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Vol. 18, Issue 5, 1657-1669, May 2007
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Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom
Submitted October 4, 2006;
Revised February 6, 2007;
Accepted February 7, 2007
Monitoring Editor: Yixian Zheng
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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).
One of the best characterized roles of the kinase Aurora B is to correct defective kinetochore-microtubule attachment before anaphase onset and therefore ensure proper chromosome biorientation (Tanaka et al., 2002; Ditchfield et al., 2003; Hauf et al., 2003; Tanaka, 2005; Pinsky et al., 2006). Aurora B is also crucial for the recruitment of some spindle checkpoint components to kinetochores (Ditchfield et al., 2003; Vigneron et al., 2004). Thus Aurora B regulates both the physical connections of chromosomes onto the spindle and mitotic progression. When Aurora B activity is compromised, chromosomes mis-segregate massively, leading to aneuploidy (reviewed in Giet et al., 2005). Defective kinetochore-microtubules attachments accumulate in these cells and chromosomes never reach a proper metaphase plate (Hauf et al., 2003).
Aurora B is one of the Chromosomal Passenger proteins, first identified in vertebrates as proteins sharing a complex and highly regulated localization pattern in mitosis (Earnshaw and Bernat, 1991). In particular they transfer abruptly from the inner-centromeres to the spindle midzone at the metaphase to anaphase transition. Each Chromosomal Passenger protein, Survivin, Borealin, TD60, INCENP, and Aurora B has long been recognized as major regulators of mitosis (see Vagnarelli and Earnshaw, 2004 for review). In fission yeast, homologues of only three of the Chromosomal Passenger proteins have been identified, Bir1/Survivin, Pic1/INCENP, and Ark1/Aurora B (Morishita et al., 2001; Petersen et al., 2001; Leverson et al., 2002; Huang et al., 2005). Their localization pattern is also carefully regulated and closely resembles their vertebrate counterparts (see below) and they are also considered as crucial players of mitosis in fission yeast. The mechanisms ensuring the localization and the transfer of the Passenger proteins from one location to another are still poorly understood, as are their exact roles at each location they visit in the cell.
The Shugoshin proteins were first identified as regulators of chromosome segregation in Drosophila meiosis (MEI-S332) and have since fuelled a great deal of interest (reviewed in Watanabe and Kitajima, 2005). Shugoshin 1 (Sgo1) protects cohesion at centromeres in the pre-anaphase stages of meiosis I, partly by regulating cohesin phosphorylation status through the recruitment of a specific phosphatase to centromeres (Kitajima et al., 2006; Riedel et al., 2006; Tang et al., 2006). Similarly, homologues of Sgo1 help protect centromeric cohesion during the pre-anaphase stages of vertebrate mitosis (reviewed in Watanabe, 2005). However, the sole budding yeast Shugoshin (ScSgo1) is required to maintain sister chromatid cohesion in meiosis I but not in mitosis (Katis et al., 2004; Marston et al., 2004; Indjeian et al., 2005), demonstrating that Shugoshin function in centromere cohesion is not universal in mitosis. Other roles have been ascribed to the Shugoshin proteins, such as monitoring tension between sister-chromatids (Indjeian et al., 2005) and regulating microtubules dynamics (Salic et al., 2004; Suzuki et al., 2006).
Fission yeast has two members of the Shugoshin family, Sgo1 and Sgo2. Sgo1 appears to have only meiotic functions, whereas lack of Sgo2 triggers both meiotic and mitotic defects. In meiosis I, Sgo2 is required to ensure mono-orientation of sister-chromatids (Rabitsch et al., 2004; Vaur et al., 2005). In mitosis, cells lacking Sgo2 show no visible defect in chromosome segregation (Rabitsch et al., 2004), but they nonetheless lose chromosomes at a significant rate and exhibit sensitivity to the microtubule-depolymerizing drug TBZ (Kitajima et al., 2006). Taken together, these data suggest an as yet unidentified role of Sgo2 in the faithful segregation of chromosomes in mitosis.
Here we demonstrate that fission yeast Shugoshin2 (Sgo2) is crucial for chromosome biorientation in the first anaphase after a prolonged spindle checkpoint arrest, likely through regulating the Passenger proteins. Indeed, Sgo2 colocalizes with the Passenger proteins in early mitosis and promotes their efficient recruitment onto centromeres and telomeres. Sgo2 also regulates the stability of the Survivin-INCENP interaction. Sgo2 localization on centromeres is itself dependent on Survivin. Taken together our results suggest that Sgo2 either regulates, or is itself, a docking site for the recruitment of the Passenger proteins on centromeres.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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). nda3-KM311 cells (
5–7.106 cells per ml of rich media) were shifted to 18°C for typically 6–8 h. The synchrony upon release was greater when cells were arrested for 6 h and released at 30°C. At 8 h, hyper-condensed chromosomes were more often individualized and had the tendency to move away from each other (especially chromosome 3, the smallest of all three chromosomes in Schizosaccharomyces pombe). In these conditions there was greater chromosome loss, even in the wild-type (see Figure 1).
Microscopy
Yeast cells with green fluorescent protein (GFP)-, cyan fluorescent protein (CFP)-, or mCherry-tagged proteins (Snaith, 2005) were grown in rich media and very briefly fixed in 100% methanol (<30 s) before observation, unless stated otherwise. Preliminary experiments have demonstrated that the methanol fixation did not affect the localization pattern of the proteins of interest (not shown). Imaging was performed using an Intelligent Imaging Innovations (3i) Marianas system (Denver, CO). This system uses a Zeiss Axiovert fluorescence microscope (Thornwood, NY), a CoolSNAP HQ charge-coupled device camera (Photometrics, Woburn, MA), and Slidebook software (3i; Photometrics). Unless otherwise stated, images were deconvolved using the no-neighbor algorithm. For immunofluorescence, cells were fixed for 5–15 min by the addition of freshly prepared paraformaldehyde solution (3%).
Coimmunoprecipitations
Coimmunoprecipitations were carried out as previously described (Vanoosthuyse et al., 2004). When stated, immunoprecipitated proteins were dephosphorylated using 
-phosphatase (New England Biolabs, Beverly, MA) according to the manufacturer's instructions. Immunoprecipitated proteins were analyzed by Western blot and when stated were quantified using the ImageQuant software (GE Healthcare, Little Chalfont, United Kingdom). In Figure 5D1, quantifications were performed on five blots corresponding to five independent experiments.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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) on chromosome segregation. As reported earlier, lack of Sgo2 has only a mild effect on chromosome segregation in cycling, unchallenged cells (roughly 1% of cells mis-segregate chromosome 2; Kitajima et al., 2004 and not shown). However, we found that sgo2 cells struggled to recover from an early mitotic arrest induced by the cold-sensitive 
-tubulin mutant nda3-KM311. Unable to form a spindle in the cold, nda3KM311 cells generate unattached kinetochores in mitosis that are recognized by the spindle checkpoint (Hiraoka et al., 1984). The viability of sgo2 nda3-KM311 cells decreased by 40–50% after 8 h at 18°C (not shown and Figure 1A). However this was not due to a spindle checkpoint defect because sgo2 nda3-KM311 cells arrested normally in early mitosis before cytokinesis (Figure 1, B and C, and not shown). Indeed chromosomes hyper-condensed in sgo2 cells as in sgo2+ cells and <2% of cells had septa (not shown). Furthermore, kinetochores recruited normal levels of the spindle checkpoint kinase Bub1 (Figure 1B), Polo and Cdc2 kinases accumulated on spindle pole body (SPBs; Figure 1C, data not shown), and cohesion at centromeres was maintained (Supplementary Figure 1). We conclude from these observations that Sgo2 is not required for a spindle checkpoint arrest triggered by the lack of attachment of kinetochores to microtubules.
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) die after the arrest, we monitored the first mitosis after the release. The nda3-KM311–dependent arrest is reversible by shifting the temperature back to 30–36°C. This allows the spindle to be reformed and the cells enter fairly synchronously into anaphase (Kanbe et al., 1990 and Materials and Methods). The timing of anaphase onset was similar in wild-type and in sgo2 cells (Supplementary Figure 2). However we observed a significant increase in chromosome mis-segregation in the absence of Sgo2 (up to 35% of cells mis-segregate chromosome II when cells were maintained in the arrest for 8 h and subsequently released at 36°C; see Figure 1D and Materials and Methods). Consistent with this, a significant proportion of cells proceeded through anaphase with clear kinetochore-microtubule attachment defects (Figure 1, E–G): 1) some cells proceeded into anaphase even though one or two chromosome(s) were still unattached and 2) some kinetochores appeared to show monotelic/syntelic attachments (because it is difficult to observe single microtubules in S. pombe, it is hard to show conclusively whether both kinetochores of a pair are attached or not). These segregation defects are consistent with inefficient chromosome biorientation.
Sgo2 Colocalizes with Chromosomal Passenger Proteins in Early Mitosis
One major regulator of both kinetochore-microtubule function and the spindle checkpoint are the Chromosomal Passenger Proteins (see Vagnarelli and Earnshaw, 2004 for review). Interestingly, sgo2 cells display a synthetic lethal interaction with cut17.275, a mutant allele of the Passenger protein Survivin, supporting the idea that Sgo2 and the Passenger proteins act together to ensure accurate chromosome segregation (not shown). To test the possibility that Sgo2 might regulate the Chromosomal Passenger proteins, we first examined their localization pattern concomitantly throughout the cell cycle. In interphase, Sgo2 usually localized at one to three distinct foci that colocalize with the DNA but not with Cut12 (Figure 2A, panel 1). These foci actually represent the heterochromatic regions present at telomeres (a detailed analysis of Sgo2 function in interphase is to be published elsewhere). A fainter signal also colocalized with the whole DAPI mass, suggesting that Sgo2 might also associate with chromatin. In interphase, Passenger proteins were enriched in the nucleolus and on the SPB-centromere cluster (Supplementary Figure 3A). In early mitosis, however, Sgo2 and Bir1/Survivin colocalized to two to three distinct foci associated with the DNA, on either side of the nucleus (Figure 2A, panel 2): one focus was in close association with the SPB-centromere cluster, whereas the other foci represented the clustered telomeres, as shown by their colocalization with the telomere component Taz1 (Figure 2C). In metaphase, Sgo2 and Bir1/Survivin still colocalized, at least partially (Figure 2A, panel 3): they were both found on centromeres and telomeres, but Bir1/Survivin also localized on SPBs, as shown by its colocalization with Cut12 and Sad1 (Figure 2B). This localization pattern has been confirmed by arresting the cells in metaphase by overexpressing the spindle checkpoint component Mad2 (He et al., 1997; Supplementary Figure 3B). In early anaphase (Figure 2A, panels 4 and 5), Bir1/Survivin and Sgo2 displayed very little colocalization: the bulk of Bir1/Survivin was found on the two SPBs and the spindle, whereas Sgo2 remained on centromeres (see blowup in Figure 2A, panel 4). Occasionally Sgo2 was also found on the spindle in the early stages of anaphase, as reported earlier (Rabitsch et al., 2004). Later in anaphase, Sgo2 left centromeres to associate with the whole chromosomes, whereas Bir1/Survivin concentrated on the spindle midzone (Figure 2A, panel 6). In septating cells (G1/S transition, Figure 2A, panel 7), Sgo2 and Bir1/Survivin both adopted their interphase localization pattern. In conclusion, Sgo2 and Bir1/Survivin have distinct and very complex localization patterns throughout the cell cycle, and they only colocalize in the early stages of mitosis before anaphase onset. A summary of their localization pattern is presented in Table 1. Note that in cycling cells, all three Passenger proteins Bir1/Survivin, Pic1/INCENP, and Ark1/ Aurora B colocalized (see Morishita et al., 2001; Huang et al., 2005; Figure 2C and not shown). This detailed in vivo analysis of Passenger proteins demonstrates that the localization pattern published previously is incomplete: our data show that in interphase they localize not only in the nucleolus but also on the SPB-centromere cluster and that they localize on centromeres, telomeres, and SPBs every mitosis.
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Previous chromatin immunoprecipitation (ChIP) studies have shown that both Sgo2 and Bir1/Survivin bind to the inner-centromere (outer heterochromatic repeats [OTR]; see Morishita et al., 2001; Kitajima et al., 2004). Therefore, we tested whether the lack of Sgo2 would affect the localization of the Passenger proteins on centromeres. Note that the localization pattern of Sgo2 and the Passenger proteins are significantly affected by prolonged activation of the spindle checkpoint: in early mitosis of a cycling population, the Passenger proteins normally localize to SPBs, centromeres, and telomeres (Table 1), whereas Sgo2 localizes to centromeres and telomeres. In nda3KM311-arrested sgo2+ cells, Sgo2 and all three Passenger proteins become enriched only on centromeres (Figure 3, A and B). Lack of Sgo2 had a dramatic effect on the localization of all three Passenger proteins: in nda3KM311-arrested cells lacking Sgo2, no more than two Bir1/Survivin foci were observed, and Bir1/Survivin was also found in the nucleus (Figure 3, C and D). The two foci corresponded to SPBs, as shown by their colocalization with the SPB marker Cut12 (Figure 3D). In 80% of the cells Pic1/INCENP and Ark1/Aurora B localized on SPBs and in the nucleus. In the remaining 20% of the cells, Pic1/INCENP and Ark1/Aurora B could still be observed on centromeres, but at a dramatically reduced level (estimated at a 10-fold reduction by quantification of the fluorescence; Figure 3E and not shown). In conclusion, Sgo2 is crucial for the localization and/or maintenance of all three Passenger proteins on centromeres upon checkpoint arrest. This is consistent with the results described in Figure 1, showing that Sgo2 is required to ensure proper chromosome biorientation upon recovery from a prolonged checkpoint arrest.
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cells (39/60 early mitotic cells analyzed after 3D capture and deconvolution), Bir1/Survivin failed to localize on centromeres and concentrated on SPBs instead (Figure 3G). In 10% of the cells, Bir1/Survivin prematurely transferred to the spindle. In the remaining 25% of the cells, Bir1/Survivin remained on centromeres, albeit at a reduced level. Interestingly, lack of Sgo2 had a less severe effect on the localization of the two other Chromosomal Passenger proteins Pic1/INCENP and Ark1/Aurora B in cycling cells: their localization at centromeres was maintained in more than 65% of the cells (Figure 3G). To determine whether Ark1/Aurora B was recruited to centromeres with the same efficiency in the absence of Sgo2, two populations of cells expressing Ark1/Aurora B-GFP (with and without a functional Sgo2+ gene) were arrested in metaphase by overexpression of the spindle checkpoint component Mad2. Both populations were mixed and imaged at the same time. To differentiate between the two types of cells, the kinetochore component Ndc80 was tagged with CFP in sgo2 cells, whereas the kinetochore component Mis13 was tagged with mCherry in Sgo2+ cells. By analyzing the fluorescence intensities of the GFP signals in both populations, imaged in the same fields of view, we were able to show that lack of Sgo2 induces a reduction of 40–50% in the amount of Ark1/Aurora B recruited on centromeres in cells arrested in metaphase by the overexpression of Mad2 (Figure 3H). However the total amount of Ark1/Aurora B in the cell was not affected by lack of Sgo2, as shown by Western blot analysis (Figure 3I and see also Figure 5). In conclusion, Sgo2 is also an important regulator of the Chromosomal Passenger proteins in cycling cells. Interestingly, lack of Sgo2 had a more penetrant effect on Bir1/Survivin localization.
To test how specific an effect the lack of Sgo2 was having on overall centromere structure, we analyzed the localization of a number of other proteins important for kinetochore-microtubule attachments. Lack of Sgo2 did not affect the localization of the centromere-specific histone H3-variant CENP-A/Cnp1 (Figure 1E), the spindle checkpoint component Bub1 (Figure 1B), the kinetochore component Ndc80 (Figures 1G and 3, C and F) and the Ask1 member of the DASH complex (Supplementary Figure 4A). Furthermore we detected no alleviation of silencing of a reporter gene inserted in the centromeric heterochromatin (outer heterochromatic repeats OTR::ade6+) in the absence of Sgo2, suggesting that lack of Sgo2 does not affect heterochromatin structure and function (Supplementary Figure 4B). Taken together, these data show that lack of Sgo2 does not affect centromere and kinetochore functions per se and demonstrate that the sgo2 effect on the localization of the Passenger proteins is specific.
Sgo2 Localization Pattern Is Affected in the Bir1/Survivin Mutant bir1.46
Recently it has been shown that centromeric localization of the Drosophila Shugoshin homologue MEI-S332 is dependent on the Passenger protein INCENP (Resnick et al., 2006). To determine whether Sgo2 localization was regulated by the Passenger proteins, we analyzed the localization pattern of Sgo2 in a Bir1/Survivin temperature-sensitive mutant (bir1.46, where Bir1/survivin is mutated on residue 976 [C976Y], Huang et al., 2005). As reported previously (Huang et al., 2005), we found that INCENP did not localize on centromeres anymore at the restrictive temperature of 36°C (not shown). In these cells, at either the permissive temperature of 25°C or the restrictive temperature of 36°C, we could not detect Sgo2 localization on centromeres (Figure 4). We conclude that Sgo2 localization on centromeres is dependent on Survivin.
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) had a far less penetrant effect on the localization of Sgo2, Aurora B, and INCENP (Supplementary Figure 5). Moreover, cut17.275 experienced a transient Mad2-dependent delay at the restrictive temperature of 36°C (Supplementary Figure 5). We suggest that cut17.275 is a weaker allele of Bir1/Survivin than bir1.46.
Sgo2 Regulates the Stability of the Pic1/INCENP-Bir1/Survivin Complex
In vertebrates the Passenger proteins form a stable complex, the CPC (reviewed in Vader et al., 2006b). However in fission yeast, the fact that lack of Sgo2 has a more penetrant effect on Bir1/Survivin localization than on the other Passenger proteins brings into question the idea that the three Chromosomal Passenger proteins form a single stable "trimeric" complex, while on centromeres. To address this issue further, we undertook a biochemical analysis of the complexes formed by the Chromosomal Passenger proteins in the presence or absence of Sgo2. To do this, we fused a C-terminal S-ZZ tag (Cheeseman et al., 2001) to all four proteins Ark1/Aurora B, Bir1/Survivin, Pic1/INCENP and Sgo2. These proteins all replace the endogenous proteins and are expressed at their native loci from their own promoters. They are stable and detectable on a Western blot, although they appear to be present at different levels, consistent with them having independent roles in the cell (Figure 5A). We cannot however rule out that these differences are not in part due to different transfer efficiencies. We noticed that Pic1/INCENP-SZZ was cleaved artifactually during immunoprecipitation (Figure 5, C and D, and not shown). Therefore coimmunoprecipitation experiments were not carried out for more than 40 min to avoid the complete cleavage of Pic1/INCENP in the extract. In these experiments, we failed to detect an interaction between Bir1/Survivin and Ark1/Aurora B (see below). However, the formation of a complex between Ark1/Aurora B and Pic1/INCENP on one hand and Pic1/INCENP and Bir1/Survivin on the other hand was clearly detectable (Figure 5B). Interestingly, lack of Sgo2 did not affect complex formation between Ark1/Aurora B and Pic1/INCENP (Figure 5C), but the complex between Pic1/INCENP and Bir1/Survivin was consistently found at reduced levels in mitotic checkpoint-arrested cells (in 10 experiments, see Figure 5D). We observed a two- to fivefold reduction in the amount of Bir1/Survivin recovered after Pic1/INCENP immunoprecipitation in the absence of Sgo2 compared with wild type. Interestingly, the effect of Sgo2 on the Survivin-INCENP complex was specific to checkpoint-arrested cells where all three proteins localize on centromeres (Figure 3). Lack of Sgo2 had no significant effect on the Survivin-INCENP complex in interphase cells (Figure 5D2), where Sgo2 does not colocalize with Survivin or INCENP (Figure 2).
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; Klein et al., 2006; Vader et al., 2006a). Our data suggest that this trimeric complex, if it exists in fission yeast, is not very abundant or stable, as we can readily detect a complex between Survivin and the full-length INCENP on one hand and an INCENP-Aurora B complex on the other hand (Figure 5). The in vitro cleavage of INCENP might also prevent us from detecting this trimeric complex, as INCENP would fail to act as a bridge between Survivin and Aurora B. Together, these observations suggest that the Passenger proteins form several independent complexes in fission yeast. To test whether Sgo2 directly interacts with Passenger proteins, we used conventional coimmunoprecipitation and tandem affinity purifications followed by mass spectrometry analysis. However, despite intense efforts, we have so far failed to find reproducible evidence of a stable Sgo2-Passenger protein complex (not shown and see Discussion).
The Conserved C-Terminus Domain of Sgo2 Is Crucial for Its Localization
Sgo2 can be divided into three domains: a conserved N-terminal coiled-coil region, an internal domain rich in hydroxylated residues, and finally a conserved C-terminus (Rabitsch et al., 2004). The conserved N-terminus of human Sgo1 has been shown to bind microtubules (Salic et al., 2004). The function of the conserved C-terminal domain of Sgo2 is unknown. We therefore deleted it (sgo2563-647). The truncated protein was stable (Figure 6A). Unlike full length Sgo2, it was not enriched on telomeres in interphase, but instead decorated all chromatin (Figure 6B). In early mitosis, sgo2563-647 still localized on centromeres in 90% of cells, but localized on telomeres in only 10% of cells (Figure 6C and not shown). Strikingly, sgo2563-647 was no longer found on centromeres upon nda3KM311-dependent arrest (Figure 6D, the arrest itself was unaffected). Instead sgo2563-647 relocalized to the whole nucleus. Furthermore, Bir1/Survivin was targeted to SPBs, suggesting that Sgo2 needs to be on centromeres for Bir1/Survivin to be targeted to centromeres (Figure 6D). Thus, upon checkpoint arrest, sgo2563-647 cells had a very similar phenotype to sgo2 cells. Consistent with this, sgo2563-647 and sgo2 cells exhibited a similar amount of chromosome loss upon release (13.9 ± 1.95% for sgo2 and 18 ± 3.95% for sgo2563-647, a minimum of 700 cells counted). Thus the conserved C-terminus is required for Sgo2 localization on centromeres in checkpoint-activated cells, but is apparently dispensable in a normal unchallenged mitosis. These observations suggest that the conserved C-terminus is required to maintain Sgo2 (and therefore the Passengers) on centromeres when mitosis is unusually delayed/arrested. Another possible interpretation is that the affinity of the centromere for Sgo2 is different in cycling cells versus checkpoint-arrested cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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How Does Sgo2 Control Passenger Protein Localization?
It has been demonstrated in vertebrates that the complex formed between Survivin, Borealin, and INCENP is crucial for the targeting of the Passenger proteins to centromeres (Klein et al., 2006). Borealin has the ability to bind to chromatin in vitro (Klein et al., 2006), and it stabilizes the complex between Survivin and INCENP in vivo (Gassmann et al., 2004; Klein et al., 2006; Vader et al., 2006a). Creating a protein fusion between Survivin and INCENP bypasses the requirement for Borealin, confirming that stability of the Survivin-INCENP complex is a key determinant of Passenger localization on centromeres (Vader et al., 2006a). We have shown that lack of Sgo2 destabilizes the Pic1/INCENP-Bir1/Survivin complex and impairs the recruitment of the Passenger proteins to centromeres. Therefore, fission yeast Sgo2 appears to play a similar role to Borealin in vertebrate cells.
In HeLa cells, Survivin forms oligomers on centromeres (Klein et al., 2006). If that were true in fission yeast, our observations could be interpreted in at least three ways: either 1) Sgo2 is a cofactor that increases the stability of the Bir1/Survivin-Pic1/INCENP complex, or 2) lack of Sgo2 prevents a core Bir1/Survivin-Pic1/INCENP-Ark1/Aurora B complex from recruiting additional Bir1/Survivin molecules, or finally 3) Sgo2 recruits Bir1/Survivin to a preformed and perhaps independently targeted INCENP-Aurora B complex to generate a trimeric complex (Survivin-INCENP- Aurora B) that is more stable on centromeres than the INCENP-Aurora B complex on its own.
As yet we have failed to demonstrate a stable protein interaction between Sgo2 and the Passenger proteins. This could be for technical reasons, as both Bir1/Survivin and Pic1/INCENP are susceptible to proteolysis. In addition, the Sgo2 interaction with the Passengers might be very dynamic and/or take place only on chromosomes, either of which would make it very difficult to analyze biochemically. FRAP (fluorescence recovery after photobleaching) experiments have been attempted to compare the dynamics of fission yeast Passengers proteins with one another and with Sgo2. Because of low signals Sgo2 has so far proven difficult to analyze. Preliminary results show that Bir1/Survivin and Pic1/INCENP both turnover rapidly at fission yeast centromeres but that they display different dynamics (not shown). Vertebrate Survivin is known to be a far more dynamic member of the Passenger proteins on centromeres than Aurora B (Delacour-Larose et al., 2004), suggesting that local regulation of individual Passenger proteins occurs on centromeres. Two distinct models take account of such properties: 1) Survivin could interact with Sgo2 and form a dynamic subcomplex of the Passenger proteins with important targeting functions, or 2) Sgo2 could influence Passenger protein localization indirectly, most simply through regulation of their centromere docking site. In this model, destabilization of the Survivin-INCENP complex in the absence of Sgo2 is a secondary consequence of docking site mis-regulation (Figure 7A).
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). However, upon checkpoint activation lack of Sgo2 has dramatic consequences (Figures 1 and 2). What is special about chromosome segregation after this spindle checkpoint arrest? In nda3KM311-arrested cells, we can distinguish three types of kinetochore pairs: 1) some have no connection with either SPB, 2) some show monotelic or syntelic attachments, and finally 3) some are already amphitelically attached (Supplementary Figure 7A). Furthermore, the duplicated SPBs often move away from each other (Grishchuk and McIntosh, 2006) and, as reported previously, the nuclear envelope can lose its circular shape and ultimately form separate vesicles (Kanbe et al., 1990; Grishchuk and McIntosh, 2006; Supplementary Figure 7B). These observations suggest that the geometry created during the nda3-KM311 arrest (kinetochore-SPBs distance + nuclear shape) constrains kinetochore capture by microtubules when the spindle reforms (see Figure 7B). We propose that during recovery from this arrest efficient Passenger proteins function is required to correct kinetochore-microtubule attachments, until proper biorientation of all chromosomes is achieved. Passenger proteins function may also be required to prolong the arrest until biorientation is achieved, but our data (Supplementary Figure 2) suggest that such a delay is brief as no significant difference in the timing of anaphase onset was observed between wild-type and sgo2 cells. In a normal mitosis there are fewer geometric constraints on kinetochore-microtubule attachments and weakened Passenger protein function would be tolerated by most cells. Therefore the role of Sgo2 in ensuring the efficient recruitment of the Passenger proteins on centromeres is especially important after a prolonged nda3KM311-dependent arrest.
Why Is the Effect of Sgo2 on Passenger Protein Localization More Pronounced in a Spindle Checkpoint Arrest?
Although lack of Sgo2 has a mild effect on the localization of the Passenger proteins in a normal mitosis, Sgo2 becomes indispensable upon checkpoint activation. We can interpret this observation in three ways that are not mutually exclusive: 1) Sgo2 could regulate the maintenance of the Passenger proteins on centromeres. Cells lacking Sgo2 might be able to maintain sufficient Passenger proteins on centromeres in a normal mitosis (which lasts only a few minutes in fission yeast), whereas Sgo2 might be required to maintain the Passengers on centromeres during a prolonged mitosis (several hours in the case of an nda3KM311-dependent arrest). 2) In addition to Sgo2 there could be another factor (referred to as X, see Figure 7A) that recruits the Passenger proteins to centromeres. Although factor X can recruit the Passenger proteins to centromeres in a normal mitosis in the absence of Sgo2, it is unable to compensate for loss of Sgo2 in an nda3KM311 arrest. To explain this, we speculate that factor X acts in a microtubule-dependent manner. In budding yeast, the Cdc14 phosphatase regulates the centromere to spindle transfer of passenger proteins in anaphase (Pereira and Schiebel, 2003), and in fission yeast, the Cdc14-related phosphatase Clp1/Flp1 has been shown to regulate Ark1/Aurora B localization on centromeres (Trautmann et al., 2004). However Clp1 localizes on centromeres in nda3KM311-arrested cells (Trautmann et al., 2004), so we think it is unlikely that Clp1 is factor X. 3) Finally the interaction of the Passenger proteins with centromeres is qualitatively different in nda3KM311-arrested cells (possibly due to chromatin modifications; see below) and Sgo2 is necessary for this interaction.
Passenger Protein Localization Is Affected in nda3KM311-arrested Cells
Interestingly, the localization pattern of the Passenger proteins is affected by activation of the spindle checkpoint. In early mitosis of a cycling population, the Passenger proteins localize to SPBs, centromeres and telomeres (Table 1); however in an nda3KM311-dependent checkpoint arrest (a prometaphase-like mitotic arrest), they concentrate exclusively on centromeres (Figure 3, A and B). This indicates that the interaction of the Passenger proteins with centromeres is enhanced relative to telomeres and SPBs in nda3KM311-arrested cells. On recovery from checkpoint arrest, the Passenger proteins relocalize to telomeres and SPBs (data not shown), suggesting that the process of "exclusion" from telomeres and SPBs is reversible. This behavior is reminiscent of the bromodomain protein BrD4 in HeLa cells. It has recently been reported that BrD4 is released from chromatin upon activation of the spindle checkpoint but relocalizes to chromatin upon recovery from the arrest (Nishiyama et al., 2006). The release of BrD4 from chromatin coincides with changes in the acetylation status of specific histone residues. These observations suggest that chromatin is modified during prolonged mitotic arrest and that these changes modify the chromatin association of a subset of proteins. It is therefore possible to envisage that the association of the Passenger proteins with telomeres is destabilized upon checkpoint activation by such chromatin modifications or that their centromere association is enhanced.
Conservation of Sgo2 Function(s)?
It would appear that Sgo2 has conflicting roles in meiosis and in mitosis in fission yeast. In meiosis I, Sgo2 is required to maintain sister-chromatid mono-orientation (Vaur et al., 2005), whereas our data demonstrate that Sgo2 helps ensure proper biorientation of sister-chromatids in mitosis. One way to reconcile these apparent differences would be to propose that in meiosis I Sgo2 actually helps to maintain biorientation of the homologues and hence mono-orientation of the sister-chromatids. Whether this is compatible with a role of Sgo2 in recruiting Passenger proteins onto meiotic centromeres remains to be elucidated.
Arabidopsis thaliana, Zebrafish, Mus muculus, and Homo sapiens also have a Shugoshin2 homologue (Rabitsch et al., 2004; Watanabe, 2005). So far there are conflicting reports regarding the role of human hSgo2: one suggests an involvement for hSgo2 in chromosome biorientation, which is consistent with our results (Watanabe and Kitajima, 2005). However, a later report argues that hSgo2 is involved in the protection of centromeric cohesion in mitosis by recruiting the phosphatase PP2A to centromeres (Kitajima et al., 2006).
The mitotic functions of fission yeast Sgo2 appear similar to the mitotic functions of budding yeast Sgo1. Indeed deletion of ScSgo1 also triggers biorientation defects upon release from a spindle checkpoint arrest (Indjeian et al., 2005). It has also been suggested that ScSgo1 is crucial to activate the spindle checkpoint in response to lack of tension at kinetochores (Indjeian et al., 2005). Interestingly, it has recently been shown in budding yeast that the Survivin-INCENP complex was required to link centromeres to microtubules, independently of Aurora B. However, that linkage could provide local regulation of Aurora B in response to lack of tension at kinetochores (Sandall et al., 2006). To explain how ScSgo1 is required to sense lack of tension at kinetochores, it would be interesting to know whether ScSgo1, like SpSgo2 in fission yeast, is able to regulate the Survivin-INCENP complex.
It has recently been demonstrated that the centromeric localization of MEI-S332, the Drosophila Shugoshin homologue, is under the control of the Chromosomal Passenger proteins INCENP and Aurora B and that this is important to protect centromeric cohesion in meiosis I (Resnick et al., 2006). Together with our results, this demonstrates that multiple members of the Shugoshin family interact with Chromosomal Passengers. We suggest that Sgo-Passenger protein interactions will regulate distinct processes, including cohesion, biorientation and microtubule dynamics, in a wide range of model systems and in different developmental contexts.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Address correspondence to: Vincent Vanoosthuyse (vvanoost{at}staffmail.ed.ac.uk)
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-GFP antibody) and the kinetochore marker Cnp1 (
