ARTICLE Received 10 Sep 2015 | Accepted 10 Feb 2016 | Published 18 Mar 2016 DOI: 10.1038/ncomms11005 OPEN A CEP215–HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer Pavithra L. Chavali1, Gayathri Chandrasekaran1, Alexis R. Barr1,w,Pe´ter Ta´trai1, Chris Taylor1, Evaggelia K. Papachristou1, C. Geoffrey Woods2, Sreenivas Chavali3 & Fanni Gergely1 Numerical centrosome aberrations underlie certain developmental abnormalities and may promote cancer. A cell maintains normal centrosome numbers by coupling centrosome duplication with segregation, which is achieved through sustained association of each centrosome with a mitotic spindle pole. Although the microcephaly- and primordial dwarfism- linked centrosomal protein CEP215 has been implicated in this process, the molecular mechanism responsible remains unclear. Here, using proteomic profiling, we identify the minus end-directed microtubule motor protein HSET as a direct binding partner of CEP215. Targeted deletion of the HSET-binding domain of CEP215 in vertebrate cells causes centro- some detachment and results in HSET depletion at centrosomes, a phenotype also observed in CEP215-deficient patient-derived cells. Moreover, in cancer cells with centrosome amplification, the CEP215–HSET complex promotes the clustering of extra centrosomes into pseudo-bipolar spindles, thereby ensuring viable cell division. Therefore, stabilization of the centrosome–spindle pole interface by the CEP215–HSET complex could promote survival of cancer cells containing supernumerary centrosomes. 1 Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Robinson Way, Cambridge CB2 0RE, UK. 2 Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK. 3 MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK. w Present address: Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK. Correspondence and requests for materials should be addressed to F.G. (email: [email protected]). NATURE COMMUNICATIONS | 7:11005 | DOI: 10.1038/ncomms11005 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11005 entrosomes act as dominant sites of microtubule assembly To this end, affinity purification tags (GsTAP containing protein in mitosis and therefore centrosome number corresponds G and streptavidin-binding protein) were inserted in-frame into Cto the number of spindle poles formed1. Because faithful both alleles of the CEP215 gene (CEP215-TAP cell line) transmission of genetic information requires a bipolar mitotic in the chicken B cell line, DT40 (refs 27,35). Following spindle, centrosome numbers must be tightly controlled in affinity purification, protein complexes were analysed by mass cells. Accordingly, centrosome numbers are regulated by two spectrometry (Fig. 1a; Supplementary Fig. 1). Proteins were mechanisms. First, centrosome duplication is limited to once per considered as hits if they were represented by one or more unique cell cycle ensuring that cells enter mitosis with two functional peptides in all three biological replicates and by four or more centrosomes2,3. Second, each centrosome associates and unique peptides in at least two replicates. We filtered out putative co-segregates with its own mitotic spindle pole causing each hits if they were represented even by a single unique peptide in daughter cell to inherit precisely one centrosome4. Centrosomes pulldowns performed from wild-type (WT) cells. Hits were and mitotic spindle poles are distinct structures, well illustrated further filtered against other GsTAP affinity purification experi- by the presence of focused spindle poles in cells lacking ments to exclude TAP tag-specific binding36. An interacting centrosomes5–7. Spindle pole formation relies on microtubule network of CEP215 was constructed based on these criteria motors and microtubule-associated proteins that crosslink and (Fig. 1b). All previously reported interacting partners have focus bundles of kinetochore-associated microtubules (k-fibres). been identified, in addition to new ones that include PCM1, In Drosophila S2 cells the key protein responsible for holding CKAP5/ch-Tog and HSET, a minus end-directed microtubule centrosomes at spindle poles is dynein, a minus end-directed motor (Fig. 1b; Supplementary Table 1; Supplementary Data 1). motor8–10. Dynactin increases the processivity of dynein and Western blot analysis confirmed interactions (Fig. 1c). Because of together they transport the spindle pole integrity protein, nuclear its roles in mitotic spindle pole organization in Drosophila and mitotic apparatus (NuMA) to the minus ends of spindle cancer cells, we have decided to focus on HSET for the purpose of microtubules11,12. In NuMA-deficient mammalian cells, k-fibres this study. lose focus and centrosomes detach from the poles13. Similar phenotypes have been documented in Drosophila cells and CEP215 and HSET bind directly in vertebrates. CEP215 embryos upon disruption of the minus end-directed kinesin-14 interacts with the microtubule motor dynein and its adaptor, motor protein, non-claret-disjunctional (ncd)10,14. By contrast, dynactin37. To establish if HSET, dynein and CEP215 exist in the the mammalian homologue HSET is largely dispensable for same complex, CEP215-TAP-containing protein complexes k-fibre focus. Instead, HSET contributes to spindle elongation were fractionated on a sucrose gradient. CEP215-bound HSET through crosslinking and sliding microtubules, functions sedimented at a lower sucrose concentration than CEP215-bound dependent on its C-terminal motor domain and the additional dynein, indicative of separate complexes (Fig. 2a). Gel filtration microtubule-binding site in its N-terminal tail15. Both ncd experiment yielded similar results (Supplementary Fig. 2a). and HSET have been implicated in survival of cells with To further characterize the CEP215-HSET interaction, we centrosome amplification16–19. In particular, the orthologues elucidated the respective binding domains in human CEP215 and mediate clustering of supernumerary centrosomes into pseudo- HSET. CEP215 fusion products were expressed in HeLa cells bipolar spindles, a role essential for continued proliferation of constitutively depleted of CEP215 (Supplementary Fig. 2b). The cells with centrosome amplification. HSET also promotes HSET-binding region was mapped to the two overlapping regions clustering of acentrosomal spindle poles17. in the N terminus of CEP215: amino acids (aa) 500–700 and 300– The centrosome comprises a pair of centrioles embedded 600 (Fig. 2b,c). In HSET it is aa1–150 at the N terminus (that is, in the pericentriolar matrix (PCM), the site of microtubule the tail domain) that binds CEP215 (Fig. 2d). The CEP215–HSET nucleation. CEP215 is an evolutionarily conserved PCM interaction is direct, as suggested by yeast two-hybrid assays and protein present in microtubule-organizing centres from yeast to surface plasmon resonance (SPR) (Fig. 2e; Supplementary human; the centrosomin motif 1 (CM1) in its N terminus binds Fig. 2c). In SPR aa500–700 of CEP215 displayed an B2.5-fold the g-tubulin complex20–23. CEP215 organizes several PCM greater binding to HSET when compared with aa300–600. components including pericentrin and AKAP450 (refs 24–30). We therefore consider aa500–700 of CEP215 as the minimal Deletion of centrosomin (cnn), its Drosophila orthologue, HSET-binding region (HBR). Sequence analysis of HBR of disruption of the CM1 domain of chicken CEP215 and human CEP215 revealed three helical regions that are conserved depletion of CEP215 in HeLa cells all cause centrosome in vertebrates. Remarkably, the tail of HSET also shows a high detachment from mitotic spindle poles27,31,32. However, spindle degree of conservation in the vertebrate lineage, raising the pole focus is maintained in CM1-deficient cells, consistent with possibility that the interaction between HSET and CEP215 arose normal localization of NuMA and dynactin27. Mutations in in this lineage (Fig. 2f; Supplementary Figs 2d, 3 and 4). Indeed, CEP215 are associated with congenital diseases such as primary we could not detect binding between Drosophila cnn and ncd, the microcephaly and primordial dwarfism33,34. respective homologues of human CEP215 and HSET, whereas Here we set out to identify the molecular mechanism by which the two proteins co-immunoprecipitated in human HeLa cells CEP215 maintains centrosome attachment to spindle poles. We (Fig. 2g,h). The ancestral cnn gene underwent a duplication identify HSET as a direct interactor of CEP215 and demonstrate event in cephalochordates producing CEP215 and another that HSET binding by CEP215 is crucial for its role in this CM1-containing gene, myomegalin. Unlike CEP215, myomegalin process. We further show that cancer cells with centrosome lacks an HBR and, accordingly, failed to interact with HSET amplification rely on the CEP215–HSET complex for centrosome (Supplementary Fig. 2e). clustering and survival. CEP215-HSET complex connects centrosomes to spindle poles. Results We next wanted to address the functional significance of the Identification of CEP215-interacting partners in DT40 cells. CEP215–HSET interaction. Using gene targeting we created To establish the molecular basis for CEP215 function in chicken DT40 cell lines in which either HSET or the HBR of centrosome–spindle pole attachment, we employed an unbiased CEP215 was disrupted.