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The Plant Journal (2006) 48, 572–580

doi: 10.1111/j.1365-313X.2006.02893.x

Aurora kinase is required for chromosome segregation in tobacco BY-2 cells Daisuke Kurihara, Sachihiro Matsunaga, Akira Kawabe†, Satoru Fujimoto‡, Masanori Noda, Susumu Uchiyama and Kiichi Fukui* Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Osaka, Japan Received 15 May 2006; revised 21 July 2006; accepted 26 July 2006. *For correspondence (fax þ81 6 6879 7441; e-mail [email protected]). Present addresses: †Institute of Evolutionary Biology, University of Edinburgh, Ashworth Laboratory, King’s Buildings, West Mains Road, Edinburgh EH9 3JT, UK; ‡ Research Center for Ultra-High Voltage Electron Microscopy, Osaka University, 7-1, Mihogaoka, Ibaraki 565-0871, Osaka, Japan.

Summary Post-translational modifications of core histone tails play crucial roles in chromatin structure and function. Although phosphorylation of Ser10 and Ser28 (H3S10ph and H3S28ph) of histone H3 is ubiquitous among eukaryotes, the phosphorylation mechanism during the cell cycle remains unclear. In the present study, H3S10ph and H3S28ph in tobacco BY-2 cells were observed in the pericentromeric regions during mitosis. Moreover, the Aurora kinase inhibitor Hesperadin inhibited the kinase activity of Arabidopsis thaliana Aurora kinase 3 (AtAUR3) in phosphorylating both Ser10 and Ser28 of histone H3 in vitro. Consistently, Hesperadin inhibited both H3S10ph and H3S28ph during mitosis in BY-2 cells. These results indicate that plant Aurora kinases phosphorylate not only Ser10, but also Ser28 of histone H3 in vivo. Hesperadin treatment increased the ratio of metaphase cells, while the ratio of anaphase/telophase cells decreased, although the mitotic index was not affected in Hesperadin-treated cells. These results suggest that Hesperadin induces delayed transition from metaphase to anaphase, and early exit from mitosis after chromosome segregation. In addition, micronuclei were observed frequently and lagging chromosomes, caused by the delay and failure of sister chromatid separation, were observed at anaphase and telophase in Hesperadin-treated BY-2 cells. The data obtained here suggest that plant Aurora kinases and H3S10ph/H3S28ph may have a role in chromosome segregation and metaphase/anaphase transition. Keywords: Aurora kinase, Hesperadin, histone H3 phosphorylation, lagging chromosome, chromosome segregation.

Introduction Chromosomes change their morphology and position dynamically during mitosis, and the processes of chromosome condensation and segregation play crucial roles in equal separation of genetic information to both daughter cells. Mitosis depends mainly on two post-translational mechanisms: protein phosphorylation and proteolysis. Cell division is regulated by mitotic kinases, such as cyclindependent kinase (CDK), Polo-like kinase (Plk), never in mitosis A (NIMA) and Aurora kinase families as well as kinases implicated in mitotic checkpoints, mitotic exit and cytokinesis (Nigg, 2001). Aurora kinases belong to a cell cycle-dependent serine/ threonine protein kinase family and are highly conserved 572

from yeast to humans. Two members of this kinase family have been identified as Aurora A and B in higher eukaryotes. Aurora kinases share a similar primary structure, but their functions differ. Aurora A regulates centrosome segregation and maturation and stabilizes spindle microtubules, while Aurora B is associated with chromosome condensation and segregation, establishment of microtubule–kinetochore attachment and cytokinesis (Carmena and Earnshaw, 2003). There is an additional member in mammals, Aurora C, which is specifically expressed in testis (Kimura et al., 1999). In addition, Aurora C is a novel chromosomal passenger protein that coordinates with Aurora B to regulate mitotic chromosome dynamics (Li et al., 2004). Aurora B is ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd

Aurora kinase in chromosome segregation 573 involved in chromosome condensation through interaction with the histone H3 tail (Carmena and Earnshaw, 2003). Aurora B also phosphorylates Ser28 of histone H3 from prophase to metaphase, and is thought to be associated with chromosome condensation (Goto et al., 1999, 2002). In Arabidopsis thaliana three genes have been identified as Aurora kinases: A. thaliana Aurora kinases 1, 2 and 3 (AtAUR1, 2 and 3; Demidov et al., 2005; Kawabe et al., 2005). All three AtAURs possess the same kinase activity, phosphorylating Ser10 of histone H3 in vitro (Kawabe et al., 2005). In plants, H3S10ph occurs from prophase until telophase in pericentromeric regions (Houben et al., 1999), and H3S28ph occurs from late prophase to early telophase in the central part of pericentromeric regions (Gernand et al., 2003). Previous studies have suggested that phosphorylation of histone H3 is involved in sister chromatid cohesion, but not chromosome condensation (Gernand et al., 2003; Manzanero et al., 2000). Dynamic analyses of GFP-tagged AtAUR fusion proteins revealed that AtAUR1 and AtAUR2 are localized on the nuclear membrane at interphase and on the mitotic spindles during mitosis. Moreover, AtAUR1 was also found to be localized in the cell plates. AtAUR3 was shown to be localized at the nuclear periphery during interphase, and shows a speckled distribution on condensed chromosomes at prophase before localizing at the metaphase plate. At late anaphase, AtAUR3 is evenly localized on chromosomes (Demidov et al., 2005; Kawabe et al., 2005). Studies on the localization of AtAURs in mitotic cells and the ability of recombinant AtAURs to phosphorylate Ser10 of histone H3 suggest that AtAUR3 phosphorylates Ser10 of histone H3 in vivo (Kawabe et al., 2005).

Hesperadin is a small molecule that inhibits Aurora B kinase activity and has been used in recent studies on mammalian cells. H3S10ph is greatly reduced in Hesperadin-treated cells, and loss of Aurora B kinase activity by Hesperadin causes an accumulation of malorientated chromosomes in dividing cells, wherein the kinetochore–microtubule fibres attach both sister kinetochores to the same pole (Hauf et al., 2003; Lampson et al., 2004). In the present study, the functional role of plant Aurora kinases in mitosis was examined using Hesperadin in tobacco BY-2 cells. Our study shows that Hesperadin inhibits both H3S10ph and H3S28ph by plant Aurora kinases in mitosis, and induces aberrant chromosome segregation in BY-2 cells. These data suggest that Aurora kinases are required for regular chromosome segregation in plants. Results Phosphorylation of histone H3 at Ser10 and Ser28 during the cell cycle To investigate the localization of H3S10ph in Nicotiana tabacum cv. Bright Yellow-2 (tobacco BY-2) cultured cells, we performed indirect immunofluorescence using the antiH3S10ph antibody. As shown in Figure 1, when chromosomes began to condense at prophase, dot-like signals of H3S10ph were detected in the pericentromeric regions. Dotted signals on mitotic chromosomes were observed at the metaphase plate then, after segregation of mitotic chromosomes to opposite poles, the signals dispersed along the sister chromatids at late anaphase. The signals

Figure 1. Phosphorylation of histone H3 at Ser10 and Ser28 during the cell cycle. BY-2 cells immunostained using antiH3S10ph or anti-H3S28ph antibodies. DNA was stained with DAPI. Merged images of DNA (blue); H3S10ph (red); H3S28ph (green). Scale bar, 10 lm.

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574 Daisuke Kurihara et al. were drastically reduced and then disappeared at telophase. The H3S10ph distribution is consistent with other plant species with monocentric chromosomes (Gernand et al., 2003). In plants, it has been found that phosphorylation of histone H3 occurs not only at Ser10, but also at Ser28 (Gernand et al., 2003; Zhang et al., 2005). H3S28ph occurs in pericentromeric regions during mitosis in A. thaliana, Luzula luzuloides, barley, rye (Gernand et al., 2003) and maize (Zhang et al., 2005). We confirmed the H3S28ph distribution pattern in BY-2 cells by indirect immunofluorescence using the anti-H3S28ph antibody. As with H3S10ph, H3S28ph was also detected in the pericentromeric regions, although it appeared later and disappeared earlier (Figure 1). The localization of H3S28ph was also very similar to that of AtAUR3 (Kawabe et al., 2005). Hesperadin inhibits the activity of AtAUR3 in vitro To analyse the function of plant Aurora kinases in detail, we used an Aurora kinase inhibitor, Hesperadin. In mammalian cells, H3S10ph is greatly reduced after Hesperadin treatment (Hauf et al., 2003). To investigate whether Hesperadin can inhibit AtAUR3 kinase activity, we performed an in vitro kinase assay using purified recombinant GST-tagged AtAUR3 with A. thaliana histone H3 as a substrate. Phosphorylation of histone H3 was examined by immunoblotting using anti-H3S10ph and anti-H3S28ph antibodies. Not only Ser10 of histone H3, but also Ser28 were phosphorylated by GST-AtAUR3 (Figure 2a), whereas H3T3ph and H3T11ph were not detected (data not shown). This result indicates that AtAUR3 phosphorylates not only Ser10 of histone H3, but also Ser28, making it different from AtAUR1, which phosphorylates only Ser10 (Demidov et al., 2005). Levels of H3S10ph and H3S28ph were drastically reduced with GSTAtAUR3 in the presence of Hesperadin, indicating that Hesperadin acts as an Aurora kinase inhibitor, as in mammals (Figure 2a; Hauf et al., 2003).

The efficiency of Hesperadin inhibition of AtAUR3 kinase activity in vitro was examined by changing the concentration from 0.1 to 10 lM Hesperadin (Figure 2b). The ability of GSTAtAUR3 to phosphorylate Ser10 of histone H3 was reduced by about 50% at Hesperadin concentrations >1 lM. However, even with 10 lM Hesperadin treatment, kinase activity of AtAUR3 could not be completely inhibited. In mammals, 250 nM concentration is enough for half-maximal inhibition of Aurora kinases (Hauf et al., 2003). Thus, the efficiency of Hesperadin as a kinase inhibitor in plants is not as strong as in animals. Effect of Hesperadin on BY-2 cells In Hesperadin-treated BY-2 cells, H3S10ph, as well as H3S28ph, was greatly reduced in mitotic cells (Figure 3a,b). These results suggest that in mitosis, Ser10 and Ser28 of histone H3 are phosphorylated by an Aurora kinase in BY-2 cells, and Hesperadin prevents these phosphorylations by inhibiting the activity of the endogenous tobacco Aurora kinase (N. tabacum Aurora kinase, NtAUR). Phosphorylation sites in histone H3 at Thr3 and Thr11 have been reported for mammals and plants (Dai et al., 2005; Houben et al., 2005; Preuss et al., 2003). However, as expected from the in vitro experiments, localization and levels of neither H3T3ph nor H3T11ph were changed in Hesperadin-treated BY-2 cells (Figure 3c,d), although H3S28ph was greatly reduced, as confirmed by double indirect immunofluorescence. These results suggest that Hesperadin specifically inhibits NtAUR in vivo. To analyse the effect of Hesperadin on cell growth, we counted the number of BY-2 cells every day after Hesperadin treatment. The growth rate of Hesperadin-treated BY-2 cells was similar to that of control cells (Figure 4a). Also, no significant difference was detected between the mitotic indexes of Hesperadin-treated and control BY-2 cells (Figure 4b). However, the percentage of metaphase cells was higher, while that of anaphase/telophase cells was lower in

Figure 2. Hesperadin-induced inhibition of histone H3 phosphorylation by AtAUR3 in vitro. An in vitro kinase assay was performed using recombinant GST-AtAUR3 with Arabidopsis histone H3 as a substrate. GST-AtAUR3 was incubated with histone H3 for 10 min at 30C. (a) The kinase reaction mixtures were incubated with 10 lM Hesperadin (middle lane) or DMSO (left lane), and without GST-AtAUR3 as negative control (right lane). Total histone H3 was stained with Coomassie brilliant blue (upper panel) and phosphorylated histone H3 was immunostained using anti-H3S10ph and H3S28ph antibodies (lower panels). (b) Kinase reactions were examined after addition of various concentrations of Hesperadin (10, 5, 1, 0.5 and 0.1 lM; lanes 2–6, respectively) or DMSO (lane 1), and without GST-AtAUR3 as negative control (lane 7). Phosphorylated histone H3 was detected by immunoblotting using the anti-H3S10ph antibody.

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Aurora kinase in chromosome segregation 575

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Figure 3. Mitotic phosphorylations of Ser10 and Ser28 of histone H3 were reduced in Hesperadin-treated BY-2 cells. (a–d) BY-2 cells were treated with 5 lM Hesperadin for 24 h, then isolated nuclei were immunostained using anti-H3S10ph (a) or anti-H3S28ph (b) antibodies; and double immunostained using anti-H3S28ph (c, d) and antiH3T3ph (c) or anti-H3T11ph (d) antibodies. DNA was stained with DAPI. Merged images of DNA (blue); H3S10ph (green, a); H3S28ph (green, b–d); H3T3ph and H3T11ph (red, c and d). Scale bars, 10 lm.

Hesperadin-treated BY-2 cells than in control cells (Figure 4c). These results suggest that Hesperadin has no effect on proliferation, but affects cell cycle progression during metaphase/anaphase transition in BY-2 cells. During interphase, micronuclei were frequently observed in Hesperadin-treated BY-2 cells, while control untreated cells showed almost no such abnormality (Figure 5a,b). This formation of micronuclei in Hesperadin-treated BY-2 cells might be accompanied by aberrant chromosome segregation. In mammalian cells, Hesperadin induces defects in spindle organization and improper kinetochore–microtubule attachments (Hauf et al., 2003; Lampson et al., 2004; Rosa et al., 2006). To analyse the microtubule and chromosome dynamics in BY-2 cells, we performed indirect immunoflu-

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Figure 4. Cell growth, mitotic index and frequencies at each cell stage after Hesperadin treatment. (a) Cell numbers were counted using a Burker–Turk chamber every day after Hesperadin treatment. The difference in cell numbers between Hesperadintreated and control cells was not significant. (b and c) BY-2 cells were treated with 0 (control) and 5 lM Hesperadin for 24 h. (b) The mitotic index was calculated in 1000 cells, revealing no statistically significant differences between Hesperadin-treated and control cells (t-test, P > 0.1). (c) Mitotic cells were classified into each mitotic phase by the distinctive pattern of DNA staining. The increase in metaphase cells and decrease in anaphase/telophase cells were significant at P > 0.05 (t-test) when comparing 5 lM Hesperadin-treated cells with the control. Data were averaged from three independent measurements (SD).

orescence using the anti-a-tubulin antibody after Hesperadin treatment. As shown in Figure 5(c), in Hesperadin-treated BY-2 cells the chromatin fibres were condensed and the pre-

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576 Daisuke Kurihara et al. Figure 5. Hesperadin-induced micronuclei at interphase and lagging chromosomes at anaphase/telophase in BY-2 cells. (a–d) BY-2 cells were treated with 5 lM Hesperadin for 24 h. (a) DNA was stained with DAPI. (b) Cells containing micronuclei were counted in 1000 cells. The increase in cells containing micronuclei was statistically significant at P < 0.05 (t-test) when 5 lM Hesperadin-treated cells were compared with the control. (c) Hesperadin-treated BY-2 cells were immunostained using the anti-a-tubulin antibody; DNA was stained with DAPI. Arrows indicate lagging chromosomes. (d) Cells containing lagging chromosomes were counted in 500 anaphase/telophase cells. The increase in cells containing lagging chromosomes was statistically significant at P < 0.05 (t-test) when comparing 5 lM Hesperadin-treated cells with the control. Data were averaged from three independent measurements (SD). Merged images in (c) of DNA (blue); a-tubulin (green). Scale bars (a and c), 10 lm.

prophase bands appeared at prophase. As the cells reached metaphase, chromosomes aligned along the metaphase plate and the kinetochore microtubules were organized. At telophase, sister chromatids separated and phragmoplasts appeared in Hesperadin-treated BY-2 cells. Thus, Hesperadin did not affect microtubule dynamics in BY-2 cells. However, lagging chromosomes were observed at anaphase/telophase (Figure 5c) and the frequency of anaphase/ telophase cells containing lagging chromosomes was greatly increased in Hesperadin-treated BY-2 cells (Figure 5d). Thus, Hesperadin appears to induce the occurrence of lagging chromosomes at anaphase/telophase, possibly causing formation of micronuclei in daughter cells. Discussion In this study we showed that, in tobacco BY-2 cells, the Aurora kinase inhibitor Hesperadin reduced both H3S10ph and H3S28ph, which are localized at pericentromeric regions similarly to AtAUR3. The BY-2 expressed sequence tag (EST) database (Matsuoka et al., 2004) contains one clone that possibly encodes a protein homologous to that of AtAUR3 (80% identity with the 128 C-terminal amino acid sequence of AtAUR3). No previously identified kinase has been shown to phosphorylate Ser28 of histone H3 in plants; however, the results presented here clearly indicate that AtAUR3 and

NtAUR phosphorylate not only Ser10, but also Ser28 of histone H3. In contrast, AtAUR1 is able to phosphorylate only Ser10 of histone H3, not Ser28 in vitro (Demidov et al., 2005), suggesting that each AtAUR plays a distinct role through different phosphorylation modes for histone H3. Hesperadin inhibited H3S10ph and H3S28ph, but not H3T3ph and H3T11ph, in BY-2 cells. This result suggests that Hesperadin specifically inhibits Aurora kinase activity in plants. Recently, crystallization studies of the binding form of Hesperadin to the Xenopus laevis Aurora B:INCENP complex showed that Hesperadin is an ATP-competitive inhibitor (Sessa et al., 2005). Because the Hesperadin-binding sites of Aurora B are conserved between humans and A. thaliana (AtAUR1–3), Hesperadin potentially inhibits plant Aurora kinases in the same manner as ATP-competitive inhibitors in animals. Furthermore, Hesperadin probably inhibits AtAUR1–3 kinase activities in vitro. To date, the BY-2 EST database contains only one homologue of AtAUR3, but not of AtAUR1 and 2. However, if such homologues exist for AtAUR1 and 2, it is highly possible that Hesperadin can also inhibit the kinase activities of these tobacco homologues. In mammals, 250 nM Hesperadin was required to inhibit the Aurora kinase activity in vitro, and severe defects were observed in 50 nM Hesperadin-treated HeLa cells (Hauf et al., 2003). However, the amount of Hesperadin required to inhibit the kinase activity of AtAUR3 in vitro was 1 lM, and

ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 48, 572–580

Aurora kinase in chromosome segregation 577 chromosome defects were observed in BY-2 cells treated with over 5 lM Hesperadin. Considering that the effective Hesperadin concentration was different between mammals and plants in vitro, the difference of Hesperadin effect in vivo may be due to the subtle difference of the protein structure of Aurora kinases. Inhibition of histone deacetylation by trichostatin A (TSA) in Nicotiana sylvestris protoplasts during mitosis induces the accumulation of metaphase cells, and reduces H3S10ph at anaphase and telophase. In addition, the mitotic index is reduced and abnormal chromosomes are observed in TSAtreated N. sylvestris cultured cells (Li et al., 2005). The effects of Hesperadin on BY-2 cells – the accumulation of metaphase cells and lagging chromosomes – were similar to those of TSA on N. sylvestris protoplasts and cultured cells. However, the mitotic index was not significantly different between Hesperadin-treated BY-2 cells and controls. Although Hesperadin increased metaphase cells, it reduced anaphase/telophase cells, suggesting Hesperadin-induced aberrant metaphase/anaphase transition, and early exit from mitosis after chromosome segregation. Aurora B is known to play roles in chromosome alignment and segregation, and studies in various organisms have shown that Aurora B promotes chromosome bi-orientation by monitoring incorrect kinetochore–microtubule attachments. The function of Aurora B is associated with its ability to sense tension between sister centromeres, which is established once sister kinetochores are attached to opposite poles (Andrews et al., 2003). In Xenopus, microinjection of anti-Xaurora B antibody blocked chromosome alignment and segregation, and overrode the spindle-attachment checkpoint (Kallio et al., 2002). In human cells, depletion of Aurora B by RNAi and Aurora kinase inhibitor treatment altered chromosome alignment (Ditchfield et al., 2003; Hauf et al., 2003). Furthermore, Hesperadin treatment also caused an elevated frequency of chromosomes showing syntelic attachment, where both sister kinetochores attach to microtubules from the same spindle pole in mammalian cells (Hauf et al., 2003; Lampson et al., 2004). Thus, Aurora B defects result in a greater proportion of chromosomes with incorrect kinetochore–microtubule attachments and a corresponding increase in chromosome misalignment. In contrast, because chromosome misalignment was not observed in Hesperadin-treated cells, plant Aurora kinase is not required for regular chromosome alignment through regulation of kinetochore–microtubule attachment. Sister chromatid separation requires the removal of cohesin from chromosomes (Haering and Nasmyth, 2003). Inhibition of Aurora B by Hesperadin reduces dissociation of cohesin from chromosome arms in human cells (GimenezAbian et al., 2004). Caenorhabditis elegans Aurora B kinase, AIR-2, regulates the release of chromosome cohesion via phosphorylation of meiotic cohesin subunit REC8 (Rogers et al., 2002). Therefore, inhibition of plant Aurora kinase by

Hesperadin is thought to prevent cohesin dissociation, followed by arrest at metaphase and an increase in lagging chromosomes in BY-2 cells. The formation of micronuclei in Hesperadin-treated BY-2 cells should be caused by the presence of lagging chromosomes. Surprisingly, the formation of micronuclei by Hesperadin had no effect on cell growth in BY-2 cells. This may be explained by the redundancy of gene function in the polyploid genome because N. tabacum is an allotetraploid (2n ¼ 4x ¼ 48). In the future it is therefore necessary to observe a longer period and/or use a species with a small diploid genome, such as A. thaliana. In AtAUR3-overexpressing BY-2 cells, spindle microtubules become disassembled and the orientation of cell division is altered (Kawabe et al., 2005). In contrast, no aberrations in microtubule dynamics were observed in Hesperadin-treated BY-2 cells. Aberrations in cell division were thus caused by the presence of elevated AtAUR3 kinase activity, not by the inhibition of NtAUR kinase activity. Although it is necessary to analyse cell division in NtAURoverexpressing BY-2 cells, AtAUR3 and NtAUR are thought to regulate cell division through interaction with other proteins. In plants, H3S10ph and H3S28ph are restricted to the pericentromeric regions during mitosis and meiosis II, while during meiosis I both S10 and S28 of histone H3 are phosphorylated along the entire chromosomes (Gernand et al., 2003; Kaszas and Cande, 2000; Manzanero et al., 2000). Single chromatids resulting from equational segregation of univalents at anaphase I are normally condensed without H3S10ph during meiosis II (Manzanero et al., 2000). These observation indicate that H3S10ph and H3S28ph are involved in sister chromatid cohesion, rather than chromosome condensation (Gernand et al., 2003; Kaszas and Cande, 2000; Manzanero et al., 2000). Consistently, chromatids were normally condensed without H3S10ph and H3S28ph during mitosis in Hesperadin-treated BY-2 cells. However, chromosome misalignment caused by defective chromatid cohesion was not observed in Hesperadin-treated BY-2 cells. This result suggests that H3S10ph and H3S28ph are not necessary for the establishment and maintenance of chromatid cohesion during mitosis, although it is possible that the low level of H3S10ph and H3S28ph, not detected by indirect immunofluorescence, is sufficient for chromatid cohesion. Recently, the ‘ready production-label’ model has been proposed (Hans and Dimitrov, 2001; Prigent and Dimitrov, 2003). According to this model, H3S10ph and H3S28ph in mitosis serve as a signal for metaphase/ anaphase transition. Based on our results, the Hesperadininduced aberrant metaphase/anaphase transition suggests that H3S10ph and H3S28ph may be concerned with the ready-production label in plants. In mammals, Aurora kinases also phosphorylate CENP-A at Ser7 (Zeitlin et al., 2001). Phosphorylation of CENP-A at Ser7 is required for kinetochore function, such as chromo-

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578 Daisuke Kurihara et al. some alignment (Kunitoku et al., 2003). In plants, centromeric histone H3 (CENH3), which is homologous to CENP-A in mammals, has been identified in A. thaliana (Talbert et al., 2002); maize (Zhong et al., 2002); rice (Nagaki et al., 2004); sugarcane (Nagaki and Murata, 2005) and Luzula nivea (Nagaki et al., 2005). In addition, recent studies showed that maize CENH3 is phosphorylated in mitosis and meiosis (Zhang et al., 2005). These findings raise the possibility that CENH3 is phosphorylated by Aurora kinases in plants. Therefore, the phenotypes in Hesperadin-treated BY-2 cells may be due to the defect of phosphorylation of CENH3. Further analyses will be required to determine whether phosphorylation of CENH3 plays a role in the kinetochore function in plants. In conclusion, we found that Hesperadin induces aberrant chromosome segregation in BY-2 cells. Hesperadin prevents H3S10ph and H3S28ph, probably by inhibiting Aurora kinase activity in BY-2 cells. Moreover, micronuclei and lagging chromosomes were observed in Hesperadin-treated BY-2 cells. Considering that chromosome misalignment was not observed in Hesperadin-treated BY-2 cells, Aurora kinases may be concerned with cohesin dissociation rather than regulation of kinetochore–microtubule attachment in plants. However, given that Aurora B has various roles in chromosome alignment and segregation in mammals, and forms a complex with INCENP, Survivin and Borealin/DasraB (socalled chromosomal passenger complex; Vagnarelli and Earnshaw, 2004), further analyses of interacting proteins and substrate of plant Aurora kinases would provide insights into chromosome alignment and segregation. The current results on Aurora kinases using Hesperadin showed that they have an important role in chromosome segregation in plants. Experimental procedure Culture of tobacco BY-2 cells and experimental treatments Tobacco BY-2 cells were maintained as described previously by Nagata et al. (1992). BY-2 cells were cultured in modified Linsmaier and Skoog (LS) medium in a rotary shaker at 25C in the dark. For Hesperadin treatment, tobacco BY-2 cells were used 2 days after subculture. Five micromolar Hesperadin (Boehringer Ingelheim, Vienna, Austria) was added to 2-days-old BY-2 cells and cultured for another 24 h. For measurements of cell number, a portion of the culture was sampled every day after Hesperadin treatment, and the cell number was counted under a light microscope (TMS; Nikon, Tokyo, Japan) in a Burker–Turk chamber (Erma Optical Works, Tokyo, Japan). For determination of the mitotic index, cells were stained with 4¢,6-diamidino-2-phenylindole (DAPI) and the number of mitotic cells was counted under a fluorescence microscope (AxioPlan II, Zeiss, Jena, Germany).

Indirect immunofluorescence BY-2 cells were fixed with 4% (w/v) paraformaldehyde for 20 min in phosphate-buffered saline (PBS). Cell collection was performed by

gravity-dependent sedimentation. Fixed cells were washed in PBS and placed on coverslips treated with poly-L-lysine (Sigma, St Louis, MO, USA). Cell walls were briefly digested with cell wall-degrading enzyme mixture for 10 min at room temperature. After being washed twice in PBS, cells were permeabilized by 15 min incubation with PBS containing 0.5% Triton X-100 at room temperature, and washed twice more in PBS. After blocking in PBS containing 1% (w/v) BSA, cells were incubated with respective primary antibodies at the following dilutions in PBS overnight at 4C: 1:500 dilutions of rabbit polyclonal antibodies against H3S10ph, H3T3ph and H3T11ph ( Upstate Biotechnology, Lake Placid, NY, USA); rat polyclonal antibodies against H3S28ph (HTA28, Goto et al., 1999) and 1:20 dilution of mouse monoclonal antibodies against a-tubulin (Calbiochem, La Jolla, CA, USA). After being washed twice in PBS, cells were incubated with respective secondary antibodies at the following dilutions in PBS for 3 h at room temperature: 1:200 dilutions of Alexa 488-conjugated goat antirabbit IgG antibodies; Alexa 488-conjugated goat antimouse IgG antibodies ( Invitrogen Molecular Probes, Eugene, OR, USA); FITC-conjugated goat antirat IgG antibodies (Chemicon, Temecula, CA, USA) and 1:50 dilution of rhodamine-conjugated goat antirabbit IgG antibodies ( Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Cells were washed twice in PBS then mounted with DAPI solution (2 lg ml)1). For isolation of nuclei, fixed cells were incubated in an enzyme mixture for 30 min at 37C to digest the cell wall. Cells were resuspended in Galbraith buffer [45 mM MgCl2, 30 mM sodium citrate, 20 mM MOPS (3-(N-morpholino)propanesulfonic acid), 0.1% Triton-X 100 pH 7.3; Galbraith et al., 1983] and filtered through a 30-lm nylon mesh. Isolated nuclei were centrifuged onto microscope slides (s8111; Matsunami Glass, Tokyo, Japan) using Cytospin (Cytospin 3; Shandon Scientific Ltd, Runcorn, UK) at 72 g for 5 min. Indirect immunofluorescence was performed as described above.

Microscopy Images were taken with a fluorescence microscope (AxioPlan II) equipped with a cooled charged-coupled device (CCD) camera (MicroMax; Roper Scientific, Trenton, NJ, USA). Image processing was performed with the following software: IPLAB (Visitron Systems, Eichenau, Germany), SCION IMAGE ( Scion Corporation, Frederick, MD, USA) and ADOBE PHOTOSHOP 7.0 (Adobe Systems, San Jose, CA, USA).

Preparation of recombinant proteins Cloning of the expression vectors for AtAUR3 and A. thaliana histone H3 was conducted as described previously, except for cloning of the AtAUR3 gene into the pDEST15 expression vector (Kawabe et al., 2005). A GST-tagged recombinant AtAUR3 fusion protein was expressed in Escherichia coli strain BL21 (DE3) pLysS. Cells were grown to OD600 ¼ 0.6 at 37C in 1 l LB medium then, after further incubation overnight at 18C with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG), cells were harvested and lysed in PBS containing 1 mM 2-mercaptoethanol, 1 mM EDTA, 1 mM phenylmethansulfonyl fluoride (PMSF) and complete protease inhibitor cocktail (Roche, Mannheim, Germany). GST fusion proteins were purified from the supernatants by affinity chromatography using a GSTrap column (Amersham Biosciences, Uppsala, Sweden) and gel filtration chromatography (Amersham Biosciences) in 50 mM Tris–HCl buffer pH 7.7 containing 1 mM 2-mercaptoethanol and 1 mM EDTA.

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Aurora kinase in chromosome segregation 579 His-tagged recombinant histone H3 was expressed in E. coli BL21 (DE3). Cells were grown to OD600 ¼ 0.6 at 37C in 1 l LB medium then, after further incubation for 3 h at 37C with 1 mM IPTG, His-histone H3 was obtained in the form of an inclusion body. The inclusion body was washed with Tris–HCl-buffered saline (TBS) and TBS containing 2 M urea, then dissolved in TBS containing 6 M urea. His-histone H3 was purified by Ni chelate affinity chromatography using a HiTrap chelating column (Amersham Biosciences), and separated on a Protein C4 reverse-phase chromatography column (Vydac, Hysperia, CA, USA) using an HPLC system (Tosoh, Tokyo, Japan). The elutions were collected, frozen and lyophilized.

In vitro kinase assay In vitro kinase assay was performed with purified recombinant proteins with histone H3 as the substrate and 0.25 lM ATP in kinase buffer (10 mM K  HEPES, 50 mM sucrose, 100 mM KCl, 20 mM MgCl2, 0.1 mM CaCl2, 2 mM K  EGTA pH 7.7) for 15 min at 30C. Phosphorylated histone H3 was determined by immunoblotting using 1:1000 dilutions of rabbit polyclonal antibodies against H3S10ph, H3T3ph and H3T11ph (Upstate Biotechnology), and rat polyclonal antibodies against H3S28ph (HTA28; Goto et al., 1999). Immunoreactive proteins on the gel blots were reacted with 1:10 000 dilution of alkaline phosphatase (AP)-labelled goat antirabbit IgG antibodies ( Vector Laboratories, Burlingame, CA, USA), AP-labelled rabbit antirat IgG antibodies (Sigma), and developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Roche) as substrate.

Acknowledgements We are grateful to Dr H. Goto and Dr M. Inagaki (Aichi Cancer Center Research Institute, Japan) for providing the antibody against phosphorylated histone H3 at Ser28, Dr N. Kraut (Boehringer Ingelheim) for providing Hesperadin, Dr A. Houben (IPK, Germany) for advice on the isolation of nuclei and Reiko Isobe for technical assistance. This study was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and the Industrial Technology Research Grant Program in 2003 from the New Energy and Technology Development Organization (NEDO) of Japan. D. K. was supported by the Research Fellowships of the Japanese Society of the Promotion of Science for Young Scientists.

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