Research Article 4321 Cep68 and Cep215 (Cdk5rap2) are required for cohesion

Susanne Graser1, York-Dieter Stierhof2 and Erich A. Nigg1,* 1Max-Planck-Institute for Biochemistry, Department of Cell Biology, Am Klopferspitz 18, D-82152 Martinsried, Germany 2Electron Microscopy Unit, Center for Plant Molecular Biology, University of Tübingen, Auf der Morgenstelle 5, D-72076 Tübingen, Germany *Author for correspondence (e-mail: [email protected])

Accepted 22 September 2007 Journal of Cell Science 120, 4321-4331 Published by The Company of Biologists 2007 doi:10.1242/jcs.020248

Summary The centrosome duplicates during the cell cycle but Cep68 is readily recruited to ectopic fibres. These functions as a single -organising centre until data suggest that Cep68 cooperates with rootletin and C- shortly before mitosis. This raises the question of how Nap1 in centrosome cohesion. By contrast, Cep215 centrosome cohesion is maintained throughout interphase. associates with throughout the cell cycle and One dynamic model proposes that parental are does not appear to interact with Cep68, rootletin or C- held together through -associated, entangling Nap1. Instead, our data suggest that Cep215 functionally filaments. Central to this model are C-Nap1, a putative interacts with pericentrin, suggesting that both centriolar docking and rootletin, a fibrous influence centrosome cohesion through an indirect component. Here we identify two novel proteins, Cep68 and mechanism related to cytoskeletal dynamics. Cep215, as required for centrosome cohesion. Similar to rootletin, Cep68 decorates fibres emanating from the proximal ends of centrioles and dissociates from Supplementary material available online at centrosomes during mitosis. Furthermore, Cep68 and http://jcs.biologists.org/cgi/content/full/120/24/4321/DC1 rootletin depend both on each other and on C-Nap1 for centriole association. Unlike rootletin, overexpression of Key words: Cep68, Cep215, Centrosome cohesion, Rootletin, Cell Cep68 does not induce extensive fibre formation, but cycle

Introduction (Bettencourt-Dias and Glover, 2007; Nigg, 2007). As a result The centrosome is the major microtubule-organising centre of this duplication, a G2 cell harbours two centrosomes, each

Journal of Cell Science (MTOC) of animal cells (Bornens, 2002; Doxsey, 2001; Luders comprising two closely associated centrioles. Remarkably, the and Stearns, 2007; Nigg, 2004). The single centrosome present attachment between parent and progeny centriole within each in a mammalian G1-phase cell comprises two centrioles centrosome – termed ‘engagement’– persists throughout G2 surrounded by a fibrous protein matrix, the so-called and early M phase, until the two centrioles separate at the end (PCM). The PCM is responsible for the of mitosis. This ‘disengagement’ is then thought to constitute nucleation and anchoring of (MTs) (Bornens, a necessary licensing step for a further round of duplication 2002; Job et al., 2003). It comprises several large coiled-coil (Tsou and Stearns, 2006a; Tsou and Stearns, 2006b). proteins (Doxsey et al., 2005; Ou et al., 2002) and provides At the onset of mitosis, the activation of several protein docking sites for ␥- ring complexes (Luders and kinases triggers centrosome separation, leading to the Stearns, 2007) as well as cell-cycle-regulatory proteins formation of a bipolar spindle (Berdnik and Knoblich, 2002; (Doxsey, 2001; Fry and Hames, 2004). The two centrioles Blangy et al., 1995; Fry et al., 1998a; Giet et al., 1999; Glover present within a G1 centrosome are structurally and et al., 1995; Hannak et al., 2001; Lane and Nigg, 1996; Sawin functionally distinct. Only the mature centriole carries and Mitchison, 1995). However, before late G2 phase, the two appendages at its distal end and is competent to function as a centrosomes function as a single MTOC. This raises the basal body in ciliogenesis (Sorokin, 1968). It is also this mature question of how this ‘centrosome cohesion’ is maintained centriole that anchors the bulk of the MTs, whereas the other throughout the major part of interphase. Two prominent centriole is relatively free to move (Piel et al., 2000). The models for centrosome cohesion have been proposed. The first average distance between these two centrioles is variable, cell- model emphasises the role of cytoskeletal dynamics and type dependent and influenced by cytoskeletal dynamics and proposes that the close proximity between parental centrioles physiological conditions (Buendia et al., 1990; Euteneuer and is primarily a consequence of forces exerted by the Schliwa, 1985; Holy et al., 1997; Jean et al., 1999; Rodionov cytoskeleton (Euteneuer and Schliwa, 1985; Jean et al., 1999; and Borisy, 1997; Schliwa et al., 1983; Schliwa et al., 1982; Thompson et al., 2004). The second model postulates the Sherline and Mascardo, 1982). existence of dedicated ‘linker proteins’ that function to provide During S phase, a single new centriole forms at an a dynamic connection between parental centrioles. Clearly, the orthogonal angle close to the proximal end of each parental two models are not mutually exclusive. Importantly, the centriole, suggesting that the wall of the parental centriole influence of cytoskeletal alterations on centrosome cohesion is favours the formation of exactly one progeny centriole not necessarily independent of linker structures, because 4322 Journal of Cell Science 120 (24)

interference with the cytoskeleton may well affect the transport proteins previously implicated in centrosome cohesion, namely of structural or regulatory components that are important for C-Nap1, rootletin and pericentrin (Bahe et al., 2005; Jurczyk the functionality of the putative linker (Meraldi and Nigg, et al., 2004; Mayor et al., 2000; Yang et al., 2006). Centrosome 2001). One major attraction of the ‘dynamic linker model’ is splitting in response to depletion of the above proteins was also that it readily explains how cell-cycle-dependent observed in two other cell lines, A549 and hTERT-RPE1 phosphorylation can regulate centrosome cohesion (reviewed (RPE1), although effects were quantitatively reduced in RPE1, by Meraldi and Nigg, 2001). The linker model is further presumably because of lower transfection efficiency (data not supported by electron microscopy (Bornens et al., 1987; shown). The above results indicate that splitting is not an Paintrand et al., 1992), and the identification of two specific inevitable consequence of interference with overall centrosome proteins, C-Nap1 (Fry et al., 1998a; Mayor et al., 2000) and structure. Yet, the available evidence indicates that centrosome rootletin (Bahe et al., 2005; Yang et al., 2006), that are required cohesion is affected through multiple mechanisms and its for the maintenance of centrosome cohesion. Remarkably, both regulation is likely to be complex (Meraldi and Nigg, 2001). proteins dissociate from the centrosome when the activity of In this regard, two observations made during the above screen the protein kinase Nek2 exceeds that of a counteracting type 1 are noteworthy. First, depletion of caused centrosome phosphatase, concomitant with centrosome separation at the splitting only in RPE1 cells, and depletion of Cep164 caused G2-to-M transition (Bahe et al., 2005; Fry et al., 1998b; Helps splitting only in serum-deprived RPE1 cells (data not shown). et al., 2000; Yang et al., 2006). Additional proteins, notably This confirms that the strength of centrosome cohesion differs pericentrin (Jurczyk et al., 2004) and dynamin-2 (Thompson et between cell types and growth conditions. Second, a al., 2004), have also been implicated in centrosome cohesion, quantitative analysis of centrosome splitting revealed that, of but these latter proteins are known to play multiple roles. all proteins examined so far, depletion of C-Nap1 and rootletin Depletion of all the above proteins causes centrosome splitting produced the most drastic phenotype, both with regard to but whether they function through a common mechanism is not penetrance – the percentage of cells showing split centrosomes presently clear. (supplementary material Fig. S1) – and the extent of separation We emphasise that the term ‘centrosome splitting’ is used between centrioles (see below). here to describe the separation of parental centrioles. From a mechanistic perspective, this event is expected to be intimately Characterisation of Cep68 and Cep215 related to centrosome separation, which occurs under The two proteins newly identified here as being involved in physiological conditions at the G2-to-M transition. It is centrosome cohesion, Cep68 and Cep215, were originally important to distinguish centrosome splitting from centriole described in the course of a proteomic characterisation of the disengagement (which is occasionally also called ‘centriole human centrosome (Andersen et al., 2003). Orthologues of splitting’), a term that describes the separation of parent and both proteins are readily detectable in other mammalian progeny centrioles (within the same centrosome) that normally genomes. Cep68 is encoded on human 2p14 and occurs at the end of mitosis (see Nigg, 2006). alternative splicing is thought to give rise to two isoforms In this study, we used a siRNA screen to search for proteins comprising 757 and 620 amino acids, respectively (Fig. 1A).

Journal of Cell Science involved in centrosome cohesion. In addition to confirming Here, these isoforms are distinguished by using the suffix L or the roles of C-Nap1, rootletin and pericentrin, this screen S (referring to long and short isoforms, respectively). Both identified only two additional proteins, Cep68 and Cep215, isoforms are predicted to share a globular domain at the C- whose depletion triggered substantial centrosome splitting. terminus (Fig. 1A). Cep215 is also known as Cdk5rap2 (Cdk5 This indicates that centrosome splitting is not a common regulatory subunit associated protein 2) and has attracted consequence of interference with centrosome structure but considerable interest because of its implication in the control rather reflects the impairment of specific protein functions. of brain size. In fact, Cep215/Cdk5rap2 is encoded on human Both Cep68 and Cep215 localised to centrosomes, but chromosome 9q33.2 and homozygous mutations in this differed in their precise localisations during both interphase have been linked to primary microcephaly (Bond et al., 2005). and mitosis. Our functional and cytological studies indicate As in the case of Cep68, alternative splicing is known to that Cep68 contributes to a dynamic linker structure, whereas produce two isoforms, but these differ only in one relatively the role of Cep215 in centrosome cohesion is likely to be small region (Fig. 1B). Sequence analysis using the Pfam more indirect, possibly related to centrosome-MT database (Finn et al., 2006) predicts several coiled-coil interactions. domains as well as an N-terminal ‘microtubule association region’ in both isoforms. The latter region is conserved in Results and Drosophila centrosomin, but its functional siRNA screen to identify proteins involved in centrosome significance is not presently known. cohesion Rabbit antibodies were raised against both Cep68 (residues To search for proteins that might play a role in centrosome 1-497) and Cep215 (residues 503-1010) expressed in E. coli. cohesion, we performed a siRNA screen focused on In western blots performed on total lysates of HeLaS3, U2OS centrosomal proteins (supplementary material Table S1) and and 293T cells anti-Cep68 antibodies recognised two bands of monitored centrosome splitting in U2OS cells. Centrosomes ~90 and 67 kDa, respectively, in good agreement with the were counted as split whenever the distance between the two predicted sizes of the two Cep68 isoforms (81 and 67 kDa for parental centrioles was >2 ␮m. Of 38 proteins tested, depletion Cep68L and -S, respectively), whereas pre-immune serum of only five proteins resulted in substantial centrosome showed no specific bands (Fig. 1C, left panels). Furthermore, splitting (supplementary material Fig. S1, Table S1). These ectopic expression of Cep68L in 293T cells enhanced the include two new proteins Cep68 and Cep215, as well as three expected signal, giving confidence that the 90 kDa band Novel proteins for centrosome cohesion 4323

Fig. 1. Characterisation of anti-Cep68 and anti-Cep215 antibodies. (A) Primary structure of human Cep68L/S. Note that Cep68L comprises a 137 aa insertion compared with Cep68S (at residue 492) and both isoforms are predicted to form a C- terminal globular domain (at aa 618-757 in Cep68L and at aa 454-620 in Cep68S; grey). The horizontal line (antigen) denotes the region used for antibody production. The sequence data for Cep68 are available from Ensembl [accession no. ENST00000377990 (Cep68L) and ENST00000260569 (Cep68S)]. (B) Primary structure of human Cep215. Two isoforms with 1893 and 1814 residues, respectively, have been described; they are identical between residues 1 and 1396, followed by non-identical stretches of 259 (isoform 1) and 180 residues (isoform 2), and identical C-termini (starting at residue 1655 in isoform 1 and 1576 in isoform 2). Both isoforms are predicted to contain several coiled-coil regions (black, CC) and a microtubule association region (grey). The horizontal line (antigen) denotes the region used for antibody production. The sequence data for Cep215 cDNAs are available from NCBI [accession no. NM_018249 (isoform 1) and NM_001011649 (isoform 2)]. (C) Antibodies directed against Cep68 (R170) and corresponding pre-immune serum were tested on western blots of total cell lysates of HeLaS3, U2OS and 293T cells (left) and centrosome preparations from KE37 cells (centre). In the latter case, proteins show a retarded electrophoretic Journal of Cell Science mobility because of the high sucrose concentration present in centrosome preparations. Arrowheads indicate Cep68L and -S. Right, western blot showing efficient depletion of Cep68 from U2OS cells by 48 hours siRNA, using oligonucleotide duplexes 262 or 263. (D) Antibodies directed against Cep215 (R174) and corresponding pre-immune serum were tested on western blots of total cell lysates of HeLaS3, U2OS and 293T cells (left) and centrosome preparations from KE37 cells (centre). In the latter case, proteins show a retarded electrophoretic mobility because of the high sucrose concentration present in centrosome preparations. Arrowheads indicate Cep215. Right, western blot showing efficient depletion of Cep215 from U2OS cells by 72 hour siRNA, using oligonucleotide duplexes 282 or 283. GL2 treatment is used for control and blotting for ␣-tubulin illustrates equal loading.

represents Cep68L (data not shown). Similarly, anti-Cep215 Endogenous Cep68 and Cep215 could readily be visualised antibodies recognised a band of the expected size, ~210 kDa, at centrosomes by immunofluorescence microscopy (Fig. 2), on western blots of HeLaS3, U2OS and 293T cells, whereas confirming and extending previous data (Andersen et al., 2003; pre-immune serum showed no specific bands (Fig. 1D, left Bond et al., 2005). Attesting to staining specificity, the panels). Because the two isoforms of Cep215 are expected to corresponding pre-immune sera did not produce any specific display very similar migration behaviour, these data do not signals and siRNA-mediated depletion of the antigens provide information about isoform expression. Anti-Cep68 essentially abolished the staining (Fig. 2). Furthermore, and anti-Cep215 antibodies also detected their antigens in siRNA-mediated depletion of either Cep68 or Cep215 caused isolated centrosomes, purified from human KE37 T- centrosome splitting (Fig. 2A,B, compare ␥-tubulin staining in lymphoblastoid cells (Fig. 1C,D, central panels). In the case bottom panels with upper panels), confirming the results of the of Cep68, the larger version was predominant, indicating that siRNA screen and the role of these proteins in centrosome KE37 cells predominantly express the Cep68L isoform. cohesion. Although Cep68 and Cep215 both associate with Finally, antibody specificity was confirmed by siRNA. centrosomes, they clearly display distinct localisations. Cep68 Transfection of U2OS cells with siRNA oligonucleotides localised to thin fibres protruding away from the two centrioles targeting either Cep68 (for 48 hours) or Cep215 (for 72 hours), (Fig. 2A), highly reminiscent of rootletin (Bahe et al., 2005; using two different duplexes to target each protein, resulted in Yang et al., 2006; Yang et al., 2002). By contrast, Cep215 the (near-)complete loss of Cep68 or Ce215, respectively (Fig. displayed a rather compact localisation at the centrosome (Fig. 1C,D, right panels). 2B). The two proteins also displayed distinct behaviours during 4324 Journal of Cell Science 120 (24)

Fig. 2. Distinct associations of Cep68 and Cep215 with centrosomes. Antibodies directed against Cep68 (A) or Cep215 (B) and corresponding pre-immune sera were used for immunofluorescence on U2OS cells. The antibodies (green) recognise the centrosome as indicated by colocalisation with ␥-tubulin (red). Insets show enlarged areas to better visualise the centrosomes; note filamentous staining produced by anti- Cep68 antibody and compact staining produced by anti-Cep215 antibody. Pre-immune sera did not show any specific staining (top panels) and 48 hour siRNA treatment using duplex 262 targeting Cep68 or duplex 283 targeting Cep215 abolished Cep68 (A, bottom) and Cep215 signals (B, bottom), respectively. Note the centrosome splitting induced by siRNA-mediated depletion of either Cep68 or Cep215. Bars, 10 ␮m.

cell cycle progression. Whereas Cep68 staining was the centriolar cylinders, often embedding them and progressively reduced during prophase, resulting in Cep68 occasionally joining them (Fig. 4B,C, lower panels). being undetectable on mitotic spindle poles (Fig. 3A), Cep215 clearly persisted on centrosomes throughout mitosis, in Interdependency of proteins implicated in centrosome agreement with previous data (Bond et al., 2005). We cannot cohesion rigorously exclude the fact that the disappearance of Cep68 To explore the functions of Cep68 and Cep215 in centrosome from mitotic spindle poles might reflect epitope masking, but cohesion, we used siRNA to determine to what extent these

Journal of Cell Science emphasise that a similar cell-cycle-regulated displacement proteins were required for centrosomal localisation of other from mitotic centrosomes has previously been documented for proteins implicated in centrosome cohesion. When compared C-Nap1 and rootletin (Bahe et al., 2005; Fry et al., 1998a; with GL2-treated control cells, where all proteins showed the Mayor et al., 2002; Mayor et al., 2000; Yang et al., 2006; Yang expected centrosomal localisation (supplementary material Fig. et al., 2002), two proteins with which Cep68 interacts S2), depletion of Cep68 mostly abolished rootletin staining, but functionally (see below). did not produce any significant effects on the localisation of Cep215, C-Nap1 or pericentrin (Fig. 5A and Table 1). Depletion Subcellular localisation of Cep68 and Cep215 at high of Cep215, on the other hand, exerted at most a marginal resolution influence on any of the other proteins examined (Fig. 5B and To corroborate the above results, we carried out both high- Table 1). In reciprocal experiments, we examined the fate of resolution fluorescence microscopy, using a Deltavision Cep68 and Cep215 in response to depletion of rootletin, C-Nap1 deconvolution instrument, and pre-embedding immuno- or pericentrin. Depletion of rootletin caused a loss of Cep68 from electron microscopy (immuno-EM). Both approaches centrosomes but did not detectably influence either Cep215 or confirmed that Cep68 localises to striking fibres originating any of the other cohesion proteins [Fig. 6A and Table 1; for C- from centrioles (Fig. 4A; Fig. 4C, upper panel). These fibres Nap1 see Bahe et al. (Bahe et al., 2005)]. Following C-Nap1 were clearly associated with the proximal ends of centrioles, depletion, Cep68 staining at centrosomes was mostly abolished, as inferred from EM images revealing the position of whereas the localisation of the other proteins was largely appendages (Fig. 4C; upper panel; arrowhead) and/or the unaltered [Fig. 6B and Table 1; for rootletin see Bahe et al. (Bahe presence of nascent pro-centrioles next to Cep68 fibres et al., 2005)]. Finally, depletion of pericentrin caused an almost (arrow). In general, several (most often two to four) Cep68- complete loss of Cep215 from centrosomes, a detectable positive fibres emanated from individual centrioles and the reduction in centrosomal levels of Cep68 and rootletin, but no length of some fibres exceeded 0.5 ␮m. Control experiments significant effect on C-Nap1 (Fig. 6C and Table 1). Taken (without primary antibody) showed background labelling but together, these results point to functional (and perhaps no staining of fibres (Fig. 4C, right panels). Clearly, the molecular) interactions between (1) Cep68 and rootletin and (2) localisation of Cep68 is remarkably similar to that reported Cep215 and pericentrin. previously for rootletin (Bahe et al., 2005; Yang et al., 2006; Next, we performed overexpression experiments to assess Yang et al., 2002). By contrast, Cep215 was associated with the ability of the above proteins to colocalise in vivo. In Novel proteins for centrosome cohesion 4325 Journal of Cell Science

Fig. 3. Cell cycle analysis of centrosome localisation of Cep68 and Cep215. Asynchronously growing U2OS cells were co-stained with antibodies against ␥-tubulin (central panels) and either Cep68 (A; upper panels) or Cep215 (B; upper panels). Lower panels show merged images including DNA staining by DAPI (Cep68 or Cep215 in green; ␥-tubulin in red). Bars, 10 ␮m.

particular, we exploited the previous observation that excess protein could be seen throughout the cytoplasm (Fig. overexpression of GFP-rootletin causes the extensive 7B). Interestingly, endogenous Cep68, but not Cep215, was formation of both centrosome-associated and cytoplasmic also recruited to conspicuous filaments that formed when Myc- filaments (Bahe et al., 2005; Yang et al., 2002). When Myc- rootletin was expressed ectopically (Fig. 7C). These results Cep68 was coexpressed with GFP-rootletin, extensive demonstrate that although Cep68 is not able to form fibres on colocalisation could be seen, whereas Myc-Cep215 showed no its own, it is readily recruited to rootletin fibres. Thus, Cep68, significant association with filamentous structures (Fig. 7A). but not Cep215, is able to interact with rootletin in vivo. This When expressed alone, Myc-Cep68 associated with suggests that Cep68 cooperates with C-Nap1 and rootletin in centrosomes, but did not produce any filaments, even though forming a dynamic linker structure, whereas Cep215 is likely 4326 Journal of Cell Science 120 (24) Journal of Cell Science

Fig. 4. High-resolution analysis of Cep68 and Cep215 localisation. (A,B) U2OS cells were co-stained with antibodies against ␥-tubulin (middle columns) and either Cep68 (A; left column) or Cep215 (B; left column) and examined using deconvolution on a Deltavision instrument. Right columns show merged images with Cep68 or Cep215 in green and ␥-tubulin in red. Bars, 2 ␮m. (C) U2OS cells were subjected to pre- embedding immuno-gold labelling EM. Cells were labelled with anti-Cep68 (top left) or anti-Cep215 (bottom left) antibodies, followed by nanogold-coupled secondary antibody. Controls (right) show centrioles stained with secondary antibody only. Note that Cep68 decorates striking fibres protruding away from the proximal ends of centrioles; proximal ends are identified by the proximity of fibres to nascent pro- centrioles (white arrow) and their distance to distal appendages (white arrowhead). Bars, 250 nm.

Table 1. Dependency of centrosome localization on siRNA-mediated protein depletion siRNA Cep68 Cep215 Rootletin C-Nap1 Pericentrin Cep68 Good depletion – Displaced from centrosome – – Cep215 – Some residual protein – – – Rootletin Displaced from centrosome – Good depletion – – C-Nap1 Displaced from centrosome* – Displaced from centrosome* Some residual protein – Pericentrin Diminished Strongly reduced Diminished – Good depletion

–, no significant effect on localization. *Depending on the extent of C-Nap1 depletion, Cep68 and rootletin were either displaced from the centrosome or formed fewer and longer filaments at centrioles, exactly as reported previously (Bahe et al., 2005). Novel proteins for centrosome cohesion 4327

Fig. 5. Effect of Cep68 and Cep215 depletion on other cohesion proteins. U2OS cells were transfected for 48 hours with siRNA duplexes

Journal of Cell Science specific for Cep68 (A) or Cep215 (B) and then co-stained with the antibodies indicated (left columns) and antibodies against ␥-tubulin (centre columns). Columns on the right show merged images with DAPI staining of DNA in blue, ␥-tubulin in red and the various proteins in green. Bars, 10 ␮m.

to affect centrosome cohesion through a distinct, more indirect from the proximal ends of parental centrioles. Depletion of mechanism. Cep68 caused a loss of rootletin from centrioles and vice versa, and depletion of C-Nap1 often caused a loss of both Cep68 and Discussion rootletin. Moreover, as described previously for C-Nap1 (Fry Here, we have performed a siRNA screen (encompassing 38 et al., 1998a; Mayor et al., 2002) and rootletin (Bahe et al., proteins) to search for centrosomal proteins that play a role in 2005; Yang et al., 2006; Yang et al., 2002), Cep68 was centrosome cohesion. Under the conditions of this assay, the displaced from centrosomes at the onset of mitosis and absent depletion of only five proteins (including three previously from mitotic spindle poles, consistent with the notion that a known ones) caused robust centrosome splitting. This is an linker structure needs to be dismantled for centrosome important finding because it indicates that centrosome splitting separation at the onset of mitosis. In future studies, it will thus is not a common consequence of interfering with centrosome be interesting to explore whether, similarly to C-Nap1 and integrity. Instead, our data indicate that only few proteins are rootletin, Cep68 is also regulated by an antagonism between critically required for centrosome cohesion, consistent with the Nek2 kinase and a type 1 phosphatase (Bahe et al., 2005; Fry existence of specific cohesion structures. Because the extent of et al., 1998b; Helps et al., 2000; Yang et al., 2006). centrosome cohesion depends on cell type and physiological Overexpression of Cep68 in U2OS cells did not induce the conditions (Euteneuer and Schliwa, 1985; Jean et al., 1999; formation of extended fibres, suggesting that Cep68 is not able Meraldi and Nigg, 2001), we would expect that additional to form polymers on its own. However, Cep68 was readily proteins will be found to affect centrosome cohesion under recruited to fibrous structures formed by overexpressed appropriate circumstances. rootletin. It is possible that the two proteins co-polymerise, but The two proteins newly identified here as being required for considering that only rootletin (but not Cep68) comprises coiled- centrosome cohesion, Cep68 and Cep215, were studied further. coil domains, it is perhaps more likely that Cep68 stabilises Like rootletin, endogenous Cep68 localises to fibres emanating rootletin homopolymers. Taken together, our data strongly 4328 Journal of Cell Science 120 (24)

Fig. 6. Mutual dependencies of cohesion-regulatory centrosomal proteins. U2OS cells were transfected for 48 hours with siRNA duplexes specific for rootletin (A), C-Nap1 (B) or pericentrin (C) and then co-stained with the antibodies indicated (left columns) and antibodies against ␥-tubulin (centre columns). Columns on the right show merged images with DAPI staining of DNA in blue, ␥-tubulin in red and the various proteins in green. Bars, 10 ␮m.

indicate that Cep68 cooperates with C-Nap1 and rootletin in the Cep215 is of considerable medical interest because

Journal of Cell Science formation of a dynamic linker structure connecting parental mutations in the corresponding gene are linked to autosomal centrioles. So far, no conclusive biochemical evidence could be recessive primary microcephaly (MCPH) (Bond et al., 2005). obtained for a molecular interaction between Cep68 and either Whether the pathology resulting from mutations in Cep215 rootletin or C-Nap1, presumably because of the small amounts relates to the function of this protein in centrosome cohesion of the endogenous proteins and antibody-related limitations. will require further study. However, it is intriguing that Cep68 Thus, it will be interesting to examine the expression and might also potentially be related to human disease. The localisation of Cep68 in ciliated cells that express higher levels corresponding gene in fact maps to a locus on chromosome of rootletin and harbour prominent striated fibrous networks 2p14 that is frequently mutated in retinitis pigmentosa (Kumar known as ciliary rootlets. Moreover, studies involving et al., 2004). Specifically, haplotype analysis of an Indian recombinant proteins will be required to explore whether Cep68 family identified Cep68 (KIAA0582) as one of only 14 interacts with rootletin directly or indirectly. expressed in the eye or retina that map to a critical 1.06 cM In contrast to Cep68, we obtained no evidence for a direct region (Kumar et al., 2004). Thus, it may be rewarding to functional interaction between Cep215 and any of the proteins further explore the possibility that Cep68, much like Cep215, implicated in forming a dedicated linker structure. Cep215 is implicated in human disease. showed no obvious fibre localisation and no mutual In conclusion, the data reported here support the view that dependency with C-Nap1, rootletin or Cep68. Furthermore, centrosome cohesion is determined through both direct and Cep215 persisted at the centrosome throughout mitosis, indirect mechanisms. A prominent direct mechanism is likely making it unlikely that it is part of a cell-cycle-regulated linker to involve entangling filaments and requires the proteins C- structure. In all these aspects, Cep215 more closely resembles Nap1 (Fry et al., 1998a; Mayor et al., 2000), rootletin (Bahe et pericentrin, the one other protein implicated in centrosome al., 2005; Yang et al., 2006) and, as shown here, Cep68. All cohesion in this study as well as previously (Jurczyk et al., three proteins show not only interdependencies in their 2004). Interestingly, pericentrin depletion caused not only localisations but also a common cell-cycle-regulated centrosome splitting but also a strong reduction of Cep215 association with the centrosome (Bahe et al., 2005; Mayor et levels at the centrosome, suggesting that displacement of al., 2000; Yang et al., 2006; Yang et al., 2002) (this study). By Cep215 contributes to explain this phenotype. How exactly contrast, pericentrin and Cep215 appear to function primarily Cep215 and pericentrin contribute to centrosome cohesion in a distinct context. Both proteins remain associated with remains to be clarified. spindle poles throughout mitosis and they do not appear to Novel proteins for centrosome cohesion 4329

view of the reported functions of pericentrin (Dictenberg et al., 1998; Jurczyk et al., 2004; Miyoshi et al., 2006; Takahashi et al., 2002), it seems likely to relate to centrosome architecture and positioning, microtubule organisation and/or intracellular transport.

Materials and Methods Plasmid preparation and recombinant proteins Polymerase chain reaction was used to amplify full-length human Cep68 from KIAA0582 clone (from Kazusa DNA Research Institute, Kisarazu, Japan). The cDNA was then subcloned into a mammalian expression vector providing a C- terminal Myc-tag. The Cep215 cDNA sequence was obtained from KIAA1633 clone (from Kazusa DNA Research Institute). Using PCR, a frameshift within the sequence was corrected and missing 5Ј coding information was obtained by PCR amplification from Marathon cDNA library (Clontech). Constructs were fused to yield the complete coding sequence of Cep215, and subcloned into a mammalian expression vector providing a C-terminal Myc-tag. All constructs were confirmed by sequencing. For expression of recombinant protein fragments, bp 1-1491 of Cep68 and bp 1508-3030 of Cep215 were PCR amplified, inserted into the expression vector pGEX-5X-2 (Stratagene) and confirmed by sequencing. GST- tagged fragments were expressed in E. coli strain BL21(DE3) and purified under denaturing conditions using standard protocols (QIAexpressionist system, Qiagen).

Antibody production Rabbit anti-Cep68 antibodies (R169 and R170) were raised against an N-terminal fragment (aa 1-497) and anti-Cep215 antibodies (R173 and R174) against an internal fragment (aa 503-1010) (both Charles River Laboratories, Chatillon-sur- Chalaronne, France). Similar results were obtained with both antibodies against the respective proteins. Antibodies R170 (anti-Cep68) and R174 (anti-Cep215) were affinity purified in a two-step process using Affigel (Bio-Rad) according to standard protocols: first GST-protein bound to Affigel-10 was used to remove any anti-GST antibodies, followed by purification of specific antibodies via antigen bound to Affigel-15. R170 and R174 were used for all experiments shown in this study.

Cell culture and transfections Cells were grown at 37°C under 5% CO2. For U2OS, HeLaS3, A549 and 293T cells, DME medium was used, supplemented with 10% FCS and penicillin- streptomycin (100 IU/ml and 100 ␮g/ml, respectively). hTERT-RPE1 cells were grown in DMEM nutrient mixture F-12 Ham supplemented with 10% FCS, penicillin/streptomycin, 2 mM glutamine and 0.348% sodium bicarbonate. 293T cells were transfected using the calcium phosphate precipitation method (Krek and Journal of Cell Science Nigg, 1991). U2OS cells were transfected using FUGENE6 reagent (Roche) according to the manufacturer’s protocol. Cells were analysed 24–36 hours post transfection.

Immuno-electron microscopy For pre-embedding immuno-EM of whole cells, U2OS cells, grown on coverslips, were fixed with 4% formaldehyde for 10 minutes, permeabilised with PBS + 0.5 % Fig. 7. Recruitment of Cep68 but not Cep215 to rootletin fibres. Triton X-100 for 2 minutes. Blocking and primary antibody incubations were Tagged constructs of rootletin, Cep68 and Cep215 were performed as described for immunofluorescence microscopy, followed by overexpressed in U2OS cells and filament formation as well as the incubation with goat anti-rabbit IgG-Nanogold (1:50; Nanoprobes). Nanogold was recruitment of endogenous proteins to induced filaments was silver-enhanced with HQ Silver (Nanoprobes). For controls, the primary antibody examined by immunofluorescence microscopy. Bars, 10 ␮m. was omitted. Cells were further processed as described (Fry et al., 1998a). (A) GFP-rootletin (left) was coexpressed with Myc-Cep68 or Myc- Cep215 (centre); merged images are shown on the right (GFP- Immunofluorescence microscopy and immunoblotting Cells were grown on coverslips, washed once in PBS and fixed in –20°C methanol rootletin in green; Myc in red). Note that Myc-Cep68 but not Myc- for 10 minutes. Then, coverslips were washed in PBS and blocked in 1% bovine Cep215 colocalises with rootletin fibres. (B) Overexpressed Myc- serum albumin (BSA) in PBS for 30 minutes, before incubation with primary Cep68 (centre) colocalises with endogenous rootletin at the antibodies (diluted in 3% BSA-PBS) for 1 hour at room temperature. After three centrosome (centre) but does not form polymers; merged image on washes in PBS for 10 minutes each, incubations with secondary antibodies and the right shows Myc-Cep68 in green and rootletin in red. subsequent washes were done the same way. Coverslips were mounted on slides using glycerol-based mounting medium containing p-phenylenediamine as anti- (C) Endogenous Cep68 (centre) colocalises with Myc-rootletin (left) fading agent. Primary rabbit antibodies were anti-Cep68 affinity-purified IgG overexpressed to low (top) and high (middle) levels, but endogenous (R170, 1 ␮g/ml) or corresponding pre-immune serum (1:1000), anti-Cep215 affinity Cep215 is not recruited to Myc-rootletin filaments (bottom); merged purified IgG (R174, 1 ␮g/ml) or corresponding pre-immune serum (1:1000), anti- images on the right show Cep68 and Cep215 in green and Myc- rootletin serum (R145, 1:1000), anti-C-Nap1 affinity purified IgG [R63 (Mayor et rootletin in red. al., 2000)] and anti-pericentrin (1 ␮g/ml, ab4448, Abcam). Primary mouse monoclonal antibodies were anti-␥-tubulin (1:1000, GTU-88, Sigma), anti-␣- tubulin (1:5000, DM1A, Sigma) and anti-Myc [1:3, hybridoma tissue culture supernatant, 9E10 (Evan et al., 1985)]. Secondary antibodies were Alexa Fluor 488 form part of a linker structure. Our observation that Cep215 or 555-conjugated donkey IgGs (1:1000, Molecular Probes) and Cy2-/Cy3- may function downstream of pericentrin suggests that the two conjugated donkey IgGs (1:1000, Dianova). DNA was stained with 4,6-diamidino- 2-phenylindole (DAPI; 2 ␮g/ml). proteins affect centrosome cohesion through a common Immunofluorescence microscopy was performed using a Zeiss Axioplan II mechanism. This mechanism remains to be elucidated, but in microscope (Carl Zeiss, Jena, Germany) equipped with an Apochromat 63ϫ oil 4330 Journal of Cell Science 120 (24)

immersion objective, and images were acquired using a Micromax charge coupled References device (CCD) camera (model CCD-1300-Y; Princeton Instruments) and MetaView Andersen, J. S., Wilkinson, C. J., Mayor, T., Mortensen, P., Nigg, E. A. and Mann, software (Visitron Systems). Alternatively (Fig. 4A,B), a Deltavision microscope M. (2003). Proteomic characterization of the human centrosome by protein correlation on a Olympus IX71 base (Applied Precision) equipped with an Apo 100ϫ/1.35 oil profiling. Nature 426, 570-574. immersion objective and a CoolSnap HQ camera (Photometrics) was used for Bahe, S., Stierhof, Y. D., Wilkinson, C. J., Leiss, F. and Nigg, E. A. (2005). Rootletin collecting 0.2 ␮m distanced optical sections in the z-axis. Images at single forms centriole-associated filaments and functions in centrosome cohesion. J. Cell focal planes were processed with a deconvolution algorithm (100ϫ: Biol. 171, 27-33. Olympus_100X_140_10103.otf). 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