Kendrin Is a Novel Substrate for Separase Involved in the Licensing of Centriole Duplication

Current Biology 22, 915–921, May 22, 2012 ª2012 Elsevier Ltd All rights reserved

DOI 10.1016/j.cub.2012.03.048

Report Kendrin Is a Novel Substrate for Separase Involved in the Licensing of Centriole Duplication Kazuhiko Matsuo,1,2 Keita Ohsumi,4 Mari Iwabuchi,4 Toshio Kawamata,5 Yoshitaka Ono,2,3,6 and Mikiko Takahashi1,2,3,* 1Faculty of Pharmaceutical Science, Teikyo Heisei University, Ichihara 290-0193, Japan 2Graduate School of Science 3Biosignal Research Center Kobe University, Kobe 657-8501, Japan 4Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan 5Graduate School of Health Science, Kobe University, Kobe 654-0142, Japan

Summary The centrosome, consisting of a pair of centrioles surrounded by pericentriolar material, directs the formation of bipolar spindles during mitosis. Aberrant centrosome number can promote chromosome instability, which is implicated in tumorigenesis [1, 2]. Thus, centrosome duplication needs to be tightly regulated to occur only once per cell cycle. Separase, a cysteine protease that triggers sister chromatid separation [3], is involved in centriole disengagement, which licenses centrosomes for the next round of duplication [4–8]. However, at least two questions remain unsolved: what is the substrate relevant to the disengagement, and how does separase, activated at anaphase onset, act on the disengagement that occurs during late mitosis [6, 7, 9, 10]. Here, we show that kendrin, also named pericentrin, is cleaved by activated separase at a consensus site in vivo and in vitro, and this leads to the delayed release of kendrin from the centrosome later in mitosis. Furthermore, we demonstrate that expression of a noncleavable kendrin mutant suppresses centriole disengagement and subsequent centriole duplication. Based on these results, we propose that kendrin is a novel and crucial substrate for separase at the centrosome, protecting the engaged centrioles from premature disengagement and thereby blocking reduplication until the cell passes through mitosis. Results and Discussion Kendrin Is Specifically Cleaved at a Consensus Site for Separase during Mitosis Kendrin is a giant coiled-coil protein localized in the pericentriolar material as an integral component [11] where it serves as a multifunctional scaffold by anchoring numerous proteins and protein complexes [12–14]. Our anti-kendrin antibody detected two discrete bands on immunoblots: a slowmigrating band corresponding to the full-length protein and a fast-migrating band (Figure 1A). Both bands decreased after treatment with kendrin small interfering RNA (siRNA)

6Deceased on August 2, 2011 *Correspondence: [email protected]

(Figure 1A), confirming that they were translated from kendrin messenger RNA. Furthermore, the intensity of the two bands fluctuated during the cell cycle (Figure 1B). The full-length band was detected mainly from the G1/S boundary through early mitosis (at 0–8 hr in Figure 1B and Figure S1A available online), whereas the fast-migrating band became prominent from late mitosis through early G1 (at 10–12 hr in Figure 1B and Figure S1A). In addition, the full-length band was shifted by phosphorylation when arrested in early mitosis by nocodazole treatment (Figure S1B). To examine whether the fast-migrating band was generated by limited proteolysis, we expressed full-length kendrin double-tagged with hemagglutinin (HA) and Flag at the N and C termini, respectively (Figure 1C), in HeLa cells, and collected the cells at four time points to detect typical changes in the kendrin immunoreactive bands: (1) asynchronous, (2) G1/S, (3) prometaphase, and (4) late mitosis (Figure 1D). Anti-HA antibody detected all the bands detected by the anti-kendrin antibody, whereas anti-Flag antibody detected only the full-length band, indicating that the fast-migrating band lacked the C terminus and was thus generated by limited proteolysis of the C-terminal region. By using C-terminal truncated mutants and internal deletion mutants, we narrowed the potential cleavage site to the region between amino acids 2136 and 2427 (see Figures S1C–S1E). We then searched for protease recognition sites in this region and identified a consensus sequence for separase cleavage (SxExxR) [15] at amino acids 2226–2231 (Figure 1E), which is well conserved among various vertebrate orthologs of kendrin. We generated a mutant of this potential cleavage site from SxExxR to SxRxxE (E2228R/R2231E), named kendrinERRE, and examined its cleavage during late mitosis. Immunoblotting with anti-HA antibody showed that HA-kendrinERRE-Flag was not cleaved (Figure 1F, middle). However, the fast-migrating band derived from endogenous kendrin was still detected (Figure 1F, left), confirming that separase activity was not suppressed in these cells. Furthermore, the mutant localized to the centrosomes, as does the wild-type (WT) (Figure 1G) excluding the possibility that kendrinERRE escaped cleavage via mislocalization. These results indicate that kendrin is cleaved at a consensus site for separase at the centrosome. Kendrin Is a Novel Centrosomal Substrate for Separase We next asked whether kendrin cleavage is dependent on separase by suppressing separase expression using RNA interference techniques. As shown in Figure 2A, the amount of the fast-migrating band decreased in parallel with the decrease in separase protein (both the full-length and autocleavage products), indicating that cleavage of kendrin was dependent on the presence of separase. We next examined the effect of separase coexpression on the change in the kendrin bands (Figure 2B). Catalytically inactive separase (separaseCA) [16] had no effect on the fluctuation of kendrinWT bands. By contrast, coexpression of separasewt with kendrinWT resulted in an increase in intensity of the fast-migrating band, even at prometaphase (Figure 2B, condition 3), when kendrinwt usually remains intact (see

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Figure 1. Kendrin Is Specifically Cleaved at a Consensus Site for Separase during Mitosis (A) HeLa cells treated with control or kendrin siRNA (#1 and #2) were immunoblotted with anti-kendrin antibody. The bands labeled by black and white arrowheads indicate full-length kendrin and the fast-migrating band, respectively. The filter was reblotted with antip150Glued antibody as a loading control. (B) HeLa cells were arrested at the G1/S boundary by double thymidine treatment and subsequently released in fresh medium for the indicated time periods. Cells lysates were then analyzed by immunoblotting with antibodies against kendrin and the indicated proteins. (C) Schematic representation of kendrin and its mutant. Mutated residues (E2229 and R2231) are indicated by asterisks. (D) HeLa cells expressing HA-kendrinWT-Flag were synchronized at various time points and the cell lysates were analyzed by immunoblotting with the indicated antibodies. Numbers 1–4 correspond to the conditions described above the blots. The full-length and cleaved-kendrin bands are indicated by black and white arrowheads, respectively. (E) Sequence homology around the potential cleavage sites for separase among kendrin orthologs from various species (Homo sapiens: NP_ 006022.3, Canis lupus familiaris: XP_548735.2, Mus musculus: NP_032813.3, Gallus gallus: XP_421895.2, Danio rerio: XP_699814.3). The alignment was obtained from HomoloGene (NCBI). The position of the consensus sequence is boxed, with an arrow showing the potential cleavage site. (F) HeLa cells expressing HA-kendrinERRE-Flag were synchronized under the same conditions as in (D), and analyzed by immunoblotting. (G) HeLa cells expressing HA-kendrinWT or HA-kendrinERRE were fixed and double-stained with anti-HA and anti-C-Nap1. Centrosomes are indicated by arrows. Scale bar represents 10 mm. Asterisks in (D) and (F) indicate crossreactive bands. See also Figure S1.

Figure 1D, condition 3). We next performed in vitro cleavage assay using the purified proteins. Active and inactive mutants of separase, separaseSA [16], and separaseCA, respectively, were prepared by treatment with a mitotic extract of Xenopus eggs [17] (Figure 2Ca) as described in Supplemental Experimental Procedures [18]. Figure 2Cb shows that the fast-migrating band was generated by separaseSA, but not by separaseCA, indicating that activated separase can cleave kendrin in vitro at the same site identified in vivo. Furthermore, the timing of endogenous kendrin cleavage (1.5 hr after release from nocodazole arrest) coincided with the appearance of the autocleavage product of separase (Figure 2D), an indicator of endogenous separase activation [16]. Taken together, these results indicate that kendrin is cleaved at the consensus site (R2231) by activated separase in vivo as well as in vitro, and thus is a novel substrate for separase at the centrosome.

Cleaved Kendrin Is Released from Centrosomes after a Short Lag We next followed the fate of kendrin after cleavage. Although we tried to detect the C-terminal cleavage product generated from HA-kendrinWT-Flag, it could not be detected in either detergent-soluble or -insoluble fractions (Figure S2A). This fragment may be degraded soon after the cleavage because the newly generated N-terminal lysine is a destabilizing residue according to the N-end rule [19]. On the other hand, HA-kendrin1-2231, corresponding to the N-terminal cleavage product, lacks the C-terminal centrosome-targeting domain [20] and, therefore, localized in the cytosol (Figure 3A). Because the epitope recognized by the anti-kendrin antibody lies within kendrin1-2231 (Figure 1C), the immunofluorescence of endogenous kendrin may be dissociated from the centrosomes after cleavage. As shown in Figure 3B and Figure S2B, the kendrin immunofluorescence at the centrosome increased until metaphase. Surprisingly, however, the signal

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Figure 2. Kendrin Is a Novel Substrate for Separase (A) HeLa cells treated with control or separase siRNA (iSep1-3) were analyzed by immunoblotting with anti-kendrin and anti-separase antibodies. The full-length and cleavage products of kendrin or separase are indicated by arrowheads. The filter (anti-kendrin) was reblotted with anti-p150Glued as a loading control. The anti-cyclin B1 blot revealed that suppression of kendrin cleavage was not caused by cell cycle arrest at early mitosis. (B) HeLa cells expressing HA-kendrinWT (WT) or HA-kendrinERRE (ERRE) in combination with Flag-tagged WT or an inactive mutant (CA) of separase were synchronized as in Figure 1D, and the cell lysates were analyzed by immunoblotting. Black and white arrowheads indicate anti-HA- and anti-Flag-specific bands, respectively. The intensity of the fast-migrating bands in the anti-HA blot was quantified as described in Supplemental Experimental Procedures. The cell-cycle progression of these cells was normal, as detected by phosphorylation of MPM2 epitopes. WCL, whole cell lysates. Asterisks indicate cross-reactive bands. (Ca) Flag-tagged separaseSA and separaseCA expressed in 293T cells were immunoprecipitated with anti-Flag (IP), incubated with mitotic extract of Xenopus eggs (M.E.) to degrade securin, and then eluted with 3 3 Flag peptide as described in Supplemental Experimental Procedures. The samples were immunoblotted with antiFlag. The full-length and cleavage products of separase are indicated by arrowheads. (Cb) Purified HA-kendrin-Flag was incubated with purified Flag-separaseSA or Flag-separaseCA for 0 and 90 min, and kendrin cleavage was analyzed by immunoblotting with anti-kendrin. The full-length and cleavage products of kendrin are indicated by arrowheads. (D) Time course of kendrin cleavage and separase activation during mitosis. HeLa cells synchronized at prometaphase were released into fresh medium for the indicated time periods. Cell lysates were then analyzed by immunoblotting for kendrin and indicated proteins. The anti-separase antibody detected the full-length 220 kDa protein and the 65 kDa autocleavage product, an indicator of separase activation. AS, asynchronous culture.

remained strong at the spindle poles in anaphase, and then decreased from telophase to cytokinesis. Kendrin is a coiled-coil protein that can form a homodimer or an oligomer (Figure S2C); thus, the N-terminal cleavage product might be retained at the centrosomes via interactions with either the C-terminal cleavage product or uncleaved kendrin. The interaction of kendrin1-2231 with full-length kendrin but not with kendrin2232-3245 was detected by immunoprecipitation (Figure 3C) and by immunofluorescence (Figure 3D), in which HA-kendrin1-2231 was recruited to the centrosome only when Flag-kendrinWT was coexpressed (compare with Figure 3A). These results suggest that kendrin1-2231 remains attached to the centrosome until late mitosis via interaction with as-yet uncleaved kendrin. Centriole Disengagement Coincides with a Decrease in the Kendrin Signal at the Centrosome Despite its dependence on separase activation, centriole disengagement occurs from late mitosis to G1 [9, 10]. Therefore, we hypothesized that this time lag may be attributed to a delay in the release of cleaved kendrin from the centrosomes. We compared the time course of these events using immunofluorescence studies. Engaged and disengaged centrioles can be distinguished by the number of C-Nap1 (free proximal-end marker of centriole) [21] and centrin2

(distal-end component of centriole) [22] foci: engaged centriole pair, 1:2; disengaged centriole pair, 2:2 [6] (see Figure S4A). Consistent with previous observations [9, 10], engaged centriole pairs were still detected at telophase and early cytokinesis, whereas disengaged centriole pairs were detected only at late cytokinesis (Figure S2D, arrows). Disengaged centrioles (identified by the presence of 2 C-Nap1 foci/centrosome) were associated with faint kendrin signals (Figure 3E, arrows), suggesting that centriole disengagement occurs when kendrin is dissociated to a certain extent. Next, to examine the relationship between the amount of kendrin at the centrosome and centriole disengagement, kendrin expression was suppressed by RNA interference. Figure S3A shows that the frequency of disengaged centrioles and multipolar spindles increased at prometaphase in kendrin-suppressed cells; the latter may be a consequence of centriole overduplication and/or splitting of prematurely disengaged centrioles. These phenotypes agree with those observed in cells derived from patients with Majewski/microcephalic osteodysplastic primordial dwarfism type II, recently identified as harboring mutations in the kendrin gene, PCNT [23, 24]. These results suggest that loss of kendrin from engaged centrioles (to a certain extent) causes centriole disengagement, which may account for the time lag between separase activation and disengagement.

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Figure 3. Kendrin Is Released from Centrosomes upon Limited Cleavage by Separase after a Short Time Lag (A) Subcellular localization of kendrin1-2231. HeLa cells transiently expressing HA-kendrin1-2231 were double-stained with anti-HA and anti-C-Nap1. Centrosomes are indicated by arrows. (B) Changes in kendrin immunofluorescence during the cell cycle. HeLa cells were triple-stained for kendrin (red, upper panel), a-tubulin (green), and DNA (blue). Z series images taken across the cells were projected onto a single view. The centrosomes of the cells at telophase and cytokinesis are indicated by arrows. (C) Interaction between kendrin fragments. Cos7 cells expressing the indicated combinations of the kendrin fragments were immunoprecipitated with antiFlag or anti-HA antibodies and analyzed by immunoblotting. (D) HeLa cells coexpressing HA-kendrin1-2231 and Flag-kendrinwt were triple-stained with anti-HA, anti-Flag, and DAPI. Centrosomes are indicated by arrows. (E) Relationship between kendrin immunofluorescence intensity and the number of C-Nap1 foci was examined at late mitosis. Arrows indicate disengaged centrioles (2 C-Nap1 foci/centrosome). Scale bars in (A), (B), (D), and (E) represent 10 mm. See also Figure S2.

Expression of the Noncleavable Kendrin, KendrinERRE, Suppresses Centriole Disengagement and Subsequent Duplication To ascertain whether kendrin is a crucial substrate in the separase-dependent disengagement process, we examined the effects of noncleavable mutant (kendrinERRE) expression on centriole disengagement. U2-OS cells stably expressing centrin2-GFP were transfected with mCherry-tagged g-tubulin, kendrinWT, or kendrinERRE expression plasmid, and then fixed at the G1/S phase for immunostaining. About 70% of the control cells (Figure 4A), g-tubulin-expressing cells (Figure 4B), or kendrinWT-expressing cells (Figure 4C) contained one disengaged centriole pair per cell (Figure 4E). By contrast, about 70% of the cells expressing kendrinERRE contained

one engaged centriole pair per cell (Figures 4D and 4E). These results indicate that centriole disengagement can be suppressed by expression of kendrinERRE. We then examined whether centriole duplication occurs in these cells at a later stage of the cell cycle. U2-OS cells arrested at the G1/S phase as described above were released for 8 hr to pass through S phase. Nearly 80% of the control cells (Figures 4F), g-tubulin-expressing cells (Figures 4G), or kendrinWT-expressing cells (Figures 4H) contained two engaged centriole pairs (Figure 4J), indicating that these cells underwent centriole duplication. By contrast, about 70% of the cells expressing kendrinERRE contained a single engaged centriole pair (Figures 4I and 4J), and fewer than 20% of the cells underwent centriole duplication (these

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Figure 4. Expression of a Noncleavable Mutant, KendrinERRE, Suppresses Centriole Disengagement and Subsequent Centriole Duplication (A–D and F–I) U2-OS cells stably expressing centrin2-GFP were nontransfected (A and F) or transfected with g-tubulin-mCherry (B and G), mCherrykendrinWT (C and H), or mCherry-kendrinERRE (D and I) expression plasmid. The cells were then synchronized at G1/S phase by double thymidine block (A–D) or released for a further 8 hr to pass through S phase (F–I). Nontransfected cells were immunostained for C-Nap1 and kendrin (A and F), whereas cells expressing mCherry-tagged proteins were stained for C-Nap1 (B–D and G–I). Centrosomes marked by squares are magnified and presented on the right. Dashed circles indicate the positions of the nuclei. C-Nap1 immunofluorescence and mCherry fluorescence are visualized as pseudocolors red and blue, respectively. Scale bar represents 5 mm. (E and J) Quantitative analysis of centriole configurations in the cells shown in (A)–(D) and (F)–(I), respectively. Error bars indicate the SD of triplicate experiments. At least 90 cells were scored per condition. Statistical significance was assessed by analysis of variance and Tukey-Kramer tests. **p < 0.001 versus normal and versus WT are indicated. (K) U2-OS cells stably expressing centrin2-EGFP were immunostained for C-Nap1 (blue) and kendrin (red). Localization of kendrin before (a) and after (b) centriole duplication was examined by taking z series sections. Images of a typical single z section are shown. Arrowheads indicate the location of centrin2 detected outside of the kendrin cylinder. Scale bar represents 1 mm. See also Figure S3.

cells may be derived from cells that possessed disengaged centriole pairs at the G1/S phase [Figures 4E]). The cells expressing either kendrinERRE or kendrinWT incorporated BrdU

under this condition (Figure S3B), excluding the possibility that the kendrinERRE-expressing cells were delayed in G1 phase.

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Taken together, these results indicate that expression of noncleavable kendrinERRE suppresses centriole disengagement and subsequent centriole duplication. What, then, is the role of kendrin in the centriole disengagement? One possibility is that kendrin is directly involved in the linkage between engaged centrioles. We carefully observed kendrin localization both before and after centriole duplication by collecting Z-series sections. Figure 4K shows typical images of a single Z-section. Before duplication (C-Nap1: centrin2 = 2:2), kendrin was detected as a ring and a pair of parallel lines (Figure 4Ka), which may correspond to a cross section and an axial section, respectively, of a hollow cylinder-like structure. C-Nap1 and centrin2 were detected near the opposite ends of the cylinder, suggesting that kendrin encircles the walls of disengaged centrioles. After duplication (C-Nap1: centrin2 = 2:4), kendrin was still detected as cylindrical structures (Figure 4Kb), maintaining a similar relationship with C-Nap1 and one of the two centrin2 foci. The other centrin2 focus was detected outside each kendrin cylinder (Figure 4Kb, arrows) where no break was observed. These results suggest that kendrin, encircling the walls of mother centrioles, may be involved in the linkage between mother and daughter centrioles. Consequently, cleavage and delayed release of kendrin may liberate daughter centrioles from mother centrioles, leading to centriole disengagement. In addition, an appreciable amount of kendrin was already recruited to disengaged centrioles at the G1/S boundary (Figure 4A), when centriole duplication initiates. It is therefore possible that kendrin could be involved in the assembly of new daughter centrioles in the canonical pathway, as is observed during de novo centriole generation in cells overexpressing kendrin/pericentrin [25]. Another possibility is that the cleavage and release of kendrin may lead to the dissociation of protein(s) involved in centriole engagement. CDK5RAP2, a centrosomal protein (defects in which cause autosomal recessive primary microcephaly [26]), is involved in the maintenance of engagement [27] and interacts with kendrin [28, 29]. We found that the intensity of the CDK5RAP2 signal at the centrosomes decreased at late mitosis concurrently with that of kendrin (our unpublished data). Kendrin and CDK5RAP2 have also been implicated in cohesion during interphase [14, 30, 31], suggesting that these two proteins contribute to the formation of both mother-daughter centriole linkage (i.e., engagement) and mother-mother centriole linkage (i.e., cohesion). On the basis of our results, we propose a model for the possible behavior of kendrin in the centriole cycle (Figure S4B). At early G1 phase, newly synthesized kendrin is recruited to the disengaged centrioles; then, the amount of kendrin gradually increases until early mitosis. At anaphase onset, kendrin is cleaved by activated separase and the resultant N-terminal fragment is retained at the centrosomes for a period of time through interaction with as-yet uncleaved kendrin. It is then released from the centrosomes. After, or at the same time as, release of the cleaved kendrin at late mitosis, the tight connection between engaged centrioles is relieved to yield the disengaged configuration, which is then ready for the next round of centriole duplication. There still remains a short time lag between the substantial decrease in kendrin signals (during telophase) and the appearance of two C-Nap1 foci (disengagement at late cytokinesis) (Figures 3B and 3E). One possibility is that the amount of kendrin required to maintain centriole engagement is relatively low; thus, disengagement occurs at late cytokinesis. Another possibility is that it reflects the time lag between the dissociation of the mother-daughter

connection (disengagement) and visualization of the two C-Nap1 foci. Recently, it was shown that cleavage of the cohesin ring is also required for centriole disengagement [32, 33]. The relationship between kendrin and the cohesin ring, which is transiently recruited to the centrosome during mitosis, in the regulation of centriole disengagement remains to be investigated. The timing of centriole disengagement must be tightly regulated to restrict centriole duplication to once per cell cycle. We identified the centrosomal protein kendrin as a novel substrate for separase in vertebrates. Expression of a noncleavable kendrin mutant suppressed centriole disengagement and subsequent centriole duplication. Furthermore, sustained association of the kendrin cleavage product with the centrosomes may account for the time lag between separase activation and disengagement. Kendrin may play a pivotal role in the licensing of centriole duplication by protecting the engaged centrioles from disengagement until late mitosis. Supplemental Information Supplemental Information includes four figures and Supplemental Experimental Procedures and can be found with this article online at doi:10. 1016/j.cub.2012.03.048. Acknowledgments We thank E.A. Nigg and J.L. Salisbury for antibodies against C-Nap1 and centrin2, respectively, and E.G. McGeer for critical reading of the manuscript. This work was supported in part by the ‘‘Global Center of Excellence’’ program of the Ministry of Education, Culture, Sports, Science and Technology, Japan, a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, Japan, and by a research grant from the Mitsubishi Foundation. Received: June 7, 2011 Revised: February 13, 2012 Accepted: March 16, 2012 Published online: April 26, 2012 References 1. Nigg, E.A., and Raff, J.W. (2009). Centrioles, centrosomes, and cilia in health and disease. Cell 139, 663–678. 2. Fukasawa, K. (2007). Oncogenes and tumour suppressors take on centrosomes. Nat. Rev. Cancer 7, 911–924. 3. Nasmyth, K., Peters, J.M., and Uhlmann, F. (2000). Splitting the chromosome: cutting the ties that bind sister chromatids. Science 288, 1379– 1385. 4. Wong, C., and Stearns, T. (2003). Centrosome number is controlled by a centrosome-intrinsic block to reduplication. Nat. Cell Biol. 5, 539–544. 5. Tsou, M.F., and Stearns, T. (2006). Controlling centrosome number: licenses and blocks. Curr. Opin. Cell Biol. 18, 74–78. 6. Tsou, M.F., and Stearns, T. (2006). Mechanism limiting centrosome duplication to once per cell cycle. Nature 442, 947–951. 7. Nigg, E.A. (2007). Centrosome duplication: of rules and licenses. Trends Cell Biol. 17, 215–221. 8. Tsou, M.F., Wang, W.J., George, K.A., Uryu, K., Stearns, T., and Jallepalli, P.V. (2009). Polo kinase and separase regulate the mitotic licensing of centriole duplication in human cells. Dev. Cell 17, 344–354. 9. Kuriyama, R., and Borisy, G.G. (1981). Centriole cycle in Chinese hamster ovary cells as determined by whole-mount electron microscopy. J. Cell Biol. 91, 814–821. 10. Piel, M., Nordberg, J., Euteneuer, U., and Bornens, M. (2001). Centrosome-dependent exit of cytokinesis in animal cells. Science 291, 1550–1553. 11. Doxsey, S.J., Stein, P., Evans, L., Calarco, P.D., and Kirschner, M. (1994). Pericentrin, a highly conserved centrosome protein involved in microtubule organization. Cell 76, 639–650. 12. Delaval, B., and Doxsey, S.J. (2010). Pericentrin in cellular function and disease. J. Cell Biol. 188, 181–190.

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13. Takahashi, M., Yamagiwa, A., Nishimura, T., Mukai, H., and Ono, Y. (2002). Centrosomal proteins CG-NAP and kendrin provide microtubule nucleation sites by anchoring gamma-tubulin ring complex. Mol. Biol. Cell 13, 3235–3245. 14. Matsuo, K., Nishimura, T., Hayakawa, A., Ono, Y., and Takahashi, M. (2010). Involvement of a centrosomal protein kendrin in the maintenance of centrosome cohesion by modulating Nek2A kinase activity. Biochem. Biophys. Res. Commun. 398, 217–223. 15. Hauf, S., Waizenegger, I.C., and Peters, J.M. (2001). Cohesin cleavage by separase required for anaphase and cytokinesis in human cells. Science 293, 1320–1323. 16. Stemmann, O., Zou, H., Gerber, S.A., Gygi, S.P., and Kirschner, M.W. (2001). Dual inhibition of sister chromatid separation at metaphase. Cell 107, 715–726. 17. Ohsumi, K., Katagiri, C., and Kishimoto, T. (1993). Chromosome condensation in Xenopus mitotic extracts without histone H1. Science 262, 2033–2035. 18. Kudo, N.R., Anger, M., Peters, A.H., Stemmann, O., Theussl, H.C., Helmhart, W., Kudo, H., Heyting, C., and Nasmyth, K. (2009). Role of cleavage by separase of the Rec8 kleisin subunit of cohesin during mammalian meiosis I. J. Cell Sci. 122, 2686–2698. 19. Varshavsky, A. (1996). The N-end rule: functions, mysteries, uses. Proc. Natl. Acad. Sci. USA 93, 12142–12149. 20. Gillingham, A.K., and Munro, S. (2000). The PACT domain, a conserved centrosomal targeting motif in the coiled-coil proteins AKAP450 and pericentrin. EMBO Rep. 1, 524–529. 21. Fry, A.M., Mayor, T., Meraldi, P., Stierhof, Y.D., Tanaka, K., and Nigg, E.A. (1998). C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2. J. Cell Biol. 141, 1563–1574. 22. Salisbury, J.L., Suino, K.M., Busby, R., and Springett, M. (2002). Centrin2 is required for centriole duplication in mammalian cells. Curr. Biol. 12, 1287–1292. 23. Rauch, A., Thiel, C.T., Schindler, D., Wick, U., Crow, Y.J., Ekici, A.B., van Essen, A.J., Goecke, T.O., Al-Gazali, L., Chrzanowska, K.H., et al. (2008). Mutations in the pericentrin (PCNT) gene cause primordial dwarfism. Science 319, 816–819. 24. Griffith, E., Walker, S., Martin, C.A., Vagnarelli, P., Stiff, T., Vernay, B., Al Sanna, N., Saggar, A., Hamel, B., Earnshaw, W.C., et al. (2008). Mutations in pericentrin cause Seckel syndrome with defective ATRdependent DNA damage signaling. Nat. Genet. 40, 232–236. 25. Loncarek, J., Hergert, P., Magidson, V., and Khodjakov, A. (2008). Control of daughter centriole formation by the pericentriolar material. Nat. Cell Biol. 10, 322–328. 26. Bond, J., Roberts, E., Springell, K., Lizarraga, S.B., Scott, S., Higgins, J., Hampshire, D.J., Morrison, E.E., Leal, G.F., Silva, E.O., et al. (2005). A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size. Nat. Genet. 37, 353–355. 27. Barrera, J.A., Kao, L.R., Hammer, R.E., Seemann, J., Fuchs, J.L., and Megraw, T.L. (2010). CDK5RAP2 regulates centriole engagement and cohesion in mice. Dev. Cell 18, 913–926. 28. Hubner, N.C., Bird, A.W., Cox, J., Splettstoesser, B., Bandilla, P., Poser, I., Hyman, A., and Mann, M. (2010). Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J. Cell Biol. 189, 739–754. 29. Buchman, J.J., Tseng, H.C., Zhou, Y., Frank, C.L., Xie, Z., and Tsai, L.H. (2010). Cdk5rap2 interacts with pericentrin to maintain the neural progenitor pool in the developing neocortex. Neuron 66, 386–402. 30. Jurczyk, A., Gromley, A., Redick, S., San Agustin, J., Witman, G., Pazour, G.J., Peters, D.J., and Doxsey, S. (2004). Pericentrin forms a complex with intraflagellar transport proteins and polycystin-2 and is required for primary cilia assembly. J. Cell Biol. 166, 637–643. 31. Graser, S., Stierhof, Y.D., and Nigg, E.A. (2007). Cep68 and Cep215 (Cdk5rap2) are required for centrosome cohesion. J. Cell Sci. 120, 4321–4331. 32. Scho¨ckel, L., Mo¨ckel, M., Mayer, B., Boos, D., and Stemmann, O. (2011). Cleavage of cohesin rings coordinates the separation of centrioles and chromatids. Nat. Cell Biol. 13, 966–972. 33. Wang, X., Yang, Y., Duan, Q., Jiang, N., Huang, Y., Darzynkiewicz, Z., and Dai, W. (2008). sSgo1, a major splice variant of Sgo1, functions in centriole cohesion where it is regulated by Plk1. Dev. Cell 14, 331–341.