1 School of Biological Sciences, 2.205 Stopford Building, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, UK
2 Universite de Rennes I, CNRS UMR 6061, Avenue Pr Léon Bernard, 35043 Rennes Cédex, France
*Author for correspondence (e-mail: iain.hagan{at}man.ac.uk)
Accepted September 6, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Aurora, S. pombe, INCENP, Mitosis, Histone H3
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Members of the aurora kinase family regulate a range of mitotic processes. The link between altered expression of human aurora kinases and cancer suggest that these proteins play a vital role in maintaining genome integrity (Adams et al., 2001a; Giet and Prigent, 1999). Metazoans appear to contain three distinct aurora-related kinases, each one of which regulates a different set of mitotic events. In each system, aurora A kinase controls spindle formation while aurora B kinase regulates chromosome disjunction and cytokinesis. Less is known about the function of the aurora C kinase.
Consistent with its role in spindle formation, aurora A kinase associates with the centrosome and microtubules. Mutation of the gene encoding Drosophila aurora A, results in the formation of monopolar rather than bipolar mitotic spindles (Glover et al., 1995). A similar defect in spindle formation occurs when the Xenopus aurora A, Eg2, is depleted from Xenopus egg extracts (Roghi et al., 1998). The Xenopus protein Eg5 is a member of the BimC family of kinesin-related proteins that are required for bipolar spindle formation in a range of systems (Walczak and Mitchison, 1996). Eg5 inhibition, like loss of aurora A function, results in the formation of monopolar rather than bipolar spindles (Kapoor et al., 2000). The ability of Eg2 to bind and phosphorylate Eg5 suggests that one aspect of Eg2 function may be to control the anti-parallel sliding of microtubules during mitosis (Giet et al., 1999). Aurora A may control additional events during spindle formation because the Xenopus and mammalian molecules associate with centrosomes in the absence of microtubules (Gopalan et al., 1997; Roghi et al., 1998).
Aurora B kinases associate with chromatin and the spindle mid-zone (Adams et al., 2001a). This association requires the function of two different conserved molecules, INCENP and a member of a distinct sub-class of the proteins that share a common motif called the BIR domain, which we shall refer to as the BIRMs (for BIR domain proteins of mitosis) (Silke and Vaux, 2001). Mammalian INCENP was first identified as a chromosome-associated antigen that accumulated with the inner centromere regions of mammalian chromosomes (Cooke et al., 1987). Like aurora B, INCENPs and BIRM proteins move from the centromere to the overlap zone during anaphase B and are required for cytokinesis.
Recent data have highlighted a correlation between disruption of aurora B, INCENP or BIRM function and a decrease in chromosome condensation in a number of different systems. The loss of chromosome condensation correlates with the reduction in the level of phosphorylation of serine 10 of the N-terminal tail of histone H3 (Adams et al., 2001b; Giet and Glover, 2001; Hsu et al., 2000). Phosphorylation of histone H3 at this site was first shown to be associated with mitotic chromosome condensation in Tetrahymena (Wei et al., 1998). Mutation of serine 10 to alanine in this system resulted in perturbation of mitotic chromosome transmission and reduced chromosome condensation (Wei et al., 1999). Immunofluorescence revealed a correlation between the location of regions where histone H3 was phosphorylated on serine 10 and chromosome condensation (Schmiesing et al., 2001). This suggested that this phosphorylation event may drive chromosome condensation by recruiting condensin. Consistently depletion of aurora B from a Drosophila tissue culture cell line reduced serine 10 histone H3 phosphorylation and resulted in a concomitant reduction in chromatin association of a condensin sub-unit, Barren (Giet and Glover, 2001).
We describe the characterisation of the S. pombe aurora-related kinase Ark1. We describe the association of Ark1 with mitotic structures and show that Ark1 is required for spindle formation, kinetochore microtubule interactions and chromosome resolution during anaphase. We show that the chromosome resolution defects correlate with a reduction in the level of phosphorylation of histone H3 on serine 10 and that immunoprecipitates containing Ark1 can phosphorylate histone H3 on serine 10 in vitro. Ark1 protein levels do not change as cells progress through the cell cycle.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Western blotting to monitor Ark1.PkC levels
Total cell extracts were prepared by TCA precipitation (Caspari et al., 2000), run on 10% SDS-PAGE and blotted as described previously (Mulvihill et al., 1999) using alkaline phosphatase coupled secondary antibodies.
Kinase assays
A cell pellet containing 1.2x108 cells was washed once in stop buffer and snap frozen. The pellet was resupended in 100 µl HEN buffer (50 mM Hepes (pH 8.0), 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 1% NP-40, 50 mM ß-glycerophosphate, 50 mM NaF, 1 mM Na3VO4, 1 mM leupeptin, 1 µg/ml aprotinin, 1 mM PMSF) (Cueille et al., 2001). 1 ml ice-cold glass beads was added to the suspension and the tube was shaken in a Ribolyser at 4°C for 4 seconds at full speed. The cell lysate was cleared by spinning at 16,000 g for 10 minutes at 0°C. 336 anti-PK epitope monoclonal antibody (Serotec MCA 1360) (Southern et al., 1991) was covalently coupled to sepharose beads (Harlow and Lane, 1988) and the beads were then equilibrated with HEN buffer. 95 µl of soluble extract was added to 10 µl of packed beads and the mixture was incubated at 4°C for 30 minutes. The beads, with the associated immunoprecipitate, were washed three times in 0.5 ml HEN buffer and three times in 0.5 ml KAB buffer (50 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM DTT, 10 mM MgCl2) before final resuspension in 20 µl KAB buffer. After incubation at 32°C for 5 minutes, 5 µl of substrate mix containing 20 µM ATP and 5 µg purified Histone H3 (Roche Molecular Biochemicals) were added to the IP complex. After a further 20 minutes at 32°C the reaction was stopped by the addition of 10 µl of 3x SDS-page loading buffer and boiling for 5 minutes. A nitrocellulose blot of a 15% SDS-PAGE gel was cut in half and the upper part (30-90 kDa) was probed with -PK antibodies to detect Ark1.PkC. The lower part (10-30 kDa) of the same membrane was probed with rabbit poly-clonal anti-phospho-serine 10-histone H3 (H3SP) (cat. no. 06-570 lot no:19633; Upstate Biotechnology, NY). Both primary antibodies were detected with SuperSignal ULTRA (Pierce).
Fluorescence microscopy
Immunofluorescence was as described (Hagan and Asycough, 2000). For anti-tubulin immunofluorescence with TAT1 antibodies (Woods et al., 1989) standard combined aldehyde fixation was used. For other staining cells were fixed with 3% formaldehyde alone. For localisation of Pk-tagged Ark1 with the monoclonal antibody 336, which recognises the PK epitope (Southern et al., 1991), and Cut7 with cAP5LB antibodies (Hagan and Yanagida, 1992) fixation was carried out for 2 minutes. With the 2 minutes fixation it was vital to incubate in antibodies at 4°C. Cells were fixed for 5 minutes in preparation for H3SP (Upstate biotechnology, NY). Images were acquired with a Quantix (Photometrix) slow scan CCD camera and processed with Metamorph (Universal Imaging).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In order to identify molecules with aurora-related functions we isolated multi-copy plasmids that could suppress the lethal consequences of Xenopus aurora A overproduction. Two plasmids were retrieved that contained the S. pombe aurora-related kinase, which we will refer to as ark1+ (aurora-related kinase; SPCXC320.13C at Sanger centre; CAA18315 at NCBI). Increasing ark1+ gene dosage suppressed both the spindle formation and chromosome segregation defects arising from Xenopus aurora A expression (data not shown). We conclude that Xenopus aurora A generated mitotic defects because it perturbed Ark1 function. Searching the S. pombe genome database has failed to identify a second aurora-related kinase suggesting that S. pombe, like S. cerevisiae, contains a single aurora kinase. Using the Jotun Heim alignment algorithm to compare ark1+ with the two Drosophila aurora kinases gave virtually identical scores suggesting that ark1+ is no more closely related to the aurora A or aurora B isoforms (data not shown; for a full alignment see Giet and Prigent) (Giet and Prigent, 1999).
Cells lacking ark1+ exhibit defects in spindle formation and chromosome segregation
The region of the genome encoding one of the two ark1+ genes present in the diploid strain IH1877 was replaced with LEU2+ encoding sequences. The deletion allele this generated was called ark1.1. Tetrad dissection of IH1877 gave two viable leu spores indicating that the ark1+ gene is essential for viability. Expression of ark1+, but not Eg2, from a heterologous locus supported growth of ark1.
1 cells (see below). In order to determine the consequences of loss of Ark1 function a preparation of spores from IH1877 was grown in the absence of leucine to specifically favour for growth of only ark1.
1 spores and not ark1+ spores. The distribution of microtubules and chromatin revealed four phenotypes. The most prevalent phenotype at early time points following germination at 25°C was a stretching of chromatin along an elongating anaphase B spindle (Fig. 2A). This phenotype suggested a defect in chromosome condensation in prophase or resolution during the metaphase/anaphase transition. In the second phenotypic class, condensed chromosomes were seen alongside robust short spindles. The chromosomes in these cells often failed to associate with the spindle or were found at one end of the spindle (Fig. 2B, arrow). The latter phenotype suggested that kinetochore function is perturbed by loss of Ark1 function while the former is reminiscent of the dis phenotype (Ohkura et al., 1988). (dis mutants form a mitotic spindle that elongates but does not completely disappear at the end of anaphase B. As sister chromatids do not separate in a dis mitosis, the unsplit chromosomes randomly associate with either of the two poles.) The third phenotype that was very rare, but consistently seen at 30°C, was a spindle formation defect as microtubules emanated from a single focus of SPB staining (Fig. 2C). The final phenotype that became apparent 36 hours after germination at 30°C was the appearance of exceptionally elongated cells (Fig. 2D). The chromatin of these cells was either compact (Fig. 2D) or a diffuse amorphous staining.
|
Logarithmically growing wild-type S. pombe cells have fulfilled the size requirement for passage through START when mitosis is complete. Cells therefore commit to the next cell cycle before cytokinesis has produced two daughter cells (Baum et al., 1997). G1 phase is therefore very short in logarithmically growing cultures. Binucleate, post-mitotic cells are therefore in either an extremely short G1 or S phase of the cell cycle, whereas all uni-nucleate interphase cells in a culture are in G2. Thus cell 1 in Fig. 3A is in G2, cell 2 and 3 are in mitosis while cell 4 is in G1 or S phase. Comparison of the H3SP staining patterns (Fig. 3A, top) in these cells shows little difference in the intensity of H3 staining between cells 1-3 but a considerable decrease in cell 4. In order to determine whether the reduction in staining in cell 4 corresponded to a reduction of staining in G1 or S phase we stained for H3SP following arrest of cell cycle progression in G1 or S phase of the cell cycle. The nuclei of cells that had been arrested in early S phase by incubation with hydroxyurea stained strongly with H3SP (Fig. 3B). By contrast, staining in cdc10.v50 cells that had been arrested in G1 before START (Fig. 3C, uninucleate cells) was much lower than that of the control cdc7.A20 mutant cells that had been mixed into the culture to provide an internal reference for the basal level of H3SP staining (Fig. 3C, multinucleate cells). We conclude that H3SP reactivity was lowest in G1 phase of the cell cycle.
|
|
The levels of epitope tagged Ark1 remain constant as cells progress through the cell cycle
Having established the phenotypic consequences of deleting the ark1+ gene we tagged the gene at its C-terminus with three copies of the Pk epitope (Southern et al., 1991) in order to initiate a biochemical and cytological characterisation of Ark1 protein. This C-terminally tagged protein will be referred to as Ark1.PkC. Because we experienced repeated difficulties in directing integration to the ark1+ locus throughout the course of this study the tagged gene was integrated at the leu1 locus. The expression of this ark1.PkC allele at the leu1 locus (leu1::ark1.PkC ura4+) was regulated by the endogenous ark1+ promoter. The ark1.PkC allele was combined with the ark1.1 allele in strain IH2036 so that the tagged Ark1.PkC was the only Ark1 protein in the cell. These cells undergo a normal mitosis (see below) (Fig. 8) indicating that the protein is fully functional. Small G2 IH2036 cells were isolated from an asynchronous culture by centrifugal elutriation and Ark1.PkC levels were monitored as the cells progressed through two successive cell cycles. No appreciable changes in protein level were detected (Fig. 5A,B). As discussed above, when cells are growing in logarithmic phase, G1 events are initiated upon commitment to mitosis (Baum et al., 1997). Thus G1 phase is effectively absent from the synchronous culture shown in Fig. 5. The constant level of Ark1.PkC throughout the time course may be seen because the phase of the cell cycle in which the protein is unstable, G1 is effectively missing. We therefore used a different approach to ask whether Ark1.PkC levels in G1 phase cells were lower than those in a log phase or an M phase population. Protein extracts were prepared from a culture of cdc10.v50 cells grown at the permissive temperature of 25°C or from a portion of the same culture that had been incubated at the restrictive temperature of 37°C for 5.5 hours to arrest cell cycle progression in G1 phase. To prepare a mitotic extract, strain IH2117 was incubated for 4.25 hours at 36°C to arrest cells in G2 and then the temperature was returned to 25°C to induce a synchronous mitosis. The M phase population was harvested 45 minutes after the return to 25°C. There was no appreciable difference in Ark1.PkC levels between these samples or those in mitotic cells (Fig. 5C,D). These data suggest that, unlike other aurora-related kinases, Ark1.PkC is not subject to bulk degradation after mitosis.
|
|
|
|
Ark1.PkC was present in discrete nuclear foci throughout interphase (Fig. 8, cells 1-3). These interphase foci did not associate with the single spot of Nuf2.GFP staining. When cells reached the critical length for commitment to mitosis the entire nucleus became more reactive to anti-Pk antibodies and a bright spot of Ark1.PkC staining was seen in tight association with a single Nuf2-GFP dot (Fig. 8, cells 4-7). Spots were still seen in other regions of the nucleus, but they were fainter than this Nuf2.GFP-associated signal. From this point on until anaphase the strongest regions of Ark1.PkC staining were associated with a Nuf2.GFP signal. The two signals often overlapped, but on occasion it was possible to see two spots of Nuf2.GFP staining either side of an Ark1 spot. A good example of such a pattern is seen in Fig. 8, cell 8 and the enlarged version of this image in Fig. 9A. The kinetochores in this cell are neither maximally separated nor aligned in the linear fashion that would indicate spindle association. Given the striking resemblance between this pattern and the Nuf2.GFP staining in Fig. 7C, we conclude that these cells have very short spindles and that the kinetochore in question is not yet associated with it. This relationship between the Ark1.PkC and Nuf2.GFP signals shown by the arrows in Fig. 9A is consistent with Ark1.PkC localisation to the inner centromere region. As the kinetochores moved towards the SPBs during anaphase A, Ark1.PkC association with the kinetochores became less pronounced (Fig. 8, cells 9,10; Fig. 9B,C). Several kinetochores did not have any associated Ark1 signals. At this level of resolution it is not possible to determine whether this was due to the disassociation of Ark1.PkC from the kinetochores or because of deformation of the kinetochore/centromere complex prior to sister chromatid separation. After anaphase A was complete and the chromosomes had reached the SPBs, Ark1.PkC was seen along the short spindle (Fig. 8, cell 11). The protein accumulated in the mid-zone during spindle elongation (Fig. 8, cell 12). The region of Ark1.PkC staining in the spindle extended as the spindle extended (Fig. 8, cells 13,14) until late anaphase B when it was once more a restricted region of punctate staining (Fig. 8, cell 16). Once the spindle had broken down and cytokinesis had started, punctate Ark1.PkC staining, which was not associated with the SPB/centromere complex, was seen once more (Fig. 8, cell 17).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mitotic distribution of Ark1 is reminiscent of the distribution of metazoan aurora B (Adams et al., 2001a). Before mitosis it was localised to between one to three nuclear foci. The nature of these foci and whether they are reflecting an important role for Ark1 in interphase remains unclear at present. It is clear that they do not represent the centromere/SPB complex (Funabiki et al., 1993). It is unclear whether they are stable structures that persist at particular sites throughout interphase, or whether Ark1 is accumulating in different regions of the nucleus at different times. What could Ark1 be doing at these sites? A strong link between aurora B function and phosphorylation of histone H3 on serine 10 during mitosis has been established (Adams et al., 2001a). Phosphorylation at this site also plays a critical role in interphase when it is important to modulate transcriptional activity by controlling chromatin architecture (Cheung et al., 2000). We show that Ark1 can phosphorylate serine 10 of histone H3 in vitro and that this modification is virtually absent from mitotic cells from which ark1+ has been deleted. It is therefore possible that during interphase Ark1 is conducting functions that are analogous to those carried out by other histone H3 kinases such as JIL1 (Wang et al., 2001), which control transcription by regulating chromatin architecture. This would be entirely consistent with the similarity between the distribution of interphase Ark1 spots and the foci of sites at which histone H3 is phosphorylated on serine 10 during interphase. The slight enhancement of H3SP reactivity in interphase extracts over the background level seen in control samples may represent an interphase Ark1 kinase activity. However, although the current assay does demonstrate a very strong mitotic activation of Ark1 activity, it is not sensitive enough to draw firm conclusions about a possible interphase activity at present.
The progressive acquisition of phosphorylation of histone H3 on serine 10 shows a strong correlation with progressive chromosome condensation during mitotic commitment in mammalian cells (Hendzel et al., 1997). Moreover, the reduction in this phosphorylation event that occurs after RNAi-mediated ablation of Drosophila aurora B is accompanied by an inability to recruit the condensin sub-unit Barren (Giet and Glover, 2001). Ark1 also appears to play a crucial role in both chromosome condensation and phosphorylation of histone H3 (H3SP) on serine 10 as chromosome segregation fails and antibodies that recognise H3SP stain wild-type, but not ark1.1 cells and Ark1 immunoprecipitates can phosphorylate this serine residue. Punctate interphase staining for both Ark1 and H3SP was succeeded by staining throughout the early mitotic nucleus and strong association of both epitopes with the centromeres. Pronounced H3SP reactivity of centromeres is reminiscent of the accumulation of H3SP staining in pericentromeric regions in late G2 of mammalian cells (Hendzel et al., 1997). H3SP staining of centromere/kinetochore persisted throughout mitosis, but Ark1 lost its affinity for these regions during anaphase A and associated with the spindle microtubules after anaphase A was complete. Ark1 maintained its association with the central spindle to varying degrees for the duration of anaphase B. After spindle dissolution Ark1 was once more localised to discrete nuclear spots.
Both aurora A and B have been reported to associate with the microtubules of the mitotic spindle (Adams et al., 2001a; Giet and Prigent, 1999). Xenopus aurora A has been shown to associate with and phosphorylate the motor protein Eg5 (Giet et al., 1999). This raised the possibility that in addition to a direct association with microtubules (Roghi et al., 1998) aurora A associates with microtubules via an interaction with Eg5-related molecules. However, we failed to find a strong correlation between the distribution of the S. pombe Eg5 homologue Cut7 and the distribution of Ark1 suggesting that Ark1 is not associating with the spindle as a consequence of an interaction with Cut7. However, Ark1 does resemble aurora A in being required for spindle formation. Aurora A associates with the spindle pole in a microtubule independent manner (Gopalan et al., 1997; Roghi et al., 1998). Despite a concerted effort we were unable to detect Ark1 on the SPB from prometaphase to telophase. However, we cannot rule out a transient association of Ark1 with the SPB in the very early stages of mitosis because the proximity of the centromere associated Ark1 to the SPB at this time would obscure such a signal.
Aurora B behaves like a classical chromosomal passenger protein in that it leaves the centromeres and associates with the spindle microtubules in anaphase B (Earnshaw and Bernat, 1991). Abolition of the function of aurora B or its binding partners INCENP or BIRM blocks cytokinesis (Giet and Glover, 2001; Kaitna et al., 2000; Severson et al., 2000a; Uren et al., 2000). It has been proposed that this effect is due to the weakening of the integrity of the spindle mid-zone that occurs when this complex is not operative. These abnormalities in spindle structure correspond to the loss of a conserved kinesin-related protein that is required to locate the mitotic kinase Polo to the mid-zone where it is thought to control cytokinesis (Adams et al., 1998). Alternatively, because INCENP binds to the cleavage furrow (Eckley et al., 1997) the INCENP/aurora B complex may play a direct role in regulating cytokinesis. What are the parallels with fission yeast? It seems unlikely that Ark1 is required to maintain the integrity of the central spindle as the morphology of anaphase B spindles was normal when the chromosomes were clearly not being resolved. As in other systems, S. pombe polo kinase Plo1 is required to define the architecture of the acto-myosin ring; however, Plo1 executes this role much earlier than in higher systems and it is complete by metaphase (Bähler et al., 1998). Although S. pombe does then use Plo1 to regulate the contraction of the actin ring during cell division this appears to be mediated through the control of the SPB-located septum initiation network rather than an effect in the cleavage furrow (Tanaka et al., 2001). Despite these differences does it remain possible that Ark1 is associating with the spindle to directly regulate aspects of cytokinesis? Because S. pombe has a closed mitosis, spindle associated Ark1 is shielded from directly influencing the pre-assembled cleavage furrow by the nuclear membrane. Also, Ark1 still accumulates in the central overlap zone of both meiotic spindles, neither of which is followed by cytokinesis. Although this may rule out an influence on the acto-myosin ring, it is worth noting that, when aurora B is depleted from C. elegans or Drosophila by RNAi, it is the late, rather than early, stages of cytokinesis that fail (Adams et al., 2001b; Giet and Glover, 2001; Kaitna et al., 2000; Schumacher et al., 1998; Severson et al., 2000a). Fusion of the plasma membrane to generate two separate membranes is an integral part of the later stages of cytokinesis. A similar problem confronts systems with a closed mitosis, but it is more complex as they must resolve both the nuclear and plasma membranes. It is therefore possible that Ark1 associates with the spindle to get to the regions where it can regulate membrane fusion events in telophase.
In line with our ability to detect Ark1 in interphase cells by immunofluorescence, microscopy western blot analysis of synchronous cultures and cells arrested in G1, S and G2, as well as an M phase population, indicated that Ark1 levels did not change appreciably as cells progressed through the mitotic cell cycle. This contrasts with mammalian aurora A whose abundance is highest during mitosis (Gopalan et al., 1997). While the protein levels of aurora B have not been characterised, transcription of the mammalian aurora B AIM-1 peaks in mitotic cells (Kawasaki et al., 2001). The levels of the S. cerevisiae protein Ipl1p are lower in G1 arrested cells than in actively cycling cells or cells arrested in mitosis (Biggins et al., 1999). It has been proposed that the presence of a KEN box in aurora-related molecules may target them for cell cycle stage-specific destruction and so account for fluctuations in protein level (Pfleger and Kirschner, 2000). However, if there is a relation between the KEN box and cyclical degradation of aurora-related kinases it is likely to be more complex than currently appreciated as Ark1 contains a KEN box but does not appear to be degraded in a cell cycle-dependent fashion in logarithmically dividing cells.
In many respects other than protein stability, Ark1 resembles the single S. cerevisiae aurora-related kinase Ipl1p. This kinase associates with the anaphase B spindle and is required for chromosome segregation and, like Ark1, has a minor defect in spindle architecture (Chan and Botstein, 1993; Kim et al., 1999). Errors in chromosome disjunction are common in ipl1.2 mutant cells and probably arise from a reduction in the affinity of kinetochores for microtubules (Biggins et al., 1999). This effect is likely to be mediated by a direct regulation of kinetochore function as both Ipl1p and its partner Bir1p bind to the kinetochore protein Ndc10 and a GST-Ipl1p fusion protein can phosphorylate an GST-Ndc10p fusion protein in vitro (Biggins et al., 1999; Yoon and Carbon, 1999). A role for aurora kinases in regulating kinetochore activity may well be conserved as depletion of aurora B from Drosophila cell lines leads to lagging chromosomes in anaphase B (Adams et al., 2001b; Giet and Glover, 2001). It is therefore significant that a subset of ark1.1 cells have defects in chromosome association with an apparently normal spindle. In addition we observed several cells with the dis phenotype in which chromosomes with unseparated chromatids are seen at the spindle poles (Ohkura et al., 1988). One of the dis mutants dis2.11 encodes a protein phosphatase 1 catalytic subunit (Ohkura et al., 1989). Significantly, mutations in the budding yeast PP1 GLC7 or overproduction of a Glc7p inhibitor can suppress the chromosome segregation defects of Ipl1 mutants (Francisco et al., 1994; Sassoon et al., 1999). Along with the hyperphosphorylation of Ndc10p in glc7.10 mutants these data indicate that Glc7p antagonises the activity of Ipl1p. If the defect in ark1.
1 cells is just a defect in kinetochore activity one is forced to ask why the sister chromatids are not separating in anaphase ark1.
1 cells that have the dis phenotype (Fig. 2B). One explanation may lie in a recently postulated role for aurora-related kinases in the regulation of sister chromatid cohesion (Adams et al., 2001a).
The localisation and functional interactions between Ipl1p, the INCENP-related protein Sli15p, Bir1p and kinetochore proteins suggests that Ipl1p is more akin to an aurora B than an aurora A. A similar image is likely to emerge from S. pombe as cells lacking the BIRM protein Pbh1 have the same chromosome segregation defects as ark1.1 cells (Rajagopalan and Balasubramanian, 1999). However, mutation of both Ark1 and Ipl1p results in the formation of monopolar rather than bipolar spindles, a feature most commonly attributed to aurora A molecules. Intriguingly, in experiments in which the kinase domain was swapped with that from aurora A and aurora B it was only the catalytic domain of aurora A that was able to support growth of Ipl1 mutants (Bischoff et al., 1998). Ark1 and Ipl1p may therefore constitute a distinct class of aurora-related kinase. The divergence of function may reflect differences in the biology of these two systems from higher eukaryotes. Because the actin ring forms before anaphase in each yeast (in budding yeast when the bud emerges) the cell does not need to use auroras to control ring formation. Similarly, the inner face of the SPB is only used for nucleating spindle microtubules. It does not therefore require an aurora to drive the upregulation of microtubule nucleating activity that is integral to converting the interphase centrosome to an active mitotic pole in higher systems. Further study of these two proteins and their partner molecules will therefore clearly complement the extensive body of data that is rapidly accumulating from the analysis of the two main higher eukaryotic isoforms aurora A and B.
![]() |
ACKNOWLEDGMENTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, R. R., Tavares, A. A. M., Salzberg, A., Bellen, H. J. and Glover, D. M. (1998). pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis. Genes Dev. 12, 1483-1494.
Adams, R. R., Carmena, M. and Earnshaw, W. C. (2001a). Chromosomal passengers and the (aurora) ABCs of mitosis. Trends Cell Biol. 11, 49-54.[Medline]
Adams, R. R., Maiato, H., Earnshaw, W. C. and Carmena, M. (2001b). Essential roles of Drosophila inner centromere protein (INCENP) and aurora B in histone H3 phosphorylation, metaphase chromosome alignment, kinetochore disjunction, and chromosome segregation. J. Cell Biol. 153, 865-880.
Bähler, J., Steever, A. B., Wheatley, S., Wang, Y. L., Pringle, J. R., Gould, K. L. and McCollum, D. (1998). Role of polo kinase and Mid1p in determining the site of cell division in fission yeast. J. Cell Biol. 143, 1603-1616.
Baum, B., Wuarin, J. and Nurse, P. (1997). Control of S-phase periodic transcription in the fission yeast mitotic cycle. EMBO J. 16, 4676-4688.
Biggins, S., Severin, F. F., Bhalla, N., Sasoon, I., Hyman, A. A. and Murray, A. W. (1999). The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast. Genes Dev. 13, 532-544.
Bischoff, J. R., Anderson, L., Zhu, Y., Mossie, K., Ng, L., Souza, B., Shryver, B., Flanagan, P., Clairvoyant, F., Ginther, C. et al. (1998). A homologue of the Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers. EMBO J. 17, 3052-3065.
Caspari, T., Dahlen, M., Kanter-Smoler, G., Lindsay, H. D., Hofmann, K., Papadimitriou, K., Sunnerhagen P. and Carr, A. M. (2000). Characterization of Schizosaccharomyces pombe Hus1: a PCNA-related protein that associates with Rad1 and Rad9. Mol. Cell Biol. 20, 1254-1262.
Chan, C. S. M. and Botstein, D. (1993). Isolation and characterization of chromosome-gain and increase-in- ploidy mutants in Yeast. Genetics. 135, 677-691.
Cheung, P., Allis, C. D. and Sassone-Corsi, P. (2000). Signaling to chromatin through histone modifications. Cell 103, 263-271.[Medline]
Cooke, C. A., Heck, M. M. and Earnshaw, W. C. (1987). The inner centromere protein (INCENP) antigens: movement from inner centromere to midbody during mitosis. J. Cell Biol. 105, 2053-2067.[Abstract]
Craven, R. A., Griffiths, D. J. F., Sheldrick, K. S., Randall, R. E., Hagan, I. M. and Carr, A. M. (1998). Vectors for the expression of tagged proteins in Schizosaccharomyces pombe. Gene 221, 59-68.[Medline]
Cueille, N., Salimova, E., Esteban, V., Blanco, M., Moreno, S., Bueno, A. and Simanis, V. (2001). Flp1, a fission yeast orthologue of the S. cerevisiae CDC14 gene, is not required for cyclin degradation or rum1p stabilisation at the end of mitosis. J. Cell Sci. 114, 2649-2664.[Medline]
Drummond, D. R. and Hagan, I. M. (1998). Mutations in the bimC box of Cut7 indicate divergence of regulation within the bimC family of kinesin related proteins. J. Cell Sci. 111, 853-865.
Earnshaw, W. C. and Bernat, R. L. (1991). Chromosomal passenger proteins: Towards an integrated view of mitosis. Chromosoma. 100, 139-146.[Medline]
Eckley, D. M., Ainsztein, A. M., Mackay, A. M., Goldberg, I. G. and Earnshaw, W. C. (1997). Chromosomal proteins and cytokinesis: patterns of cleavage furrow formation and inner centromere protein positioning in mitotic heterokaryons and mid-anaphase cells. J. Cell Biol. 136, 1169-1183.
Francisco, L., Wang, W. F. and Chan, C. S. M. (1994). Type-1 protein phosphatase acts in opposition to Ipl1 protein-kinase in regulating yeast chromosome segregation. Mol. Cell Biol. 14, 4731-4740.[Abstract]
Funabiki, H., Hagan, I., Uzawa, S. and Yanagida, M. (1993). Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast. J. Cell Biol. 121, 961-976.[Abstract]
Giet, R. and Glover, D. M. (2001). Drosophila aurora B kinase is required for histone H3 phosphorylation and condensin recruitment during chromosome condensation and to organize the central spindle during cytokinesis. J. Cell Biol. 152, 669-682.
Giet, R. and Prigent, C. (1999). Aurora/Ipl1p-related kinases, a new oncogenic family of mitotic serine-threonine kinases. J. Cell Sci. 112, 3591-3601.
Giet, R., Uzbekov, R., Cubizolles, F., Le Guellec, K. and Prigent, C. (1999). The Xenopus laevis aurora-related protein kinase pEg2 associates with and phosphorylates the kinesin-related protein XlEg5. J. Biol. Chem. 274, 15005-15013.
Glotzer, M. (1997). The mechanism and control of cytokinesis. Curr. Opin. Cell Biol. 9, 815-823.[Medline]
Glover, D. M., Leibowitz, M. H., McLean, D. A. and Parry, H. (1995). Mutations in Aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell. 81, 95-105.[Medline]
Gopalan, G., Chan, C. S. M. and Donovan, P. J. (1997). A novel mammalian, mitotic spindle-associated kinase is related to yeast and fly chromosome segregation regulators. J. Cell Biol. 138, 643-657.
Hagan, I. and Yanagida, M. (1992). Kinesin-related Cut7 protein associates with mitotic and meiotic spindles in fission yeast. Nature 356, 74-76.[Medline]
Hagan, I. and Yanagida, M. (1995). The product of the spindle formation gene sad1+ associates with the fission yeast spindle pole body and is essential for viability. J. Cell Biol. 129, 1033-1047.[Abstract]
Hagan, I. M. and Asycough, K. R. (2000). Fluorescence microscopy in Yeast. In Protein Localisation by Fluorescence Microscopy. (ed. V. J. Allan), pp. 179-206. Oxford University Press, Oxford.
Harlow, E. and Lane, D. (1988). Antibodies: a Laboratory Manual. Cold Spring Harbor Laboratory, New York.
Hendzel, M. J., Wei, Y., Mancini, M. A., Van Hooser, A., Ranalli, T., Brinkley, B. R., Bazett-Jones, D. P. and Allis, C. D. (1997). Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106, 348-360.[Medline]
Hsu, J. Y., Sun, Z. W., Li, X., Reuben, M., Tatchell, K., Bishop, D. K., Grushcow, J. M., Brame, C. J., Caldwell, J. A., Hunt, D. F. et al. (2000). Mitotic phosphorylation of histone H3 is governed by Ipl1/aurora kinase and Glc7/PP1 phosphatase in budding yeast and nematodes. Cell 102, 279-291.[Medline]
Kaitna, S., Mendoza, M., Jantsch-Plunger, V. and Glotzer, M. (2000). Incenp and an aurora-like kinase form a complex essential for chromosome segregation and efficientcompletion of cytokinesis. Curr. Biol. 10, 1172-1181.[Medline]
Kapoor, T. M., Mayer, T. U., Coughlin, M. L. and Mitchison, T. J. (2000). Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5. J. Cell Biol. 150, 975-988.
Kawasaki, A., Matsumura, I., Miyagawa, J., Ezoe, S., Tanaka, H., Terada, Y., Tatsuka, M., Machii, T., Miyazaki, H., Furukawa, Y. and Kanakura, Y. (2001). Downregulation of an AIM-1 kinase couples with megakaryocytic polyploidization of human hematopoietic cells. J. Cell Biol. 15, 275-288.
Kim, J., Kang, J. and Chan, C. S. M. (1999). Sli15 associates with the Ipl1 protein kinase to promote proper chromosome segregation in Saccharomyces cerevisiae. J. Cell Biol. 145, 1381-1394.
Maundrell, K. (1990). Nmt1 of Fission Yeast a Highly Transcribed Gene Completely Repressed By Thiamine. J. Biol. Chem. 265, 10857-10864.
Maundrell, K. (1993). Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 123, 127-130.[Medline]
Moreno, S., Klar, A. and Nurse, P. (1991). Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795-823.[Medline]
Mulvihill, D. P., Petersen, J., Ohkura, H., Glover, D. M. and Hagan, I. M. (1999). Plo1 kinase recruitment to the spindle pole body and its role in cell division in Schizosaccharomyces pombe. Mol. Biol. Cell. 10, 2771-2785.
Nasmyth, K., Peters, J. M. and Uhlmann, F. (2000). Splitting the chromosome: cutting the ties that bind sister chromatids. Science. 288, 1379-1385.
Nigg, E. A. (2001). Mitotic kinases as regulators of cell division and its checkpoints. Nat. Rev. Mol. Cell Biol. 2, 21-32.[Medline]
Ohkura, H., Adachi, Y., Kinoshita, N., Niwa, O., Toda, T. and Yanagida, M. (1988). Cold-sensitive and caffeine-supersensitive mutants of the schizosaccharomyces-pombe dis genes implicated in sister chromatid separation during mitosis. EMBO J. 7, 1465-1473.[Abstract]
Ohkura, H., Kinoshita, N., Miyatani, S., Toda T. and Yanagida, M. (1989). The fission yeast dis2+ gene required for chromosome disjoining encodes one of 2 putative type-1 protein phosphatases. Cell. 57, 997-1007.[Medline]
Petersen, J., Nielsen, O., Egel, R. and Hagan, I. M. (1998). F-actin distribution and function during sexual differentiation in Schizosaccharomyces pombe. J. Cell Sci. 111, 867-876.
Pfleger, C. M. and Kirschner, M. W. (2000). The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. Genes Dev. 14, 655-665.
Rajagopalan, S. and Balasubramanian, M. K. (1999). S. pombe Pbh1p: an inhibitor of apoptosis domain containing protein is essential for chromosome segregation. FEBS Lett. 460, 187-190.[Medline]
Reymond, A., Schmidt, S. and Simanis, V. (1992). Mutations in the cdc10 START gene of Schizosaccharomyces pombe implicate the region of homology between cdc10 and sw16 as important for p85cdc10 function. Mol. Gen. Genet. 234, 449-456.[Medline]
Roghi, C., Giet, R., Uzbekov, R., Morin, N., Chartrain, I., Le Guellec, R., Couturier, A., Doree, M., Philippe, M. and Prigent, C. (1998). The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to spindle microtubules and is involved in bipolar spindle assembly. J. Cell Sci. 111, 557-572.
Sassoon, I., Severin, F., Andrews, P. D., Taba, M.-R., Kaplan, K. B., Ashford, A. J., Stark, M. J. R., Sorger, P. K. and Hyman, A. A. (1999). Regulation of Saccharomyces cerevisiae kinetochores by the type 1 phosphatase Glc7p. Genes Dev. 13, 545-555.
Schmiesing, J. A., Gregson, H. C., Zhou, S. and Yokomori, K. (2001). A human condensin complex containing hCAP-C hCAP-E and CNAP1, a homolog of Xenopus XCAP-D2, colocaliszes with phosphorylated histone H3 during the early stage of mitotic chromosome condensation. Mol. Cell Biol. 20, 6996-7006.
Schumacher, J. M., Gloden, A. and Donovan, P. J. (1998). AIR-2: an aurora/Ipl1-related kinase associated with chromosomes and midbody microtubules is required for polar body extrusion and cytokinesis in Caenorhabditis elegans. J. Cell Biol. 143, 1635-1646.
Severson, A. F., Hamill, D. R., Carter, J. C., Schumacher, J. and Bowerman, B. (2000a). The aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis. Curr. Biol. 10, 1162-1171.[Medline]
Severson, A. F., Hamill, D. R., Carter, J. C., Schumacher, J. and Bowerman, B. (2000b). The aurora-related kinase AIR-2 recruits ZEN-4/CeMKLP1 to the mitotic spindle at metaphase and is required for cytokinesis. Curr. Biol. 10, 1162-1171.[Medline]
Silke, J. and Vaux, D. L. (2001). Two kinds of BIR-containing protein - inhibitors of apoptosis, or required for mitosis. J. Cell Sci. 114, 1821-1827.
Southern, J. A., Young, D. F., Heaney, F., Baumgartner, W. K. and Randall, R. E. (1991). Identification of an epitope on the p-proteins and v-proteins of simian-virus 5 that distinguishes between 2 isolates with different biological characteristics. J. Gen. Virol. 72, 1551-1557.[Abstract]
Tanaka, K., Petersen, J., MacIver, F., Mulvihill, D. P., Glover, D. M. and Hagan, I. M. (2001). The role of Plo1 kinase in mitotic commitment and septation in Schizosaccharomyces pombe. EMBO J. 20, 1259-1270.
Uren, A. G., Wong, L., Pakusch, M., Fowler, K. J., Burrows, F. J., Vaux, D. L. and Choo, K. H. (2000). Survivin and the inner centromere protein INCENP show similar cell cycle localization and gene knockout phenotype. Curr. Biol. 10, 1319-1328.[Medline]
Walczak, C. E. and Mitchison, T. J. (1996). Kinesin related proteins at mitotic spindle poles - function and regulation. Cell 85, 943-946.[Medline]
Wang, Y., Zhang, W., Jin, Y., Johansen, J. and Johansen, K. M. (2001). The JIL-1 tandem kinase mediates histone H3 phosphorylation and is required for maintenance of chromatin structure in Drosophila. Cell 105, 433-443.[Medline]
Wei, Y., Mizzen, C. A., Cook, R. G., Gorovsky, M. A. and Allis, C. D. (1998). Phosphorylation of histone H3 at serine 10 is correlated with chromosome condensation during mitosis and meiosis in Tetrahymena. Proc. Natl. Acad. Sci. USA 95, 7480-7484.
Wei, Y., Yu, L., Bowen, J., Gorovsky, M. A. and Allis, C. D. (1999). Phosphorylation of histone H3 is required for proper chromosome condensation and segregation. Cell. 97, 99-109.[Medline]
Wigge, P. A. and Kilmartin, J. V. (2001). The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation. J. Cell Biol. 152, 349-360.
Wittmann, T., Hyman, A. and Desai, A. (2001). The spindle: a dynamic assembly of microtubules and motors. Nat. Cell Biol. 3, E28-E34.[Medline]
Woods, A., Sherwin, T., Sasse, R., Macrae, T. H., Baines, A. J. and Gull, K. (1989). Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal-antibodies. J. Cell Sci. 93, 491-500.[Abstract]
Yoon, H.-J. and Carbon, J. (1999). Participation of Bir1p, a member of the inhibitor of apoptosis family, in yeast chromosome segregation events. Proc. Natl. Acad. Sci. 96, 13208-13213.