are present in the lysate (input). amount centrosomal β-catenin compared to sh3-transfected cells Furthermore, neither β-catenin nor ODF2 bound unspecifically to the (P<0.001; sh3+hCenexin: n=25) but an amount that is similar to the beads (Fig. 6A, co-IP, −). We, additionally, excluded the possibility amount of centrosomal β-catenin in K07-transfected cells (P>0.1) that binding to β-catenin could be mediated by the GFP fusion tag by (Fig. 3D). Our results, therefore, demonstrate that ODF2 suppresses immunoprecipitation of β-catenin from lysate from cells expressing accumulation of β-catenin at the centrosome. GFP (Fig. S3). The co-IP data are corroborated by pulldown assays in which bacterially expressed ODF2 fused to 6×His (13.8NC-6×His) ODF2 inhibits β-catenin-mediated reporter gene activation was incubated with HEK293 cell lysates and affinity purified on To further corroborate our previous results indicating a negative Ni-NTA agarose beads. As shown in Fig. 6B, β-catenin was trapped effect of ODF2 on β-catenin accumulation or stability, we took in the presence of ODF2–His but not in its absence (control). advantage of the canonical Wnt reporter gene assay. The assay was To further elucidate whether ODF2 is a component of the performed using the reporter gene OT, which expresses firefly β-catenin destruction complex, we co-transfected HEK293 cells Luciferase under the control of three copies of the wild-type Tcf-4- with both the ODF2 construct 13.8NC-GFP and Axin1-Myc, binding sequence (Fig. 4). Co-transfection of expression plasmid and immunoprecipitated the ODF2–GFP fusion protein. encoding β-catenin activated the reporter resulting in a ∼2.5-fold Immunoblotting revealed expression of ODF2–GFP and Axin1– increase compared to the control transfected only with the reporter Myc (Fig. 6C, input; Axin1–Myc ∼130 kDa), and co-precipitation Journal of Cell Science

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Fig. 2. See next page for legend. Journal of Cell Science

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Fig. 2. Depletion of ODF2 affects centrosomal β-catenin. ODF2 was Centrosomal ODF2 decreases from interphase to mitosis depleted by means of RNAi (Odf2 siRNA), and ODF2 or β-catenin was ODF2 depletion caused an increase in centrosomal β-catenin and detected immuno-cytologically (red). To identify the centrosome but to omit consequently centrosome splitting. We thus questioned whether dual antibody incubation cells were first transfected with Centrin2–GFP (green). All experiments were compared to the control in which a non-target centrosome splitting at the onset of mitosis is accompanied by a siRNA was transfected (control siRNA). Transfection of Odf2 siRNA decreased decrease in ODF2. Cycling NIH3T3 cells were immunostained for the amount of centrosomal ODF2 (E–H compared to the control A–D). The γ-tubulin and ODF2, and the level of centrosomal ODF2 quantified amount of centrosomal β-catenin is increased by Odf2 knockdown (M–P with ImageJ. Interphase centrosomes were identified either through compared to I–L). (Q) Quantification of centrosomal β-catenin revealed an the presence of a single γ-tubulin spot per cell (G1 centrosome) or ∼ β 2-fold increase of -catenin upon ODF2 depletion. The box represents the the presence of twin γ-tubulin spots (G2 centrosome). Mitotic 25–75th percentiles. The median is indicated by a line, and the mean by a centrosomes were identified through the presence of distant cross. The whiskers show the minimum and maximum values inside the range γ given by Q1–1.5×IQR and Q3+1.5×IQR. Individual data points are also shown. -tubulin spots in conjunction with chromosome condensation. **P<0.01. DNA was counterstained with DAPI (blue). Scale bars: 10 µm. We found a significant decrease of centrosomal ODF2 on the Enlargements show the centrosomal areas (highlighted in boxes) at a higher mitotic centrosome (n=27) compared to the interphase centrosome magnification. (n=53) (P<0.001, Student’s t-test) indicating that centrosome splitting at the onset of mitosis occurs at the same time as a of both proteins [Fig. 6C, +ODF2 (13.8NC-GFP), Axin1–Myc]. decrease in ODF2 (Fig. 7). Neither unspecific binding to beads, by omitting the precipitating antibody (Fig. 6C, −control), nor unspecific binding to control IgG DISCUSSION was found (Fig. 6C, +control IgG) although both ODF2 (13.8NC ODF2 is a component of the insoluble centrosomal scaffold, and without the GFP-tag) and the endogenous Axin1 are present in the beyond that specifically associates with the subdistal appendages of lysate (input control, without Axin1-Myc transfection). Collectively, the mother centriole (Lange and Gull, 1995; Nakagawa et al., 2001; our results support the view that ODF2 coexists in a complex with Tateishi et al., 2013). Moreover, since appendage formation is β-catenin and Axin1. crucially dependent on ODF2, they are lacking in ODF2-deficient

Fig. 3. Increase of centrosomal β-catenin upon shRNA-mediated depletion of ODF2. NIH3T3 cells were transfected with Centrin2–Cherry (red) and either the non- targeting control plasmid K07 (A), the short hairpin plasmid sh3, specifically targeting mouse ODF2 (B), or sh3 and the expression plasmid encoding human Cenexin (hCenexin) for rescue (C). Immunocytological detection of β-catenin (green) is shown. Nuclear counterstain (DAPI) is in blue. Scale bars: 5 µm. The insets show a higher magnification of the centrosomal areas with Centrin2–Cherry- decorated centrosomes. (D) Quantification of ODF2 [αODF2] or β-catenin [αβ-catenin] at the centrosome in cells transfected with K07, sh3,orsh3 and hCenexin (sh3+hCenexin) in conjunction with Centrin2–Cherry. The box represents the 25–75th percentiles. The median is indicated by a line, and the mean by a cross. The whiskers show the minimum and maximum values inside the range given by Q1–1.5×IQR and Q3+1.5×IQR. Individual data points are also shown. ***P<0.001. Journal of Cell Science

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Fig. 4. ODF2 inhibits the canonical Wnt pathway. Overexpression of ODF2 (13.8NC fused to GFP, 13.8NC-GFP) inhibited canonical Wnt signaling as determined by reporter gene assays. NIH3T3 cells were transfected with the reporter vector OT, the control vector phRL and expression plasmids encoding the indicated proteins 24 h before assay. Transfection of Odf2 siRNA was performed 24 h after seeding and 48 h before reporter gene assay. The relative activity was calculated from the firefly luciferase (OT) and the Renilla luciferase ( phRL) activity of each individual experiment. Cumulative result of two independent experiments each done in triplicate are shown. Overexpression of ODF2 (13.8NC-GFP) significantly inhibited β-catenin mediated activation of the reporter OT. The box represents the 25–75th percentiles. The median is indicated by a line, and the mean by a cross. The whiskers show the minimum and maximum values inside the range given by Q1–1.5×IQR and Q3 +1.5×IQR. Individual data points are also shown. ***P<0.001.

F9 cells (Ishikawa et al., 2005). In epithelial cells, appendages on the mother centriole persist throughout the cell cycle, including mitosis, whereas they are formed on the immature former daughter centriole not earlier than through the second half of mitosis. Throughout G2, when the recruitment of appendage proteins has already started, and the first half of mitosis, appendages are not Fig. 5. ODF2 promotes β-catenin degradation. (A) Immunoblotting for visible on the immature centriole (Vorobjev and Chentsov, 1982; β-catenin, α-tubulin and ODF2–GFP (13.8NC-GFP). Cell lysates of Kobayashi and Dynlacht, 2011; Lee and Rhee, 2015). In G0/G1 untransfected HEK293 cells, as well as of those transfected with expression phase, ODF2 preferentially associates with the mother centriole, but plasmids encoding β-catenin, ODF2 (13.8NC-GFP) or both were separated on becomes detectable in both mother and daughter centrioles towards a denaturing polyacrylamide gel, and β-catenin and α-tubulin immunologically the G1/S transition, just before centriole duplication (Nakagawa detected. Transfection of plasmids is indicated by +. Splicing of multiple β et al., 2001). High-resolution imaging in ciliated cells has revealed a images are indicated by spacing between panels. (B) Quantity of -catenin related to the quantity of α-tubulin displayed as fold change of the control (either ring-like arrangement of ODF2 at the distal part of the basal body, untransfected or transfected with β-catenin, respectively). Quantification was indicating recruitment of ODF2 to additional sites during the performed on three or four independent experiments. The 25–75th percentile centriole to basal body transition (Herawati et al., 2016). Moreover, (box), mean (cross) and median (line) are shown. *P<0.05; **P<0.01. these results illustrate the promising power of new imaging tools. Since for most centrosomal proteins, including the Wnt pathway leads to activation of downstream kinases to phosphorylate linker components, only a rather imprecise centrosomal location has been proteins eventually resulting in dissolution of the proteinaceous reported, super-resolution microscopy techniques are important linker and centrosome disjunction (Soung et al., 2006; Soung et al., to clarify the exact location of centrosomal proteins during cell 2009; Mardin et al., 2011; Nam and van Deursen, 2014; Joukov cycle progression. et al., 2014). The ultimate kinase responsible for linker protein Centrosomes are unique cellular organelles in cycling cells that phosphorylation is Nek2, which, in addition, phosphorylates are duplicated once per cell cycle. However, duplicated centrosomes β-catenin, resulting in its stabilization (Bahmanyar et al., 2008; stick together to function as a single entity until onset of mitosis Mbom et al., 2014). when the proteinaceous linker is dissolved to allow bipolar spindle β-catenin, as well as components of the β-catenin degradation formation. The serine/threonine kinase Plk1 plays a critical role in complex, are located at the centrosome, and stabilization of centrosome separation and maturation by mediating the recruitment β-catenin promotes centrosome splitting (Fumoto et al., 2009; of PCM components. Plk1 itself is recruited to the centrosome by Itoh et al., 2009; Kim et al., 2009; Hadjihannas et al., 2010; Mbom the ODF2 isoform known as cenexin, which is a marker protein of et al., 2013). Moreover, besides its well-known effect on β-catenin the mature centriole. Cenexin provides the docking site for the Polo- stabilization, aberrant activation of Wnt signaling also caused box domain when phosphorylated by CDK1. Activated Plk1 then centrosome splitting that is eventually followed by centrosome Journal of Cell Science

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Fig. 6. ODF2 is a component of a β-catenin–Axin1-containing multi- protein complex. (A,B) Co-precipitation of ODF2 and β-catenin. (A) Cells were transfected with the indicated expression plasmids and endogenous β-catenin immunoprecipitated from cell lysates. Co-immunoprecipitation was found for each ODF2 construct analyzed, including the full-length [+ODF2 (13.8NC)], as well as the C-terminally and N-terminally truncated proteins (+NC2-GFP and +N2C-GFP, respectively). Neither ODF2 (fused to GFP, transfection of plasmid 13.8NC-GFP) nor endogenous β-catenin was precipitated with control IgGs [+control IgG; ODF2 (13.8NC-GFP)] and no unspecific binding of β-catenin to beads was detected (−). The input demonstrates the presence of the proteins in the cell lysates. Immunoblot detection of proteins was performed with the indicated antibodies.+indicates the presence of the antibody for immunoprecipitation. The control IgG blot was first probed for ODF2 and afterwards for β-catenin. (B) Affinity purification of His-tagged ODF2 (13.8NC-6xHis) demonstrating conjoint pulled down of endogenous β-catenin. In control experiments without His-tagged ODF2 no β-catenin is found. (C) Co-precipitation of ODF2 and Axin1. Cells were either co-transfected with expression plasmids encoding the ODF2–GFP fusion (13.8NC-GFP)in conjunction with a plasmid encoding Axin1–Myc [input,−control, and +ODF2 (13.8NC-GFP), Axin1-Myc] or with ODF2/13.8NC (without GFP) exclusively (input control and +control IgG). ODF2–GFP was immunoprecipitated with anti-GFP antibodies [+ODF2 (13.8NC-GFP), Axin1-Myc] and co-precipitation of both ODF2 and Axin1 was confirmed. Neither ODF2 (13.8NC-GFP) nor Axin1 bound unspecifically to beads when omitting antibody incubation of the lysate (−control). Additionally, neither ODF2 (13.8NC) nor endogenous Axin1 was precipitated with anti-GFP IgG (+control IgG) although both proteins are still present in the lysate (input control). Target proteins were detected on western blots with anti-ODF2 and anti-Axin1 antibodies, respectively. + or − indicates the presence or absence of IgG in the lysates, respectively. Splicing of multiple images are indicated by spacing between panels.

Previously, the HeLa human cancer cell line was used to examine the relevance of the Plk1-recruiting protein ODF2. The results, obtained by using a stable knockdown cell line, indicated that ODF2 is involved in bipolar spindle formation and normal mitotic progression. However, up to now, the effect appeared to be unique for the HeLa cancer cell line since the data could not be reproduced in the ODF2-deficient F9 cell line (Ishikawa et al., 2005; Soung et al., 2006, 2009). Here, by undertaking a transient depletion of ODF2 in the NIH3T3 mouse fibroblast cell line, we show that ODF2 is required to prevent premature β-catenin accumulation at the centrosome and centrosome disjunction. Quantitative immunoblotting and reporter gene assays using the canonical Wnt signaling pathway indicated that ODF2 affected the abundance of β-catenin. However, since ODF2 is a coiled-coil protein without any known enzymatic activity, its negative effect on β-catenin accumulation is most probably mainly indirect. Given that ODF2 is a microtubule- associated protein, one likely explanation is that it might prevent β-catenin accumulation by either stabilizing the β-catenin destruction complex or promoting β-catenin degradation (Donkor et al., 2004) (Fig. 8). In fact, co-precipitation of ODF2 together with β-catenin and Axin1 corroborates this view. Taken together, our data aberrations, a hallmark of cancer cells (Kaplan et al., 2004; suggest that ODF2 controls the amount of β-catenin at the Hadjihannas et al., 2010). The significance of β-catenin centrosome. Decreasing the level of ODF2 by means of both accumulation for centrosome disjunction inspired a new model in siRNA and shRNA caused an increase in the amount of centrosomal which β-catenin has been attributed a central role. In this model, β-catenin and centrosome disjunction. License of centrosome centrosome cohesion is promoted by phosphorylation-mediated disjunction at the onset of mitosis could, therefore, be granted by β-catenin degradation, whereas stabilized β-catenin promotes a decrease in the amount of endogenous ODF2 that, in turn, would centrosome splitting (Hadjihannas et al., 2010). However, the enable accumulation of β-catenin. This view is corroborated by the Nek2 kinase, which appeared responsible for both, linker observation that the amount of ODF2 fluctuates within the cell dissolution and β-catenin stabilization, seems to be dispensable cycle, revealing a significant higher amount of ODF2 in interphase for centrosome disjunction and cell cycle progression (Fletcher centrosomes than in mitotic centrosomes. et al., 2004; Mardin and Schiebel, 2012). Accordingly, another Our data indicate that ODF2 plays a central role in centrosome pathway for linker dissolution, in addition to the Nek2 pathway, cohesion besides acting solely as a docking site for the recruitment must operate, which credits the prominent role β-catenin plays. of Plk1 at mitosis. In interphase centrosomes, ODF2 keeps Journal of Cell Science

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Fig. 7. Decrease of ODF2 in M-phase centrosomes. (A) Centrosomes in NIH3T3 cells immunostained for γ-tubulin (green) and ODF2 (red). Interphase centrosomes are identified by twin γ-tubulin positive spots, M-phase centrosomes are identified by chromosome condensation and detached discrete γ-tubulin spots. The nucleus is counterstained with DAPI (blue). Scale bars: 2 µm. (B) Fold change of centrosomal ODF2 quantity. The box represents the 25–75th percentiles. The median is indicated by a line, and the mean by a cross. The whiskers show the minimum and maximum values inside the range given by Q1–1.5×IQR and Q3+1.5×IQR. Individual data points are also shown. ***P<0.001.

centrosomal β-catenin low to maintain centrosome cohesion. At the microtubule stability. Depletion of ODF2 thus disturbs asymmetric onset of mitosis, accumulation of β-catenin is supported by a cell division, resulting in a depletion of the stem cell pool during decrease of ODF2, thus acting in line with the activation of the Nek2 neurogenesis (Wang et al., 2009; Tylkowski et al., 2014; Gasic pathway to finally promote centrosome splitting (Fig. 8). However, et al., 2015; Hung et al., 2016). The preimplantation lethality given that ODF2 serves as a docking site for the recruitment of the observed in ODF2-deficient mice may therefore be caused by Plk1 kinase to activate Nek2, a decrease of ODF2 is likely to also perturbing replenishing of the stem cell pool (Salmon et al., 2006). counteract Nek2 activation. Nevertheless, supposing that ODF2 The establishment and the orientation of a bipolar mitotic spindle, as plays an important role in preventing β-catenin accumulation at the well as the anchoring of MTs at the centrosome, are also controlled interphase centrosome, in the G2/M centrosome, β-catenin by β-catenin. Likewise, β-catenin is of utmost importance for accumulation could be maintained even in the absence of sustaining a stem cell pool (Huang et al., 2007; Chilov et al., 2011). activated Nek2 by just decreasing the amount of ODF2. Thus, However, beyond experimental evidence, a clear picture of its depletion of ODF2 would not only abrogate recruitment of Plk1 and molecular function is still missing (Bryja et al., 2017). Although we subsequent activation of the Nek2 pathway to promote linker have shown that ODF2 counteracts β-catenin accumulation at the dissolution and β-catenin stabilization but, additionally, also abolish centrosome, future work has to elucidate whether and how both restraint β-catenin accumulation (Fig. 8). proteins converge in MT formation and spindle positioning. Owing to the nature of centriole duplication, the two spindle To sum up, our results corroborate the data obtained by Soung poles of the bipolar spindle are not identical but comprise either the et al. (2006, 2009) using the HeLa cancer cell line suggesting that old mother centriole or the newly formed mature centriole, finally ODF2 is involved in bipolar spindle formation. Furthermore, we resulting in asymmetric centrosome inheritance. Stem cells, demonstrate that ODF2 restricts β-catenin accumulation at the stereotypically, inherit the centrosome that nucleates more-stable centrosome, which, in turn, prevents aberrant centrosome splitting microtubules, and that is, in most cases, the old centrosome. ODF2 and spindle formation. Since experimental depletion of ODF2 causes not only defines the old centrosome but is also responsible for centrosome splitting and fosters the formation of tripolar spindles, Journal of Cell Science

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For detection of acetylated tubulin [anti-acetylated α-tubulin (clone 6- 11B-1), sc-23950 Santa Cruz Biotechnology] cells were fixed in 3.7% paraformaldehyde in PBS and processed as described above. DNA was counterstained with DAPI. Images were taken by confocal microscopy (LSM 510, Zeiss), and processed using Adobe Photoshop 5.0. ImageJ was used for quantification of the centrosomal area. Amira 5.3.2 or ImageJ were used for measuring centrosomal distances (Stalling et al., 2005). For statistical analyses, a one-tailed, type 2 Student’s t-test was used.

Reporter gene assay Reporter gene assays were performed in NIH3T3 or HEK293 cells. Cells were seeded at a density of 1.5×105 or 1×106 cells, respectively, per well of a 12- well plate. After 24 h Odf2 siRNA (stealth siRNA ODF2MSS207236; Life Technologies; 12 nmol per well) was transfected, when required. Transfection of reporter gene constructs (1 µg/well) together with expression plasmids (each 100 ng/well) was performed 24–48 h before luciferase assays. As reporter genes for the canonical Wnt assay, we used plasmids OT (containing three copies of the wild-type Tcf-4 binding sequence) and OF (containing the mutant Tcf-4 binding sequence) (Shih et al., 2000). For activation of Wnt- Fig. 8. Model depicting the proposed effect of ODF2 on centrosome β cohesion. In interphase, ODF2 prevents β-catenin accumulation, thus reporter genes we used a plasmid encoding -catenin (Morin et al., 1997; OT OF maintaining centrosome cohesion. At G2/M ODF2 is phosphorylated by Cdk1 Korinek et al., 1997). Reporter gene plasmids and , and the expression β- to act as a docking site for the recruitment of Plk1, which, in turn, activates plasmid for catenin were kindly provided by Bert Vogelstein, The Howard the Nek2 pathway for linker displacement and β-catenin stabilization. Hughes Medical Institute & Sidney Kimmel Comprehensive Cancer Center, Concurrently, a decrease in ODF2 enables β-catenin accumulation to promote Baltimore, MD. Constructs were co-transfected with phRL-SV40 (Promega, centrosome disjunction. The mutual relationship between the amount of ODF2 Madison, USA), used as internal control. The Dual-Glo Luciferase Assay and β-catenin is depicted by the size of the text. Curved blue arrow: System (Promega, USA) was used for measuring firefly and Renilla luciferase phosphorylation. Arrows: activation, bars: inhibition. activity using the Centro LB 960 luminometer (Berthold Technologies, Germany). Fold changes were calculated based on the relative luminescence (firefly luminescence/Renilla luminescence). Each experiment was aberrant expression of ODF2 launches a link to chromosomal performed in triplicate and repeated at least 3 times. instability (CIN), which is a hallmark of tumors (de Cárcer and Malumbres, 2014). Pull down assay ODF2 isoform 13.8NC (Hüber et al., 2008) was subcloned into pET28a(+) MATERIALS AND METHODS using EcoRI and NotI to yield p13.8NC-6xHis. E. coli BL21, inoculated Cell culture with p13.8NC-6His, was pre-cultured in 5 ml lysogeny broth (LB) medium NIH3T3 or HEK293 cells (ATCC; regularly authenticated and tested for at 37°C. Large-scale expression cultures were inoculated with pre-culture contamination) were cultured in Dulbecco’s modified Eagle’smedium diluted 1:500 and expression was induced by auto induction in ZYM 5052 (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 1000 U/ml overnight at 37°C (Studier, 2005). The inclusion bodies of the 13.8NC- penicillin, 1000 µg/ml streptomycin and 20 mM L-glutamine at 37°C and 6xHis fusion protein were dissolved in wash buffer [50 mM Tris-HCl pH 5% CO2. NIH3T3 cells were transfected using Transfectin (BioRad) or 7.5, 100 mM NaCl, 1 mM EDTA, 0.2 mM PMSF and 1 mM DTT] and Lipofectamin 2000 (ThermoFisher Scientific Biosciences); HEK293 cells purified by three repetitive sonication and centrifugation steps (5 s pulse at were transfected with XtremeGene HP (Roche). Odf2 siRNA (stealth siRNA 50 W followed by centrifugation at 13,000 g for 30 min). The pellet was ODF2MSS207236; Life Technologies) and control siRNA (siGenome Non- additionally washed and sonicated two times in wash buffer containing 1% targeting siRNA #1; ThermoFisher Scientific Biosciences) were transfected Triton X-100 (v/v). The pellet was then dissolved in 100 mM Tris-HCl (pH with silentFect (BioRad). At 24 h before siRNA transfection cells, were 8.0), 50 mM glycine and 8.5 M urea. 5 mM reduced glutathione (GSSH) initially transfected with an expression plasmid encoding Centrin2–GFP to and 0.5 mM oxidized glutathione (GSSG) were added and stirred overnight highlight centrosomes. Short hairpin constructs sh3 (specifically targeting at 4°C. Refolding of the protein was performed by dialyzing against the sequence GAACTCCTCCAGGAGATAC of mouse ODF2; Tylkowski refolding buffer (100 mM Tris-HCl, 400 mM L-arginine, 1 mM EDTA and et al., 2014) or K07 (Origene), which functions as a control since 0.2 mM PMSF, pH 8.0) with decreasing urea concentrations (4 M, 2 M, 1 M homology to any known mRNA is missing, and the expression plasmids and 0.5 M urea) over several days at 4°C. The penultimate dialyzing step encoding human cenexin (hCenexin) (Soung et al., 2006), human centrin 2 was performed against refolding buffer without urea, and the final step was fused to Cherry (Centrin2-Cherry), and histone H2A fused to Emerald performed with 1×PBS (pH 7.6). Each dialyzing step was performed (Addgene #54110) were transfected with Lipofectamin 2000 overnight at 4°C. The amount of 6×His-13.8NC fusion protein was (ThermoFisher Scientific Biosciences). Eg5-inhibitor VS-83 was used at quantified by comparing it to BSA standards via Coomassie Blue staining. a final concentration of 5 µM for 2 h (Millipore-Calbiochem). For For in vitro interaction experiments, 6×His-13.8NC was added to washed immunocytology, NIH3T3 cells grown on coverslips were fixed in Ni-NTA agarose (Qiagen GmbH, Hilden) [wash buffer at pH 7.6; 50 mM methanol, pre-incubated in 0.3% Triton X-100 in PBS for 10 min, washed NaPi, 500 mM NaCl, 30 mM imidazole, 0.2 mM PMSF and protease in PBS and then incubated in 1% bovine serum albumin, 0.5% Tween-20 inhibitors (Protease Inhibitor Cocktail Tablets, complete Mini, Roche, in PBS (PBT) for blocking unspecific binding sites. Incubation with the Mannheim, Germany)] and incubated for 2 h at 4°C with constant agitation. antibodies was performed at 37°C for 1 h. Primary antibodies used were NIH3T3 cell lysates were added to the resin and incubated for at least 2 h at against β-catenin (ab79089, Abcam), ODF2 (ESAP15572, Antibodies 4°C. The resin was then washed four times with wash buffer, followed by a online; Brohmann et al., 1997), γ-tubulin (T6557; Sigma-Aldrich) and final washing step performed overnight. Proteins were eluted with 50 mM pericentrin (ab4448, Abcam), all diluted 1:100. Secondary antibodies NaPi, 500 mM NaCl, 500 mM imidazole, 0.2 mM PMSF. Finally, probes were goat anti-mouse-IgG antibody conjugated to Alexa Fluor 488 were analyzed by SDS-PAGE and western blotting (Laemmli, 1970; (Molecular Probes, Eugene), goat anti-rabbit IgG antibody conjugated to Towbin et al., 1979) using mouse anti-His tag (1:1000, #70796-3, MFP-488 (Mobitec, #MFP-A1008), goat anti-mouse-IgG antibody Novagen) and rabbit anti-β-catenin (1:1000, ab79089, Abcam) antibodies. conjugated to Alexa Fluor 555 (Invitrogen) and goat anti-rabbit IgG As a negative control, Ni-NTA agarose was incubated with NIH3T3 cell antibodyconjugatedtoAlexaFluorMFP590 (Molecular Probes, Eugene). lysates without 6×His-13.8NC and processed as described. Journal of Cell Science

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Co-immunoprecipitation Chang, J., Seo, S. G., Lee, K. H., Nagashima, K., Bang, J. K., Kim, B. Y., NIH3T3 cells or HEK293 cells were transfected with expression plasmids Erikson, R. L., Lee, K.-W., Lee, H. J., Park, J.-E. et al. (2013). Essential role of using either Transfectin reagent (BioRad, Munich, Germany) for NIH3T3 Cenexin1, but not Odf2, in ciliogenesis. Cell Cycle 12, 655-662. Chilov, D., Sinjushina, N., Rita, H., Taketo, M. M., Mäkelä, T. P. and Partanen, J. cells or X-tremeGene HP (Roche Diagnostics) for HEK293 cells. (2011). Phosphorylated ß-catenin localizes to centrosomes of neuronal Generation of ODF2 truncations (NC2-GFP and N2C-GFP) is described progenitors and is required for cell polarity and neurogenesis in developing in Donkor et al. (2004). At 24 h post transfection, cells were trypsinized and midbrain. Dev. Biol. 357, 259-268. washed two times in PBS. Cells were then lysed in lysis buffer [1×PBS Dawe, H. R., Farr, H. and Gull, K. (2007). Centriole/basal body morphogenesis and containing 1% Nonidet P40, 100 µg/ml (w/v) PMSF and protease inhibitor migration during ciliogenesis in animal cells. J. Cell Sci. 120, 7-15. mix (protease inhibitor cocktail tablets, complete Mini, Roche, Mannheim, de Cárcer, G. and Malumbres, M. (2014). A centrosomal route for cancer genome Germany)] for 20 min on ice using 1 ml buffer per 107 cells followed by instability. Nat. Cell Biol. 16, 504-506. Donkor, F. F., Mönnich, M., Czirr, E., Hollemann, T. and Hoyer-Fender, S. (2004). passing it at least 10 times through a syringe with a 21-gauge needle. After Outer dense fibre protein 2 (ODF2) is a self-interacting centrosomal protein with centrifugation (20,000 g for 15 min at 4°C), the supernatant was incubated affinity for microtubules. J. Cell Sci. 117, 4643-4651. with 2 µg/ml of an anti β-catenin antibody for 2 h at room temperature. Not Doxsey, S. (2001). Re-evaluating centrosome function. Nat. Rev. Mol. Cell. Biol. 2, adding antibody and adding an unrelated IgG were performed as negative 688-698. controls. Immobilized protein G–agarose (Thermo Fisher Scientific Elia, A. E. H., Cantley, L. C. and Yaffe, M. B. (2003). Proteomic screen finds pSer/pThr- Biosciences) was washed three times with lysis buffer and added to the binding domain localizing Plk1 to mitotic substrates. Science 299, 1228-1231. supernatant. Incubation was carried out overnight at 4°C. Afterwards, Faragher, A. J. and Fry, A. M. (2003). Nek2A kinase stimulates centrosome disjunction and is required for formation of bipolar mitotic spindles. Mol. Biol. Cell. the resin was washed four times in lysis buffer. Proteins were finally 14, 2876-2889. extracted in SDS sample buffer by boiling for 5 min. Proteins were analyzed Fletcher, L., Cerniglia, G. J., Nigg, E. A., Yend, T. J. and Muschel, R. J. (2004). by SDS-PAGE and immunoblotting using rabbit anti-GFP (1:1000, Inhibition of centrosome separation after DNA damage: a role for Nek2. Radiat. ab26422; Abcam), rabbit anti-ODF2 (1:1000, Brohmann et al., 1997), Res. 162, 128-135. and mouse anti β-catenin (1:1000, 7F7.2; Millipore) antibodies, Fry, A. M., Mayor, T., Meraldi, P., Stierhof, Y.-D., Tanaka, K. and Nigg, E. A. respectively. For co-immunoprecipitation of ODF2 with Axin1, cells (1998). C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate were co-transfected with ODF2 (construct 13.8NC-GFP)andAxin1-Myc of the cell cycle-regulated kinase Nek2. J. Cell Biol. 141, 1563-1574. Fumoto, K., Kadono, M., Izumi, N. and Kikuchi, A. (2009). Axin localizes to (Addgene #21287; Zeng et al., 1997). Proteins were co-precipitated using the centrosome and is involved in microtubule nucleation. EMBO Rep. 10,606-613. anti GFP antibody. Omitting anti-GFP antibody or incubation with an Gasic, I., Nerurkar, P. and Meraldi, P. (2015). Centrosome age regulates unrelated IgG, respectively, served as controls. Detection was achieved by kinetochore-microtubule stability and biases chromosome mis-segregation. using rabbit anti-GFP (1:1000, ab26422, Abcam) and rabbit anti-Axin1 eLIFE 4, e07907. (1:1000, C76H11; Cell Signaling) antibodies, respectively. Gerdes, J. M., Davis, E. E. and Katsanis, N. (2009). The vertebrate primary cilium in development, homeostasis, and disease. Cell 137, 32-45. Acknowledgements Hadjihannas, M. V., Brückner, M. and Behrens, J. (2010). Conductin/axin2 and For the kind gift of plasmids, we thank our colleagues Kyung S. Lee (National Wnt signalling regulates centrosome cohesion. EMBO Rep. 11, 317-324. Institutes of Health, Bethesda), Jeffrey Salisbury (Mayo Clinic, Rochester), Roger Hardy, T., Lee, M., Hames, R. S., Prosser, S. L., Cheary, D.-M., Samant, M. D., Tsien (Howard Hughes Medical Institute, La Jolla), Bert Vogelstein (The Howard Schultz, F., Baxter, J. E., Rhee, K. and Fry, A. M. (2014). Multisite Hughes Medical Institute & Sidney Kimmel Comprehensive Cancer Center, phosphorylation of C-Nap1 releases it from Cep135 to trigger centrosome Baltimore) and Terry Yamaguchi (National Institutes of Health, Frederick, MD). disjunction. J. Cell Sci. 127, 2493-2506. Herawati, E., Taniguchi, D., Kanoh, H., Tateishi, K., Ishihara, S. and Tsukita, S. Competing interests (2016). Multiciliated cell basal bodies align in stereotypical patterns coordinated The authors declare no competing or financial interests. by the apical cytoskeleton. J. Cell Biol. 214, 571-586. Hoyer-Fender, S. (2010). Centriole maturation and transformation to basal body. Author contributions Sem. Cell Dev. Biol. 21, 142-147. Hoyer-Fender, S. (2013). Primary and motile cilia: their ultrastructure and Conceptualization: S.H.-F.; Methodology: K.Y., M.A.T., D.H.; Formal analysis: S.H.-F.; ciliogenesis. pp. 1-53. In Cilia and Nervous System Development and Function Investigation: K.Y., M.A.T., D.H., C.T.C., S.H.-F.; Writing - original draft: S.H.-F.; (ed. K. L. Tucker and T. Caspary). Springer. Supervision: D.H., S.H.-F.; Project administration: S.H.-F. Hoyer-Fender, S., Neesen, J., Szpirer, J. and Szpirer, C. (2003). Genomic organisation and chromosomal assignment of Odf2 (outer dense fiber 2), Funding encoding the main component of sperm tail outer dense fibers and a This research received no specific grant from any funding agency in the public, centrosomal scaffold protein. Cytogenet. Genome Res. 103, 122-127. commercial or not-for-profit sectors. Huang, P., Senga, T. and Hamaguchi, M. (2007). A novel role of phospho-ß- catenin in microtubule regrowth at centrosome. Oncogene 26, 4357-4371. Supplementary information Hüber, D. and Hoyer-Fender, S. (2007). Alternative splicing of exon 3b gives rise to Supplementary information available online at ODF2 and Cenexin. Cytogenet. Genome Res. 119, 68-73. http://jcs.biologists.org/lookup/doi/10.1242/jcs.220954.supplemental Hüber, D., Geisler, S., Monecke, S. and Hoyer-Fender, S. (2008). Molecular dissection of ODF2/Cenexin revealed a short stretch of amino acids necessary for References targeting to the centrosome and the primary cilium. Eur. J. Cell Biol. 87, 137-146. Anderson, C. T. and Stearns, T. (2009). Centriole age underlies asynchronous Hung, H.-F., Hehnly, H. and Doxsey, S. (2016). The mother centriole appendage primary cilium growth in mammalian cells. Curr. Biol. 19, 1498-1502. protein Cenexin modulates lumen formation through spindle orientation. Curr. Bahe, S., Stierhof, Y.-D., Wilkinson, C. J., Leiss, F. and Nigg, E. A. (2005). Biol. 26, 793-801. Rootletin forms centriole-associated filaments and functions in centrosome Ishikawa, H., Kubo, A., Tsukita, S. and Tsukita, S. (2005). Odf2-deficient mother cohesion. J. 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