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Received 10 Sep 2015 | Accepted 10 Feb 2016 | Published 18 Mar 2016

DOI: 10.1038/ncomms11005 OPEN

A CEP215HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer

Pavithra L. Chavali1, Gayathri Chandrasekaran1, Alexis R. Barr1,w, Pter Ttrai1, 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 dwarsm-linked centrosomal protein CEP215 has been implicated in this process, the molecular mechanism responsible remains unclear. Here, using proteomic proling, 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-decient patient-derived cells. Moreover, in cancer cells with centrosome amplication, the CEP215HSET complex promotes the clustering of extra centrosomes into pseudo-bipolar spindles, thereby ensuring viable cell division. Therefore, stabilization of the centrosomespindle pole interface by the CEP215HSET 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: mailto:[email protected]

Web End [email protected] ).

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Centrosomes act as dominant sites of microtubule assembly in mitosis and therefore centrosome number corresponds to the number of spindle poles formed1. Because faithful

transmission of genetic information requires a bipolar mitotic spindle, centrosome numbers must be tightly controlled in cells. Accordingly, centrosome numbers are regulated by two mechanisms. First, centrosome duplication is limited to once per cell cycle ensuring that cells enter mitosis with two functional centrosomes2,3. Second, each centrosome associates and co-segregates with its own mitotic spindle pole causing each daughter cell to inherit precisely one centrosome4. Centrosomes and mitotic spindle poles are distinct structures, well illustrated by the presence of focused spindle poles in cells lacking centrosomes57. Spindle pole formation relies on microtubule motors and microtubule-associated proteins that crosslink and focus bundles of kinetochore-associated microtubules (k-bres).

In Drosophila S2 cells the key protein responsible for holding centrosomes at spindle poles is dynein, a minus end-directed motor810. Dynactin increases the processivity of dynein and together they transport the spindle pole integrity protein, nuclear mitotic apparatus (NuMA) to the minus ends of spindle microtubules11,12. In NuMA-decient mammalian cells, k-bres lose focus and centrosomes detach from the poles13. Similar phenotypes have been documented in Drosophila cells and embryos upon disruption of the minus end-directed kinesin-14 motor protein, non-claret-disjunctional (ncd)10,14. By contrast, the mammalian homologue HSET is largely dispensable for k-bre focus. Instead, HSET contributes to spindle elongation through crosslinking and sliding microtubules, functions dependent on its C-terminal motor domain and the additional microtubule-binding site in its N-terminal tail15. Both ncd and HSET have been implicated in survival of cells with centrosome amplication1619. In particular, the orthologues mediate clustering of supernumerary centrosomes into pseudo-bipolar spindles, a role essential for continued proliferation of cells with centrosome amplication. HSET also promotes clustering of acentrosomal spindle poles17.

The centrosome comprises a pair of centrioles embedded in the pericentriolar matrix (PCM), the site of microtubule nucleation. CEP215 is an evolutionarily conserved PCM protein present in microtubule-organizing centres from yeast to human; the centrosomin motif 1 (CM1) in its N terminus binds the g-tubulin complex2023. CEP215 organizes several PCM components including pericentrin and AKAP450 (refs 2430). Deletion of centrosomin (cnn), its Drosophila orthologue, disruption of the CM1 domain of chicken CEP215 and depletion of CEP215 in HeLa cells all cause centrosome detachment from mitotic spindle poles27,31,32. However, spindle pole focus is maintained in CM1-decient cells, consistent with normal localization of NuMA and dynactin27. Mutations in CEP215 are associated with congenital diseases such as primary microcephaly and primordial dwarsm33,34.

Here we set out to identify the molecular mechanism by which CEP215 maintains centrosome attachment to spindle poles. We identify HSET as a direct interactor of CEP215 and demonstrate that HSET binding by CEP215 is crucial for its role in this process. We further show that cancer cells with centrosome amplication rely on the CEP215HSET complex for centrosome clustering and survival.

ResultsIdentication of CEP215-interacting partners in DT40 cells. To establish the molecular basis for CEP215 function in centrosomespindle pole attachment, we employed an unbiased proteomic approach to isolate and identify CEP215 interactors.

To this end, afnity purication tags (GsTAP containing protein G and streptavidin-binding protein) were inserted in-frame into both alleles of the CEP215 gene (CEP215-TAP cell line) in the chicken B cell line, DT40 (refs 27,35). Following afnity purication, protein complexes were analysed by mass spectrometry (Fig. 1a; Supplementary Fig. 1). Proteins were considered as hits if they were represented by one or more unique peptides in all three biological replicates and by four or more unique peptides in at least two replicates. We ltered out putative hits if they were represented even by a single unique peptide in pulldowns performed from wild-type (WT) cells. Hits were further ltered against other GsTAP afnity purication experiments to exclude TAP tag-specic binding36. An interacting network of CEP215 was constructed based on these criteria (Fig. 1b). All previously reported interacting partners have been identied, in addition to new ones that include PCM1, CKAP5/ch-Tog and HSET, a minus end-directed microtubule motor (Fig. 1b; Supplementary Table 1; Supplementary Data 1). Western blot analysis conrmed interactions (Fig. 1c). Because of its roles in mitotic spindle pole organization in Drosophila and cancer cells, we have decided to focus on HSET for the purpose of this study.

CEP215 and HSET bind directly in vertebrates. CEP215 interacts with the microtubule motor dynein and its adaptor, dynactin37. To establish if HSET, dynein and CEP215 exist in the same complex, CEP215-TAP-containing protein complexes were fractionated on a sucrose gradient. CEP215-bound HSET sedimented at a lower sucrose concentration than CEP215-bound dynein, indicative of separate complexes (Fig. 2a). Gel ltration experiment yielded similar results (Supplementary Fig. 2a).

To further characterize the CEP215-HSET interaction, we elucidated the respective binding domains in human CEP215 and HSET. CEP215 fusion products were expressed in HeLa cells constitutively depleted of CEP215 (Supplementary Fig. 2b). The HSET-binding region was mapped to the two overlapping regions in the N terminus of CEP215: amino acids (aa) 500700 and 300 600 (Fig. 2b,c). In HSET it is aa1150 at the N terminus (that is, the tail domain) that binds CEP215 (Fig. 2d). The CEP215HSET interaction is direct, as suggested by yeast two-hybrid assays and surface plasmon resonance (SPR) (Fig. 2e; Supplementary Fig. 2c). In SPR aa500700 of CEP215 displayed an B2.5-fold greater binding to HSET when compared with aa300600. We therefore consider aa500700 of CEP215 as the minimal HSET-binding region (HBR). Sequence analysis of HBR of human CEP215 revealed three helical regions that are conserved in vertebrates. Remarkably, the tail of HSET also shows a high degree of conservation in the vertebrate lineage, raising the possibility that the interaction between HSET and CEP215 arose in this lineage (Fig. 2f; Supplementary Figs 2d, 3 and 4). Indeed, we could not detect binding between Drosophila cnn and ncd, the respective homologues of human CEP215 and HSET, whereas the two proteins co-immunoprecipitated in human HeLa cells (Fig. 2g,h). The ancestral cnn gene underwent a duplication event in cephalochordates producing CEP215 and another CM1-containing gene, myomegalin. Unlike CEP215, myomegalin lacks an HBR and, accordingly, failed to interact with HSET (Supplementary Fig. 2e).

CEP215-HSET complex connects centrosomes to spindle poles. We next wanted to address the functional signicance of the CEP215HSET interaction. Using gene targeting we created chicken DT40 cell lines in which either HSET or the HBR of CEP215 was disrupted. The HSET knockout line (HSETKO) was generated by replacing the exons encoding the tail and stalk

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CM1 CM2 CEP215

GsTAP

CM1 CEP215

CM2

WT cells

CEP215-TAP cells

LCMS analysis

Cell lysate

Streptavidin affinity purification

Biotin elution (Elu)

Protein interaction map

Western blot

TUBG1

CEP215-TAP

kDa

HSET DYNC1H1

IST1

WCE Elu WCE Elu

250

Strep HRP (CEP215)

Negative control

ACAP2

CEP152

WDR67

DCTN

PRKACB

410

250

HSET

SEPT6

SEPT9

EB1

DIC

CLASP2

CKAP5

MT motors

Kinase

Centrosomal proteins

SEPT7

150

p150

SEPT2

CEP215

PLK1

AKAP450

PCM1

EB1

Centrin-1

-tubulin

AKAP9

MT end-binding protein

PRKAR2A

LOC101750034

CCDC77

PCM1

AZI1

Endosome

Actin

Satellite

Centrosome

Microtubule Golgi

Figure 1 | Protein interaction network of CEP215. (a) Schematic representation of the workow used to identify interacting partners of CEP215. (b) The interactome map was constructed based on the mass spectrometric analysis of afnity-puried TAP-CEP215-containing protein complexes. GsTAP tag consists of protein G and streptavidin-binding protein. Each node represents a binding partner of CEP215 identied in all three biological replicates, but absent in WT cells and detected by a minimum of four unique peptides in at least two replicates (Supplementary Table 1). Actual or predicted subcellular localization of proteins are colour coded. The greater a Mascot score (best of three replicates), the darker the corresponding line. Dashed line for CEP152 refers to protein being found only in two experiments. Blue dashed lines mark previously reported binding between interactors of CEP215. (c) Whole-cell extracts (WCE) of WT or TAP-CEP215 cells were subjected to afnity purication (Elu) and immunoblotted with the indicated antibodies. DIC, dynein intermediate chain; MT, microtubule.

domains (aa1345) with antibiotic resistance genes38 (Supplementary Fig. 5a). Using western blots and immunouorescence, we conrmed that HSETKO cells were protein null (Fig. 3a,b).

The HBR in chicken CEP215 maps to aa482663. The CEP215DHBR cell line was generated through an in-frame fusion of exons 11 and 17, resulting in deletion of aa468665 (Supplementary Fig. 5b). Since the genomic sequence encoding for HBR spans 12.8 kb, we performed sequential targeting: rst, exons 1316 were removed followed by exon 12. Antibiotic resistance genes were excised using cre recombinase after each round (Supplementary Fig. 5b). As expected, CEP215DHBR cells expressed a truncated CEP215 mRNA in which exons 11 and 17 are fused (Supplementary Fig. 5c,d). The corresponding protein product (termed CEP215(DHBR)) showed similar expression levels and localization to the wild-type protein, suggestive of normal folding, yet did not interact with HSET (Fig. 3ce). In addition to CEP215DHBR, an intermediate cell line called CEP215DN was included in our study. In this case exons 1316 were replaced by antibiotic resistance genes, but these were not excised by cre recombinase (Supplementary Fig. 5b). An

antibody against aa40375 of CEP215 revealed no product in CEP215DN cells (Fig. 3c). Thus, even if a truncated protein is produced from the mutant alleles, this product lacks both the CM1 (aa83141) and HBR domains. mRNA analysis of CEP215DN showed a truncated transcript with low expression levels (Supplementary Fig. 5d). All three lines were viable, but exhibited a mild proliferation defect and an elevated mitotic index (Supplementary Fig. 6a,b).

Centrosome detachment was observed in HSETKO, CEP215DN and CEP215DHBR cells (Fig. 3f,g). The category detached includes cells with one or two partially or completely detached centrosomes. Over 30% of CEP215DHBR mitotic cells displayed centrosome detachment, suggesting that HSET binding by CEP215 is vital to maintain centrosomes at spindle poles in DT40 cells. The centrosome detachment phenotype reached B60% in HSETKO and CEP215DN cells. A further 10%

of the mutants displayed multipolar spindles with an additional B510% of cells showing abnormal spindle morphology ranging from unfocussed spindle to monopolar/collapsed spindles in HSETKO (Fig. 3g). To better understand these phenotypes,

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Sucrose gradient

40%

CM1 CEP215

CM2

GS-TAP

19S

CEP215-TAP cells

kDa Fraction no.

Streptavidin affinity purification

Biotin elution

Sucrose gradient

190

55 70

CEP215

HSET

DIC

b c d

FLAG

1 1,893CEP215

Bioease

1 1,190

CEP215 fragments

CEP215

1 673 Bioease HSET

1673

CEP215 fragments

HSET fragments

11,893

11,190

1,1901,893

HSET

HSET(WCE)

1300

300600

500700

700900

9001,190

1300

300673

1150

151300

kDa

250

110

72 55

190

kDa

72 55 72

kDa

190

IP:FLAG

Strep HRP

CEP215

CEP215 (WCE)

FLAG

Strep pull down

HSET

HSET (WCE)

e f g

HSET

HeLa

WCE

220

Con

Ligand:

IP: CEP215

CEP215

MBP

Mammals (35)

Vertebrates Invertebrates

CEP215

kDa

MBP CEP215 (300600) MBP CEP215 (500700)

Analytes

Aves (1)

Relative response units

300

250

200

150

100

Reptilia (2)

HSET

Amphibia (1)

Coelacanthimorpha (1)

Teleostei (10)

Cephalochordata (1)

DMel-2

Tunicata (2)

Nematoda (3)

KDa

WCE

Con

IP: Cnn

GST-HSET

GST (1150)

150

Arthropoda (4)

Annelida (1)

Cnn

Ncd

GST-HSET

(150300)

GST-HSET

(1300)

GST-HSET

(300673)

Mollusca (1)

Figure 2 | CEP215 and HSET interact through vertebrate-specic binding domains. (a) Left panel depicts the workow for separation of TAP-CEP215-bound complexes on a 540% sucrose gradient. Western blots of sucrose fractions probed with antibodies as indicated. (b) Whole-cell extracts (WCE) of HeLa cells expressing FLAG-tagged CEP215 fragments were subjected to FLAG pull-down followed by western blotting with the indicated antibodies. (c) WCE of CEP215-depleted HeLa cells expressing Bioease-tagged CEP215 fragments as indicated were subjected to streptavidin (strep) pull-down followed by western blotting with the indicated antibodies. (d) WCE of HeLa cells expressing Bioease-tagged HSET fragments were subjected to streptavidin (strep) pull-down followed by western blotting with the indicated antibodies. (e) HSET and CEP215 bind directly. Graph depicts qualitative analysis of binding between MBP-tagged CEP215 fragments (substrates) and GST-tagged HSET fragments (ligands) using surface plasmon resonance plotted as relative response units. GST and MBP proteins were used as controls. MBP shows background response for each analyte. Values for three technical replicates are shown. Error bars correspond to standard deviation. (f) Sequences of HBR of CEP215 and aa1150 of HSET have been analysed across 97 organisms (Supplementary Fig. 2d). Dark grey cells indicate high sequence conservation within HBR of CEP215 and aa1150 of HSET. Light grey cells depict lesser conservation of aa1150 of HSET. Compared with human HSET aa1150, invertebrates showed an average sequence identity of 12% in contrast to 54% among vertebrates. White cells depict the absence of HBR in CEP215 orthologues. Numbers in parentheses represent the number of organisms per class for which CEP215 and/or HSET sequences are available. (g) WCE of mitotic HeLa cells were subjected to immunoprecipitation by an anti-CEP215 antibody or random IgG (con) followed by western blotting with the indicated antibodies. (h) WCE of Drosophila Dmel2 cells were subjected to immunoprecipitation by an anti-centrosomin (Cnn) antibody followed by western blotting with the indicated antibodies.

mitosis was followed live using GFP-EB3 in HSETKO and CEP215DHBR cells (Fig. 3h; Supplementary Movies 17). Partial and/or complete centrosome detachment was seen in both

HSETKO and CEP215DHBR. Furthermore, 24% of HSETKO cells showed a transient collapse of the spindle into a monopole soon after nuclear envelope breakdown, revealing a role for HSET in

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HSET -tubulin

HSETWT/KO1

CEP215

CM1 HBR CM2

kDa

HSETKO1

HSETWT/KO2

HSETKO2

CEP215HBR

(468665) CEP215N

(1665)

HSET

p150

CEP215N1

150

HSETKO

CEP215N2

CEP215HBR1

CEP215HBR2

kDa

250

CEP215

CEP215 -tubulin

CEP215

-tubulin

Prometaphase and metaphase cells (%)

CEP215NWT

CEP215HBR

-tubulin

WT (n=412)

0 10 20 30 40 50 60

CEP215HBR1

(n=465)

CEP215HBR2

(n=406)

Detached

Disorganized

Multipolar

CEP215N1

(n=425)

CEP215N2

(n=485)

CEP215HBR

WCE

Con

CEP215

kDa

Prometaphase and metaphase cells (%)

250

CEP215

0 10 20 30 40 50 60

HSET

WT (n=455)

HSETKO1

(n=516)

HSETKO2

(n=508)

-tubulin

Centrin-2

-tubulin

Centrin-2

CEP215NWT

CEP215HBR

Phenotypes

CEP215HBR

(n=30)

HSETKO

(n=42)

Partially detached centrosome Fully detached centrosome

Loss of spindle pole focus Transient monopolar/ collapsed spindle

Multipolar spindle

63% 13%

10%

62% 26%

24%

10%

HSETKO2+HSETN593K#2

Prometaphase and metaphase cells (%)

0 20 40 80 100

(n=332)

HSETKO

HSETKO2+HSET#1

HSETKO2+HSET#2

HSETKO2+HSETN593K#1

HSETKO2

HSETGFP-HSET

HSETKO2+

HSET

P=3.1109

(n=303)

P=1.21032

P=1.41066

kDa

110

(n=389)

(n=321)

HSETKO2+

HSETN593K

(n=420)

HSET

150

p150

Figure 3 | HSET binding by CEP215 is required for association between centrosomes and spindle poles. (a) Whole-cell extracts (WCE) of wild-type (WT) DT40, heterozygous and homozygous clones of HSETKO are immunoblotted with an anti-HSET antibody recognizing aa300673.(b) Immunouorescence images show WTand HSETKO1 cells stained for HSET (red) and a-tubulin (green). DNA is in blue. Scale bar, 3 mm. (c) Schematics of expected truncations are shown on top. Note that an N-terminally truncated product may be expressed in CEP215DN. At the bottom, WCE of WT and homozygous clones of CEP215DHBR and CEP215DN are immunoblotted with an N-terminal anti-CEP215 antibody. (d) Representative images show WT, CEP215DHBR and CEP215DN cells stained for CEP215 (red) and a-tubulin (green). DNA is in blue. Scale bar, 4 mm (e) WCE of WT and CEP215 DHBR cells were subjected to immunoprecipitation (IP) by an anti-CEP215 antibody or random IgG (con) followed by western blotting. Antibodies for immunoblotting are indicated. CEP215DHBR does not interact with HSET. (f) Representative images illustrate mitotic phenotypes in CEP215DN, CEP215DHBR and HSETKO cells stained for centrin-2 (red) and a-tubulin (green). DNA is in blue. Arrows indicate completely or partially detached centrosomes. Bottom panel shows collapsed spindle in HSETKO. Scale bar, 4 mm. (g) Graph depicts quantication of phenotypes as percentage of total mitotic cells in two independent clones of CEP215DHBR, CEP215DN and HSETKO cells (4500 mitotic cells per clone). (h) Table summarizes mitotic phenotypes of CEP215DN and HSETKO cells from time-lapse experiments. (i) WCE from HSETKO2 cells stably transfected with GFP-tagged wild-type HSET (HSETKO2-HSET) or mutant HSETN593K (HSETKO2-HSETN593K) were subjected to western blotting with the indicated antibodies. Graph on right depicts quantication of phenotypes as percentage of total mitotic cells (colours as in g). P values were obtained by Fishers exact test. In the graph P values are shown for the detachment phenotype. P values for the second clones: HSETKO versus HSETKO HSET#2: P 1.02 10 69; HSETKO versus HSETKO HSET(N593K)#2: P 1.03 10 43;

HSETKO HSET#2 versus HSETKO HSET(N593K)#2: 2.77 10 6. P values for disorganized spindle are shown in the text.

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maintenance of bipolarity at the early stages of spindle assembly (Fig. 3h). Nevertheless, all HSETKO cells subsequently regained bipolarity and initiated normal anaphase. Importantly, we found no evidence for loss of centrosome integrity in the mutants: normal PCM organization was conrmed by confocal and 3D-structured illumination microscopy both in spindle pole-associated and detached centrosomes (Supplementary Fig. 6ce). Consistently, microtubule-nucleating capacity of isolated centrosomes was preserved when tested in Xenopus egg extracts (Supplementary Fig. 6f).

Ncd/HSET contains separate microtubule-binding and motor domains that permit microtubule crosslinking and sliding, respectively3941. To address which function is responsible for linking centrosomes with spindle poles, we made use of the N593K point mutation in HSET, which markedly decreases the ATPase and sliding activities of the motor without impacting on its crosslinking function15. HSETKO cells were transfected with GFP fusions of wild-type or N593K-mutant human HSET. Single clones (called HSETKO-HSET and HSETKOHSETN593K) were selected with transgene expression levels comparable to endogenous HSET. GFP-HSET almost fully rescued centrosome detachment and disorganized spindles in HSETKO cells (Fig. 3i). By contrast, GFP-HSET(N593K) reduced centrosome detachment to B15%, a signicant, but nonetheless inferior rescue when compared with GFP-HSET. Therefore, microtubule crosslinking appears to be the more dominant role of HSET in attaching centrosomes to spindle poles, but sliding also plays a part. Interestingly, GFP-HSET(N593K) was unable to prevent formation of disorganized spindles, suggesting that the motor activity is crucial for HSET function in spindle organization (P values for disorganized spindle phenotype: HSETKO versus HSETKO HSET#1: 1.07 10 7; HSETKO

versus HSETKO HSET(N593K): 0.7821486; Fishers exact tests).

CEP215 is responsible for centrosomal accumulation of HSET. We next asked whether CEP215 could inuence localization of HSET to the spindle or centrosomes. HSET localized normally to spindles of CEP215DHBR cells (Supplementary Fig. 6g). To measure the centrosomal pool of HSET specically, microtubules were depolymerized with nocodazole in WT and CEP215DHBR DT40 cells (Fig. 4a). HSET signal intensity was then quantied in mitotic centrosomes as dened by the volume of g-tubulin staining. While centrosome volumes were similar between WT and CEP215DHBR, HSET levels were signicantly reduced at centrosomes (Fig. 4a). Likewise, when centrosomes were isolated by sucrose sedimentation from WT and CEP215DHBR cells, a marked decrease in HSET was seen in the latter (Fig. 4b). These ndings raised the possibility that the CEP215HSET interaction might occur at centrosomes. We tested the idea using the STILKO DT40 cell line that lacks functional centrosomes7. In STILKO cells HSET is present, whereas CEP215 is absent from the spindle apparatus (Fig. 4c)7. Strikingly, immunoprecipitation of CEP215 in STILKO cells revealed loss of interaction with HSET, implying that intact centrosomes are a prerequisite of CEP215HSET complex formation (Fig. 4d). We conclude that CEP215 is likely to bind HSET at centrosomes, which in turn increases centrosomal levels of HSET.

HBR and CM1 domains of CEP215 scaffold distinct interactions. Our group previously reported centrosome detachment in a cell line where the rst 140 aa of CEP215, containing the centrosomin motif 1 (CM1), were deleted (called CEP215DCM1)27. Because disruption of CM1 decreases centrosomal levels of CEP215 by nearly 70%, the observed centrosome detachment phenotype (B50%) could reect the combined effect of CM1 deletion and

reduced centrosomal accumulation of CEP215. These ndings have nonetheless raised the question of how the CM1 and HBR domains contribute to the function of CEP215 at the centrosomespindle pole interface. To address this point, CEP215DCM1-TAP and CEP215DHBR-TAP cells were generated through biallelic insertion of GsTAP tags into the respective mutant CEP215 loci (Fig. 5a). As in Fig. 1, we employed TAP afnity purication to uncover binding partners of the truncated proteins. Remarkably, except for HSET, CEP215(DHBR)-

TAP precipitated every interactor from Fig. 1c. By contrast, CEP215(DCM1)-TAP could bind HSET, but failed to precipitate g-tubulin, dynein, PCM1 and Plk1 kinase amongst others (Fig. 5b).

Sequences within CM1 have been shown to activate g-tubulin complexes in vitro, albeit this interaction does not seem relevant to the mitotic role of CEP215 (refs 21,22,30). Therefore, we wondered if this highly conserved domain could also bind microtubules. Bacterially expressed aa1300 of CEP215 co-pelleted with microtubules, indicative of direct binding (Fig. 5c). Moreover, microtubule spin-down experiments from cell lysates revealed a 3.4-fold reduction in microtubule binding of CEP215(DCM1)-TAP when compared with CEP215-TAP and

CEP215(DHBR)-TAP (Fig. 5d). Collectively, our data demonstrate that CEP215 utilizes HBR exclusively for HSET binding, whereas the CM1 domain mediates microtubule association and a host of other interactions.

CEP215 and HSET co-localize on pericentrosomal particles. We showed that binding between CEP215 and HSET requires intact centrosomes (Fig. 4d). However, the CEP215HSET complex was isolated from afnity purication experiments performed on cytoplasmic lysates, and not on centrosomal fractions, indicating that some of the complex is associated only loosely with centrosomes and/or may even leave the organelle. In y embryos GFP-fused Cnn/CEP215 appear on centrosome ares, PCM particles that detach from centrosomes42. We therefore wondered if similar structures existed in vertebrate cells, and if so, whether these contained HSET. Flare-like CEP215 staining was detected in B8% of WT mitotic DT40 cells (Fig. 5e,f). Treatment with the proteasome inhibitor MG132 raised centrosomal CEP215 levels and concomitantly increased the percentage of cells with pericentrosomal CEP215 particles to over 70% both in WT and CEP215DHBR cells (Fig. 5e,f). As in ies, these particles decreased upon depolymerization of microtubules by nocodazole (Supplementary Fig. 7a)42. HSET was visible in these structures, suggesting that CEP215HSET may travel on these pericentrosomal particles in a microtubule-dependent fashion (Fig. 5g). Interestingly, such particles were absent in CEP215DCM1 cells, although this could be due to lower levels of CEP215(DCM1) at centrosomes both in DMSO- and

MG132-treated cells (Fig. 5e)27.

Pericentriolar satellites are small granules that surround the centrosome in interphase and are thought to disperse during mitosis43. Since the core satellite component, PCM1, was present in the CEP215 interaction network (Fig. 1b), we tested if ares in mitotic DT40 cells could correspond to satellites. However, this is unlikely to be the case, since we found no evidence for PCM1 enrichment in the ares (Supplementary Fig. 7b).

Reduced HSET in centrosomes of CEP215 mutant patient cells. Mutations in CEP215 cause autosomal recessive primary micro-cephaly33. We have derived parent-of-patient and patient B lymphocytes (CEP215 / and CEP215 / , respectively) that carry the premature stop codon 243 T4A (S81X) in exon 4 of CDK5RAP2/CEP215 (ref. 33). On western blots of CEP215 /

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Centrosome volume HSET intensity at centrosome

-tubulin

HSET Merge

P = 0.31

P = 3.7 1023

CEP215HBR

n=39

1.8

0.3

Mean intensity of HSET (a.u.)

WTCEP215 HBR

-tubulin volume (a.u.)

0.6

1.5

1.2

0.9

WT n=40

CEP215HBR

n=39

10 WT n=40

HSET -tubulin

WT CEP215HBR

kDa

inp

HSET

Centrin1

-tubulin

STILKO

HSET

DT40 cells

kDa

70% 40% inp 1 2 3 4 5 6 7 8 9

CEP215 -tubulin

210

40 19

CEP215

-tubulin

CEP215

HSET

-tubulin Centrin1

DT40 cells

CEP215HBRWT

210

40 19

CEP215

HSET

Centrin1

STILKO

WT CEP215HBR

-tubulin

HSET/Cen1 ratio

WCE IP:con IP:CEP215

STILKO

kDa

STILKO

250

CEP215

HSET

0 Fraction 3 Fraction 4 Fraction 5

Figure 4 | CEP215 promotes association of HSET with centrosomes. (a) Images show WT and CEP215DHBR cells in which microtubules were depolymerized by nocodazole. Cells are stained for HSET (green) and g-tubulin (red). Dot plots on right depict the volume of centrosomes (that is, measured as the volume of g-tubulin-positive structures) and the mean signal intensity of HSET in centrosomes. Note that each dot represents a cell;

centrosome volumes and mean HSET intensities were averaged across the two centrosomes in each cell (WT: n 40; CEP215DHBR: n 39). P values are

obtained by Fishers test. Scale bar, 3 mm. (b) Representative western blots of centrosomes isolated from WT and CEP215DHBR cells. Western blot on top shows cell lysates before and after centrifugation onto a 50% sucrose cushion to enrich for centrosomes (CE and inp, respectively). This input (inp) was further centrifuged through a discontinuous sucrose gradient (% sucrose is indicated above blots) with results shown on western blots below. Frame depicts centrin-rich fractions corresponding to centrosomes. Antibodies for immunoblotting are indicated. Note reduction of HSET in CEP215DHBR

centrosomes. Graph below shows quantication of the HSET to centrin-1 signal ratio in centrin-rich fractions; n 3 biological replicates. Error bars

correspond to standard deviation. (c) WTand STILKO cells in top panels are stained for HSET (green) and a-tubulin (red), and in bottom panels for CEP215 (green) and g-tubulin (red). DNA is in blue. Scale bar, 4 mm. (d) WCE of WT and STILKO cells were subjected to immunoprecipitation (IP) by random IgG (con) or anti-CEP215 antibody, followed by western blotting using indicated antibodies.

cells an antibody against the C terminus of CEP215 revealed a 78% reduction in the intensity of a band similar in size to full-length CEP215 (Fig. 6a). As in chicken cells, centrosomes isolated from patient-derived CEP215 / B cells contained less

HSET than their CEP215/ counterparts (Fig. 6b).

Although only 2% of CEP215 / lymphocytes showed centrosome detachment, 24% exhibited centrosomes that appeared at an angle greater than 15 with respect to the spindle axis (3% in CEP215 / ; Fig. 6c). We also measured the distance between centrosomes and spindle poles and found it increased in

CEP215 / cells (Fig. 6d). Moreover, we noted that whereas centrosomes were contained within the spindle pole in almost all

CEP215 / cells, they seemed to be outside the spindle poles in

nearly 25% of CEP215 / , indicating an outward displacement in the mutants.

Depletion of HSET or CEP215 in HeLa cells also produced centrosome displacement phenotypes, but none replicated the complete centrosome detachment seen in DT40 cells15,37 (Supplementary Fig. 8a). Several not mutually exclusive explanations exist for the milder phenotype seen in human cells. First, residual CEP215 might be sufcient to maintain centrosome attachment to spindle poles. Second, there may be a partially redundant pathway to CEP215HSET in human cells, such as that mediated by spindle pole component WDR62, which has no obvious orthologues in chicken44. Third, forcesexternal or internal to the spindlecould contribute to the phenotype and

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Genotype Cell line

MBP-CEP215 (1300)

S P S P S P S P

+ + + + Taxol

250

90 70

150

MBP

CM1 HBR CM2

kDa

GsTAP CEP215-TAP

GsTAP

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CEP215CM1-TAP

MBP-CEP215 (1300)

Tubulin (polymerized)

CEP215CM1-TAP

CEP215HBR-TAP

CEP215-TAP

kDa

Strep HRP(detecting CEP215-TAP)

-tubulin

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CEP215WT-TAP

CEP215HBR-TAP

CEP215CM1-TAP

+ + + + + +

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Taxol

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p150

-tubulin

EB1

kDa S P S P S P S P S P S P

Strep HRP (CEP215)

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CEP215CM1-TAP

kDa

WCE IP WCE IP 250

WCE IP

460

Strep HRP (CEP215)

DIC

EB1

AKAP450

70 250

HSET

-tubulin

PLK1

Cells with pericentrosomal CEP215 particles (%)

0 20 40 60 80 100

CEP215HBR

CEP215CM1 MG132

DMSO

PCM1

HAUS6

CEP215 -tubulin

CEP215

CEP215 -tubulin

-tubulin

Hoescht -tubulin

CEP215 HSET

DMSO

CEP215 HSET

Merge

CEP215HBR

TAP

CEP215CM1-

TAP

DMSO MG132

CEP215

MG132

Figure 5 | HBR of CEP215 mediates HSET binding exclusively, whereas its CM1 domain is responsible for multiple interactions. (a) Table depicts summary of TAP-tagged cell lines. The panel below shows the expression of protein products from CEP215-TAP, CEP215DHBR-TAP and CEP215DCM1-TAP cell lines. (b) CEP215-containing protein complexes were afnity puried from CEP215-TAP, CEP215DHBR-TAP and CEP215DCM1-TAP cells, followed by western blotting for indicated antibodies. (c) Binding of MBP-CEP215 (1300) to microtubules was assayed using microtubule spin-down in the presence of tubulin ( taxol) or taxol-stabilized ( taxol) microtubules. MBP served as a negative control. Following centrifugation supernatants (S) and pellets (P)

were loaded on gel and stained with Coomassie blue. (d) Microtubule spin-down assays were performed from lysates of CEP215-TAP, CEP215DHBR-TAP and CEP215DCM1-TAP cells in the presence of tubulin ( taxol) or taxol-stabilized microtubules ( taxol). Following centrifugation supernatants (S) and

pellets (P) were subjected to western blotting. Antibodies for immunoblotting are indicated. Arrowhead marks the panel depicting the reduction of CEP215(DCM1)-TAP binding to microtubules. (e) Pericentrosomal CEP215 particles are visualized in DMSO- and MG132-treated WT, CEP215DHBR and

CEP215DCM1 cells. Cells were stained for CEP215 (green) and g-tubulin (red). DNA is in blue. Arrow highlights a particle. Insets show higher magnication of CEP215 and g-tubulin stainings corresponding to framed areas. Scale bar, 4 mm. (f) Graphs show quantitation of pericentrosomal CEP215 particles as percentage of mitotic cells in the presence of DMSO or MG132. P values of paired t-tests (*Po0.05, **Po0.005); n 3 biological replicates. Error bars

correspond to standard deviation. (g) DMSO- and MG132-treated WT cells were stained for CEP215 (green) and HSET (red). DNA is in blue. Scale bar, 4 mm. Insets show higher magnication of CEP215 and HSET stainings corresponding to framed areas. Note co-localization of the two proteins on pericentrosomal particles.

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CEP215

b 70% 40%

CEP215/CEP215+/

+/ /

kDa

inp 1 2 3 4 5 6 7 8 9

kDa

HSET

-tubulin

Centrin3

HSET

-tubulin

Centrin3

CEP215

-tubulin

250

Human B cells

-tubulin

-tubulin -tubulin

30 1530

Deviation of centrosome from spindle axis

CEP215+/

CEP215/

100

0.3

0.6

1.0

CEP215+/

n =104

P =1.26105

CEP215/

n =100

Centrosomes (%)

0 015 1530 >30

Distance between spindle pole and centrosome

CEP63 -tubulin

CEP63 P=3.06105

d(m)

CEP215+/

CEP215/

d=distance between spindle pole and centrosome

CEP215+/

n=42

CEP215/

n=44

Figure 6 | Centrosomes from CEP215 mutant patient cells contain reduced levels of HSET and show mild displacement from spindle poles. (a) Whole-cell extracts were prepared from CEP215/ and CEP215 / human B lymphocytes followed by western blotting with the indicated antibodies. CEP215 was detected by an antibody against aa900950. (b) Representative western blots of centrosomes isolated from CEP215 / and CEP215 / human B lymphocytes. Cell lysates were enriched for centrosomes by centrifugation onto a 50% sucrose cushion (inp) followed by centrifugation through a discontinuous sucrose gradient (% sucrose is indicated above blots). Antibodies for immunoblotting are indicated. (c) CEP215/ and CEP215 /

human lymphocytes were sequentially stained for a-tubulin (green) and g-tubulin (red). Spindle axis (marked as white dotted line) was determined using automated image analysis (see Methods for details). Position of centrosomes with respect to the axis was determined manually as depicted in schematics and data is shown in a bar chart. Arrow points to a centrosome positioned over 30 from spindle axis. P values were obtained by Fishers exact test for n 100 cells. Scale bar, 3 mm. (d) Images show CEP215 / and CEP215 / human lymphocytes stained for the centrosomal protein CEP63 (red) and

a-tubulin (green). Dot plot depicts distribution of distance between centrosomes and corresponding spindle poles (CEP215/ : n 42 and CEP215 / :

n 44 cells). P values are obtained by Wilcoxon-rank sum test. Scale bar 3 mm.

these may vary between species and cell types45,46. The ratio of centrosomal microtubules versus k-bres could inuence internal forces; this may be skewed in DT40 cells, which have a diploid chromosome number of 78 (normal genome size in chicken), accompanied by weak astral microtubules in mitosis. In addition, external forces could also vary due to differences in cortical organization and cell adhesion45. We found that depolymerization of actin in HSETKO and CEP215DHBR cells by cytochalasin D reduced the incidence of centrosome detachment in both mutants (Supplementary Fig. 8b). Thus, actomyosin

contributes to the centrosome detachment phenotype, probably by increasing forces on the centrosomespindle pole interface.

CEP215HSET promotes centrosome clustering in cancer cells. Cells with centrosome amplication must cluster their super-numerary centrosomes into a pseudo-bipolar spindle for survival, and HSET plays a vital role in this process18,47. Since our study has identied a functional interaction between HSET and CEP215 in centrosomespindle pole attachment, we reasoned

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that CEP215 could also be involved in centrosome clustering. We have therefore examined loss-of-function phenotypes of CEP215 in two cell lines with centrosome amplication: the mouse neuroblastoma line N1E-115 and the human breast cancer cell line BT459, with respective incidences of 499% and B25%

supernumerary centrosomes. Both BT459 and N1E-115 cells depend on HSET for survival17,18.

Because small interfering (si) RNAs were ineffective, we used retroviral small hairpin (sh) RNA to deplete CEP215 in N1E-115 cells, achieving 64% depletion after 72 h (Fig. 7a; Supplementary Fig. 9a). In both CEP215- and HSET-depleted cells we noted an increase in multipolar spindles along with a range of aberrant spindle conformations (Fig. 7b). Multipolar anaphases in live cells were used as a measure of inefcient centrosome clustering. Nearly all N1E-115 cells exhibit bipolar anaphases after resolving multipolar spindle intermediates into pseudo-bipolar spindles. In line with previous reports, time-lapse analysis of N1E-115 siRNA-mediated depletion of HSET caused multipolar anaphases in B70% of cells18,48, whereas 22% of CEP215-depleted cells displayed multipolar anaphases (Fig. 7c; Supplementary Movies 8 and 9). Consistently, cell survival was reduced in both cases (Fig. 7d).

We next asked if HSET binding by CEP215 contributed to its function in centrosome clustering. To this end, we generated single clones of N1E-115 cells by stably expressing Flag only or Flag fusions of human CEP215 or CEP215(DHBR). These clones were then transduced with control shRNAs or shRNAs specic to mouse CEP215 (Fig. 7e; Supplementary Fig. 9b). While both FLAG fusion products localized to centrosomes, Flag-CEP215(DHBR) exhibited reduced efcacy in centrosome clustering (Fig. 7f). Because Flag-CEP215(DHBR) can still mediate some clustering, other sequences in CEP215 such as CM1 might also contribute to CEP215 function in this process (Fig. 7g).

In BT-549 breast cancer cells a 94% depletion of CEP215 levels was achieved by siRNA (Fig. 8a). Cells were analysed with immunouorescence and time-lapse microscopy. Both revealed an increase in multipolar spindles as well as multipolar anaphases upon CEP215 knockdown with a concomitant reduction in cell survival (Fig. 8b,c). While analysing centrosome clustering, we noted centrosome detachment in BT-549 cells (Fig. 8d). Detachment was seen in cells with bipolar and multipolar spindles. However, due to the prevalence of acentrosomal spindle poles in these cells17, we scored centrosome detachment only in cells that contained a bipolar spindle and two centrosomes. As in DT40 cells, depletion of CEP215 and HSET both triggered centrosome detachment (Fig. 8e).

DiscussionCentrosomes and spindle poles harbour distinct microtubule populations: the former contains predominantly astral micro-tubules, whereas the latter contains k-bres and interpolar microtubules49,50. Therefore, centrosomes and spindle poles experience different forces, calling for an active mechanism to link the two structures during mitosis4. Here we describe a vertebrate-specic interaction between CEP215 and the motor protein HSET, which is required for connecting centrosomes with mitotic spindle poles. Formation of the CEP215HSET complex requires intact centrosomes and CEP215 promotes centrosomal accumulation of HSET.

Our current understanding of how centrosomes and spindle poles are connected stems from experiments in Drosophila S2 cells, where dynein plays a central role by transporting microtubules as well as crosslinking k-bres with astral micro-tubules10,51,52. In vertebrate cells removal of astral microtubules

does not trigger centrosome detachment, and instead centrosomes move closer to spindle poles, suggesting a nonessential role for astral microtubules in maintaining centrosomes at spindle poles (Supplementary Fig. 10). In mammalian cells centrosome detachment has been observed upon loss of spindle pole focus (that is, disruption of NuMA13) or following depletion of the spindle pole protein WDR62 or the centromere component CENP-32, although in these cases the molecular mechanisms are still unclear44,53,54. Intriguingly, CENP-32 depletion leads to a reduction in CEP215 and AKAP450 at mitotic centrosomes53. Moreover, like CEP215, mutations in WDR62 cause micro-cephaly, indicating that an impaired spindle polecentrosome interface could preclude normal brain development55,56.

What could be the molecular mechanism by which the CEP215HSET complex holds centrosomes at spindle poles? We propose a model whereby CEP215 through its HBR captures HSET-bound microtubules, resulting in centrosomal anchoring of k-bres and interpolar microtubules by CEP215HSET (Fig. 8f). NuMA and dynein have been shown to accumulate on free microtubule minus ends and facilitate the processive poleward movement of these microtubules57. Interestingly, our mass spectrometry analysis of CEP215-binding partners has identied not only dynein but also NuMA, albeit the latter was present in only two experiments. Therefore, CEP215 may also contribute to capturing dynein/NuMA-bound microtubule ends, perhaps through the CM1 domain. This could explain why centrosome detachment is less frequent in CEP215DHBR cells than in CEP215DN cells where both CM1 and HBR domains are missing. Within the centrosome CEP215 appears to be positioned with its N terminus pointing towards the cytoplasm; such conguration is ideal for the CM1 and HBR domains to capture motors and incoming microtubules28.

Impaired centrosomespindle pole attachment can cause abnormal centrosome segregation, which can lead to super-numerary centrosomes. Indeed, HSET and CEP215 knockout cells displayed an increase in spindle multipolarity (Fig. 3gi). A hypomorphic mouse model of CEP215 also exhibits centro-some amplication and multipolar spindles in the developing brain, phenotypes observed upon in utero siRNA-mediated depletion of CEP215 as well29,58. Likewise, CEP215-decient mouse embryonic broblasts contain extra centrosomes26.

Centrosome clustering in cancer cells with centrosome amplication relies on a range of processes that include the spindle assembly checkpoint, matrix adhesion, microtubule minus end motors dynein and HSET, the chromosome passenger complex and various microtubule-associated proteins18,5961. Microtubule attachment and spindle tension seem a prerequisite for efcient clustering59. Since centrosome clustering also requires cortical actomyosin forces that act on astral microtubules, these forces must be transmitted from the spindle pole to the centrosome and vice versa18. By stabilizing the centrosomespindle pole connection, CEP215HSET may coincidentally increase the efciency of centrosome clustering. In fact, multipolar spindle arrangements could pose the ultimate challenge for centrosome and spindle pole connection. In these unbalanced and asymmetric spindle congurations k-bre numbers, spindle forces and geometries can differ from pole to pole, as can centrosome size and microtubule nucleation capacity.

In N1E-115 and BT-549 cells depletion of HSET triggers a more severe declustering phenotype than that observed upon CEP215 knockdown. Moreover, ncd/HSET is required for centrosome clustering in ies and also for focusing acentrosomal spindle poles in ies and mammals15,41,60. In these cases the complex is probably irrelevant, because CEP215 and ncd do not seem to interact in ies and require centrosomes to interact in vertebrates. These ndings indicate that HSET has

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Luminescence (x103 RLU)0 2 6 10

4 8

Unt

shCon

siHSET

shCEP215

HSET

kDa

Unt

shCon

siHSET

250

CEP215

-tubulin

* **

shCEP215

-tubulin centrin-2

Flag-CEP215

Flag-CEP215 (HBR)

Flag

shCon

shCEP215

Parental

Flag-CEP215 (HBR)

Flag-CEP215

Flag

shCon

siHSET

centrin-2 -tubulin

shCEP215

+ + + +

Low exp

High exp

250

CEP215

-tubulin

FLAG

FLAG -tubulin

shRNA/siRNA

Time-lapse microscopy

Flag

Time (hours)

48 72

Time (hours:min)

shCon

shCEP215

siHSET

0:00

0:45

0:55

0:45

1:15

2:00

1:40

1:25

2:10

1:50

Flag-CEP215

Flag-CEP215 (HBR)

0 25

5 10 15 20

(n=517)

Multipolar anaphase (%) 0

Multipolar anaphase (%)

(n=568)

20 40 60 80 100

Unt n=394

ShCon n=408

siHSET n=258

Flag

Flag-CEP215

Flag-CEP215(HBR) shCon shCEP215

(n=609)

(n=476)

ShCEP215 n=1708

n.s

(n=475)

(n=601)

Figure 7 | CEP215 facilitates centrosome clustering in mouse neuroblastoma cells via its HBR domain. (a) Western blots of whole-cell extracts of N1E-115 cells untreated (unt) or transfected with HSET siRNA (siHSET), retroviral control shRNA (shCon) or CEP215 shRNA (shCEP215) with the indicated antibodies. (b) Images show siRNA/shRNA-treated N1E-115 cells stained for centrin-2 (red) and a-tubulin (green)) Scale bar, 8 mm. (c) Experimental timeline is shown in schematic. Still frames from time-lapse experiments depict mitosis in untreated (unt) or siRNA/shRNA-treated N1E-115 cells. Graph below shows percentage of mitotic cells with multipolar anaphases from time-lapse experiments. Total number of mitoses analysed per treatment is shown. Two-way ANOVA followed by Tukeys test were performed (**Po0.005); n 3 biological replicates. Error bars correspond to standard deviation. (d) Graph

depicts viability of untreated (unt) or siRNA/shRNA-treated N1E-115 cells as a function of relative light units (RLU) using CellTiter-Glo assay. P values of paired t-tests (**Po0.005); n 3 biological replicates. (e) N1E-115 cells stably expressing Flag, Flag-CEP215 or Flag-CEP215(DHBR) were transduced with a

control (shCon) or CEP215 shRNA (shCEP215) and 72 h later immunoblotted for indicated antibodies. Both low and high exposures of the blot are presented. (f) Images show N1E-115 cells stably expressing Flag, Flag-CEP215 or Flag-CEP215(DHBR) stained for FLAG (green) and a-tubulin (red). DNA is in blue. Scale bar, 8 mm. (g) Parental N1E-115 cells or those stably expressing Flag, Flag-CEP215 or Flag-CEP215(DHBR) were transduced with control (shCon) or CEP215 shRNA (shCEP215) and followed live with same timeline as in panel c. Graph shows the percentage of mitotic cells with multipolar anaphases from time-lapse experiments. Total number of mitoses analysed per treatment is shown. Two-way ANOVA followed by Tukeys test were performed (**Po0.005); n 4 biological replicates. Error bars correspond to s.d.

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a d

-tubulin

CPAP -tubulin

CPAP

siHSET

siCEP215

siCon

kDa

Unt

250

CEP215

siCon

HSET

-tubulin

siCEP215

siHSET

siRNA Time-lapse

microscopy

0 48 72

Time (h)

Time (h:min)

siHSET

0:00 1:15 3:00 3:05 3:10

siCon

siCEP215

0:00 0:15 1:25 1:30 1:40

0:00 0:50 2:00 2:05 2:25

Detached Multipolar

Bipolar Clustered

Multipolar anaphases (%)

0 10 20 30 40

Unt n =101

siCEP215 n =117

Prometaphase and metaphase cells (%)

siCon n =86

siHSET n =140

Luminescence (104 RLU)

Unt

siCon

siCEP215

si HSET

0 10 20 30 40 50

siCon siHSET siCEP215

Centrosomalmicrotubule Kinetochore

HBR CM1

k-fibre

CEP215

HSET

Dynein

Centrosome

Centriole

PCM

Figure 8 | CEP215 and HSET promote centrosome association with mitotic spindle poles and centrosome clustering in human breast cancer cells. (a) Western blots of whole-cell extracts of BT-549 cells prepared 72 h after siRNA transfections. Untreated (unt) cells are included as controls. Antibodies for immunoblotting are indicated. (b) Still frames from a time-lapse experiment show mitosis in BT-549 cells untreated (unt) or treated with control (siCon), CEP215 (siCEP215) or HSET (siHSET) siRNAs. Graph depicts the number of multipolar anaphases in cells treated with the indicated siRNAs. Total number of mitoses analysed per treatment is shown. (c) Graph shows viability of untreated (unt) or siRNA-treated BT-549 cells as a function of relative light units (RLU) using CellTiter-Glo assay. n 3 biological replicates, where error bars denote standard deviation and statistical signicance was computed

using paired t-test. (d) Images of siRNA-treated BT-549 cells stained for the centriolar marker CPAP (green) and a-tubulin (red). DNA is in blue. Arrows mark detached centrosomes. Scale bar, 6 mm. (e) Graphs show quantications of centrosome and spindle phenotypes (as depicted in schematics) in siRNA-treated BT-549 cells. Two-way ANOVA followed by Tukeys test were performed (*Po0.05, **Po0.005); n 3 biological replicates. Error bars

correspond to standard deviation. (f) Schematic representation of the proposed function of CEP215 at the centrosomespindle pole interface. Briey, through HBR CEP215 captures HSET- bound minus ends of k-bres and interpolar microtubules, thereby anchoring these at the centrosome. CEP215 may also capture dynein-associated microtubules through the CM1 domain.

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CEP215-independent functions in centrosome clustering that are likely to involve sliding and crosslinking of parallel microtubules.

Nonetheless, an interesting conclusion of our study is the vertebrate lineage-specic interaction between CEP215 and HSET, raising the question as to why vertebrate cells have acquired new complexes to secure the connection between centrosomes and spindle poles. With larger genome sizes chromosome numbers often increase, leading to an increase in k-bre numbers and possibly greater forces at the centrosome spindle pole interface. Furthermore, whereas in Drosophila centrosomes are nonessential for development beyond the syncytial stages, loss of centrosomes causes embryonic lethality in mice, and absence of centrosomes triggers p53-dependent apoptosis in cultured mammalian somatic cells6,6264. By facilitating a stable association of centrosomes with spindle poles and thereby correct centrosome segregation, the CEP215 HSET complex could promote cell survival in vertebrates; if so, CEP215 deciency is expected to cause cell loss, consistent with the primordial dwarsm phenotype seen in patients with mutations in CEP215 (ref. 34).

Methods

Cell culture and drug treatments. DT40 and human B cells were cultured as described previously27,36. BT-549 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum (FBS) and 0.023 IU insulin. HeLa and ecotropic Phoenix cells were cultured in DMEM medium with 10% FBS. HeLa cells were obtained from Jonathan Pines (Gurdon Institute, Cambridge, UK) over 10 years ago, whereas BT-549 cells were a gift by Carlos Caldas (CRUK CI, Cambridge, UK). Identities of these cells lines were conrmed by STR genotyping. Our original stock of DT40 cells was obtained from Julian Sale (MRC-LMB, Cambridge, UK) over 10 years ago. Dmel2 cells from David Glover (University of Cambridge, UK) were cultured in Serum free medium (GIBCO) with 110 U ml 1 penicillin, 10 mg ml 1 streptomycin. CytochalasinD (Sigma-Aldrich) was used at 1 mg ml 1. To obtain mitotic extracts of HeLa cells, 9 mM RO3306 was added for 20 h (h), then washed three times and incubated for 15 min.

Homologous gene targeting in DT40 cells. Gene targeting was performed according to standard protocol38. Briey, homology arms were cloned into pJET or PGEMT-Easy and subcloned into pBluescript II SK (pSK). The primers used to amplify homology arms of each construct are listed in Supplementary Table 2.

A drug resistance cassette (neomycin/Neo, blasticidin/Blasti or puromycin/Puro) was cloned into pSK between BamHI sites65. The two alleles of HSET and CEP215 were targeted sequentially: for HSETKO, the rst allele was targeted with blasticidin and the second with puromycin. For CEP215, the rst allele was targeted with neomycin and the second with blasticidin. All nal constructs were linearized and transfected as described previously27. Targeted integration of the resistance cassettes was screened by PCR. Primers used for PCR reactions are listed in the Supplementary Table 3. To generate CEP215DHBR cell lines, CEP215DN was subjected to cre recombinase-mediated excision of the antibiotic resistance cassette27. This was further targeted to remove exon 12, subjected to another round of cre-mediated excision of antibiotic resistance cassettes. C-terminal TAP tagging of CEP215 was performed as described previously27. CEP215-TAP cells were shown to display normal mitotic spindle morphology. For random integration of GFP-HSET and GFP-HSETN593K, 10 mg of linearized plasmid was electroporated into HSETKO cells using a gene pulser (Bio-Rad Laboratories) at 250 V and 950 mF. Cells were plated into three 96-well plates and selected by 1.5 mg ml 1 neomycin.

Drug-resistant colonies were selected and screened for the expression of GFP-tagged proteins. mRNA was isolated using RNAeasy minikit (Qiagen). One microgram of total RNA was reverse transcribed using Super Script II reverse transcriptase and used for PCR analysis.

Plasmid constructs and transfection in mammalian cells. For testing interactions in Fig. 2, different fragments of human HSET and CEP215 were cloned into pcDNA6-Bioease vectors using Gateway technology (Life Technologies). Primers used to clone into pDONR221 are listed in Supplementary Table 3. Positive clones from pDONR221 were exchanged into Bioease for afnity purications and MBP and GST for recombinant protein production in bacteria. Primers used to generate Flag-CEP215(DHBR) construct are listed in Supplementary Table 3. Flag-CEP215 (ref. 27) was used as template for Q5 site-directed mutagenesis kit (NEB) to introduce deletion of aa500700 (HBR) of CEP215. Stable cells expressing Flag, Flag-CEP215 and Flag-CEP215(DHBR), 1.5 106 N1E-115 cells were generated by

selecting transfected cells in 96-well plates containing 0.5 mg ml 1 Neomycin. After 1012 days, colonies were picked and screened for the expression of Flag-tagged proteins. HuSH pRS plasmids-encoding CEP215 shRNA or control shRNA (Origene technologies) were transfected in ecotropic Phoenix cells by the calcium

phosphate method, and viral supernatants were collected 48 h after transfection and were added to N1E-115 cells (1:1 ratio of carrier to target cells). Polybrene was added to 5 mg ml 1 and 72 h after infection of cells, depletion of CEP215 was assessed by immunoblotting. CEP215 (AM16708, Life Technologies) and HSET siRNAs (AM51331, Life Technologies) were transfected using Lipofectamine RNAiMax following manufacturers instructions. After 72 h of transfection, depletion of the respective proteins was assessed by immunoblotting. Patient B lymphocytes were isolated from blood of affected patient and parent and were immortalized by EBV transformation36.

Yeast two-hybrid assay. Yeast two-hybrid analysis was performed using Gateway-based yeast two-hybrid system. Briey, truncations of CEP215 and HSET were cloned into PDEST 32 (bait-GAL4 DNA binding domain) or PDEST22 (prey-DNA activation domain) vectors, transformed into yeast and analysed for growth in medium lacking histidine (SCTLH ) supplemented with 50 mM 3-aminotriazole

(3AT). Growth in SCTLH in the presence of 3AT indicates an interaction

between proteins that are fused to activation domain and binding domain.

Recombinant proteins and HSET antibody generation. Recombinant proteins of different truncations of human HSET and CEP215 were cloned using GST and MBP vectors using Gateway technology. Primers used are listed in Supplementary Table 3. Proteins were induced with 1 mM IPTG and puried using Glutathione Sepharose (GE Healthcare) or Amylose resin (NEB) as described earlier66. Antibodies were raised in rabbits against bacterially expressed and puried glutathione S-transferase fusion proteins that contained aa300673 of the human HSET protein. Antibodies were produced by Eurogentec and were subsequently afnity puried against fusion proteins for use in western blotting in chicken. Additional afnity purication against aa625673 of HSET was carried out for use in immunostainings in chicken.

Surface plasmon resonance. The binding of the HSET truncations to MBP-tagged CEP215 truncations was determined using the SPR-based biosensor BiacoreT200 (Biacore). Experiments were performed in 10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% (v/v) Tween-20 at 25 C. About 1000 RUs of each of the MBP-CEP215 truncations was immobilized on test ow cells (Fc-3, Fc-4) of a CM5 sensor chip using amine-coupling chemistry and non-immobilized ow cell (Fc-1) served as the control ow cell and (Fc-2) was MBP protein alone. One micromolar of each of the truncations was own over the chip at 30 ml min 1 for 120 s and dissociation was followed for an additional 180 s. The chip was regenerated by injecting brief pulses of 0.2 M sodium carbonate, pH 9.5. Data obtained for the control ow cell were subtracted from those obtained for test ow cell and binding evaluated using BIAevaluation software.

Antibodies and immunostainings. Primary antibodies used in this studywere CEP215 (ref. 27, 1:700 or Bethyl laboratories A300554A 1:500), FLAG (Cell Signaling #2368 1:1000 or Sigma-Aldrich F3165 1:2000); HSET (Bethyl laboratories A300-952A 1:1000 or our own 1:500); centrin-1 (Sigma-Aldrich C7736 1:500); centrin-2 (Biolegend poly6288 1:300); centrin-3 (Abnova H00001070-M01 1:500); Streptavidin HRP (Cell Signaling #3999 1:1000); PCM1 (Abcam ab154142 1:1000); PLK1 (BD biosciences #558446 1:1000); CEP63 (ref. 36), a-tubulin (Dm1a

T9026 1:1000 or Dm1a-FITC F2168 1:500 both Sigma-Aldrich); g-tubulin (GTU88; Sigma-Aldrich 1:1000); dynein intermediate chain (DIC; Abcam ab23905 1:1000) and p150 dynactin (BD Biosciences 610473 1:2000). DNA was stained with Hoechst 33258 (Sigma-Aldrich). DT40 and B cells were processed as described in (ref. 36).

Afnity purication and immunoprecipitation. For afnity purication of CEP215-TAP complexes, 2 109 cells were pelleted and lysed in 5 ml of lysis buffer

containing 10 mM Tris-HCl (pH8), 100 mM KCl, 1.5 mM MgCl2, 0.5% Triton-X 100, 5% Glycerol and 10 mM b-mercaptoethanol supplemented with protease inhibitor cocktail (Sigma-Aldrich). Cleared whole-cell extracts were obtained by centrifuging cell lysates at 16,000g for 15 min at 4 C and incubated with 200 ml of

Streptavidin Dynabeads. After 3 washes with lysis buffer containing 0.2% Triton-X-100 and 3 washes with 25 mM ammonium bicarbonate, samples were subjected to tryptic digestion and mass spectrometry or western blot analysis. Lysates prepared as above were subjected to immunoprecipitation with Dynabead coupled CEP215 antibody for 4 h as described66 and processed for western blotting.

Western blotting and intensity measurements. Whole-cell extracts for western blotting were prepared by lysing cells in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail). Lysates were separated on 38% Tris-acetate or 412% Bis-Tris SDSpolyacrylamide gel electrophoresis gels (Life Technologies) and transferred onto nitrocellulose for western blot analysis. Image J was used to quantify signal intensities normalized against appropriate loading controls. Full scans of western blots are included in Supplementary Figs 1117.

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Sucrose gradient ultracentrifugation and gel ltration. CEP215-TAP-tagged complexes were puried as described before from 5 109 cells and eluted in 800 ml

of 2 mM Biotin. This was layered onto a 5 to 40% (w/v) continuous sucrose gradient prepared in the lysis buffer minus detergent (900 ml each) and layered onto a 60% w/w cushion. The complexes were then loaded on the gradient and subjected to ultracentrifugation using SW40Ti rotor at 46,600g for 16 h at 4 C. Three hundred microlitre fractions were collected from bottom and TCA-precipitated before being subjected to western blot analyses. Cytoplasmic extracts preparedas above were subjected to gel ltration analysis on a Superose 6 10/300 GL(GE healthcare). High molecular weight kit (Sigma-Aldrich) was used to calibrate the column before analysing samples. Fractions of 250 ml were collected and subjected to Streptavidin afnity purication followed by western blot analysis.

Centrosome isolation and microtubule nucleation. Centrosomes from the indicated cells were isolated as described38. Briey, cells were treated with1 mg ml 1 cytochalasin D and 3.3 mM nocodazole for 1 h before harvesting. A total of 2 108 cells were lysed in hypotonic lysis buffer (1 mM Tris pH 8.0, 0.1%

b-mercaptoethanol (freshly added before use), 0.5% NP-40, 0.5 mM MgCl2, 150 ml of 20,000 U DNaseI), and centrifuged through a 2 ml 50% w/w sucrose cushion. The cushion-lysate interface (4 ml) was further subjected to a discontinuous gradient sucrose centrifugation (70, 50 and 40% w/w sucrose). Isolated centrosomes from each of the fractions were pelleted through 10 mM PIPES and subjected to western blot analysis. For microtubule nucleation assays, the peak centrosome fractions were pooled and 5 ml of this was added to 20 ml of Xenopus egg extracts and incubated for 10 min and xed by adding 500 ml of aster xation solution (BRB80, 10% glycerol, 0.25% glutaraldehyde and 0.1% Triton X-100). This was layered onto a 40% glycerol cushion and centrifuged onto coverslips. The asters were visualized by staining for DM1a-FITC.

Image acquisition, processing and analysis. Imaging of xed cells was performed on Nikon Eclipse A1 Ti-E scanning confocal microscope or Leica IR confocal microscope. Images shown here represent 3D projections of z-sections taken every 0.3 mm across the cell. Images represented as a single experiment were acquired using the same settings and were imported into Volocity (6.3; PerkinElmer) and Photoshop (CS6; Adobe) and were adjusted to use the full range of pixel intensities. Super-resolution microscopy was carried out using a Structured Illumination Microscope (SIM) by API OMX Deltavision. Cells were imaged with 100 1.4 numerical aperture Olympus objective. Data was reconstructed using

API SoftWorx software. For time-lapse imaging of DT40 cells expressing GFP-EB3 cells were settled onto concanavalin A-coated glass bottom dishes (Mat Tek). Cells were kept at 40 C in a humidied incubation chamber (Tokai) with 5% CO2 and were imaged using a spinning-disc confocal system (PerkinElmer) equipped with an electron microscopy charge-coupled device digital camera (C9100-13; Hamamatsu Photonics mounted on an inverted microscope (Eclipse TE2000-S; Nikon). Imaging was carried out with a frame rate of 5 min with z-steps of 1.5 mm using

Volocity 2D. N1E-115 and BT-549 cells were seeded into Ibidi 8 well chamber dish and imaging was conducted every 5 min in a humidied chamber with 37 C and 5% CO2, using a Nikon Eclipse TE2000-E microscope, and analysed with

NIS-Elements software (Nikon).

HSET levels at centrosomes were determined by measuring mean uorescence intensity of HSET in g-tubulin-positive volumes of mitotic cells. Volumes were selected in an automated fashion by applying appropriate intensity thresholding in Volocity 6.3 (PerkinElmer). Identical settings were used on all cells from one experiment regardless of genotype. In the dot plot each dot corresponds to a cell, because in each cell we averaged the mean uorescence intensity of HSET obtained from the two centrosomes.

For spindle angles, cells were selected in which the two spindle poles fell within1.8 mm in z (that is, maximum 6 z-steps). Based on intensity thresholding of a-tubulin staining, the centroids of opposite spindle poles were identied by Volocity 6.3 and connected by a line (providing the spindle axis) in an automated fashion. Maximum projections showing the spindle axis were exported into Adobe Illustrator, where position of each centrosome with respect to this axis was determined. For calculating centrosome distance from spindle poles, the centroid of centrosomes (g-tubulin staining) and the back edge of spindle poles (a-tubulin staining; longest axis points) were identied using intensity thresholding in Volocity 6.3. Coordinates of these points were exported to MATLAB, where distances between centrosome centroid and back edge of pole were calculated.

Microtubule pelleting assay. DT40 extracts from WT-TAP, CEP215(DHBR)-TAP and CEP215(DCM1)-TAP were lysed in a buffer containing 50 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 0.1 mM EGTA, and 0.5% Triton X-100, supplemented with protease and phosphatase inhibitor cocktails, and passed through a 26-Gauge needle 10 times. Extracts were precleared at 67,700g in an MLA130 rotor for 20 min at 4 C. After addition of 0.5 mM MgGTP and 2 mM MgATP, extracts were warmed to room temperature before sequential addition of 5 and 15 mM taxol.

Around 2 mg ml 1 of these extracts were mixed with taxol-stabilized micro-tutubules (0.2 mg ml 1) or nontaxol-treated tubulin (0.2 mg ml 1) and incubated at 30 C for 30 min before layering onto a 1 M sucrose cushion in BRB80 buffer (80 mM Pipes, pH 6.8, 1 mM MgCl2, and 1 mM EGTA) supplemented with 0.5 mM

ATP and with or without 10 mM taxol. Microtubules were pelleted at 67,700g in MLA130 rotor for 20 min at 22 C. Supernatants were saved for immunoblotting. Pellets were washed twice in BRB80 and re-suspended in 1 SDSPAGE loading

buffer to one fth of the volume of supernatant. Equal volume of pellets and supernatants were loaded on gel. In the case of MBP-CEP215 (1300), a total of 500 ng of dialyzed protein in PBS was incubated with microtubules or tubulin and pelleting carried out as before.

Cell viability assay. To assess cell survival following sh/siRNA, 1 105 cells were

seeded in a 48-well plate and subjected to shRNA/siRNA treatments. Six days post transfection, CellTiter-Glo substrate was added to cells as recommended by the manufacturer (Promega) and after 10 min of incubation transferred to standard opaque 96-well standard plate and luminescence assayed using PHERAStar.

NanoLCMS/MS analysis and data processing. Bead-bound proteins were digested by the addition of 10 ml trypsin solution 15 ng ml 1 (Roche) in 100 mM ammonium bicarbonate. The beads were then incubated at 37 C overnight. A second step digestion was performed the following day for 4 h. Sample tubes were placed on a magnetic rack and the supernatant solution was collected and acidied by the addition of 2 ml 5% formic acid. The samples were then cleaned using

Ultra-Micro C18 Spin Columns (Harvard Apparatus) prior to the mass spectrometry (MS) analysis according to manufacturers instructions. The liquid chromatographyMS (LCMS) analysis was performed on the Dionex Ultimate 3,000 UHPLC system coupled with the Orbitrap Velos mass spectrometer (Thermo Scientic). Digested peptides were re-suspended in 30 ml of 0.1% Formic acid for injection and a 5 ml volume was loaded on the Acclaim PepMap 100, 100 mm 2

cm C18, 5 mm, 100 trapping column with the mlPickUp Injection mode using the loading pump at 7 ml min 1 ow rate for 10 min. For the analytical separation the

Acclaim PepMap RSLC, 75 mm 25 cm, nanoViper, C18, 2 mm, 100 column

retrotted to the nanospray source was used for multi-step gradient elution. Solvent A was composed of 0.1% formic Acid, 2% MeCN and 5% DMSO with and solvent B was composed of 80% acetonitrile, 0.1% formic acid, 5% DMSO. The gradient elution method at ow rate 300 nl min 1 was as follows: for 60 min gradient up to 45% (B), for 10 min gradient up to 95% (B), for 10 min isocratic 95%

(B), for 5 min down to 5% (B), for 10 min isocratic equilibration 5% (B) at 40 C. Separated peptides were transferred to the gaseous phase with positive ion electrospray ionization applying a voltage of 2.0 kV. Targeted ions already selected for MS/MS were dynamically excluded for 40 s. Top 20 multiply charged precursor isotopic clusters with m z 1 value between 400 and 1,600 m z 1 were selected with FT mass resolution 60K and isolated for CID fragmentation within a mass window of 2.0 m z 1 and collision energy 28. The CID tandem mass spectra were processed using the SequestHT and Mascot search engines implemented on the

Proteome Discoverer software version 1.4 for peptide and protein identications. All spectra were searched against a UniProtKB/Swiss-Prot and UniProtKB/ TrEMBL fasta le. The Nodes for SequestHT and Mascot included the following parameters: Precursor Mass Tolerance 10 p.p.m., Fragment Mass Tolerance 0.5 Da, Dynamic Modications were Oxidation of M ( 15.995 Da) and Deamidation of

N, Q ( 0.984 Da). The level of condence for peptide identications was esti

mated using the Percolator node with decoy database search. FDRo1% was applied in all the experiments.

For network construction, we performed the following workow. We extracted a non-redundant list of interactors identied from pulldowns of CEP215-TAP cells that were present in at least 2 experiments. To minimize non-specic binders (that is, proteins that bind streptavidin beads or the TAP tag) we removed proteins that were represented even by a single peptide in pulldowns from untagged WT cells and other TAP-tagged cell lines generated in the group such as TAP-CEP63 and TAP-CEP135. Next, we used this dataset to screen for proteins represented by at least 4 unique peptides in 2 experiments (worksheet Filtered_2 in Supplementary Table 2). Finally, based on the Filtered_2 dataset, we shortlisted proteins present in all three experiments and included these in the worksheet called Filtered_3 in Supplementary Table 2. To represent our nal network, we shortlisted 23 proteins using GO analysis, excluding proteins limited to nucleus, spliceosome or membrane in their localization (Supplementary Table 1).

Sequence orthology detection and conservation analysis. Sequence orthologues of CEP215 and HSET were obtained from OMA orthology database and EnsemblCompara, which adopt complementary approaches for sequence orthology detection. We considered only one-to-one orthologues and disregarded any paralogues (gene-duplicates). OMA detects orthologues using an inference algorithm, which rst infers homologous sequences by performing all-against-all Smith-Waterman alignments between all sequences and retain signicant matches. Subsequently, orthologous pairs (the subset of homologues related by speciation events) were inferred using mutually closest homologues based on evolutionary distances, taking into account distance inference uncertainty and the possibility of hidden paralogy due to differential gene losses. On the other hand, Ensembl-Compara uses maximum likelihood phylogenetic gene trees obtained from the protein-based multiple alignments and reconciles them with established species tree and permits duplication calls on internal nodes. In addition, we have also included experimentally determined homologues of CEP215 (centrosomin/cnn in

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms11005 ARTICLE

fruity and Mto1 and pcp1 in ssion yeast)67,68 and HSET (Kar3 family protein pkl in ssion yeast)16. Sequence alignments for CEP215-HBR and HSET aa1150 were generated using MAFFT from EMBL-EBI web server. Pairwise sequence identity (in percentage) between human HSET aa1150 and orthologues was estimated using ClustalW server69.

Statistical analyses. Statistical analysis and graphs were carried out using Microscoft Excel or R. The numbers of experimental repeats or cells scored are reported in gures and gure legends. Data are presented as means.d. unless stated otherwise. Statistical test used for each experiment is stated in the legend.

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Acknowledgements

We thank Nimesh Joseph, S. Balaji, Magdalini Rapti for technical advice and members of the Gergely lab for helpful suggestions. We thank Isabelle Vernos for Xenopus egg extracts, Claire Walczak for GFP-HSET constructs and Jordan Raff for centrosomin antibodies, Eric Miska for GST/MBP vectors and David Glover for Dmel2 cells. We are grateful for the expert help provided by members of the Proteomics and Microscopy Core facilities of CRUK CI, especially Clive dSantos and Jeremy Pike. We also thank the families for their participation. S.C. is supported by UK Medical Research Council (MC_U105185859). This work was made possible by funding from Cancer Research UK

(C14303/A17197). We acknowledge the support of the University of Cambridge and Hutchison Whampoa Ltd.

Author contributions

P.L.C. and F.G. conceived the study. P.L.C. performed most experiments with contribution from G.C. A.R.B. and P.T. generated valuable tools. C.G.W. provided clinical material. C.T. and E.P. assisted with generation and analysis of proteomic data. S.C. performed bioinformatic analyses. P.L.C. and F.G. wrote the manuscript with comments from all authors.

Additional information

Accession codes: Proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identier PXD00338270.

Supplementary Information accompanies this paper at http://www.nature.com/naturecommunications

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Competing nancial interests: The authors declare no competing nancial interests.

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How to cite this article: Chavali, P. L. et al. A CEP215HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer. Nat. Commun. 7:11005 doi: 10.1038/ncomms11005 (2016).

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16 NATURE COMMUNICATIONS | 7:11005 | DOI: 10.1038/ncomms11005 | http://www.nature.com/naturecommunications

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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 centrosome 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.

Details

Title

A CEP215-HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer

Author

Chavali, Pavithra L; Chandrasekaran, Gayathri; Barr, Alexis R; Tátrai, Péter; Taylor, Chris; Papachristou, Evaggelia K; Woods, C Geoffrey; Chavali, Sreenivas; Gergely, Fanni

Pages

11005

Publication year

2016

Publication date

Mar 2016

Publisher

Nature Publishing Group

e-ISSN

20411723

Source type

Scholarly Journal

Language of publication

English

DOI

https://doi.org/10.1038/ncomms11005

ProQuest document ID

1774171527

A CEP215-HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer

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