A nucleolar protein RRS1 contributes to ... - Wiley Online Library

FEBS Letters 583 (2009) 1951–1956

journal homepage: www.FEBSLetters.org

A nucleolar protein RRS1 contributes to chromosome congression Arni E. Gambe a, Sachihiro Matsunaga a, Hideaki Takata b, Rika Ono-Maniwa a, Akiko Baba a, Susumu Uchiyama a, Kiichi Fukui a,* a b

Laboratory of Dynamic Cell Biology, Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, 565-0871 Osaka, Japan National Institute of Genetics, 1111 Yata, Mishima, 411-8540 Shizuoka, Japan

a r t i c l e

i n f o

Article history: Received 7 March 2009 Revised 12 May 2009 Accepted 15 May 2009 Available online 22 May 2009 Edited by Angel Nebreda Keywords: Nucleolar protein RRS1 Aurora B Sgo1 Centromeric cohesion Chromosome congression

a b s t r a c t We report here the functional analysis of human Regulator of Ribosome Synthesis 1 (RRS1) protein during mitosis. We demonstrate that RRS1 localizes in the nucleolus during interphase and is distributed at the chromosome periphery during mitosis. RNA interference experiments revealed that RRS1-depleted cells show abnormalities in chromosome alignment and spindle organization, which result in mitotic delay. RRS1 knockdown also perturbs the centromeric localization of Shugoshin 1 and results in premature separation of sister chromatids. Our results suggest that a nucleolar protein RRS1 contributes to chromosome congression. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Faithful chromosome congression requires the stable attachment of the chromosomes to microtubules to facilitate chromosome alignment by mitotic spindles [1]. Defects in kinetochore–microtubule attachments lead to the activation of the spindle checkpoint machinery to arrest cells in mitosis. A regulator of the spindle checkpoint, Aurora B, responds to tension changes caused by abnormal microtubule–kinetochore attachments [2]. Bub1 is also essential for the checkpoint control of correct chromosome congression, by regulating the localization of Aurora B [3,4]. Accurate segregation of sister chromatids in mitosis is preceded by two steps of cohesin removal [5]. At prophase, cohesin along the chromosome arms is removed through phosphorylation by Plk1/Aurora B. In the metaphase-to-anaphase transition, cohesin in the centromeric region is removed by separase. Separase is inhibited by securin and Cdk1/ cyclin B1, whose expression levels are under the control of the spindle checkpoint [6]. The untimely separation of sister chromatids is prevented by Shugoshin (Sgo) through the protection of centromeric cohesin [7]. In this report, we show that Regulator of Ribosome Synthesis 1 (RRS1) protein contributes to chromosome congression. RRS1 was Abbreviations: RNAi, RNA interference; siRNA, small interfering RNA; GFP, green fluorescent protein; H3S10ph, phospho-histone H3 serine 10 * Corresponding author. Fax: +81 6 6879 7442. E-mail address: [email protected] (K. Fukui).

found as a regulatory protein required for ribosome biogenesis in yeast [8]. Yeast RRS1 is required for export of the 60S ribosomal subunit from the nucleolus to the cytoplasm [9]. It also acts as an assembly factor with Rpf2, which recruits rpL5, rpL11, and 5S rRNA into nascent ribosomes [10,11]. Consistent with its ribosome assembly function in yeast, a human RRS1 ortholog was identified in a proteome analysis of nucleolar extracts [12,13]. Moreover, RRS1 was also found in a proteome analysis of highly purified human chromosomes [14,15]. Thus, we embarked on the functional analysis of human RRS1 during mitosis. 2. Materials and methods 2.1. Cells and transfection HeLa cells were maintained in Dulbecco’s Modified Eagle’s Medium (Gibco BRL), supplemented with 5–10% fetal bovine serum (Equitech-Bio Inc.). To analyze RRS1 localization during the cell cycle, pEGFP-C1/RRS1 was transfected into HeLa cells using FuGene6 (Roche). Geneticin (Sigma) at 800 lg/ml was added 36 h after transfection to select for stably-expressing cells. 2.2. Antibodies The following antibodies against the indicated proteins, with their corresponding dilutions and manufacturers, were used:

0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.05.033

1952

A.E. Gambe et al. / FEBS Letters 583 (2009) 1951–1956

Abnova: RRS1 (mouse polyclonal, 1:200), Santa Cruz Biotechnology: nucleolin (mouse monoclonal, 1:100), B23 (mouse monoclonal, 1:200), fibrillarin (mouse monoclonal, 1:200), Sigma: green fluorescent protein (GFP) (mouse monoclonal, 1:200), a-tubulin (rabbit monoclonal, 1:1000), cyclin B1 (rabbit polyclonal, 1:500), BD Laboratories: BubR1 (mouse monoclonal, 1:500), Aurora B (mouse monoclonal, 1:200), Chemicon: Bub1 (mouse monoclonal, 1:50), Cortex Biochem: anticentromere autoimmune serum (CREST; human monoclonal, 1:100), Abcam: centrin1 (rabbit polyclonal, 1:200), CDC27 (mouse monoclonal, 1:2000), CDC20 (mouse monoclonal, 1:1000), securin (mouse monoclonal, 1:50), Smc1 (rabbit polyclonal, 1:200), Calbiochem: Plk1 (mouse monoclonal, 1:1000), Upstate Biotechnology: phospho-histone H3 serine 10 (H3S10ph, rabbit monoclonal, 1:500), and Sgo1 (from Prof. Y. Watanabe, University of Tokyo) [16]. 2.3. Immunofluorescence Cells were fixed using 4% PFA in PBS, permeabilized with 0.2% Triton X-100 in PBS and blocked in 1% BSA for 30 min at 37 °C. Cells were incubated with the primary and secondary antibodies for 1 h each with three PBS washes. DNA was counterstained with 1 lg/ml DAPI. Spread chromosomes were prepared as described [14].

3. Results and discussion 3.1. RRS1 is localized in nucleoli and in the chromosome periphery RRS1 co-localized with other nucleolar proteins, such as nucleolin [20] and fibrillarin [21] at interphase (Fig. 1A) and mitosis (Fig. 1B). During prophase, RRS1 signals were observed on the disintegrating nucleoli (Fig. 1C). At prometaphase, metaphase and anaphase, RRS1 was mainly localized in the chromosome periphery (Fig. 1C). During telophase, RRS1 formed part of the nucleolus-derived foci and localized on the reforming nascent nucleoli at cytokinesis. The localization of RRS1 with respect to centromeric heterochromatin, inner kinetochore and chromosome periphery was elucidated by staining spread chromosomes with antibodies against CREST, Aurora B [22] and Ki-67 [23] (Fig. 1D). At the onset of mitosis, some nucleolar proteins, including Ki-67 [23], nucleolin [20] and fibrillarin [21], are known to disperse in the cytoplasm, while a subset associates with the condensing chromosomes. These nucleolar proteins around the chromosome periphery have been proposed to function in mitotic congression [24,25]. RRS1 also localized in the chromosome periphery around kinetochores. 3.2. RRS1 depletion resulted in defects of chromosome congression

2.4. RNA interference (RNAi) and rescue experiments Two small interfering RNA (siRNA) sequences were chemically synthesized: siRNA-1: 50 -CUACCGGACACCAGAGUAA-30 and siRNA-2: 50 -CCAAAGAAUGGCUGAUUGAdTdT-30 . As described previously [17], 100 nM of the siRNA duplexes were transfected into HeLa cells using Lipofectamine 2000 (Invitrogen). A firefly luciferase siRNA sequence (50 -CGUACGCGGAAUACUUCGAdTdT-30 ) was used for control. The Aurora-B siRNA sequence was previously described [18]. Cells were assayed 72 h after RNAi. A rescue vector against siRNA-1 was constructed. Primers were designed to create three silent point mutations by site-directed mutagenesis. The resulting RRS1 rescue vector (RRS1rv) contained the following sequence: 50 -CTACAGGCCACCAGAGCAA-30 (silent mutations are underlined). Cells were transfected with either the pEGFP-C1 vector without RRS1 or with RRS1rv using FuGene6 (Roche), RNAi was performed 12 h later. Cells were harvested and assayed 72 h after RNAi. Alternatively, cells were treated with 20 lM MG132 or with 0.1 lg/ml colcemid 3 h before harvest. 2.5. FACS analysis Approximately 5 104 HeLa cells were seeded on a 90 mm dish. A day after seeding, the cells were transfected with either control or RRS1 siRNAs. The cells were incubated with trypsin–EDTA at 37 °C for 5 min after which the aspirated medium was used to reconstitute the cells. After fixation in ice-cold 70% ethanol and incubation with 0.2 mg/ml RNaseA, propidium iodide (PI) was added to the cell suspension at the final concentration of 50 lg/ml. After 30 min of PI staining at 4 °C, the cells were subjected to EPICS ALTRA cell sorting system (Beckman Coulter). 2.6. Microscopy and image analysis Cell images were obtained using deconvolution (Delta Vision, Applied Precision Instruments) and fluorescence microscopy (Olympus IX81) with a cooled CCD camera (CoolSnapHQ2, Photometrics Inc.) and Metamorph image processing software (v.6.2r6, Universal Imaging Corporation Inc.). The images were further processed using ImageJ (http://rsb.info.nih.gov/ij/download.html) and Adobe Photoshop. Time-lapse analysis using HeLa-GFP-H1.2 cells was performed as described [19].

The expression level of RRS1 protein was decreased by RNAi with siRNA-1 (Fig. 2A) and siRNA-2 (Fig. S1A). In contrast, the expression level of cell-cycle related proteins was not changed. The mitotic index was increased 72 h after RRS1 knockdown, relative to the control (Fig. 2B and Fig. S1B). A significant increase of cells at prometaphase (Fig. 2C and D) and mitotic cells with aberrant chromosome alignment along the metaphase plate was observed (Fig. 2E and F). These defects were categorized as misaligned, where a few chromosomes are not aligned along a discernable plate, or non-aligned, where the chromosomes appeared scattered with no obvious congression (Fig. 2E and F). Similar phenotypes were reproduced using siRNA-2 (Fig. S1C and D). Thus, we used siRNA-1 for further knockdown analyses. Transfection with the RRS1 siRNA-resistant rescue vector (RRS1rv) resulted in lower numbers of misalignment and non-alignment phenotypes, relative to cells transfected with the pEGFP-C1 vector (Fig. 2D), suggesting specificity of these aberrations to RRS1 knockdown. Live cell imaging analyses using cells stably-expressing GFPH1.2 revealed that RRS1 knockdown induced the delay in progression from prometaphase to anaphase but results in normal segregation (Fig. 2G). The time from nuclear envelope breakdown to anaphase initiation of most control cells was within 60 min while that of RRS1-deficient cells was 75–180 min (Fig. 2G). Moreover, FACS analyses demonstrated that RRS1 depletion resulted in an increase of cells with 4N DNA contents (Fig. S1E). Taken together these results suggest that RRS1 depletion resulted in aberrant chromosome congression which contributed to the delay in mitotic progression. Mitotic cells with elongated spindle poles and multi-polar spindles with more than two spindle poles with a-tubulin foci were observed after RRS1 depletion (Fig. S2A and B). Multi-polar spindles can arise by aberrant amplification of centrosome components [26,27]. Aberrant spindle morphologies induce abnormal kinetochore–microtubule interactions, which in turn trigger spindle checkpoint activation [27]. Bub1 and BubR1 were localized on the kinetochores of aberrant chromosomes (Fig. 2E and F). Furthermore, the depletion of Aurora B rescued the mitotic delay (Fig. S3). These aberrations were therefore accompanied by spindle checkpoint activation, which is consistent with the delay at the transition time from NEBD to anaphase (Fig. 2G).


1953

Fig. 1. RRS1 localization analysis. (A) and (B) Immunostaining images of nucleolar proteins. The localization patterns of RRS1 at interphase (A) and mitosis (B) using the GFPRRS1 stably-expressing cell line. Signals of DAPI, RRS1 and other nucleolar proteins (nucleolin or fibrillarin) appear in blue, green and red, respectively. Bar = 5 lm. (C) Dynamic localization of RRS1 during mitosis using the GFP-RRS1 stably-expressing cell line. Signals of DAPI and RRS1 appear in blue and green, respectively. White arrows indicate a nucleolus-derived foci while the red arrows indicate a pre-nucleolar body. The yellow arrowheads indicate a reforming nascent nucleolus at cytokinesis. Bar = 10 lm. Insets, 4 mag. (D) RRS1 localization on the spread chromosomes. Bar = 1 lm. The Right graphs show corresponding the fluorescence intensity profiles of the line scans of regions within white boxes in 5 mag. panels.

3.3. Precocious sister chromatid separation in RRS1-depleted cells Chromosome congression requires the centromeric cohesion of sister chromatids until all chromosomes are properly attached to the spindle microtubules [5]. We therefore examined whether sister chromatids retained centromeric cohesion after RRS1 depletion. The positive staining signals for cyclin B1 and securin indicated that the RRS1-depleted cells were still before anaphase (Fig. S4A). Spread chromosomes were prepared after addition of colcemid and a proteasome inhibitor, MG132, for prometaphase–metaphase and metaphase–anaphase arrests, respectively. A significant increase in separation of sister chromatids was observed in RRS1-depleted

cells in both colcemid and MG132 treatments (Fig. 3A and B). Since Sgo1 protects the centromeric cohesion of sister chromatids [7,16], we investigated Sgo1 localization after RRS1 depletion (Fig. 3C). The expression level of Sgo1 in RRS1-depleted extracts was similar to control (Fig. S4B), however, Sgo1 signals flanking the centromere significantly decreased after RRS1 depletion (Fig. 3C). These results indicate that RRS1 depletion led to premature sister chromatid dissociation due to the perturbation of Sgo1 localization at the centromeric region. To examine the cause of Sgo1 localization defects, we performed immunostaining with Aurora B which regulates the centromeric localization of Sgo1 [28,29]. Unexpectedly, Aurora B was

1954


Fig. 2. RRS1 knockdown analyses. (A) The protein expression level in RRS1-depleted cells with siRNA-1. The Western blot analysis using HeLa cell extracts was performed with antibodies against RRS1, CDC27, CDC20, Plk1 and b-actin. (B) The mitotic index after RRS1 depletion with siRNA-1. n = 5, >1000 cells counted. indicates statisticallysignificant difference. (C) Classification of mitotic phases in control and RRS1-depleted cells with siRNA-1. DAPI-stained mitotic cells were classified into cells at prophase, prometaphase, metaphase, anaphase and telophase/cytokinesis. n = 3, >1000 cells counted. (D) Rescue assay with the RRS1 siRNA-resistant rescue vector (RRS1rv) and the GFP control vector (pEGFP-C1) after RNAi with siRNA-1. Mitotic cells with condensed chromosomes were classified into cells at prometaphase or metaphase and cells with non-aligned or misaligned chromosomes. n = 2, >1000 cells counted. (E) and (F) Immunostaining images of two spindle checkpoint proteins, Bub1 (E) and BubR1 (F). Signals of DAPI, a-tubulin and spindle checkpoint protein appear in blue, green and red, respectively. Bub1 and BubR1 signals were detected on kinetochores of prometaphase chromosomes in control cells and misaligned and non-aligned chromosomes in RNAi-depleted cells. Bar = 5 lm. (G) Live cell imaging using a GFP-histone H1 stablyexpressing cell line. White arrows indicate misaligned chromosomes. The upper panels show time-lapse images of mitotic cells. The lower panel shows the transition time from nuclear envelope breakdown (NEBD) to anaphase initiation in control (blue dots) and RRS1-depleted cells (red dots). A white arrow indicates a misaligned chromosome. Fifty cells were counted for each condition. Bar = 10 lm.

localized on the chromosome arms after RRS1 depletion, whereas Aurora B was normally localized at the centromeric region of mitotic chromosomes (Figs. 1D and 3D). In contrast, an outer kinetochore protein CENP-E, which is a kinesin-like protein required for chromosome congression [30], retained its exclusive centromeric localization (Fig. 3D). The mis-localization of Aurora B was previ-

ously observed after Bub1 depletion in Xenopus laevis egg extracts [4]. Moreover, proper centromeric localization of Sgo1 requires Bub1 [31]. We, therefore, investigated whether RRS1 depletion affected the expression levels of Bub1 at the transition from prometaphase to metaphase. The Bub1 expression level was not changed after RRS1 depletion (Fig. S4B). Furthermore, Bub1 was


1955

Fig. 3. Precocious sister chromatid separation after RRS1 knockdown. (A) Chromosomes with separated and unseparated centromeric regions. Blue and red signals represent DAPI and CREST signals, respectively. Bar = 5 lm. Inset, 2 mag. (B) Quantification of the phenotypes in A after colcemid or MG132 addition. n = 3, >500 cells counted. (C) Aberrant localization patterns of Sgo1 after RRS1 knockdown. Blue, red and green signals represent DAPI, CREST and Sgo1 signals, respectively. Bar = 5 lm. (D) Localization patterns of Aurora B and CENP-E on spread chromosome preparations. Blue and red signals represent DAPI and CREST signals, respectively. Green signals indicate Aurora B or CENP-E localization. Bar = 1 lm.

still targeted to aberrantly aligned chromosomes (Fig. 2E). Consistent with previous results [4], Aurora B mis-localization did not influence its kinase activity since the phosphorylation of its substrate, H3S10, was still observed after RRS1 depletion (Fig. S4C). These findings suggest an upstream event whereby RRS1 is involved in proper targeting and exclusive localization of Aurora B at the centromeric region. However, we cannot exclude the possibility that the mis-localization of Aurora B is a consequence of the precocious sister chro-

matid separation. The knockdown of cohesin subunit Scc1/Rad21 in HeLa cells showed similar pattern where Aurora B spreads along the chromosome arms [32]. At least, the reduction of cohesin expression is not a direct cause of the precocious sister chromatid separation in RRS1-depleted cells because the expression level of a cohesin subunit Smc1 was not changed (Fig. S4B). Further analyses of RRS1 functions will help elucidate the centromeric cohesion puzzle consisting of Sgo1, Aurora B and cohesin subunits.

1956


Acknowledgments We thank Prof. Yoshinori Watanabe (University of Tokyo) for the generous gift of the Sgo1 antibody. Mr. Akihiro Morimoto and Mr. Shinichi Aikawa (Osaka University) facilitated our FACS analysis. A.E.G. thanks MEXT (Ministry of Education, Culture, Sports, Science and Technology of Japan) for scholarship support. This work was supported by Special Coordination Funds of MEXT to K.F., by Grants-in-Aid for Scientific Research from MEXT to K.F., S.U. and S.M., and by the Japan Science and Technology Agency (BIRD to S.M., SENTAN to K.F.).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2009.05.033. References [1] Tanaka, T.U., Stark, M.J. and Tanaka, K. (2005) Kinetochore capture and biorientation on the mitotic spindle. Nat. Rev. Mol. Cell Biol. 6, 929–942. [2] Ruchaud, S., Carmena, M. and Earnshaw, W.C. (2007) Chromosomal passengers: conducting cell division. Nat. Rev. Mol. Cell Biol. 8, 798–812. [3] Meraldi, P. and Sorger, P.K. (2005) A dual role for Bub1 in the spindle checkpoint and chromosome congression. EMBO J. 24, 1621–1633. [4] Boyarchuk, Y., Salic, A., Dasso, M. and Arnaoutov, A. (2007) Bub1 is essential for assembly of the functional inner centromere. J. Cell Biol. 176, 919–928. [5] Losada, A. (2008) The regulation of sister chromatid cohesion. Biochim. Biophys. Acta 1786, 41–48. [6] Musacchio, A. and Salmon, E.D. (2007) The spindle-assembly checkpoint in space and time. Nat. Rev. Mol. Cell Biol. 8, 379–393. [7] Watanabe, Y. (2005) Shugoshin: guardian spirit at the centromere. Curr. Opin. Cell Biol. 17, 590–595. [8] Tsuno, A., Miyoshi, K., Tsujii, R., Miyakawa, T. and Mizuta, K. (2000) RRS1, a conserved essential gene, encodes a novel regulatory protein required for ribosome biogenesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 20, 2066– 2074. [9] Miyoshi, K., Shirai, C., Horigome, C., Takenami, K., Kawasaki, J. and Mizuta, K. (2004) Rrs1p, a ribosomal protein L11-binding protein, is required for nuclear export of the 60S pre-ribosomal subunit in Saccharomyces cerevisiae. FEBS Lett. 565, 106–110. [10] Nariai, M., Tanaka, T., Okada, T., Shirai, C., Horigome, C. and Mizuta, K. (2005) Synergistic defect in 60S ribosomal subunit assembly caused by a mutation of Rrs1p, a ribosomal protein L11-binding protein, and 30-extension of 5S rRNA in Saccharomyces cerevisiae. Nucleic Acids Res. 33, 4553–4562. [11] Zhang, J., Harnpicharnchai, P., Jakovljevic, J., Tang, L., Guo, Y., Oeffinger, M., Rout, M., Hiley, S., Hughes, T. and Woolford Jr., J. (2007) Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes. Genes Dev. 21, 2580–2592. [12] Andersen, J.S., Lyon, C.E., Fox, A.H., Leung, A.K., Lam, Y.W., Steen, H., Mann, M. and Lamond, A.I. (2002) Directed proteomic analysis of the human nucleolus. Curr. Biol. 12, 1–11. [13] Scherl, A., Couté, Y., Déon, C., Callé, A., Kindbeiter, A., Sanchez, J., Greco, A., Hochstrasser, D. and Diaz, J. (2002) Functional proteomic analysis of human nucleolus. Mol. Biol. Cell 13, 4100–4109.

[14] Uchiyama, S., Kobayashi, S., Takata, H., Ishihara, T., Hori, N., Higashi, T., Hayashihara, K., Sone, T., Higo, D., Nirasawa, T., Takao, T., Matsunaga, S. and Fukui, K. (2005) Proteome analysis of human metaphase chromosomes. J. Biol. Chem. 280, 16994–17004. [15] Takata, H., Uchiyama, S., Nakamura, N., Nakashima, S., Kobayashi, S., Sone, T., Kimura, S., Lahmers, S., Granzier, H., Labeit, S., Matsunaga, S. and Fukui, K. (2007) A comparative proteome analysis of human metaphase chromosomes isolated from two different cell lines reveals a set of conserved chromosomeassociated proteins. Gene Cell 12, 269–284. [16] Kitajima, T.S., Kawashima, S.A. and Watanabe, Y. (2004) The conserved kinetochore protein Shugoshin protects centromeric cohesion during meiosis. Nature 427, 510–517. [17] Takata, H., Matsunaga, S., Morimoto, A., Ono-Maniwa, R., Uchiyama, S. and Fukui, K. (2007) H1.X with different properties from other linker histones is required for mitotic progression. FEBS Lett. 581, 3783–3788. [18] Ditchfield, C., Johnson, V.L., Tighe, A., Ellston, R., Haworth, C., Johnson, T., Mortlock, A., Keen, N. and Taylor, S.S. (2003) Aurora B couples chromosome alignment with anaphase by targeting BubR1, Mad2, and Cenp-E to kinetochores. J. Cell Biol. 161, 267–280. [19] Gambe, A.E., Ono-Maniwa, R., Matsunaga, S., Kutsuna, N., Higaki, T., Higashi, T., Hasezawa, S., Uchiyama, S. and Fukui, K. (2007) Development of a multistage classifier for a monitoring system of cell activity based on imaging of chromosomal dynamics. Cytometry 71A, 286–296. [20] Ma, N., Matsunaga, S., Takata, H., Ono-Maniwa, R., Uchiyama, S. and Fukui, K. (2007) Nucleolin functions in nucleolus formation and chromosome congression. J. Cell Sci. 120, 2091–2105. [21] Amin, M.A., Matsunaga, S., Ma, N., Takata, H., Yokoyama, M., Uchiyama, S. and Fukui, K. (2007) Fibrillarin a nucleolar protein is required for normal nuclear morphology and cellular growth in HeLa cells. Biochem. Biophys. Res. Commun. 360, 320–326. [22] Yang, Y., Wu, F., Ward, T., Yan, F., Wu, Q., Wang, Z., McGlothen, T., Peng, W., You, T., Sun, M., Cui, T., Hu, R., Dou, Z., Zhu, J., Xie, W., Rao, Z., Ding, X. and Yao, X. (2008) Phosphorylation of HsMis13 by Aurora B kinase is essential for assembly of functional kinetochore. J. Biol. Chem. 283, 26726–26736. [23] Saiwaki, T., Kotera, I., Sasaki, M., Takagi, M. and Yoneda, Y. (2005) In vivo dynamics and kinetics of pKi-67: transition from a mobile to an immobile form at the onset of anaphase. Exp. Cell Res. 308, 123–134. [24] Van Hooser, A.A., Yuh, P. and Heald, R. (2005) The perichromosomal layer. Chromosoma 114, 377–388. [25] Boisvert, F.M., van Koningsbruggen, S., Navascués, J. and Lamond, A.I. (2007) The multifunctional nucleolus. Nat. Rev. Mol. Cell Biol. 8, 574–585. [26] Oshimori, N., Ohsugi, M. and Yamamoto, T. (2006) The Plk1 target Kizuna stabilizes mitotic centrosomes to ensure spindle bipolarity. Nat. Cell Biol. 8, 1095–1101. [27] Thein, K.H., Kleylein-Sohn, J., Nigg, E.A. and Gruneberg, U. (2007) Astrin is required for the maintenance of sister chromatid cohesion and centrosome integrity. J. Cell Biol. 178, 345–354. [28] Dai, J., Sullivan, B.A. and Higgins, J.M. (2006) Regulation of mitotic chromosome cohesion by Haspin and Aurora B. Dev. Cell 11, 741–750. [29] Takata, H., Matsunaga, S., Morimoto, A., Ma, N., Kurihara, D., Ono-Maniwa, R., Nakagawa, M., Azuma, T., Uchiyama, S. and Fukui, K. (2007) PHB2 protects sister-chromatid cohesion in mitosis. Curr. Biol. 17, 1356–1361. [30] Yao, X., Anderson, K.L. and Cleveland, D.W. (1997) The microtubule-dependent motor centromere-associated protein E (CENP-E) is an integral component of kinetochore corona fibers that link centromeres to spindle microtubules. J. Cell Biol. 139, 435–447. [31] Kitajima, T.S., Hauf, S., Ohsugi, M., Yamamoto, T. and Watanabe, Y. (2005) Human Bub1 defines the persistent cohesion site along the mitotic chromosome by affecting Shugoshin localization. Curr. Biol. 15, 353–359. [32] Losada, A., Yokochi, T. and Hirano, T. (2005) Functional contribution of Pds5 to cohesin-mediated cohesion in human cells and Xenopus egg extracts. J. Cell Sci. 118, 2133–2141.