© 2020. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs240267. doi:10.1242/jcs.240267

RESEARCH ARTICLE A novel -specific Cep215 domain interacts with Cep192 and phosphorylated Aurora A for organization of spindle poles Ryoko Kuriyama* and Cody R. Fisher

ABSTRACT stabilization, the PCM expands enormously during maturation (Lawo The , which consists of and et al., 2012; Sonnen et al., 2012). This expansion of the PCM is one of (PCM), becomes mature and assembles mitotic spindles by increasing the hallmarks of centrosome maturation. the number of microtubules (MTs) emanating from the PCM. Among the Molecules involved in centrosome maturation include a subset of – molecules involved in centrosome maturation, Cep192 and Aurora A core centrosomal Cep192 (Gomez-Ferreria et al., 2007; (AurA, also known as AURKA) are primarily responsible for recruitment Zhu et al., 2008), Cep215 (also known as CDK5RAP2; Fong et al., γ of γ- and MT nucleators, whereas pericentrin (PCNT) is required 2008), pericentrin (PCNT; Zimmerman et al., 2004) and -TuRC – for PCM organization. However, the role of Cep215 (also known components (Lüders et al., 2006) and two mitotic kinases, Aurora A as CDK5RAP2) in centrosome maturation remains elusive. Cep215 (AurA, also known as AURKA) and Plk1 (Lane and Nigg, 1996; possesses binding domains for γ-tubulin, PCNT and MT motors that Hannak et al., 2001; Berdnik and Knoblich, 2002; Haren et al., 2009). transport acentrosomal MTs towards the centrosome. We identify a High-resolution analysis has revealed that PCNT, an elongated mitosis-specific centrosome-targeting domain of Cep215 (215N) that scaffolding , extends toward the centrosomal periphery to interacts with Cep192 and phosphorylated AurA (pAurA). Cep192 is provide the structural frame of the expanding mitotic PCM (Lawo essential for targeting 215N to , and centrosomal et al., 2012). Cep192 is essential for MT polymerization by recruiting γ localization of 215N and pAurA is mutually dependent. Cep215 has a -TuRC to the centrosome (Gomez-Ferreria et al., 2007; Zhu et al., γ relatively minor role in γ-tubulin recruitment to the mitotic centrosome. 2008). During M phase, more binding sites for -TuRC are created on However, it has been shown previously that this protein is important for Cep192 as a result of protein phosphorylation controlled by AurA and connecting mitotic centrosomes to spindle poles. Based on the results of Plk1 (Joukov et al., 2014). In contrast to these molecules, little is rescue experiments using versions of Cep215 with different domain known of the role of Cep215 in centrosome maturation. Drosophila deletions, we conclude that Cep215 plays a role in maintaining the Cep215 is a mammalian homolog of Centrosomin structural integrity of the spindle pole by providing a platform for the (Cnn) (Zhang and Megraw, 2007), and mutations in the molecules involved in centrosome maturation. encoding human Cep215 induce primary microcephaly, a neurodevelopmental disorder (Woods et al., 2005). Members of the KEY WORDS: Centrosomes, Spindle poles, Cep215, Cep192, Cnn family are present in a wide range of species, from yeast to Phosphorylated Aurora A, NuMA humans (Verde et al., 2001; Sawin et al., 2004; Pitzen et al., 2018). Although their sizes and sequences are diverse, all members of the INTRODUCTION family contain the highly conserved Cnn motif 1 (CM1) and motif 2 The centrosome is the major microtubule (MT)-organizing center in (CM2) (Zhang and Megraw, 2007). Because CM1 is capable of animal cells and is composed of a pair of centrioles and surrounding binding γ-tubulin and γ-TuRC to initiate MT nucleation (Terada et al., pericentriolar material (PCM). During M phase, the centrosomes 2003; Choi et al., 2010), Cep215 has been predicted to be important become localized at each spindle pole to assemble the mitotic spindle for centrosome maturation by inducing MT polymerization onto the by undergoing a process called maturation, which is characterized by centrosome (Fong et al., 2008). However, this notion has been an increased number of MTs emanating from the centrosome. Some challenged (Kim and Rhee, 2014). CM2 is responsible for the MTs are directly polymerized onto MT nucleators (γ-tubulin and centrosome targeting of Cep215 (Barr et al., 2010; Wang et al., 2010) γ-TuRCs) embedded in the PCM (Kuriyama and Borisy, 1981). and for the binding of PCNT (Buchman et al., 2010; Wang et al., Other MTs are formed around the chromatin and at the walls of pre- 2010). Therefore, Cep215 is likely important for the construction of existing MTs, and are then transported toward the centrosome by MT the mitotic PCM scaffold in collaboration with PCTN (Lawo et al., motor proteins (reviewed by Meunier and Vernos, 2016; Prosser and 2012; Kim and Rhee, 2014). Indeed, Cnn is widely acknowledged to Pelletier, 2017). Stabilization of MT minus ends at the centrosome is play a major role in the assembly of mitotic centrosomes in fly also an important factor to assure the association of a large number of embryos (Conduit et al., 2010). Besides CM1 and CM2, Cep215 MTs with the centrosome. To accommodate the variety of molecules includes the binding sites of the MT motors dynein–dynactin (Jia and protein complexes required for MT nucleation, anchorage and et al., 2013) and HSET (also known as KIFC1; Chavali et al., 2016), both of which are important for transporting and focusing acentrosomal MTs to spindle poles. In fact, abnormal poles Department of Genetics, Cell Biology and Development, University of Minnesota, disconnected from mitotic centrosomes have been reported in cells Minneapolis, MN 55455, USA. lacking not only Cnn/Cep215 (Lucas and Raff, 2007; Barr et al., *Author for correspondence ([email protected]) 2010; Barrera et al., 2010; Lee and Rhee, 2010; Chavali et al., 2016), but also the Cnn/Cep215 binding partners Ncd/HSET and dynein– R.K., 0000-0002-6792-5783; C.R.F., 0000-0002-8144-9859 dynactin (Morales-Mulia and Scholey, 2005; Chavali et al., 2016).

Handling Editor: David Glover To understand how Cep215 is involved in centrosome maturation,

Received 8 October 2019; Accepted 29 October 2020 we analyzed the mouse Cep215 sequence to identify two previously Journal of Cell Science

1 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs240267. doi:10.1242/jcs.240267 unknown centrosome-targeting domains. One (215M) includes the faster than the majority of human cell lines (∼13 h versus ∼24 h dynein–dynactin-binding sequence (Jia et al., 2013) and the other doubling time) and are highly amenable to mitotic synchronization (215N) targets Cep215 to the centrosome during M phase. The 215N (∼75% mitotic index) and transfection (>50% efficiency) (Fig. S1). domain binds to Cep192 and AurA phosphorylated at T288 (pAurA), As reported previously (Barr et al., 2010; Wang et al., 2010), the both of which are essential for 215N recruitment to the centrosome. truncated polypeptide CM2, consisting of a C-terminal segment of Deletion of Cep215 does not significantly interfere with γ-tubulin Cep215 (Fig. 2A), functions as a centrosomal-targeting domain recruitment to mitotic centrosomes. However, as reported previously throughout the cell cycle (Fig. 1A, panels 1,2). By labeling Cep215 (Barr et al., 2010), the most prominent phenotype of the Cep215 with antibodies recognizing the sequence outside CM2, we found that knockout is centrosome separation from the spindle poles. We show CM2 colocalizes well with endogenous Cep215 in interphase that this phenotype is rescued by the collaborative efforts of multiple centrosomes (Fig. 1A, panels 3a,b). CM2 includes the PCNT- binding domains of Cep215 that are specific to γ-tubulin, Cep192, binding sequence (Buchman et al., 2010; Wang et al., 2010), thus pAurA, MT motor proteins and PCNT, suggesting that Cep215 PCNT shows an almost identical distribution to CM2 (Fig. 1A, panels serves as a platform for molecules important for organization of 3a,c) and endogenous Cep215 (Fig. 1A, panels 3b,c). CM1, another mitotic spindle poles during centrosome maturation. conserved domain of Cep215, is capable of γ-tubulin and γ-TuRC binding (Fong et al., 2008). Although exogenous CM1 can initiate RESULTS MT nucleation (Terada et al., 2003; Choi et al., 2010), it failed to Domain analysis of Cep215 localize to both interphase and mitotic centrosomes, as reported We first analyzed deletion constructs of the mouse Cep215 sequence previously (Fong et al., 2008; Fig. 1B). by expressing a series of c-Myc (myc)-tagged truncated polypeptides. The central region of Cep215 between CM1 and CM2 is relatively We employed Chinese hamster ovary (CHO) cells, which divide uncharacterized. Nonetheless, a unique sequence termed PReM

Fig. 1. Localization of mouse Cep215 domains at the centrosome. (A) CM2 expression. Interphase (1,1′) and mitotic (insets in 1,1′) CHO cells expressing myc-tagged CM2 were stained with anti-myc (CM2, red) and anti-α-tubulin (MT, green) antibodies. In panel 2, a mitotic cell after staining with anti-myc (CM2, red) and γ-tubulin (γ, green) antibodies is shown. High magnification images of the spindle poles (white boxes) are shown in panels 2a and 2b. Triple-staining of interphase cells for CM2 (3a), endogenous Cep215 (3b) and PCNT (3c) shows almost identical localization of three components, as highlighted by the indicated panel merge images on the right. (B) CM1 expression. Interphase and mitotic (insets) CHO cells expressing myc-tagged CM1 were stained with anti-myc (CM1, red) and either anti-γ-tubulin or anti-α-tubulin antibodies. (C) 215M expression. Interphase and mitotic (insets) CHO cells expressing myc-tagged 215M were stained with anti-myc (215M, red) and either anti-γ-tubulin or anti-α-tubulin antibodies. Arrows indicate the position of interphase centrosomes. (D) 215N expression. CHO cells expressing myc-tagged 215N were stained with anti-myc (215N, red, panels 1′,2,3,4′–6′) antibody and either anti-γ-tubulin (1), anti-pHH3 (2′,3′) or anti-α-tubulin (4–6) antibodies. 215N disperses in the entire cytoplasm of interphase cells (arrowheads in 1′ and 3), but becomes localized to the centrosomes in late G2 cells (arrow in 1′) prior to histone H3 phosphorylation (arrow in 2). In panels 7–7″, myc-tagged 215N-expressing cells depleted of Cep215 were triple-stained with anti-α-tubulin, anti-Cep215 (recognizing the sequence outside 215N, red), and anti-myc (215N) antibodies. 215N is recruited to the mitotic centrosome in the absence of endogenous Cep215. Images are representative of 83 Cep215-depleted cells. DAPI staining is shown in blue in A (panels 1,2), B, C and D (panels 1–7). Scale bars: 10 µm (A, panels 1,1′;B–D), 5 µm (A, panels 2,3; insets in A panels 1,1′, B,C), 1 µm (A, panels 2a,2b). Journal of Cell Science

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background of cytoplasmic fluorescence interfered with clear visualization of 215N signal at mitotic centrosomes.

The 215N and CM2 regions are major domains targeting Cep215 to the mitotic centrosome The presence of newly identified centrosome-targeting domains indicates that Cep215 is recruited to the mitotic centrosome via different pathways. To assess the contribution of individual domains, we quantified the fluorescence intensity of myc signal at the spindle poles (Fig. 2C). The level of 215N fluorescence corresponded ∼30% of that of the full-length protein [mean±s.e.m. of 3.11±0.15 arbitrary units (a.u.) versus 10.50±0.62 a.u., respectively; P<0.001] (Fig. 2C and Fig. 2D, panels 2,3). An 8–9% higher level of fluorescence – Fig. 2. Quantitative analysis of fluorescence intensity of Cep215 domains intensity of myc 215N was detected in cells where endogenous at the mitotic centrosome. (A) Map of full-length Cep215 (full) and the Cep215 was knocked out by introducing siRNA specific to the indicated Cep215 deletion constructs. Numbers indicate amino acid positions. sequence outside the 215N region (215N*). CM2 (∼21%) and 215M Dotted lines indicate deleted regions. (B) Western blotting analysis of myc- (∼11%) produced lower intensities of centrosomal fluorescence than tagged truncated proteins encoded by individual deletion constructs. that of 215N. The sum of these three intensity values (∼62%) is less Molecular mass markers in kDa are indicated. (C) Quantification of than 100%, possibly because adjacent targeting domains have been fluorescence intensity of Cep215 domains at spindle poles. 215N* indicates 215N-expression at the mitotic centrosome in Cep215-depleted cells. separated. When the coding sequence of 215N was removed from Individual data points are plotted in each graph, with mean±s.e.m. indicated. Cep215 (Δ215N), the spindle pole fluorescence signal fell to ∼32% of n= 171 (full), 150 (215N), 70 (215N*), 115 (215M), 119 (CM2), 166 (Δ215N), the control level (Fig. 2D, panel 4), which is a greater decrease than 191 (ΔCM2), 187 (215N+CM2) and 181 (Δ215N+ΔCM2) obtained from 2–3 that seen in cells expressing the CM2-lacking sequence (ΔCM2, independent experiments. Percentage fluorescence signal relative to ∼40% of control level) (Fig. 2C). Because the difference in the full-length protein is shown. Statistical significance was assessed by fluorescence intensity between 215N and CM2, and between comparing the mean of each treated group with the control mean by performing Δ Δ ∼ – one-tailed z-tests. P<0.001. (D) Mitotic CHO cells expressing different Cep215 215N and CM2 was 8 9%, it is reasonable to conclude that domains were either stained with anti-myc antibody alone (3–5) or double- 215N is more potent than CM2 for targeting Cep215 to the mitotic stained with anti-myc (2,6) and anti-α-tubulin (1,7) antibodies. Scale bar: centrosome in CHO cells. The contribution of 215M was significantly 10 µm. lower than that of 215N and CM2 (Fig. 2C). Statistical analysis found this difference to be highly significant (P<0.001), thus we conclude that recruitment of Cep215 to the mitotic centrosome is primarily (Conduit et al., 2014) and domains specific to several MT-interacting achieved by two domains, 215N and CM2. Indeed, a chimeric fusion proteins, including dynein light chain 8 (also known as DYNLL1; Jia protein of 215N and CM2 (215N/CM2; Fig. 2A and Fig. 2D, panel 5) et al., 2013), EB1 (also known as MAPRE1; Fong et al., 2009) and a retained nearly 80% of the targeting activity, whereas only ∼4% of minus-end-directed kinesin-like motor (HSET; Chavali et al., 2016) centrosomal fluorescence remained in cells expressing Cep215 have been identified. By cutting in the middle of this long stretch of lacking both 215N and CM2 (Δ215N/ΔCM2; Fig. 2A and Fig. 2D, ∼1300 amino acids, we generated near N-terminal (215N, amino acids panel 6). We obtained similar results of fluorescence intensity of 166–696) and mid-portion (215M, amino acids 697–1472) Cep215 domains in human cells. Bipolar spindles formed in cells polypeptides (Fig. 2A,B). 215M, which includes the sequences expressing such an extremely low level of Cep215 fluorescence at the capable of interacting with dynein and EB1, was found at the poles were thin and composed of significantly reduced amounts of interphase and mitotic centrosomes (Fig. 1C). However, its fluorescence spindle MTs (Fig. 2D, panel 7), which is further analyzed in Fig. 8. signal was found to be much weaker than that of CM2. Furthermore, interphase centrosomes labeled with 215M appeared differently in The 215N domain is different from the HSET-binding domain different cells. Some centrosomes appeared as a discrete dot, while Almost half of the C-terminal 215N region partially overlaps with others were diffuse and irregular in shape. Centrosomal localization of the sequence previously identified as an HSET-binding domain 215M was observed in HeLa and U2OS cells (Fig. S2), but we detected (amino acids 500–700 in human Cep215; Chavali et al., 2016; only a very faint signal, if any, in RPE1 cells (data not shown). Fig. S3A). To determine the relationship between 215N and the The 215N polypeptide, which consists of an ∼500-amino-acid HSET-binding domain, we cut 215N in the middle to generate sequence downstream of CM1 (Fig. 2A), was observed to evenly 215N-N2 (amino acids 166–435) and 215N-C (amino acids disperse in the entire cytoplasm of interphase cells (arrowheads in 436–696) polypeptides (Fig. S3A). Despite a significant reduction Fig. 1D). As the cell cycle proceeded, the myc signal became in myc fluorescence at the centrosomes, myc-tagged 215N-N2 was concentrated at the centrosomes in late G2 cells (arrow in Fig. 1D, still able to localize at mitotic centrosomes (fluorescence levels panel 1′) prior to histone H3 phosphorylation at Ser10 (pHH3, ∼22% of 215N; Fig. S3B, panel 4). In contrast, 215N-C totally lost arrow in Fig. 1D, panel 2). The 215N polypeptide persisted at the the ability to associate with the centrosome (Fig. S3B, panel 3 and spindle poles during mitosis, and then gradually faded away by the Fig. S3C, panel 3′). Truncation of 35 amino acids from the N end of cell division (Fig. 1D, panels 4–6). Cells depleted of terminus of 215N-N2 reduced the targeting activity of 215N-N3 endogenous Cep215 by RNAi were also capable of recruiting 215N (amino acids 201–435) to ∼16% of 215N. This difference (∼6%) to the centrosome at each spindle pole (Fig. 1D, panel 7). The 215N between 215N-N2 and 215N-N3 is more than that between 215N- fragment was targeted to the mitotic centrosome in all cell types N2 (∼22%) and 215N-N1 (∼24%). Because the addition of ∼70 tested thus far, and was better detected in cells transfected with amino acids at the C-terminal end of 215N-N2 resulted in only ∼2% significantly lower amounts of plasmid DNA than usual (see increase of the centrosomal target, it is reasonable that the

Materials and Methods). With higher amounts of DNA, a high N-terminal sequence of 215N is more crucial than the C terminus Journal of Cell Science

3 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs240267. doi:10.1242/jcs.240267 for centrosome-targeting activity. Immunostaining of 215N and targeted to the mitotic centrosomes (arrows). CM2 also localized to HSET in mitotic cells showed that the vast majority of HSET, both the centrosome in cells lacking pAurA (arrow in Fig. 4D, panel 1′). endogenous and exogenous, localizes along spindle fibers The independence of CM2 and pAurA is supported by the distinctive (Fig. S3C, panels 1–3; Kuriyama et al., 1995). In contrast, 215N distributions of these molecules (Fig. 4D, panel 2). Unlike pAurA, is found exclusively at the centrosome (Fig. S3C panels 1′,2′), which is confined to a small area of the centrosome, CM2 disperses suggesting that 215N is different from the HSET-binding domain. widely to the PCM, occasionally along the flare-like PCM particles similar to those previously reported by Megraw et al. (2002) in fly The 215N domain binds pAurA, and their centrosomal embryos. In contrast to CM2, 215N failed to localize to the mitotic localizations are mutually dependent centrosomes after depletion of detectable pAurA in AurA- Because 215N is one of the major centrosome-targeting domains, knockdown cells (arrow in Fig. 4D, panel 3′). These results we investigated this sequence further. We have previously reported indicate that pAurA is essential for centrosomal recruitment of the close relationship between Cnn and AurA (Terada et al., 2003). 215N. We noted that pAurAwas sometimes more resistant than AurA Furthermore, the timing of 215N emergence at the centrosome to exclusion from the centrosome following RNAi of AurA. It is thus resembles that of AurA phosphorylated at T288 (pAurA). Thus we possible that some of the AurA-negative cells shown in Fig. 4C, panel first compared the distributions of 215N and pAurA in mitotic 3, still had pAurA, allowing 215N to localize to the centrosome. spindles by employing two AurA antibodies: an anti-AurA Whereas Cep215 was dispensable for the centrosomal localization of monoclonal antibody (mAb) labeling non-phosphorylated AurA, AurA (arrows in Fig. 4E, panels 1,2), pAurA was no longer detected and an anti-pAurA polyclonal antibody (pAb) specific to the at the mitotic centrosome in Cep215-depleted cells (Fig. 4E, panel phospho-epitope at T288. The specificity of the pAb to pAurA was 2″). Panels 3 and 4 of Fig. 4E show results of rescue experiments. The demonstrated by loss of immunoreactivity of mitotic centrosomes 215N domain, but not myc–Cep215 lacking the 215N sequence after dephosphorylation by phosphatase treatment (Fig. S4, panels (Δ215N), was able to restore pAurA to the centrosome in Cep215- 1,2). As has been previously well-documented, non-phosphorylated depleted cells. From these results, we conclude that 215N and pAurA AurA was primarily seen at spindle poles and fibers, whereas are mutually dependent for localization to the mitotic centrosome. phosphorylated AurA appeared as a discrete dot at each spindle pole (Fig. 3A, panels 1,2; Fig. S4, panel 3). The association of pAurA Centrosomal targeting of 215N is inhibited by both AurA and AurA with spindle fibers and spindle poles was retained in and Plk1 inhibitors isolated spindles (Fig. S4, panels 4–6). It is clear that the 215N To confirm the interdependency between 215N and pAurA, we distribution is more similar to that of pAurA than that of AurA. examined the effect of MLN8237, a small molecule inhibitor of Fig. 3A, panel 3, shows GFP-tagged -1 (GFP–CETN)- AurA. Although entry into mitosis was slightly delayed, cells treated expressing cells immunostained for pAurA and Cep135 (a marker with MLN8237 entered M phase to initiate spindle assembly. Due for the proximal end of the mother ). pAurA localized at a to the lack of pole separation, a well-known phenotype of AurA position more distal than Cep135, but more proximal than CETN, inhibition, the vast majority of cells formed monopolar spindles which is known to localize in the middle to distal region of the associated with only a few spindle MTs (Fig. 5A, panel 2). In those centriole. Like pAurA, 215N displayed preferential localization at cells, the fluorescence intensity of AurA at the poles was reduced to and around the proximal end of the mother centriole. Although less than half of that of mock-treated cells [mean±s.e.m. of 2.16±0.09 similar, the distribution of the two molecules did not overlap a.u. versus 5.11±0.24, respectively; P<0.001]. The majority of entirely (arrows and arrowheads in Fig. 3A, panels 4a,b). pAurA was also removed from the spindle pole (∼32% of control The close proximity of 215N and pAurA suggests the possibility of levels remained), but a trace amount of the protein signal persisted in physical interaction between the two molecules, which we analyzed cells treated with up to 1 µM MLN8237 (Fig. 5A, panel 5′). Similar to by immunoprecipitation (Fig. 3B–D). GFP–AurA and myc–215N pAurA fluorescence, the fluorescence intensity of 215N diminished were specifically pulled down by anti-myc and anti-GFP antibodies, to ∼20% of the control level (Fig. 5A, panel 8), but centrosomal respectively (Fig. 3B). The presence of phosphorylated AurA, both targeting of CM2 was not affected by drug treatment (Fig. 5A, panel endogenous (pAurA) and exogenous (GFP–pAurA), was also 11). Because Cep215 was found to be recruited to the mitotic confirmed in the precipitated fractions (Fig. 3C). The amount of centrosomes via two independent pathways (an MLN8237-sensitive pAurA co-precipitating with 215N was elevated when exogenous 215N-mediated pathway and an MLN8237-insensitive CM2- AurA was co-expressed with 215N (lanes 3 and 4 in Fig. 3C). This mediated pathway), it is reasonable that we detected residual suggests that AurA phosphorylation is stimulated by interaction with Cep215 fluorescence (∼44% of the control level) at the mitotic 215N. Fig. 3D shows immunoprecipitation of myc–215N with centrosome in MLN8237-treated cells (Fig. 5A, panel 2′,Fig.5B). mutant AurA, in which T288 is replaced with non-phosphorylatable Mitotic cells were also treated with a Plk1 inhibitor (BI2536), alanine (T288A). GFP–AurA (T288A) was not recognized by anti- which interferes with the formation of functional bipolar spindles, as pAurA antibodies and no longer co-immunoprecipitated with 215N. does MLN8237. However, unlike AurA inhibition, BI2536 did not These results indicate that 215N specifically binds to phosphorylated significantly affect astral MTs (Fig. 5A, panels 3,6), which is in good AurA, but does not bind to non-phosphorylated AurA. agreement with previous reports (De Luca et al., 2006; Hanisch et al., To examine how Cep215/215N and pAurA relate to each other at 2006). Plk1 inhibition efficiently removed both AurA and pAurA the centrosome, we prepared RNAi cells (Fig. 4A) to analyze the from centrosomes (∼25% of AurA and 10% of pAurA remained) presence or absence of the molecules at the mitotic centrosome (see (Fig. 5A, panels 6,6′ and Fig. 5B). It also profoundly affected summary in Fig. 4B). In AurA-depleted cells, Cep215 was the centrosomal targeting of 215N and removed nearly 95% of successfully recruited to the centrosomes (arrow in Fig. 4C, panel fluorescence from the centrosome (Fig. 5A, panel 9, Fig. 5B). This is 1′). In the reverse situation, AurA was still found at the spindle fibers in striking contrast to the effect of Plk1 inhibition on CM2, which and spindle poles in Cep215-depleted cells (arrows in Fig. 4E, panels remained entirely intact, as in control cells (Fig. 5A, panel 12, 1,2). AurA-depleted cells expressing CM2 and 215N are shown in Fig. 5B). Consequently, the fluorescence intensity of Cep215 became

Fig. 4C, panels 2 and 3, where both Cep215 domains are seen to be almost halved at mitotic centrosomes in Plk1-inhibited cells (Fig. 5A, Journal of Cell Science

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Fig. 3. Interaction of 215N with pAurA. (A) In panels 1 and 2, mitotic CHO cells expressing myc-tagged 215N were stained with anti-myc (215N, grayscale) and either anti-AurA (1) or anti-pAurA (2) antibodies. Panel 3 shows RPE1 cells expressing GFP–CETN and stained with anti-pAurA and anti-Cep135 antibodies. In panel 4, RPE1 cells co-expressing GFP–CETN and myc-tagged 215N were immunostained with anti-myc (215N, red) and anti-pAurA antibodies. Each spindle pole outlined (white boxes) is shown at a high magnification (4a,4b). 215N shows localization similar but not entirely identical to that of pAurA at the centrosome (arrows and arrowheads indicate differences in localization). Scale bars: 10 µm (1,2,4), 1 µm (3,4a,4b). DAPI staining is shown in blue (panels 1,2,4). (B–D) Co-immunoprecipitation of 215N with pAurA or a non-phosphorylatable mutant of AurA (T288A). Lysates prepared from HEK293T cells co-expressing myc–215N and either GFP–AurA (B,C) or GFP–mutant pAurA (T288A) (D) were immunoprecipitated (IP) with anti-GFP or anti-myc antibodies, followed by immunoblotting with either a mixture of anti-GFP and anti-Cep215 (recognizing the coding sequence of 215N) antibodies (B), anti-pAurA antibody (C), or anti-Cep215, anti-GFP, a mixture of anti-Cep215 and anti-GFP, and anti-pAurA antibodies as indicated (D). All samples in each panel outlined by a black line were run on the same blot. White vertical lines mark the position of a lane, either blank or duplicate, removed from the figure, and black vertical lines in D indicate the separation of lanes probed with different antibodies. panel 3′, Fig. 5B). The inhibitory effect of BI2536 on Cep215 has inhibition may be due to, at least partly, the different cell types, previously been reported; Haren et al. (2009) calculated that less than species or reagents and protocols used to quantify fluorescence 20% of Cep215 fluorescence remains at mitotic centrosomes in intensities. From these results, we conclude that the 215N-dependent, BI2536-treated HeLa cells. This is much less than the 46–47% that but not the CM2-dependent, centrosome-targeting pathway is under remained in CHO cells (Fig. 5B). We repeated these experiments the control of Plk1. using RPE1 cells and found that Plk1 inhibition reduced centrosomal fluorescence of Cep215 to 32% of the control level (mean±s.e.m. of Cep192 interacts with 215N and is required for centrosomal 4.17±0.36 a.u. versus 13.02±0.81, respectively; n=54; P<0.001). localization of 215N This number is lower than that seen in CHO cells but still higher than We next examined the relationship of 215N with Cep192, a key that reported by Haren et al. (2009). The difference in the degree of molecule in centrosome maturation. Like 215N, Cep192 Journal of Cell Science

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Fig. 4. Centrosomal localizations of 215N and pAurA are mutually dependent. (A) Depletion of Cep215, AurA and pAurA by RNAi in CHO cells, compared with control siRNA treatment (mock). α-Tubulin (α-tub) is shown as a loading control. (B) Summary of centrosomal localization of Cep215, 215N, CM2, AurA and pAurA in RNAi cells. (C) AurA-depleted CHO cells, either wild type (1,1′) or expressing myc-tagged CM2 (2,2′) or myc-tagged 215N (3,3′), were stained with anti-AurA (1–3) and either anti-Cep215 (1′) or anti-myc (2′,3′) antibodies. Arrows indicate cells expressing endogenous Cep215 (1′), exogenous CM2 (2′) and 215N (3′) at mitotic centrosomes in the absence of AurA. Images are representative of n=100 (1), 34 (2) and 36 (3) AurA-depleted cells. (D) Expression of myc-tagged CM2 (1,1′) or myc-tagged 215N (3,3′) in AurA RNAi CHO cells stained with anti-pAurA (1,3) and anti-myc (1′,3′) antibodies. CM2, but not 215N, is targeted to the mitotic centrosomes in pAurA-depleted cells (arrows). In panel 2, control cells expressing myc-tagged CM2 were stained with anti-myc and anti-pAurA antibodies, and each spindle pole outlined (white boxes) is shown at a high magnification (a,b). Images are representative of n=51 (1) and 73 (3) pAurA-depleted cells. (E) Panels 1–2″ show Cep215-depleted CHO cells double (1,1′) and triple (2–2″) stained with anti-AurA (1,2), anti-Cep215 (1′,2′) and anti- pAurA (2″) antibodies. Arrows indicate Cep215-negative cells associated with AurA (1,2), but not pAurA (2″) at the centrosomes. Panels 3 and 4 show rescue experiments of Cep215-depleted CHO cells with myc-tagged 215N (3) and Δ215N (4). Centrosomal localization of pAurA is restored by introduction of 215N (arrows in 3–3″), but not 215N-lacking Δ215N (4,4′). In panels 3–3″, Cep215-depleted cells were triple-stained with anti-Cep215 (recognizing the sequence outside 215N), anti-myc (215N) and anti-pAurA antibodies. In panels 4 and 4′, Cep215-depleted cells expressing Δ215N were stained with anti-pAurA and anti- myc (Δ215N) antibodies. Because the available anti-Cep215 antibody cross-reacts with both endogenous Cep215 and Δ215N, depletion of Cep215 in siRNA-treated cells is confirmed by the absence of pAurA at mitotic centrosomes. Images are representative of n=53 (1), 55 (2), 25 (3) and 16 (4) Cep215-depleted cells. DAPI staining is shown in blue (panels C1–3, D1–3, E1–4). Scale bars: 10 µm (C; D panels 1,1′,3,3′; E), 5 µm (D, panel 2), 1 µm (D, panels 2a,b). preferentially localizes to the proximal end of the centriole (Zhu et al., Fig. 6B show colocalization of Flag–Cep192 aggregates with 2008; Lawo et al., 2012), and the two proteins indeed colocalized endogenous AurA in interphase cells, which is consistent with the quite well at each centrosome (Fig. 6A). Cells co-expressing myc– previous report of Joukov et al. (2014) showing interaction of Cep192 215N and Flag–Cep192 also revealed identical distribution of the two with AurA. proteins (Fig. 6B, panel 1). At increasing levels of protein expression, To determine whether Cep192 and 215N are required for each other Flag-tagged proteins induced cytoplasmic aggregates of various sizes to localize at mitotic centrosomes, we prepared Cep192-deleted cells and numbers throughout the cell cycle (Fig. 6B, panels 1–4). using RNAi (Fig. 6D). Without Cep192, no 215N was found at the Importantly, myc–215N became associated with each aggregate in a mitotic centrosome (Fig. 6E, panel 2), but recruitment of CM2 to the perfectly overlapping manner. This is strikingly different from cells centrosomes was not affected by the loss of Cep192 in both mitotic and expressing myc–215N alone, where no such protein aggregates were interphase cells (Fig. 6E, panels 3,4). This indicates that the 215N- induced, no matter how strongly myc–215N was expressed (arrow in dependent, but not the CM2-dependent, centrosome targeting of Fig. 1D, panel 3; Fig. S3, panel C1′). In contrast to 215N, other Cep215 is controlled by Cep192. We next analyzed centrosomal domains of Cep215 (CM1, 215M and CM2) did not appear to localization of Cep192 in Cep215-depleted cells. Among three mitotic colocalize well with Flag–Cep192 (Fig. 6B, panels 5–7). To confirm a cells included in the same frame shown in Fig. 6F, one cell expressed physical interaction between 215N and Cep192, we performed almost no Cep215 (arrow), but residual amounts of Cep215 were still immunoprecipitation experiments (Fig. 6C). Flag–Cep192 and detected in two other cells (arrowheads). Regardless of the presence or myc–215N co-expressed in cells were successfully precipitated with absence of remaining Cep215, considerable amounts of Cep192 were antibodies against the tag attached to the counterpart protein. still detected at the centrosome in all cells (Fig. 6F, panel 1″). We thus Conduit et al. (2010) have reported co-immunoprecipitation of conclude that Cep192 is recruited to the mitotic centrosome Cnn and DSpd-2 (homologs of Cep215 and Cep192, respectively) independently of Cep215. However, because no 215N-binding in fly embryos. Although the band intensity was weak, we confirmed domain of Cep192 has as yet been identified, we cannot rule out the co-precipitation of endogenous Cep215 with Flag–Cep192 in possibility that Cep192 and Cep215 are partially interdependent in mammalian cells (lower panel of Fig. 6C). Panels 8 and 8′ of localizing at the mitotic centrosome. Journal of Cell Science

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Fig. 5. Effect of MLN8237 and BI2536 on centrosomal localization of Cep215/Cep215 domains and AurA/pAurA. (A) Fluorescence staining of α-tubulin (MT, green; 1,2,3), Cep215 (1′,2′,3′), AurA (4,5,6), pAurA (4′,5′,6′), myc-215N (7–9), and myc-CM2 (10–12) in CHO cells either untreated (control) or treated with 1 µM MLN8237 or 1 µm BI2536, as indicated. The same cells were stained with anti-α-tubulin and anti-Cep215 antibodies (1–3′), and with anti-AurA and anti-pAurA antibodies (4–6′). DAPI staining is shown in blue (panels 1–3). Scale bar: 10 µm. (B) Quantitative analysis of fluorescence intensity at spindle poles in untreated (mock) and drug-treated cells. Calculated mean and s.e.m. are indicated in each frequency distribution. Below each graph is the mean centrosomal fluorescence for each treatment shown as a percentage of the untreated control value. n=91, 97, 84 (Cep215), n=64, 119, 92 (AurA), n=123, 167, 93 (pAurA); n=123, 74, 56 (215N); and n=95, 62, 63 (CM2) untreated, MLN8237-treated and BI2536-treated centrosomes, respectively, obtained from 3–6 independent experiments. Statistical significance was assessed by comparing the mean of each treated group with the control mean by performing one-tailed z-tests. P<0.001.

Cep215 does not significantly contribute to γ-tubulin The role of Cep215 in spindle pole formation recruitment to mitotic centrosomes If Cep215 is not actively involved in γ-tubulin recruitment to the The MT-nucleating activity of mitotic centrosomes is primarily centrosome, what is the role of Cep215 in centrosome maturation? controlled by Cep192 and pAurA, to which Cep215 binds via 215N. To address this question, we analyzed the mitotic phenotypes of To determine whether Cep215 is also required for promoting CHO cells in which Cep215 was deleted (Fig. 8A). Previous studies MT assembly at the centrosome, we measured the ability of have shown that the loss of Cep215 in HeLa cells induces abnormal Cep215-depleted cells to recruit γ-tubulin to mitotic centrosomes spindles, including monopolar spindles, bipolar spindles with less (Fig. 7A, panel 1). Because the RNAi treatment was not completely distance between the two poles, and a lack of prominent astral MTs efficient, a trace amount of Cep215 [1.8±0.3% of control levels emanating from the poles (Fong et al., 2008; Lee and Rhee, 2010). (mean±s.e.m.), n=68, P<0.001] was still detected at the centrosome We found a wide range of spindle abnormalities (Fig. 8A, panels in cells where a large amount of γ-tubulin (65.1±4.8% of control 2–6), including monopolar or short spindles (∼17%; Fig. 8A, panel levels) is still retained (Fig. 7B). Two different siRNAs, specific to 2 and Fig. 8B), which is consistent with previous reports. The the N- and C-terminal sequences of Cep215, yielded almost proportion of cells with bipolar spindles consisting of sparse MTs identical results. Depletion of Cep192 and AurA by siRNA (Fig. 8A, panel 3) and thin spindles (Fig. 8A, panel 4) was also treatment was less efficient than Cep215 depletion; the amount of increased after Cep215 depletion (∼22%; Fig. 8B). This type of centrosomal fluorescence quantified (as described in Materials and spindle has not previously been documented, possibly because of Methods) revealed ∼7% of Cep192 and ∼19% of pAurA the shape of mitotic spindles. It would be difficult to identify thin fluorescence still present at mitotic centrosomes in siRNA-treated spindles in cells where the distance between two poles is reduced. It cells (Fig. 7A, panels 2,3 and Fig. 7B). Nonetheless, much is likely that depletion of Cep215 induces more monopolar and lesser amounts of γ-tubulin remained in those cells (∼12% for short bipolar spindles in HeLa cells (Lee and Rhee, 2010) than in Cep192 and ∼24% for pAurA) compared with the level remaining CHO cells. in Cep215-depleted cells. Because statistical analysis showed these The most notable feature of spindles formed in Cep215 RNAi cells differences to be highly significant (P<0.001), we conclude that was the presence of ‘knots’, which were observed inside or protruding Cep215 plays a minor role in centrosomal recruitment of γ-tubulin outside the spindle depending on the direction from which the spindle during mitosis. was viewed (arrows in Fig. 8A, panels 7–10). Among antibodies tested Journal of Cell Science

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Fig. 6. Interaction of 215N with Cep192. (A) Mitotic RPE1 cells co-expressing GFP–CETN and myc–215N were immunostained with anti-myc (215N, red) and anti- Cep192 (green) antibodies. Each spindle pole outlined (white boxes) is shown at a high magnification (a,b). (B) Mitotic (1–3′) and interphase (4–7′)CHO cells co-expressing Flag–Cep192 and either myc–tagged 215N (1–4′), CM2 (5,5′), 215 M (6,6′) or CM1 (7,7′) were stained with anti-myc and anti-Flag antibodies. In panels 8 and 8′,Flag–Cep192-expressing cells were labeled with anti-AurA and anti-Flag antibodies. (C) Immunoprecipitation of Flag–Cep192 (Flag-192) with either myc–215N (upper panel) or endogenous Cep215 (lower panel). Lysates prepared from HEK293T cells expressing the indicated combinations of Flag–Cep192 and myc–215N were immunoprecipitated (IP) with anti-Flag or anti-myc mAbs and immunoblotted with Cep215 and Cep192 pAbs as indicated. The black vertical line in the top panel indicates where the membrane was cut into two pieces, which were simultaneously treated with the same anti-Cep192 antibody solution. (D) Depletion of Cep192 by RNAi in CHO cells. α-Tubulin is shown as a loading control. (E) Mitotic (1–3′) and interphase (4,4′) CHO cells expressing myc-tagged 215N (1–2′)orCM2(3–4′) were treated with mock (1,1′) and Cep192-specific siRNA (2–4′), followed by immunostaining with anti-myc (1,2,3,4) and anti-Cep192 (1′,2′,3′,4′) antibodies. 215N, but not CM2, failed to localize to the mitotic centrosome in Cep192-depleted cells. Images are representative of n=46(1),45(2), 30 (3) and 55 (4) Cep192 RNAi cells. (F) Cep192 is present in the mitotic centrosomes in Cep215-depleted CHO cells. Cep215 RNAi cells (phase) were first stained with anti-γ-tubulin (1) and anti-Cep215 (1′) antibodies to identify cells devoid of Cep215 at the mitotic centrosome (arrows), and then re-stained with anti-Cep192 antibodies (1″). Cep192 is found at mitotic centrosomes in a similar manner in cells where Cep215 is partially (arrowheads) or almost entirely (arrow) depleted. Images are representative of n=63 Cep215-depleted cells. In A,B and E, DAPI staining is shown in blue. Scale bars: 10 µm (A,B,E,F), 1 µm (A, panels a,b). thus far, we found that NuMA (nuclear mitotic apparatus protein) binding sites (Fig. 8B). Even cells with normal-looking bipolar was the best for probing these structures. As an MT-binding protein spindles revealed the presence of extra NuMA sites, and the center of interacting with dynein–dynactin, NuMA is important to focus many monopolar spindles was occupied by a cluster of NuMA dots of transported spindle MTs on the pole (Merdes et al., 1996). various sizes and shapes (arrows in Fig. 8A, panels 7′,8′). Additional Therefore, the knots labeled with NuMA are likely to correspond to locations where extra NuMA dots were commonly seen are the the dislocated centrosomes and spindle poles, which were first reported shoulder of mitotic spindles (Fig. 8A, panel 13), the tip of MT bundles by Barr et al. (2010) in Cep215 knockout chicken DT40 cells. Split shooting out from the lateral side of the spindle (Fig. 8A, panel 14) and NuMA was detected in cells with multipolar spindles (Fig. 8A, panels the end of MT bundles outspreading to form the unfocused pole at one 5,11). Some spindles were composed of totally disorganized MT side of the spindle (Fig. 8A, panel 15). arrays and randomly dispersed NuMA (Fig. 8A, panels 6,12). We Similar effects on split NuMA were also seen in cells from which observed that ∼38% of Cep215-depleted cells produced extra NuMA- Cep192 (Fig. 8C) or AurA (Fig. 8D) were depleted. These Journal of Cell Science

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Fig. 7. Cep215 has a minor role in γ-tubulin recruitment in mitotic CHO cells. (A) Centrosomal γ-tubulin in mitotic cells after depletion of Cep215 (1,1′), Cep192 (2,2′) or AurA (3,3′). Cells were double stained with anti-γ-tubulin (1′,2′,3′) with either anti-Cep215 (1), anti-Cep192 (2), or anti-pAurA (3′) antibodies. DAPI staining is shown in blue. Scale bar: 10 µm. (B) Quantitative analysis of fluorescence intensity of γ-tubulin and either Cep215, Cep192 or pAurA at mitotic centrosomes in control cells (mock) and cells treated with RNAi targeting Cep215 (left), Cep192 (middle) or AurA (right). Calculated mean and s.e.m. are indicated in each frequency distribution. Reported below each graph is the percentage of the mean centrosomal fluorescence signal in treated cells compared to control cells. n=56, 68 (Cep215); n=55, 44 (Cep192); and n=73, 74 (pAurA) mock- and RNAi-treated centrosomes, respectively, obtained from 1–2 independent experiments. Statistical significance was assessed by comparing the mean of each siRNA-treated group with that of corresponding mock cells by performing one-tailed z-tests. P<0.001.

observations are in good agreement with those of De Luca et al. specific molecules: CM1 can bind to γ-tubulin and γ-TuRC; 215N (2006), who reported that small extra poles are induced in can specifically bind to Cep192 and pAurA, and HSET-binding AurA-depleted cells. Overall, the aberrant spindle shapes and sequences overlap with 215N; 215M can interact with dynein– random distribution of NuMA were strikingly similar among the dynactin; and CM2 can associate with PCNT (Fig. 8H). We found three types of knockout cells. Furthermore, split NuMA occured at that ∼5% of mock-treated and ∼50% of RNAi-treated cells induced similar frequencies in those cells (∼32 to ∼37%) (Fig. 8F). When abnormal spindles with split NuMA. Introduction of myc–Cep215 the three proteins were simultaneously depleted (triple RNAi), a into Cep215 RNAi cells reduced the number of cells with abnormal significantly increased number of cells had mitotic spindles spindles to ∼13% (Fig. 8G). Expression of myc–Cep215 lacking consisting of highly randomized MT arrays and NuMA 215N rescued the phenotype. However, centrosome–pole separation distribution (>70%; Fig. 8E,F). The additive effect of Cep215, was still seen in almost 33% of mitotic cells, which is statistically Cep192 and AurA–pAurA on NuMA dispersion implies that these significant (P<0.05). Besides Δ215N, other deletion constructs three molecules function as a protein complex to coalesce NuMA at (ΔCM1, Δ215M and ΔCM2) also partially restored NuMA each spindle pole. coalescence at strikingly similar efficiencies (30–35%) to that of We next asked which domains of Cep215 are responsible for Δ215N. Because each domain binds the specific partner required for NuMA coalescence. A series of rescue constructs lacking one of four tethering the ends of MTs to the PCM (Fig. 8H), the role of Cep215 in domains of Cep215 (ΔCM1, Δ215N, Δ215M, ΔCM2) were prepared, centrosome–spindle pole connection is likely achieved by the and they were introduced into cells from which endogenous Cep215 collaboration of multiple binding sites spanning the entire length of was depleted by RNAi. Each deleted domain is capable of binding to the Cep215 sequence. Journal of Cell Science

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Fig. 8. See next page for legend.

DISCUSSION considered the only domain required for targeting Cep215 to The 215N domain is a newly identified mitosis-specific interphase and mitotic centrosomes (Barr et al., 2010; Wang et al., centrosome-targeting domain of Cep215 that interacts with 2010). CM2 interacts with PCNT (Buchman et al., 2010; Wang Cep192 and pAurA. Cep215 appears to be recruited to the mitotic et al., 2010), and Cep215 and PCNT require each other to localize centrosome via multiple pathways. CM2 has previously been to the centrosome (Kim and Rhee, 2014). Here, we show that Journal of Cell Science

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Fig. 8. Mitotic phenotypes and NuMA dispersion in RNAi-treated CHO mouse (amino acids 1–435) and human (amino acids 1–580) can be cells. (A) Fluorescence staining of control siRNA-treated (mock; 1,1′) and at the interphase centrosome in MEFs derived from a Cep215 – ′ α Cep215-depleted cells (2 15 ) stained with anti- -tubulin (MT, green) and knockout mouse and HeLa cells, respectively. As summarized in either anti-Cep215 (1′–6′) or anti-NuMA (7′–15′) antibodies. Loss of Cep215 induces abnormal spindles with NuMA-containing ‘knots’ at various locations Fig. S3A, the mouse clone constructed by Barrera et al. corresponds (arrows). Scale bar: 10 µm. (B) Bar chart showing percentage of normal bipolar to the sequence of the CM1 and 215N-N2 regions. The human clone spindles and abnormal spindles, classified into four categories, induced in reported by Sukumaran et al. encodes CM1 (amino acids 1–165) mock and Cep215 RNAi cells. Data are presented as mean±s.e.m. plus an additional ∼410 amino acids, a region shorter than 215N percentages of n=3 (mock; 189 cells in total) and n=5 (Cep215 RNAi; 435 cells (amino acids 166–696) but longer than 215N-N1 (amino acids in total) independent experiments. (C,D) Examples of abnormal spindles with 166–508) (Fig. S3A). We confirmed that polypeptides from three split NuMA induced in cells depleted of Cep192 (C) or AurA (D). Cells were stained with anti-α-tubulin and anti-NuMA antibodies. (E) Fluorescence constructs, consisting of CM1 plus 215N, CM1 plus 215N-N1, or staining of cells depleted of Cep215, Cep192 and AurA simultaneously (triple CM1 plus 215N-N2, are targeted to the centrosome. In contrast to RNAi) with anti-α-tubulin (1,2) and either a mixture of anti-Cep215, anti- the previous two reports, however, they are found there only in Cep192 and anti-pAurA (1′) or anti-NuMA (2′) antibodies. Scale bar: 10 µm mitotic cells, and not in interphase cells. Although the question of (C–E). (F) Quantitative analysis of mitotic spindles with split NuMA induced in cell cycle dependency is still open, these results indicate the mock, Cep215 RNAi, Cep192 RNAi, AurA RNAi and triple RNAi cells. Data are presence of multiple centrosomal targeting domains of Cep215 in – presented as the mean±s.e.m. percentage from n=7 11 independent mammalian cells other than those of mice. experiments (859, 1662, 1265, 1375 and 1542 cells in total for mock, Cep215, Cep192, AurA and triple RNAi-treated samples, respectively). Statistical PCNT plays a major role in construction of the mitotic PCM significance was assessed by comparing the mean of each treated group with lattice in mammalian cells (Lawo et al., 2012). In contrast, a role for the control mean by performing one-tailed z-tests. ***P<0.001. (G) Quantitative the Drosophila Pericentrin-like protein (D-PLP) appears to be more analysis of mitotic spindles with split NuMA in Cep215-depleted cells rescued limited than PCNT, because its localization is restricted to the by sequences encoded by the indicated Cep215 constructs. Data are outermost region of centrosomes (Fu and Glover, 2012; Mennella – presented as the mean±s.e.m. percentage from n=4 9 independent et al., 2012). The impact of its loss-of-function mutation is also experiments (1072, 806, 804, 591, 986, 435 and 744 cells in total for mock, none, full, ΔCM1, Δ215N, Δ215M and ΔCM2 samples, respectively). Statistical limited to weakening the outer structure of the PCM (Richens et al., significance was assessed by comparing the mean of each rescue group with 2015). Assembly of mature centrosomes in Drosophila embryos is the control mean by performing one-tailed z-tests. *P<0.05. (H) Schematic predominantly controlled by Cnn (Conduit et al., 2010), which diagram of Cep215 interactions with molecules functioning in the assembly of forms PCM scaffold-like micron-scale structures (Feng et al., 2017; mitotic spindles and spindle poles via different domains within the entire Citron et al., 2018). Cnn interacts with D-PLP at two distinctive sites – Cep215 sequence. HBR, HSET-binding region; Dy/Dnx, dynein dynactin (Lerit et al., 2015). In contrast, CM2 is the only PCNT-binding site complex; DBR, dynein–dynactin-binding region; γ, γ-tubulin. identified so far in Cep215 (Buchman et al., 2010; Wang et al., 2010). This difference between Cnn and Cep215 may be attributed to their distinct functions in PCM assembly. centrosome targeting of Cep215 via 215N is dependent on Cep192 AurA and Plk1 orchestrate the process of nuclear and cytoplasmic (Fig. 6E). Because no Cep215-binding domain(s) of Cep192 has as divisions by controlling a series of mitotic events, including yet been identified, it is not known whether centrosome targeting of centrosome maturation. Both Plk1 and AurA have long been Cep192 is independent or partially dependent on Cep215. In postulated to be essential for increasing the MT number associated addition, Cep192 and PCNT have been shown to be mutually with the mitotic centrosome (Lane and Nigg, 1996; Hannak et al., dependent (Gomez-Ferreria et al., 2007; Zhu et al., 2008). This 2001; Berdnik and Knoblich, 2002; Haren et al., 2009; Joukov et al., suggests that the mechanism of centrosome localization of these 2014). We showed previously that Cnn interacts with AurA (Terada three core proteins is under a complex three-way control. The et al., 2003). Here, we specify that phosphorylated AurA interacts contribution of individual pathways for targeting each molecule with Cep215 via 215N (Figs 3,4). The centrosomal targeting of 215N may vary among cell types, species, or even among the same cells depends on pAurA (Fig. 4), and MLN8237 interferes with pAurA under different physiological conditions. localization to the centrosome (Fig. 5A, panel 5′). It is thus reasonable It has recently been shown that Cnn oligomerizes through the that 215N is blocked from localizing to mitotic centrosomes in interaction of CM2 with the leucine zipper-containing sequence MLN8237-treated cells due to the absence of pAurA at the located in the middle of Cnn (Feng et al., 2017; Citron et al., 2018). centrosome (Fig. 5A, panel 8). The 215N domain is also efficiently Apparently, this central sequence of Cnn, termed PReM removed from the centrosome by treatment with BI2536 (Fig. 5A, (phosphoregulated multimerization domain; Conduit et al., 2014), panel 9). This is likely due to a loss of centrosomal pAurA caused is well-aligned to 215N of Cep215, in which the leucine zipper by Plk1 inhibition (Fig. 5A, panel 6′;DeLucaetal.,2006; motif is conserved (Feng et al., 2017). However, the probability that Hanisch et al., 2006). Plk1 is known to phosphorylate PCNT, which 215N is targeted to the mitotic centrosome via CM2 interaction is is essential for recruitment of several PCM proteins, including low, because 215N is still able to locate to mitotic centrosomes in Cep192, to the centrosome (Lee and Rhee, 2011). Because 215N cells from which CM2-containing endogenous Cep215 has been requires Cep192 to localize to the centrosome, Plk1 inactivation deleted (Fig. 1D, panel 7). Therefore, the centrosomal target of inhibits the centrosome targeting of 215N due to the absence 215N is, at least partially, independent of CM2. Is 215N, or a of Cep192. It is noteworthy that Cep215 also relies on PCNT to domain functionally equivalent to 215N, conserved among Cnn localize to the centrosome. However, this does not require Plk1 family members? Although fluorescence signals at the centrosome phosphorylation of PCNT (Lee and Rhee, 2011). This is consistent are weak, Cep215 or Cnn devoid of CM2 can localize to the spindle with our observation that the CM2-dependent centrosome targeting pole in human (Kim and Rhee, 2014), chicken (Barr et al., 2010) of Cep215 was not affected by BI2536 (Fig. 5A, panel 12 and and Drosophila (Feng et al., 2017) cells. These results indirectly Fig. 5B). Another possibility is a direct effect of Plk1 on 215N. support the presence of a centrosome-targeting domain other than It has recently been shown that Plk1 phosphorylates PReM, the CM2. More directly, Barrera et al. (2010) and Sukumaran et al. central region of Cnn, which is essential for organization of the Cnn

(2017) have reported that the N-terminal sequences of Cep215 in scaffold of fly centrosomes (Conduit et al., 2014). Because this Journal of Cell Science

11 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs240267. doi:10.1242/jcs.240267 central domain corresponds to 215N of Cep215 (Feng et al., 2017), Pelletier, 2017). It is thus plausible that CM1 participates in anchoring BI2536 may inhibit 215N recruitment to the mitotic centrosome the MT ends at the centrosome along with other domains. It is likely by interfering with oligomerization of Cep215 and assembly of that Cep215 acts to connect the mitotic centrosome to each spindle PCM scaffolds. pole by serving as a platform for the molecules involved in In mitotic cells, 215N bound to pAurA and Cep192 (Fig. 3C, centrosome maturation and bipolar spindle formation. Because Fig. 6C), and Cep192 is known to interact with both AurA and pAurA individual domains are important for the centrosome–spindle pole (Joukov et al., 2014). It is thus highly probable that Cep215 associates connection, it is reasonable to detect the same phenotype of with AurA indirectly via Cep192. However, Joukov et al. reported that centrosome–spindle pole separation in cells depleted of each Cep192, but not Cep215, is pulled down in frog extracts with beads Cep215 domain (Fig. 8G; Barr et al., 2010; Chavali et al., 2016). A coated with anti-AurA antibodies. This raises the possibility that co- lack of structural integrity of the mitotic centrosome and spindle poles precipitation of Cep215 with anti-AurA-antibody-coated beads was may disrupt centrosomal cohesion, split centrioles and displace more limited than that of Cep192 with anti-AurA beads, although it is centrioles from the PCM (Lucas and Raff, 2007; Barrera et al., 2010), unknown what percentage of beads were covered with phosphorylated all of which have been identified as unique phenotypes of Cep215 AurA. It is possible that the amount of Cep215 pulled down by beads and Cnn knockout cells. may have been below the detection level. Identification of the Cep192/ We probed the minus end of spindle MTs with NuMA, and pAurA-binding domain of Cep215 implies that Cep215 is a core Cep215 interacts with NuMA indirectly through MTs–γ-tubulin component of the signaling cascade organized by Cep192 to induce and dynein–dynactin. Haren and Merdes (2002) reported that centrosome maturation described by Joukov et al. (2014). AurA and ectopic NuMA expression induces multiple spindle poles. Plk1 are bound to Cep192 and transported to the centrosome in a Overexpression of NuMA may create a situation similar to PCNT-dependent manner, where AurA becomes activated by Cep215 depletion in cells where excess NuMA fails to tether into autophosphorylation at T288. Active AurA (pAurA) phosphorylates the centrosome due to the paucity of centrosomal Cep215. It would Plk1, which in turn causes phosphorylation of Cep192 to generate be interesting to know whether there are specific Cep215 domains multiple attachment sites for γ-TuRCs. Delivering the Cep192– that directly interact with NuMA to connect the MT minus end to kinases complex to centrosomes is achieved by Plk1-phosphorylated the mitotic centrosome. PCNT (Lee and Rhee, 2011). To the best of our knowledge, however, no evidence of direct binding of Cep192 and PCNT has been MATERIALS AND METHODS presented. Rather negative/almost negative immunoprecipitation was Cell culture, synchronization, and drug treatment reported between the Cep192 homolog DSpd-2 and the PCNT CHO and human (RPE1, U2OS, HeLa and HEK293T) cells were cultured homolog D-PLP in fly embryos (Conduit et al., 2010). It is thus in Ham’s F-10 and DMEM-GlutaMAX (Gibco) medium containing possible that Cep215 plays a role in carrying Cep192 to the 10% FBS and antibiotics. GFP tagged CETN-expressing CHO, RPE1 centrosome, either independently or in collaboration with PCNT, via and HeLa cells were described previously (Ohta et al., 2002; Steere its 215N domain for binding Cep192 and its CM2 domain for binding et al., 2012). For synchronization of mitotic cells, CHO cells were treated PCNT. Upon delivery to the mitotic centrosome, both Cep192 and with RO3306 (Alexis Biochemicals) for 5 h at a final concentration of pAurA become firmly attached to the PCM lattice through Cep215/ 10 µM, followed by incubation with fresh F-10 medium for 20–40 min to – prepare cells at different mitotic stages. To inhibit AurA and Plk1, CM2 PCNT interactions. Furthermore, assembly of Cep215 scaffolds – – promoted by Plk1 may facilitate recruitment of the Cep192–kinase MLN8237 (0.5 1.0 µM; Selleckchem) and BI2563 (0.1 1.0 µM; Axon Medchem) were added to cells at 3.5 h after incubation with RO3306 complex to the expanding PCM. Subsequent accumulation of active γ and further cultured for an additional 1.5 h. After washing out RO3306, AurA creates the -tubulin binding sites on Cep192. The 215N cells were cultured for 30–60 min in the presence of kinase inhibitors domain and pAurA are co-dependent on their localization at the before fixation. centrosome. This suggests that interaction of AurA with 215N may initiate and/or promote AurA phosphorylation at the centrosome. Plasmid preparation, RNAi and transfection Alternatively, 215N ensures preferential localization of Mouse cDNA encoding full-length Cep215 (GenBank accession: phosphorylated AurA over non-phosphorylated AurA at the mitotic AK129411) and human Flag-tagged Cep192 (NP_115518) were obtained centrosome. from Timothy Megraw (Florida State University, Tallahassee, FL) and We propose that the function of Cep215 in connecting the mitotic Laurence Pellitier (University of Toronto, Canada), respectively. Myc-tagged centrosome and spindle poles is achieved by collaborative efforts of full-coding and deletion constructs of Cep215 were generated by PCR amplification and subcloned into an N-terminally 3×myc-tagged pCS2 vector individual subdomains of Cep215 through their ability to interact obtained from Jeffrey R. Miller, Estrella Mountain Community College, with different molecules important for assembly of functional Avondale, AZ. For generation of individual constructs, we used the following spindles and spindle poles (Fig. 8H). MTs become massively internal restriction sites and primers specific to each nucleotide position: full associated with mitotic centrosomes as a result of increased MT- length (1–5,466), CM1 (1–495: XbaI), 215N (495: XbaI-2089: NheI), 215M nucleating activity of the centrosome. This activity is primarily (2089: NheI-4417: XhoI), CM2 (4417: XhoI-5466), 215N-N1 (495: XbaI- achieved by Cep192 and pAurA (Joukov et al., 2014), to which 1524: XhoI), 215N-N2 (495: XbaI-1305), 215N-N3 (603–1305), 215N-C Cep215 binds via 215N. Acentrosomal MTs are transported by motor (1305–2089: NheI), CM1+215N (1–2089: NheI), CM1+215N-N1 (1–1524: proteins, and their minus ends are coalesced to focus on the XhoI), CM1+215N-N2 (1–1305), ΔCM1 (495: XbaI-5466), Δ215N (1–495: centrosome at each spindle pole. Cep215 is able to interact with XbaI fused with 2089: NheI-5466), Δ215M (1–2089: NheI fused with 4417: Δ – dynein–dynactin and HSET, and thus is likely to anchor acentrosomal XhoI-5466), CM2 (1 4417: NheI), 215N/CM2 (495: XbaI-2089: NheI fused with 4417: NheI-5466), Δ215N/ΔCM2 (1–495: XbaI fused with 2089: MT ends to the centrosome via its binding to the motor proteins. NheI-4417: XhoI). cDNA encoding human AurA (GenBank accession: NM_ Bringing the two types of MTs close together, Cep215 next connects 001323305) was cloned as previously described (Terada et al., 2003) and all MT ends tightly to the PCM lattice by interaction with PCNT via ligated into a GFP-tagged pCS2 vector (Jeffrey R. Miller). The non- CM2. CM1 binds to γ-tubulin, which allows Cep215 to associate with phosphorylatable AurA mutant (T288A) was generated using the protocol of γ-tubulin and γ-TuRCs located at the minus ends of both centrosomal DpnI mediated site-directed mutagenesis (Steere et al., 2012). For RNAi, we and acentrosomal MTs (Meunier and Vernos, 2016; Prosser and applied the following species-specific siRNAs: Cep215 (C-terminus Journal of Cell Science

12 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs240267. doi:10.1242/jcs.240267 specific), nucleotide positions 3417–3441, 5′-GGACCAUAUUGAUGAA- Microscopy and statistical analysis of fluorescence intensity GAAGAGAGG-3′; Cep215 (N-terminus specific), nucleotide positions at mitotic centrosomes 609–627, 5′-GGACAGACUGAUUGAGGAG-3′; Cep192, nucleotide Microscopic observations were carried out using a Nikon Eclipse microscope positions 7160–7178, 5′-CUAAAGAGCCUCACAUGAA-3′;AurA, with a 100× oil immersion objective (NA 1.4) and a Photometrics CoolSNAP nucleotide positions 164–182, 5′-AGAAAGCUGUCUCAGGUCA-3′. camera. Image slices (0.2 µm) were merged using the SlideBook 4.1 program. For mock transfection, control siRNA (sc-3707, Santa Cruz Biotechn- Some images were processed through deconvolution using the program ology) was used. included in the SlideBook software. All images were exported to Adobe For transfection, cells were seeded on coverslips in a 12-well plate 1 d Photoshop (CS5) to acquire 8-bit files. before and treated with plasmid DNA and/or siRNA using Lipofectamine To measure the fluorescence intensity of the centrosomal proteins/truncated for CHO and HEK293T cells, and Lipofectamine 2000 for other types of Cep215 at spindle poles (Fig. 2C, Fig. 5B and Fig. 7B), immunostained cells human cells, as previously described (Ohta et al., 2002; Steere et al., were kept in the dark at −20°C for several days to ensure stabilization and 2012). For expression of truncated Cep215 polypeptides, plasmid DNAs equilibration of the mounting medium. Images of individual spindle poles were reduced to 20–50% (0.2–0.25 µg of 215N DNA and 0.4 µg of were captured under identical exposure conditions. During measurements of other DNA clones per well of a 12-well plate) of the normal amount γ-tubulin shown in Fig. 7B, we observed that depletion of Cep192 and pAurA (0.83 µg per well). by RNAi was less efficient than depletion of Cep215. Cells that were not obviously depleted of Cep192 were eliminated from quantification of Immunofluorescence staining fluorescence intensity. After subtraction of background fluorescence, Cells cultured on coverslips were fixed with methanol at −20°C. After fluorescence intensity was quantified in arbitrary units (a.u.) using the rehydration in phosphate-buffered saline (PBS) containing 0.05% Tween-20, ImageJ/Fiji image processing program, and individual data points were plotted cells were incubated for 30 min at 37°C with the following primary using the GraphPad Prism software. The mean and s.e.m. were calculated and antibodies: two anti-Cep215 pAbs raised against the mouse Cep215 sequence are indicated as long and short bars in each frequency distribution. The signal (amino acids 24–278; a gift from Timothy Megraw) and the human Cep215 intensity of individual Cep215 domains was lower than that of the full-coding sequence at amino acids 1307–1382 (HPA035820, Sigma-Aldrich), anti- sequence, suggesting that no inhibitory sequences are included in Cep215 Cep192 pAb (a gift from Laurence Pelletier), anti-AurA mAb (35C1, (Fig. 2C). In addition, treatment with kinase inhibitors and individual depletion Invitrogen), anti-pAurA pAb at T288 (ab83968, Abcam Biotechnology; of Cep215, Cep192 and AurA were reported to reduce the fluorescence #3091, Cell Signaling Technology), anti-NuMA (pAb, Maekawa and intensity of γ-tubulin at spindle poles (Figs 5B and 7B). Because a one-tailed Kuriyama, 1993; human auto-antibodies, Maekawa et al., 1991), anti-γ- z-test has more statistical power than a two-tailed z-test against the alternative tubulin mAb (Sigma-Aldrich, clone GTU-88), anti-PCNT (pAb, PRB-432C, hypothesis that treatments decrease signal intensity, we performed one-tailed Covance; human auto-antibodies, a gift from Stephen Doxsey, University of z-tests for assessment of statistical significance by comparing the mean of each Massachusetts Medical School, Worcester, MA), anti-Cep135 (mouse pAb treated group with the control mean. and rabbit pAb, Ohta et al., 2002), anti-HSET mAb (Kuriyama et al., 1995), anti-α-tubulin mAb (Sigma-Aldrich, clone B-5-1-2), anti-phospho-histone Acknowledgements H3 at Ser10 (pAb, #06-570, Upstate Biotechnology; mAb, #9706, Cell We thank Drs T. Megraw and L. Pelletier for sharing Cep215 and Cep192 reagents. Signaling Technology), anti-c-Myc (mAb, 9E10, Santa Cruz Biotechnology; pAb, CM-100, Gramsch Laboratories), anti-Flag mAb (M2, Sigma-Aldrich), Competing interests anti-HA mAb (12CA5, Roche Diagnostics) and anti-GFP pAbs (sc-8334, The authors declare no competing or financial interests. Santa Cruz Biotechnology; #8363, Clontech Laboratories, a gift from Ken- Ichi Takemaru, Stony Brook University, Stony Brook, NY). Primary Author contributions antibodies were used at a dilution of 1:300 to 1:5000. Fluorescein- Conceptualization: R.K.; Methodology: R.K., C.R.F.; Software: R.K., C.R.F.; Validation: R.K., C.R.F.; Formal analysis: R.K., C.R.F.; Investigation: R.K., C.R.F.; conjugated anti-mouse IgG plus IgM and Texas Red-conjugated anti-rabbit Resources: R.K., C.R.F.; Data curation: R.K., C.R.F.; Writing - original draft: R.K.; IgG antibodies were used as the secondary antibodies (Jackson Writing - review & editing: R.K., C.R.F.; Visualization: R.K.; Supervision: R.K.; ImmunoResearch). For triple staining with GFP, we used Cy3- and Project administration: R.K.; Funding acquisition: R.K. Cy5-conjugated anti-mouse and anti-rabbit secondary antibodies (Molecular Probes). Funding This work was supported by a grant to R.K. from the National Science Foundation Immunoprecipitation and western blotting (MCB1140033). Immunoprecipitation was carried out as reported previously (Steere et al., 2012). Briefly, transfected HEK293T cells with Flag–Cep192, myc–215N, Supplementary information GFP–AurA, and GFP–mutant AurA (T288A) were washed three times with Supplementary information available online at PBS and collected into ice-cold lysis buffer (20 mM Tris-HCl, pH 7.2, https://jcs.biologists.org/lookup/doi/10.1242/jcs.240267.supplemental 135 mM NaCl, 1.5 mM MgCl , 1 mM EGTA, 1% Triton X-100 and 10% 2 Peer review history glycerol) containing 1 mM Na3VO4 and a mixture of protease inhibitors μ μ – μ The peer review history is available online at (20 M leupeptin, 0.4 M chymostatin, 1 2.5 mM PMSF and 15 M https://jcs.biologists.org/lookup/doi/10.1242/jcs.240267.reviewer-comments.pdf aprotinin; Sigma-Aldrich). To enrich mitotic populations, cells were treated with 0.25 µg/ml nocodazole for 6–10.5 h. After centrifugation at 13,000 g for 10 min at 4°C, the supernatants were co-immunoprecipitated with anti-myc References and anti-Flag mAbs. 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Primary antibodies used centrosome maturation and actin-dependent asymmetric protein localization were: anti-Cep215 pAb (a gift from Timothy Megraw; 1:10,000), anti- during mitosis. Curr. Biol. 12, 640-647. doi:10.1016/S0960-9822(02)00766-2 Buchman, J. J., Tseng, H.-C., Zhou, Y., Frank, C. L., Xie, Z. and Tsai, L.-H. (2010). Cep192 pAb (a gift from Laurence Pelletier; 1:500), anti-AurA mAb Cdk5rap2 interacts with pericentrin to maintain the neural progenitor pool in the (35C1, Invitrogen; 1:3000), anti-pAurA pAb (ab83968, Abcam; developing neocortex. Neuron 55, 386-402. doi:10.1016/j.neuron.2010.03.036 1:1000), anti-GFP pAb (sc-8334, Santa Cruz Biotechnology; 1:1000) Chavali, P. L., Chandrasekaran, G., Barr, A. R., Tátrai, P., Taylor, C., and anti-c-Myc mAb (9E10, Santa Cruz Biotechnology; 1:1000). Papachristou, E. K., Woods, C. G., Chavali, S. and Gergely, F. (2016). A Journal of Cell Science

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