A study on premature segregation of unreplicated chromosomes 3

Chapter3 3.1 Premature Chromosome Segregation in cells with Unreplicated Chromosomes Background The essence of a successful mitosis is to transmit identical set of chromosomes to each of the two progeny cells. To achieve this, many eukaryotic cells assemble a bipolar apparatus, the mitotic spindle - that mediates equal partitioning of the duplicated chromosomes between the two daughters. Central to this precise segregation of the chromosomes is the amphitelic attachment of each sister-chromatid pair to the spindle such that each member of the sister-kinetochore pair is attached to the opposite pole (Dewar et al. 2004). This arrangement ensures the movement of each chromosome-set in the opposite direction, fueled by the dramatic extension of the mitotic spindle during anaphase. However, chromosome segregation is also governed by complex dynamics at another level. Prior to anaphase onset, spindle extension is resisted by the sisterchromatid cohesion mediated by the cohesin complex holding duplicated sister chromatids together. The cohesin complex in the budding yeast is composed of four subunits: Smc1, Smc3, Scc1 and Scc3 (Nasmyth et al. 2005). These subunits are assembled in a ‘ring-shaped’ structure that encircles the sister chromatids along the entire length of the chromosomes. At the onset of anaphase, sister-chromatid cohesion is dissolved by proteolytic cleavage of cohesin subunit Scc1 by separase, a caspase-like protease encoded by the ESP1 gene in budding yeast (Uhlmann et al. 1999; Uhlmann et al. 2000). However, separase Esp1 continues to be inhibited by securin, a protein encoded by the PDS1 gene (Morgan 1999; Shirayama et al. 1999) till metaphase. During metaphase to anaphase transition, the E3 ubiquitin ligase APCCdc20 mediates the proteolytic destruction of securin, freeing the separase from the inhibitory effects of securin (Visintin et al. 1997; Fang et al. 1998). This leads to the cleavage of the cohesin subunit Scc1, loss of cohesion between sister chromatids culminating in progressive separation (mediated by the mitotic spindle) of the duplicated chromosomes. Being the central act of mitosis, chromosome segregation is coordinated with other major events of the cell cycle. For instance, chromosome segregation is not initiated until DNA replication is complete. It is also transiently suppressed if cells incur DNA damage during S phase. The suppression is lifted only after the DNA lesions have been repaired. Chromosome segregation or more specifically, cohesin cleavage is also delayed when cells are unable to establish amphitelic attachment between the sister-chromatids and the mitotic spindle. Such negative regulations are imposed by various checkpoint controls that are operative during S phase and mitosis (DNA replication-, DNA damage- and spindle assembly checkpoints) (Hartwell et al. 1989; Nyberg et al. 2002). These surveillance systems ensure that chromosomes not segregate prematurely until the prior events are successfully completed. If these checkpoint controls fail or function sluggishly, cells initiate chromosome separation precociously, resulting often in unequal segregation of sister chromatids and genomic instability leading to aneuploidy. Aneuploidy is observed in many cancer cells and is thought to be due to either loss or malfunctioning of the checkpoint controls. While malfunctioning of the DNA damage checkpoint and spindle assembly checkpoint (which normally cause arrest in G2 and prometaphase, respectively) result in mis-segregation of chromosomes, untimely chromosome segregation perhaps represents the most dramatic manifestation of a defect in replication checkpoint (Krishnan et al. 2004; Krishnan et al. 2005). Wild type yeast cells treated with replication inhibitor hydroxyurea (HU) arrest in early S phase with a single nucleus, unreplicated chromosomes and a short spindle. Checkpoint defective mec1 or rad53 cells also arrest in early S phase in response to HU treatment but proceed to rapidly extend the spindle and unequally partition the largely unreplicated chromosomes (Krishnan et al. 2004). Since chromosome segregation is very conspicuously associated with mitosis (M phase), It has long been thought that precocious chromosome segregation in these cells is due a premature onset of mitosis in the absence of checkpoint control. However, it has now been shown that this unnatural chromosome segregation is not due to checkpoint deficient cells’ premature entry into mitosis but because of a combination of two events: (i) absence of chromosome biorientation due to a failure to replicate the centromeric regions caused by replication fork collapse (Krishnan et al. 2004) and (ii) deregulation of spindle dynamics in the absence of the checkpoint (Krishnan et al. 2004). These findings have put the understanding of replication checkpoint on a mechanistic footing. Temperature sensitive mutants defective in the replication protein Cdc6 also exhibit a similarly dramatic phenotype. At non-permissive temperature, the mutant cells traverse START, construct a bud but are unable to assemble a functional replication complex and therefore fail to initiate DNA replication (Piatti et al. 1995). Despite their inability to enter S phase, these cells proceed to segregate the unreplicated chromosomes unequally and undergo what has been sometimes been referred to as ‘reductional division’. Once again, because of the intimate association between chromosome segregation and mitosis, it has been proposed that Cdc6 is a component of the G1-M checkpoint whose function is to prevent onset of mitosis when cells fail to initiate DNA replication (Piatti et al. 1995). Thus far, no other component of this checkpoint pathway has been identified. This notion has gained strength from the observation that Cdc6 protein can inhibit Cdk1/Clb kinase and may facilitate mitotic exit (Calzada et al. 2001; Lau et al. 2006). However, the origin of the signal which activates the checkpoint function of Cdc6 or the exact mechanism by which it prevents mitotic entry is not understood. Clearly, the inability to initiate replication is not sensed by the replication checkpoint pathway (which detects stalled replication forks); since it is not activated in cdc6 mutant and cells are unable to prevent precocious chromosome segregation. In this chapter, we characterize the behaviour of Cdc6 deficient cells more thoroughly and explore the mechanistic underpinnings of Cdc6’s role in preventing untimely partitioning of unreplicated chromosomes. 3.2 Results 3.2.1 Cells depleted of Cdc6 undergo premature nuclear division in the absence of DNA replication Premature chromosome segregation has been reported in replication checkpoint defective mutants (such as mec1) treated with DNA replication inhibitors (hydroxyurea) (Krishnan et al. 2004). Similarly, it has also been reported that Cdc6 deficient cells fail to initiate DNA replication but proceed to segregate the unreplicated chromosomes prematurely (“reductional anaphase”) (Piatti et al. 1995; Toyn et al. 1995). Recent evidence showed that the DNA replication checkpoint thwarts untimely chromosome separation by directly regulating spindle dynamics. The role of Cdc6 or G1-M checkpoint has remained unclear since the observation was first documented (Piatti et al. 1995). The phenotype in both mec1 and cdc6 mutants suggests that the mechanisms by which the DNA replication checkpoint and Cdc6 prevent premature chromosome segregation may be similar. To pursue this question, we first re-examine if cdc6 mutant indeed exhibits premature chromosome separation. Yeast strain carrying a CDC6 deletion (cdc6Δ) and one copy of the galactoseinducible GAL-ubiCDC6 construct (US4275) was grown in YEP medium supplemented with raffinose and galactose (YEP+raff+gal). Cells were arrested in G2/M with nocodazole in YEP medium supplemented with glucose (YEPD) to repress CDC6 transcription, washed free of nocodazole and then released into YEP+Glu (YEPD) medium containing α-factor. The double synchronization afforded by the G2 and subsequent G1 (α-factor) arrest precludes assembly of pre-replication complexes (required for DNA replication) and depletes any pre-existing Cdc6. Under these conditions, Cdc6 depleted cells uniformly arrest in G1 and are unable to initiate DNA replication. Samples were collected at various time points for immunofluorescence staining and FACs analysis. As expected, all the cells arrested in G1 phase with a large bud, a long spindle, and a random segregation of unreplicated chromosome (1C DNA) (Figure 7). The presence of cells with long spindles showed that premature chromosome segregation had taken place. The wildtype cells (US1363) treated in similar manner progressed into mitosis with a large bud, a long spindle and equal segregation of duplicated sister chromatids. The kinetics of spindle elongation between wild type and cdc6Δ cells were similar with spindle elongation observed between 60 to 105 minutes after release from α factor. Moreover, the Western blot analysis confirmed that Cdc6 is completely degraded in YEPD medium. This phenotype is similar to that of the temperature-sensitive cdc6-1 mutants arrested at 37˚C (data not shown). These results confirmed that cells lacking Cdc6 are unable to replicate DNA yet they prematurely partition the unreplicated chromosomes, implying a role for Cdc6, in the G1/M checkpoint, in preventing premature mitosis or chromosome segregation. DAPI Nomarski Anti-tubulin 60min 180min 120min 90min 60min 105min noc cyc 1N 2N budding index cdc6 long spindle 80 wt budding index 60 wt long spindle 40 20 180 165 150 135 120 90 105 75 60 45 30 G6PD 100 15 Cdc6 % cells with anaphase spindles Glu RG 180min Time (mins) Figure 7. Cells depleted of Cdc6 undergo premature nuclear division in the absence of DNA replication. inducible GAL-ubiCDC6 construct was grown in YEP medium supplemented with - conditions, Cdc6 depleted cells uniformly arrest in G1 and are unable to initiate DNA replication. Samples were collected at various time points for immunofluorescence staining, FACs and Western Blot analysis. 3.2.2 Premature Nuclear Division in Cdc6 Depleted Cells is Associated with Major Mitotic Events As mentioned earlier, major mitotic events such as APC activation, destruction of securin (Pds1) and cleavage of the cohesin subunit (Scc1) precede anaphase or chromosome segregation. To determine if the premature chromosome segregation in Cdc6 deficient cells is due to premature entry into mitosis, we monitored securin (Pds1) degradation and cohesin (Scc1) cleavage. We first compared the kinetics of Pds1 degradation in a cdc6Δ mutant (US4364) and wild type cells (US3538). Both strains carrying the native promoter-driven PDS1-HA3 were synchronized as described in the previous experiment and released into YEPD medium at 25˚C. As depicted in Figure 8A, both strains showed Pds1 degradation from 105 minutes onwards. The wild type cells degraded Pds1 almost completely before entering the next G1 phase. However, Pds1 was not degraded to the same extent in cdc6Δ mutant and a significant residual amount persisted until 210 minutes (Figure 8A). Since the degree of synchrony in both strains is comparable, this indicates that the APCCdc20 activity may not be operating at its full capacity. Next we determined if nuclear division in both wild type (US3335) and cdc6Δ cells (US4344) is accompanied by Scc1 cleavage. Both strains carrying the native promoter-driven SCC1-myc18 gene were synchronized as described previously to ensure that the cdc6 mutant did not undergo S phase but instead proceeded to premature chromosome segregation. In the wild type strain, Scc1 cleavage was observed from 75 minutes onwards. However, detectable Scc1 cleavage was only noticeable after 105 minutes in cdc6Δ mutant (Figure 8B). Moreover, the abundance of Scc1 cleaved product in cdc6Δ mutant was lower compared to that in the wild type cells. This may be because Pds1 degradation is less pronounced (Figure 8A), leading to fewer available active Esp1 molecules. Besides Pds1 degradation and Scc1 cleavage, Clb2 degradation also serves as an indicator of cell cycle progression. We monitored Clb2 degradation by Western blotting in both wild type (US1363) and cdc6Δ (US4275) cells. While Clb2 degradation was prominent from 105 minutes onwards in wild type cells and diminished after 150 as cells entered the next cycle, the Clb2 proteolysis in cdc6Δ mutant was very sluggish (Figure 8C). Once again, this may be due to insufficient activation of APC in Cdc6 deficient cells. Taken together, these observations suggest that cellular events (Pds1 destruction, Clb2 proteolysis) that accompany chromosome segregation in normal cycle are significantly less pronounced in cdc6Δ cells. 160min DAPI Anti-tubulin 160 140 120 100 80 60 20 +met RG 40 +met noc -met cyc GAL-HA3-PDS1 Nomarski Pds1-HA3 G6PD 140 160 120 100 80 60 20 +met RG 40 +met noc -met cyc -PDS1 Pds1-HA3 G6PD Figure 10. APC activity is not required for the precocious chromosome segregation in Cdc6 depleted cells. -PDS1. The mutant was kept alive by MET3 promoter-driven wild type CDC6. Cdc6 can be depleted by growing these cells in methionine containing medium which represses the MET3 promoter. Cells grown in the absence of methionine were synchronized in metaphase by nocodazole treatment in +Met medium to shut off CDC6 transcription and to prevent pre-RC assembly and DNA replication. Subsequently, the cells were subjected to a second synchronization step in the ensurescence and Western Blot analysis. 3.2.4 Precocious Nuclear Division in Cdc6 Depleted Cells Can Be Prevented by Dicentric Chromosomes In a normal mitosis, chromosome segregation can be prevented by restraining cohesin cleavage. This is possible because while the microtubules emanating from opposite SPBs exert a poleward pulling force on the sister kinetochores to which they are attached, this force is opposed by the cohesins holding the two sister chromatids together, thus preventing premature chromosome segregation. In contrast, cdc6Δ mutant cells cannot undergo DNA replication and have only unreplicated chromosomes with one kinetochore monotelically attached to one SPB. Although these chromosomes have cohesion associated with them, unlike a sister-chromatid pair, they are unable to resist poleward pull exerted by any untimely extension of the spindle. It is therefore possible that premature chromosome segregation in Cdc6 deficient cells is due to unscheduled extension of the spindle. Since we have shown that premature nuclear division in cdc6Δ mutant does not appear to require the major signature-events’ of mitosis, we ask if the premature nuclear division is due to deregulation of spindle dynamics. To address this, we artificially introduced dicentric chromosomes into cdc6Δ MET-CDC6 mutant. The dicentric chromosomes comprise a circular minichromosome carrying two centromeres, one which is constitutively expressed and can support the assembly of a kinetochore and the other conditionally expressed in that it cannot support kinetochore assembly in the presence of galactose because of its juxtaposition to GAL1-10 promoter. However, in the presence of glucose that represses GAL1-10 promoter, the conditional centromere can now support kinetochore assembly (Tanaka et al., 2004). The conditional and the constitutive centromeres constitute a system that mimics the establishment of a bipolar attachment. However, since both centromeres are on a single circular vector, the bioriented minichromosomes cannot be segregated away from each other; consequently it resists spindle elongation. Therefore we introduced minichromosomes into cdc6Δ MET-CDC6 (US7007) cells where it can be propagated stably by growing cells in the absence of methionine (for Cdc6 expression) and the presence of galactose (conditional centromere inactive). In a parallel strain, we introduced a CEN4 plasmid (carrying only one functional copy of CEN4) into cdc6Δ MET-CDC6 mutant (US7008) as a control. Both strains were first arrested in +Met+galactose medium containing nocodazole to synchronize in metaphase and repress transcription of CDC6, followed by release into medium containing α-factor to impose a subsequent arrest in G1. This synchronization protocol ensures that at this juncture, both the CEN4 plasmid and dicentric chromosome have not undergone replication. Cells were then released into medium containing glucose to activate the second centromere in the dicentric plasmid and thus allow minichromosome to establish bi-orientation. We monitored the state of the nucleus and the spindle using DAPI and immunofluorescence staining, respectively. The cdc6Δ MET-CDC6 mutant carrying CEN4 plasmid equipped with only one centromere underwent premature nuclear division (Fig 11, accompanied by dramatic spindle elongation). However, the cdc6Δ MET-CDC6 mutant carrying the dicentric chromosome plasmid with two active centromeres assembled short spindles and failed to undergo premature nuclear division. From these observations, we conclude that the presence of two centromeres in the minichromosomes promotes bipolar attachment that mimics biorientation in normal duplicated chromosomes and restrains spindle elongation. Hence, it is very likely that precocious segregation of unreplicated chromosomes in cdc6Δ mutant is due to misregulation of the spindle leading to premature elongation. In the next section we explore this notion further. CEN4 URA3 Spindle elongation Centromere1 active GAL OFF Monocentric plasmid Centromere active No spindle elongation Centromere1 active Dicentric plasmid 180min DAPI Anti-tubulin CDC6 Dicentric plasmid CEN4 plasmid Nomarski Dicentric plasmid plasmid 180min 180min 120min 120min 60min 60min noc noc cyc 1N 2N cyc 1N 2N Figure 11. Precocious Nuclear Division in Cdc6 Depleted Cells Can Be Prevented by Dicentric Chromosomes. - +Met+galactose medium containing nocodazole to synchronize in metaphase and juncture, both the CEN4 plasmid and dicentric chromosome have not undergone repli- 3.2.5 Precocious Nuclear Division in Cdc6 Depleted Cells Is Due to Deregulation of Spindle Dynamics It had also been shown previously that the DNA replication checkpoint prevents precocious segregation of largely unreplicated chromosomes by regulating spindle dynamics. We suspected that Cdc6, a putative G1-M checkpoint protein, may be involved in regulating spindle dynamics through an unknown mechanism. Since the BimC family kinesins such as Cin8, Kip1 and microtubule associated proteins, Ase1 are important for the dynamic behaviour of the mitotic spindle, we compared the levels of these proteins in wild type, cdc6Δ and cdc34-1 strains. We used cdc34-1 strain as a control because, like cdc6Δ mutant, it also arrest in G1 phase at the restrictive temperature and is unable to initiate DNA replication. However, unlike cdc6Δ mutant, cdc34-1 cells not assemble a spindle although centrosomes are duplicated. To perform this study, we tagged Cin8, Kip1 and Ase1 with HA3 in both wild type (US4122, US4677 and US7009) and cdc34-1 (US5239, US6342 and US7011) strains, Cin8 and Kip1 with HA3 in cdc6Δ GAL-CDC6 strain (US4366 and US6343) and Ase1 with HA3 in cdc6Δ MET-CDC6 strain (US7010). As described earlier, all the strains were arrested in mitosis in the presence of nocodazole and subsequently in G1 by α factor treatment to ensure good synchrony as all three strains may recover from nocodazole arrest at different rate. Finally, all three strains were released into fresh medium at 36˚C and Cin8, Kip and Ase1 levels were monitored by Western Blotting. As shown in Figures 12, 13 and 14, Cin8, Kip1 and Ase1 protein levels were elevated in cdc6Δ cells compared to cdc34-1 cells. The abundance of these proteins peaked around 60 minutes after release from α-factor coinciding with that of wild type cells. The kinetics of Cin8, Kip1 and Ase1 abundance in both wild type and cdc6Δ cells are comparable, suggesting that both strains were undergoing spindle elongation and nuclear division at about the same rate after release from αfactor. The fact that Cin8, Kip1 and Ase1 levels were upregulated in cdc6Δ mutant compared to cdc34-1 cells correlates with premature nuclear division observed in cdc6Δ cells since a high abundance of Cin8, Kip1 and Ase1 has been closely associated with precocious spindle elongation (Krishnan et al. 2004). These results imply that spindle dynamics in Cdc6 depleted cells may be deregulated due to an excessive accumulation of Cin8, Kip1 and Ase1. This may implicate Cdc6’s role in regulating microtubule associated proteins and thus, in controlling spindle dynamics and spindle elongation. 180 160 140 120 100 36ºC 80 60 40 20 Glu noc Wildtype Cin8-HA3 G6PD 180 160 140 120 100 80 60 40 36ºC 20 Glu noc Cin8-HA3 G6PD 180 160 140 120 100 80 60 40 36ºC 20 Glu noc Cin8-HA3 G6PD Figure 12. Precocious Nuclear Division in Cdc6 Depleted Cells Is Due to Deregulation of Spindle Dynamics To perform this study, we tagged Cin8 with HA3 GAL-CDC6 strains. As described earlier, all the strains were arrested in mitosis ensure good synchrony as all three strains may recover from nocodazole arrest at and Cin8 levels were monitored by Western Blotting. 180 160 140 120 80 36ºC 100 40 60 20 Glu noc Wildtype Kip1-HA3 G6PD 180 160 140 120 80 100 40 60 36ºC 20 Glu noc Kip1-HA3 G6PD 180 160 140 120 100 80 40 60 36ºC 20 Glu noc Kip1-HA3 G6PD Figure 13. Precocious Nuclear Division in Cdc6 Depleted Cells Is Due to Deregulation of Spindle Dynamics To perform this study, we tagged Kip1 with HA3 GAL-CDC6 strains. As described earlier, all the strains were arrested in mitosis in good synchrony as all three strains may recover from nocodazole arrest at different levels were monitored by Western Blotting. 180 160 140 120 100 80 60 40 36ºC 20 +MET noc Wildtype Ase1-HA3 G6PD 180 160 140 120 100 80 60 20 36ºC 40 +MET noc Ase1-HA3 G6PD 180 160 140 120 80 100 60 40 36ºC 20 +MET noc Ase1-HA3 G6PD Figure 14. Precocious Nuclear Division in Cdc6 Depleted Cells Is Due to Deregulation of Spindle Dynamics To perform this study, we tagged Ase1 with HA3 MET-CDC6 strains. As described earlier, all the strains were arrested in mitosis in good synchrony as all three strains may recover from nocodazole arrest at differand Ase1 levels were monitored by Western Blotting. 3.3 Discussion Spindle elongation and chromosome segregation are events typically associated with anaphase. It is not surprising that spindle extension and segregation of haploid nuclei in Cdc6 deficient cells is believed to be a consequence of premature onset of mitosis. The Cdc6 protein is essential for the assembly of pre-replicative complexes (pre-RCs) before a “point of no return’ that occurs in late G1. This point of no return means that cells are committed to undertaking progression through the cell cycle and the process is irreversible. Therefore, if the committed cells fail to undergo DNA replication (e.g. cdc6Δ), they are presumably forced to prematurely segregate their unreplicated chromosome, resulting in daughter cells with unequal DNA content. Hence, it has been postulated that Cdc6 is a G1-M checkpoint protein that safeguards cells which are unable to undergo S phase from prematurely entering mitosis. This conclusion is especially appealing given the fact that S. pombe cells are known to regulate mitotic entry in response to fork signaling via inhibitory phosphorylation of tyrosine 15 of Cdc2. However, mitosis is associated with several other events such as APC activation, securin degradation, separase activation and cohesin cleavage. We have shown that these mitotic events occur, albeit sluggishly, in cdc6 mutant cells. Therefore, chromosome separation in cdc6 mutants is generally believed to be due to premature onset of mitosis in the absence of G1-M checkpoint control. However, our findings suggest that the major events (or regulators) are not a prerequisite for the premature spindle elongation observed in cdc6 mutant. In fact, spindle elongation can proceed in the absence of APC activity. This raises the possibility that untimely segregation of unreplicated chromosomes in Cdc6 deficient cells is not due to premature entry into mitosis but due to deregulated spindle dynamics. This notion has a parallel in a previous report documenting evidence that DNA replication checkpoint deficient cells experiencing HU mediated arrest undergo precocious chromosome segregation; not because of premature mitotic entry, but because the spindle was misregulated in the absence of the checkpoint. That the regulation of spindle dynamics is an important aspect of the S phase checkpoints is also exemplified by the fact that DNA damage checkpoint also inhibits spindle elongation to prevent segregation of the damaged chromosomes. Our observation that premature spindle elongation in cdc6 mutants can be prevented simply by expressing an uncleavable dicentric chromosome strongly suggests that Cdc6, a putative G1-M checkpoint protein, is directly regulating spindle elongation but not entry into mitosis. Hence, cdc6 mutant is perhaps a good model to study the dynamics of spindle elongation when cells fail to initiate DNA replication since the complication arising from biorientation which counters spindle extension does not exist. It should also be noted that budding yeast cell cycle varies from S. pombe or mammalian systems in that S.cerevisiae cells are able to form short spindle in mid-S phase as oppose to metaphase in other organisms. This may be because in budding yeast, unlike other organisms, Cdk1 is activated by tyrosine19 dephosphorylation to a sufficient level such that spindle assembly is mediated but not premature entry into mitosis. The short spindle formed is not deleterious to the cell as long as it is prevented from elongation. However, apparently in cdc6 mutants, the short spindle elongates untimely leading to reductional anaphase. Perhaps this is due to the upregulation of microtubule associated proteins such as Ase1, Cin8 and Kip1 observed in cdc6 mutants. There is an interesting feature to the premature segregation of unreplicated chromosomes in cdc6 mutant. Although most chromosomes in cells arrested in G1 lack sister kinetochores, and hence bipolar attachment, chromosome still managed to segregate almost equally to mother and daughter cells (55% vs 45%) in cdc6 mutant. It might be expected that, in the absence of bipolarity, all chromosomes should be attached to the mother SPB from early G1 phase and should therefore segregate into the daughter cells. The key insight came from analysis of Ipl1 function in the cdc6 cells, as they proceed to segregate the unreplicated chromosomes. In this mutant, unreplicated chromatids lack sisters and therefore not experience the tension that results from bi-orientation. The absence of Ipl1 had a dramatic effect on the segregation pattern of sisterless chromatids. While the chromatids segregate randomly to either spindle pole in the cdc6 single mutant (Ipl1 proficient), they preferentially segregate to the spindle pole in the daughter cell in ipl1 cdc6 double mutants (Stern 2002; Tanaka et al. 2002). The interpretation of this remarkable result relies on two recent discoveries: the first is that budding yeast centromeres are already attached via kinetochores and microtubules to the spindle pole in G1; and the second is that, after spindle pole body duplication and spindle formation, the ‘old’ spindle pole moves to the daughter cell, while the newly synthesized spindle pole stays in the mother cell. The observation that unreplicated chromatids in an ipl1 cdc6 double mutant remain linked to the very same pole that they were already attached to in G1 suggests that the microtubule-mediated kinetochore–spindle pole interactions are unusually stable in the absence of Ipl1. In the presence of Ipl1 the original connections are destabilized (presumably because they not generate tension), leading to random attachment to any one pole and eventually random segregation pattern. Another important factor is the monopolar attachment of kinetochores to the spindle pole bodies in cdc6 mutants. Monopolar attachment means that each chromosome is attached to only one SPB, without any ability to exert a force in opposite direction. As a consequence, it is highly possible that even a small poleward force cannot be resisted, causing the cells to be extremely vulnerable to precocious chromosome segregation. This vulnerability is demonstrated by the ability of microtubule associated proteins such as Ase1, Cin8 and Kip1 in cdc6 mutants to prematurely elongate the spindle despite the fact that cells are still in G1 phase. However this is unlikely to occur in cells arrested in G2-M where chromosomes are duplicated. This contrast is due to the fact that each duplicated chromosome (with duplicated centromeric region) is encircled by the cohesin complex, thus providing resistance to poleward forces. Thus, cdc6 mutant cells are extremely vulnerable to premature spindle elongation since spindle is formed, microtubule associated proteins are up regulated and chromosomes are able to establish monopolar attachment. The susceptibility to premature spindle elongation is not restricted to budding yeast alone. There is evidence that the Chinese hamster ovary (CHO) cells that arrest at the G1-S phase of the cell cycle with HU, proceed to segregate their chromosomes prematurely when treated with caffeine. Such mitotic cells, even in the absence of intact and replicated chromosomes, can assemble a mitotic spindle and progress through chromosome segregation (Wise et al. 1997). This emphasizes the importance of robust regulation of microtubule associated proteins such as Ase1, Cin8 and Kip1 by Cdc6 or G1-M checkpoint. In the next chapter, we explore the causal connection, if any, between the absence of Cdc6 function and the deregulation of the spindle. There is a strong likelihood that the findings presented here are applicable to other organisms, given the substantial conservation of the broad organization of the checkpoint networks across species. However, before any firm conclusion can be drawn concerning the role of Cdc6 in spindle elongation, it is necessary to investigate if premature chromosome segregation is a property associated specifically with the loss of Cdc6 function or it is a common characteristic of cells that can undergo START but unable to initiate S phase. This is the theme that we explore in Chapter 4. [...]... good synchrony as all three strains may recover from nocodazole arrest at differand Ase1 levels were monitored by Western Blotting 3. 3 Discussion Spindle elongation and chromosome segregation are events typically associated with anaphase It is not surprising that spindle extension and segregation of haploid nuclei in Cdc6 deficient cells is believed to be a consequence of premature onset of mitosis... substantial conservation of the broad organization of the checkpoint networks across species However, before any firm conclusion can be drawn concerning the role of Cdc6 in spindle elongation, it is necessary to investigate if premature chromosome segregation is a property associated specifically with the loss of Cdc6 function or it is a common characteristic of cells that can undergo START but unable... random attachment to any one pole and eventually random segregation pattern Another important factor is the monopolar attachment of kinetochores to the spindle pole bodies in cdc6 mutants Monopolar attachment means that each chromosome is attached to only one SPB, without any ability to exert a force in opposite direction As a consequence, it is highly possible that even a small poleward force cannot be... reductional anaphase Perhaps this is due to the upregulation of microtubule associated proteins such as Ase1, Cin8 and Kip1 observed in cdc6 mutants There is an interesting feature to the premature segregation of unreplicated chromosomes in cdc6 mutant Although most chromosomes in cells arrested in G1 lack sister kinetochores, and hence bipolar attachment, chromosome still managed to segregate almost equally... precocious segregation of unreplicated chromosomes in cdc6Δ mutant is due to misregulation of the spindle leading to premature elongation In the next section we explore this notion further CEN4 URA3 Spindle elongation Centromere1 active GAL OFF Monocentric plasmid Centromere 2 active No spindle elongation Centromere1 active Dicentric plasmid 180min DAPI Anti-tubulin CDC6 Dicentric plasmid CEN4 plasmid Nomarski... chromatids together, thus preventing premature chromosome segregation In contrast, cdc6Δ mutant cells cannot undergo DNA replication and have only unreplicated chromosomes with one kinetochore monotelically attached to one SPB Although these chromosomes have cohesion associated with them, unlike a sister-chromatid pair, they are unable to resist poleward pull exerted by any untimely extension of the... medium containing methionine, raffinose and galactose to suppress Cdc6 expression and to induce over-expression of Pds1 As shown in Figure 10, Pds1 overexpression is induced as early as 60 minutes after release from α-factor and peaks at around 160 minutes in cdc6Δ GAL-HA3-PDS1 strain No trace of Pds1 was detected in cdc6Δ mutant control Immunofluoresence analysis clearly showed premature chromosome segregation. .. chromosome segregation accompanied by spindle elongation in both cdc6Δ and cdc6Δ GAL-HA3-PDS1 strains (Figure 10) These results clearly suggest that premature nuclear division in cdc6Δ mutant can occur in the absence of APC activity Here, APC activity is suppressed through overexpression of Pds1; Esp1 separase is inhibited and cohesin cleavage is prevented Thus, premature chromosome segregation in Cdc6 deficient... peaked around 60 minutes after release from α-factor coinciding with that of wild type cells The kinetics of Cin8, Kip1 and Ase1 abundance in both wild type and cdc6Δ cells are comparable, suggesting that both strains were undergoing spindle elongation and nuclear division at about the same rate after release from αfactor The fact that Cin8, Kip1 and Ase1 levels were upregulated in cdc6Δ mutant compared... Onset of Mitosis APC activity is critical for progression through mitosis As Pds1 and Clb2 are degraded via APC-dependent ubiquitylation, the amount of Pds1 and Clb2 reflects the activation status of APC Destruction of Pds1 allows activated Esp1 to cleave Scc1, thus dissolving chromosome cohesion leading to partition of sister chromatids In the preceding section we observed that although cdc6Δ cells cannot . at various time points for immunofluorescence staining and FACs analysis. As expected, all the cells arrested in G1 phase with a large bud, a long spindle, and a random segregation of unreplicated. cdc6Δ GAL-HA 3 -PDS1 strain. No trace of Pds1 was detected in cdc6Δ mutant control. Immunofluoresence analysis clearly showed premature chromosome segregation accompanied by spindle elongation. exhibit a similarly dramatic phenotype. At non-permissive temperature, the mutant cells traverse START, construct a bud but are unable to assemble a functional replication complex and therefore fail