© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs220954. doi:10.1242/jcs.220954 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