A study on premature segregation of unreplicated chromosomes 4

Chapter4 Regulation of Spindle Elongation by Cdc34 4.1 Background In the previous chapter, we have explored the various reasons responsible for premature nuclear division in Cdc6 depleted cells. It has long been thought that precocious segregation of chromosomes is due to premature onset of mitosis. However, recent evidence suggests that premature chromosome segregation can occur without the onset of mitosis, APC activation, cohesion cleavage or biorientation of kinetochores. The DNA replication checkpoint has been reported to thwart untimely chromosome segregation not by inhibiting mitotic entry but by directly regulating spindle dynamics and by preventing replication fork collapse to allow duplication of centromeric DNA and hence kinetochore bi-orientation (Krishnan et al. 2004). To be more precise, the critical effectors of DNA replication checkpoint, Mec1 and Rad53, (orthologues of human ATM/ATR- and Chk2-like kinases, respectively) prevent precocious chromosome segregation by suppressing the accumulation of spindle elongation effectors such as Cin8 and Stu2, thus precluding premature induction of spindle elongation during early S phase (Krishnan et al. 2004) (Krishnan et al. 2005). A recent study in yeast suggests that concerted action by two prominent kinases Cdk1 and polo (Cdc5) are required to fully inactivate Cdh1, an activator of the E3 ubiquitin ligase APC (anaphase promoting complex) (Crasta et al. 2008). APCCdh1 is essential for proteolytic destruction of microtubule associated proteins such as Cin8, Kip1 and Ase1. Therefore, Cdh1 inactivation is required to halt the destruction of these proteins to facilitate spindle assembly and spindle elongation. These findings helped uncover an additional mechanism by which DNA damage checkpoint prevents premature chromosome segregation (Zhang et al. 2009). It was shown that activation of the DNA damage checkpoint leads to inactivation of Cdc5 polo kinase via Rad53-mediated phosphorylation (Zhang et al. 2009). Thus, inactivation of both Cdk1 and Cdc5 by the checkpoint prevents Cdh1 inactivation, which in turn continues to mediate the destruction of spindle elongation-inducing proteins Cin8, Kip1 and Ase1. Thus these studies envisaged that the DNA damage checkpoint, in addition to suppressing cohesin cleavage, maintains APCCdh1 in an active state to restrain spindle extension until the damaged chromosomes are repaired. Hence, both DNA replication and DNA damage checkpoints can prevent premature chromosome segregation by restricting the accumulation of microtubule associated proteins such as Cin8, Kip1, Ase1 and Stu2. The exact mechanism as to how DNA replication checkpoint promotes destabilization of the microtubule associated proteins remains unclear. The activated DNA damage checkpoint leads to the perturbation of Cdh1/Cdk1/polo/MAPs control circuit; Cdh1 is maintained in an active state and spindle is restrained from prematurely elongating (Crasta et al. 2008). Since cells lacking Cdc6 also encountered premature spindle elongation resulting from an accumulation of MAPs, it is possible that Cdc6 (putative G1-M checkpoint component) is able to directly regulate spindle elongation by targeting the Cdh1Cdk1/polo/MAPs control circuit. This notion would be consistent with Cdc6’s role in the inactivation of Cdk1. This chapter explores the relationship between Cdc6 and spindle dynamics. 4.2 Results 4.2.1 Premature segregation of unreplicated chromosomes in cells lacking Cdc7 and Cdc45 In the preceding chapter, we have documented that Cdc6 deficient cells fail to initiate DNA replication and proceed to undergo what-appears-to-be premature mitosis, causing a reductional anaphase. We also noted that the premature nuclear division is accompanied by high abundance of microtubule associated proteins such as Cin8, Kip1 and Ase1. Hence, it is possible that Cdc6 is involved in the regulation of effectors of spindle elongation and spindle dynamics. However, before the role of Cdc6 in spindle elongation is investigated, it is necessary to ascertain if premature chromosome segregation is a characteristic associated specifically with cdc6 mutant or it is a common characteristic of cells that can undergo START but are unable to initiate S phase. To test this notion, we monitored spindle behaviour in cells lacking Cdc7 and Cdc45. Cdc7 is a serine/threonine kinase essential for DNA replication and requires Dbf4 for its activity (hence the term Ddk for Dbf-dependent kinase) (Bousset et al. 1998) (Jares et al. 2000). Cdc45 is required for the initiation and elongation step of DNA replication (Zou et al. 1997). The CDC45 gene was shown to genetically interact with components of replication factors such as MCM and ORC. We constructed cdc7Δ GAL-CDC7 (US5582) and cdc45Δ GAL-CDC45 (US5585) strains for this study. Both strains were first arrested in metaphase by growth in galactose medium containing nocodazole. Cells were then transferred to YEPD for 30 minutes to allow depletion of Cdc7 and Cdc45 before they were released into YEPD medium containing α-factor to synchronize them in G1 phase. Once the cultures were uniformly arrested in G1, α-factor was removed and cells were released into YEPD medium at 25˚C and samples taken every 20 minutes to score the spindle lengths. As shown in Figure 15, cells lacking Cdc7 arrested in G1 with 1N DNA but proceeded to elongate the spindles from 80 minutes onwards with spindle length exceeding 4µm. Similarly, cells lacking Cdc45 also arrested in G1 with a large bud and unreplicated chromosomes but proceeded to extend the spindle from 80 minutes onwards with spindle length exceeding 4µm (Figure 16). Thus, cells defective in Cdc7 or Cdc45 traverse START, assemble a bud and fail to initiate DNA replication, but prematurely segregate the unreplicated chromosomes despite the presence of a functional CDC6. One interpretation of these results is that Cdc6, Cdc7 and Cdc45 are all involved in preventing untimely spindle elongation and segregation of unreplicated chromosomes. Alternatively, it is not inconceivable that premature segregation is not specifically due to the lack of Cdc6, Cdc7 or Cdc45 functions but is a common characteristic of cells that have traversed START but fail to initiate DNA replication. In other words, the premature partitioning of the chromosomes is regulated by some unknown mechanism and the mutations in genes such as CDC6, CDC7 and CDC45 only allow manifestation of this underlying regulation because of their common inability to initiate DNA replication. Nomarski DAPI Anti-tubulin short spindle (0-2 mm) medium spindle (2-4 mm) long spindle (>4 mm) Spindle Length 120 0-2 um 2-4 um >4 um % cells 100 80 60 40 20 0 20 40 60 80 100 120 140 160 180 Time (mins) Glu 180min Glu 120min Glu 60min Glu a Glu NOC 1N 2N Figure 15. Premature segregation of unreplicated chromosomes in cells lacking Cdc7 We constructed cdc7 strain for this study. The strain was first arrested in metaphase by growth in galactose medium containing nocodazole. Cells were then transferred to YEPD for 30 minutes to allow depletion of Cdc7 before they were released into YEPD medium containing -factor to synchronize them in G1 phase. Once the cultures were uniformly arrested in G1, -factor was removed and cells were released into YEPD medium at 25 C and samples taken every 20 minutes to score the spindle lengths and for FACS analysis. . Nomarski DAPI Anti-tubulin short spindle (0-2 mm) medium spindle (2-4 mm) long spindle (>4 mm) 120 Spindle Length 0-2 um 2-4 um % cells 100 >4 um 80 60 40 20 0 20 40 60 80 100 120 140 160 180 Time (mins) Glu 180min Glu 120min Glu 60min Glu a Glu NOC 2N Figure 16. Premature segregation of unreplicated chromosomes in cells lacking Cdc45 strain was first arrested in metaphase by growth in galactose medium containing nocodazole. Cells were then transferred to YEPD for 30 minutes to allow depletion of Cdc45 before they were released into YEPD medium and for FACS analysis. 4.2.2 Depletion of Cdc6 in cdc34-1 cells fails to promote spindle assembly or spindle elongation To directly address the possibility of a role for CDC6 in spindle regulation, we utilize cdc34-1 cells which traverse START and duplicate their centrosomes but can neither initiate S phase nor assemble a short spindle because the inter SPB bridge remains unbroken. If Cdc6 is involved in regulating spindle biogenesis and dynamics, cdc341 cells lacking Cdc6 is expected to promote spindle assembly or spindle elongation. For this experiment, we constructed cdc34-1 (US6005) and cdc34-1 cdc6Δ METCDC6 (US7012) strains expressing GFP-tagged spindle pole body component Spc42 to ascertain spindle length in both strains. To ensure Cdc6 is degraded completely, both strains were arrested in nocodazole supplemented with methionine to repress CDC6 transcription. These cells were then released into methionine medium (+Met) at non-permissive temperature of 36˚C. Samples from both cdc34-1 and cdc34-1 cdc6Δ MET-CDC6 cells were collected at 180 minutes and analyzed by immunofluorescence microscopy. As shown in Figure 17, 100% of both cdc34-1 and cdc34-1 cdc6Δ MET-CDC6 strains exhibit one Spc42-GFP dot indicating that the duplicated SPBs, have not separated and no spindle was assembled. In addition, immunofluorescence staining of 180 minutes sample showed no sign of spindle formation or spindle elongation. These results imply that Cdc6 does not influence spindle biogenesis significantly. Nomarski DAPI Spc42GFP 100% no spindle cdc34-1 Nomarski DAPI Anti-tubulin 100% no spindle Nomarski DAPI Spc42GFP 100% no spindle cdc34-1 MET-CDC6 Nomarski DAPI Anti-tubulin 100% no spindle Figure 17. Depletion of Cdc6 in cdc34-1 cells fails to promote spindle assembly or spindle elongation. cdc34-1 CDC6 cdc34-1 - 4.2.3 Ectopic expression of Sic1 and Cdh1 prevent premature spindle elongation in Cdc6 depleted cells In Chapter 3, we provided evidence that premature spindle elongation in cells lacking Cdc6 was due to the accumulation of microtubule associated proteins such as Cin8, Kip1 and Ase1. Since it has been shown previously that APCCdh1 is responsible for the ubiquitination and proteasome-mediated degradation of Cin8, Kip1 and Ase1, we therefore considered the possibility that Cdh1 inactivation may be relevant to Ase1, Cin8 and Kip1 stability in Cdc6 depleted G1 arrested cells. If this is true, then overexpression of Cdh1 would be expected to promote degradation of spindle elongation effectors such as Cin8 in Cdc6 depleted cells. To test this, we arrested two separate cultures of cdc6Δ MET-CDC6 GAL-HA3-CDH1 CIN8-HA3 (US7022) in +Met+glucose medium (to repress transcription of CDC6) containing nocodazole. Subsequently, both cultures were released from metaphase arrest into +Met medium containing α-factor to ensure that the cells were arrested in G1. One culture was then released into +Met+glucose medium at room temperature to inhibit over-expression of Cdh1. Another culture was released into medium containing Raffinose+Galactose to drive over-expression of GAL-HA3-CDH1. As shown in Figure 18A, Cdh1 expression was apparent from 80 minutes after the release. As Cdh1 over-expression peaked from 120 minutes onwards, Cin8 was concurrently degraded from 120 minutes. The unstable Cin8 was not present in sufficient amounts to allow Cdc6 deficient cells to elongate their spindles, resulting in the phenotype observed: cells with short spindles (Figure 18A). In contrast, in the control culture where Cdh1 was not over-expressed, Cin8 remained stable. The results of this experiment imply that Cdh1 may be inactive in Cdc6 depleted cells, leading to the accumulation of proteins such as Cin8 and, thus, premature spindle elongation. It is known that Sic1 is responsible for inhibition of Cdk1/Clb5, kinases in G1 phase (prior to S phase onset) (Barberis et al. 2005). Moreover, active Cdk1 is also responsible for inactivating Cdh1 via phosphorylation at multiple sites (Crasta et al. 2008). Therefore if Cdk1 activity is inhibited, Cdh1 will remain active. To further verify that premature spindle elongation in Cdc6 depleted cells is a result of Cdh1 inactivation (with accumulation of microtubule associated proteins such as Cin8), we expressed non-degradable version of Sic1 in Cdc6 depleted cells. We first arrested cdc6Δ MET-CDC6 GAL-ndSIC1 (US7013) strain in nocodazole containing medium supplemented with methionine to repress CDC6 transcription. Cells were then released into α-factor containing medium to resynchronize cells in G1. The cells were subsequently released into medium containing Raffinose and Galactose to facilitate over-expression of non-degradable Sic1. Samples were taken at 160 minutes to monitor the presence of long spindles. Almost 100% of the cells failed to elongate their spindles and arrested in G1 with large buds and short spindles (Figure 18B). This is similar to the previous experiment where Cdh1 over-expression in cells lacking Cdc6 led to arrest with short spindles. Clearly, Cdh1 inactivation is mandatory for premature spindle elongation in Cdc6 deficient cells. This also suggests that the Sic1 degradation step must be tightly regulated since untimely degradation of Sic1 promotes Cdk1-mediated inactivation of Cdh1, accumulation of Cin8 and Ase1 and hence premature spindle elongation. If SCF-mediated degradation of Sic1 is an essential step in determining the fate of spindle in Cdc6 depleted cells, then there is a strong possibility that SCF-component Cdc34 is important in the regulation of spindle elongation. Figure 28. Cdc34 can induce spindle elongation by promoting stability of microtubule associated proteins. To confirm that Cdc34 indeed induces spindle elongation by regulating microtubule associated proteins, we expressed ASE1-HA3, CIN8-HA3 or KIP1-HA3 from their respective loci in cdc34-1 cdc6Δ MET-CDC6 cdh1Δ cells. Two separate cultures of cdc34-1 cdc6Δ MET-CDC6 cdh1Δ cells expressing ASE1-HA3, CIN8-HA3 or KIP1HA3 were grown in medium containing nocodazole and methionine to repress CDC6 transcription. One culture was released at 36˚C in the presence of methionine to ensure complete depletion of Cdc6. The other culture was released into 36˚C in the presence of methionine for 210 minutes and then shifted to 24˚C to restore Cdc34 function. Samples were collected at 30 minutes intervals for Western Blot analysis. -met cyc 150 +met 36˚C 120 150 120 90 150 120 150 120 +met 36˚C 90 60 30 210 180 150 120 90 60 30 +met NOC -met cyc 150 120 90 60 30 210 180 150 120 90 60 30 +met NOC -met cyc +met 36˚C 90 60 30 210 180 150 120 90 60 30 +met NOC -met cyc 150 120 90 60 30 210 180 150 120 90 60 30 +met NOC -met cyc +met 36˚C 90 +met 36˚C 60 30 210 180 150 120 90 60 30 +met NOC -met cyc +met 36˚C 60 30 210 180 150 120 90 60 30 +met NOC Figure 28 +met 36˚C Cin8-HA3 G6PD +met 24˚C Cin8-HA3 noc G6PD cyc +met 36˚C 1N 1N 1N 2N Ase1-HA3 G6PD +met 24˚C Ase1-HA3 noc G6PD cyc 2N +met 36˚C Kip1-HA3 G6PD +met 24˚C Kip1-HA3 noc G6PD cyc 2N ASE1-HA3 - + - + Figure 29. Cdc34 can induce spindle elongation by promoting stability of microtubule associated proteins. ASE1-HA3 - 4.2.10 Microtubule associated proteins Ase1 and Cin8 are unstable in cells deficient in Cdc34 Previous experimental data suggested that microtubule associated proteins such as Ase1 and Cin8 are unstable in Cdc34 deficient cells. These proteins remained unstable even in the absence of Cdh1, suggesting that they are targets for degradation by an alternative pathway. To assess in ‘a pulse-chase experiment’ whether low abundance of these proteins is due to enhanced proteolysis, cdc34-1, cdc6Δ METCDC6, cdc34-1 cdh1Δ and cdc6Δ MET-CDC6 cdh1Δ cells carrying GAL-ASE1-myc (US7036, US7040, US7038 and US7042) or GAL-CIN8-myc (US7037, US7041, US7039 and US7043) were released into raffinose medium supplemented with methionine for hours at 36˚C and subsequently transferred to galactose medium supplemented with methionine at 36˚C for another hours to induce Ase1 or Cin8 expression. Once the cells were arrested at their respective non-permissive conditions, they were released into glucose medium supplemented with methionine and 0.1mg/ml cycloheximide (to inhibit de novo translation) and the protein pulse was monitored. As shown in Figure 30 and 31, protein pulses for Ase1 and Cin8 were not detected in cdc34-1 cells compared to cdc6Δ MET-CDC6 cells. This suggests that both Ase1 and Cin8 are very unstable in the absence of Cdc34 and explains why no spindle is assembled. However, both protein pulses for Ase1 and Cin8 were detected in cdc34-1 cdh1Δ cells but were less stable compared to protein pulses found in cdc6Δ METCDC6 cdh1Δ (Cdc34 proficient) cells. These observations are consistent with short spindle assembly in cdc34-1 cdh1Δ cells and long spindle in cdc6 MET-CDC6 cdh1Δ cells. Taken together, these results strongly support the idea that Cdc34 plays an important role in premature spindle elongation in Cdc6 deficient cells by stabilizing microtubule associated proteins Cdc34 + Cdc34 - 100 80 40 60 -MET cyc 100 80 40 60 20 -MET cyc 20 +MET Glu cycloheximide +MET Glu cycloheximide Ase1-myc G6PD 120 100 80 60 40 20 -MET cyc (Cdc34 -) Ase1-myc G6PD 120 100 80 60 40 20 -MET cyc (Cdc34 +) Ase1-myc G6PD Figure 30. Microtubule associated proteins Ase1 is unstable in cells deficient in Cdc34 To assess in ‘a pulse-chase experiment’ whether low abundance of Ase1 is due to enhanced proteolysis, , , and cells carrying were released into raffinose another hours to induce Ase1 expression. Once the cells were arrested at their respective non-permissive conditions, they were released into glucose medium supplemented with methionine and 0.1mg/ml cycloheximide (to inhibit de novo translation) and the protein pulse was monitored. Cdc34 + cdc34-1 GALCIN8-myc GALCIN8-myc 80 100 60 20 40 +MET Glu cycloheximide 36ºC -MET cyc 80 100 60 40 +MET Glu cycloheximide 36ºC 20 -MET cyc Cdc34 - Cin8-myc G6PD (Cdc34 -) 120 100 80 60 40 20 -MET cyc +MET Glu cycloheximide 36ºC Cin8-myc G6PD 120 100 80 60 40 +MET Glu cycloheximide 36ºC 20 -MET cyc (Cdc34 +) Cin8-myc G6PD Figure 31. Microtubule associated proteins Cin8 is unstable in cells deficient in Cdc34 To assess in ‘a pulse-chase experiment’ whether low abundance of Cin8 is due to enhanced proteolysis, cdc34-1, , and cells carrying GAL-CIN8-myc were released into raffinose another hours to induce Cin8 expression. Once the cells were arrested at their respective non-permissive conditions, they were released into glucose medium supplemented with methionine and 0.1mg/ml cycloheximide (to inhibit de novo translation) and the protein pulse was monitored. 4.2.11 Cdc34-mediated stabilization of microtubule associated proteins are proteasome dependent So far, we have sufficient evidence indicating that microtubule associated proteins such as Ase1, Cin8 and Kip1 are unstable in cells lacking Cdc34 function, an observation consistent with their inability to assemble a spindle. These proteins remain relatively unstable even when Cdc34 deficient cells are lacking Sic1 or Cdh1 function, suggesting that the ubiquitin-independent degradation machinery may be at play. We speculate that SCF complexes (of which Cdc34 is a component) may be responsible for the destruction of an unknown protein that mediates the degradation of microtubule associated proteins such as Cin8 and Ase1. We asked if the Cdc34mediated stabilization of microtubule associated proteins is proteasome-dependent. Two separate cultures of cdc34-1 cdc6Δ MET-CDC6 sic1Δ erg6Δ (US7034) cells were first synchronized in mitosis by nocodazole treatment in +Met medium (to repress CDC6 expression). One culture was then released into 36˚C to inactivate Cdc34 and then shifted to 24˚C to restore Cdc34 function in the absence of the proteasome inhibitor MG132, while the other was treated in the same manner but in the presence of MG132. Samples were taken for immunofluorescence staining and spindle analysis. As predicted, spindle elongation was observed in all cells where proteasome is active (-MG132). However, upon inactivation of proteasome by MG132, spindles remained short even after the restoration of Cdc34 function by a shift to 24˚C (Figure 32). This clearly implies that Cdc34 mediated stabilization of microtubule associated proteins requires proteasome activity. (-MG132) Nomarski DAPI (+MG132 ) Anti-tubulin Nomarski DAPI Anti-tubulin 100% short spindles 100% short spindles 36°C at 180min 36°C at 180min 24°C at 150min 100% long spindles 24°C at 150min 100% short spindles Figure 32. Cdc34-mediated stabilization of microtubule associated proteins are proteasome dependent. We test if the Cdc34-mediated stabilization of microtubule associated proteins is proteasome-dependent. Two separate cultures of cells were first synchronized in mitosis by nocodazole treatment in +Met medium (to repress the proteasome inhibitor MG132, while the other was treated in the same manner but in the presence of MG132. Samples were taken for immunofluorescence staining and spindle analysis. 4.3 Discussion In Chapter we presented evidence showing that the precocious segregation of unreplicated chromosomes in Cdc6 deficient cells is most likely not due to premature entry into mitosis. Instead, the untimely chromosome segregation is due to premature elongation of the spindle fueled by the accumulation of elongation conducive microtubule associated proteins (MAPs) such as Cin8, Kip1 and Ase1. In Chapter 4, we investigate the role of Cdc6 in spindle extension and the mechanism underlying the accumulation of MAPs. It has long been postulated that Cdc6 is a component of the G1-M checkpoint responsible for the inhibition of premature entry into mitosis when cells fail to initiate DNA replication (Piatti et al. 1995). However, the exact nature of this checkpoint and the role of Cdc6 has remained a mystery. Cdc6 is an unstable protein with multiple roles and its over-expression during late S phase can prevent entry into M phase by activating Chk1 kinase which is responsible for the inactivation of Cdk1/cyclinB (Synnes et al. 2002). Moreover, Cdc6 is also required for spindle formation during maturation of mouse oocytes without which meiotic progression will be disastrous (Anger et al. 2005). This supports the involvement of Cdc6 in the regulation of chromosome segregation via spindle dynamics. However, certain dbf4 and cdc7 mutants (not all) that fail to initiate S phase when grown in nonpermissive conditions have been reported to undergo premature chromosome separation requiring a functional spindle (Toyn et al. 1995). Since there is some confusion concerning the phenotype of some of the mutant alleles of these genes, we have used cdc7Δ and cdc45Δ mutants to establish that both mutants undergo premature spindle elongation and precocious segregation of unreplicated chromosomes (Figure 15 and 16). This is particularly surprising because Cdc6 is functional in these mutants. Moreover, the absence of Cdc6 does not lead to spindle formation or elongation in cdc34-1 mutants, which normally arrests in G1 with multiple buds, unreplicated DNA and duplicated but unseparated SPBs under restrictive conditions (Figure 17). Based on our observations, we suggest that premature segregation of unreplicated chromosomes in these mutants is specifically not due to the deficiency of Cdc6, Cdc7 or Cdc45 function; instead, it is a common characteristic of cells that have traversed START but have failed to initiate S phase. Thus our proposal brings into question the generally accepted belief that Cdc6 is a checkpoint protein that prevents premature entry into mitosis. Having established that precocious chromosomes segregation in Cdc6 deficient cells is not due to premature entry into mitosis but because of deregulation of the spindle, we have turned our attention to the temperature sensitive cdc34-1 cells to investigate the mechanism behind premature spindle elongation in post-START arrested, G1 cells. We specifically chose cdc34 mutant cells for this study because, like cdc6, they traverse START and arrest prior to S phase; however their arrest phenotypes are dramatically different. While cdc34 cells fail to assemble a spindle and contain a single nucleus, cdc6 cells assemble a spindle, prematurely extend it and segregate the unreplicated chromosomes. Why the mutants that arrest in the same narrow time-window between G1 and S phase (post START and prior to S phase) exhibit such dramatically different behaviour? This is the specific puzzle we have attempted to address in this chapter. It is known that cdc34-1 cells arrest with duplicated SPBs (Crasta et al. 2006). However, they are unable to break the inter-SPB bridge and therefore fail to assemble a bipolar spindle. A previous study has uncovered the following regulatory scheme which leads to the severing of the interSPB bridge in late S phase during the normal cell cycle (Crasta et al. 2006). The severing of the bridge required the bundling activity of microtubule binding proteins (MAPs) such as Cin8, Kip1 and Ase1. However, cellular levels of these proteins are kept very low by Cdh1-mediated proteolysis. In late S phase, Cdk1 and Cdc5 polo kinase collaborate to inactivate Cdh1 via multiple phosphorylations, permitting the accumulation of the MAPs which mediate the breaking of the inter-SPB-bridge and assembly of a bipolar, short spindle. Thus active Cdh1 is the critical negative regulator of spindle assembly. This is clearly the case in the present context since cdc34-1 cells are unable to assemble a spindle at restrictive temperatures, deletion of CDH1 gene allows the cells to rapidly assemble a short bipolar spindle (Figure 18 and 19). The observation that CDH1 deletion also restores the cellular levels of MAPs in these cells suggests that deficiency of MAPs is the main reason for the inability of cdc34-1 cells to assemble a spindle (Figure 19). The results documented in this chapter also bring Sic1 into the regulatory scheme that we propose for spindle biogenesis (Figure 19). While SCF-mediated destruction of Sic1 has been considered a pivotal event for the initiation of S phase because it leads to the activation of Cdk1/Clb kinase required for the onset of DNA replication, it also results in the inactivation of Cdh1 and the accumulation of MAPs. Thus, Sic1 degradation function of Cdc34 and Cdh1 inactivation are intimately linked, since upon Sic1 destruction, the activated Cdk1/Clb3/Clb4/Clb5 can now inactivate Cdh1 via multiple phosphorylations (Figure 21), a prerequisite for Clb1 and Clb2 stability that ensures full inactivation of Cdh1 and subsequently, assembly of a short spindle. The fact that SIC1 depletion also induces short spindle assembly in cdc34-1 cells strongly supports the notion that Cdc34 is an upstream regulator of spindle dynamics by promoting Cdh1 inactivation. This probably explains why cells with mutations in CDC4, CDC34 and CDC53 (components of SCF complexes) are unable to assemble a bipolar spindle and arrest with side-by-side SPBs (Mathias et al., 1996). Interestingly, CDH1 depletion can only induce short spindle assembly in cdc34-1 cells but fails to induce spindle elongation in cdc34-1 cells, unlike the cdc6 mutant cells (Figure 19). This is intriguing because it has been reported previously that over-expression of Cdc5, deficiency of Cdh1 and ectopic expression of Cin8 all lead to dramatic spindle elongation in G2-arrested DNA damaged cells once the cohesins are forcibly removed (Zhang et al. 2009). At one level, this should not be surprising because the cellular context in G1 is very different from that in G2; hence, cells’ response to perturbations is expected to be different. However, given almost identical cellular context, the difference between the state of the spindle in cdc6 cells (long spindle) and cdc34-1 cdh1Δ cells (short spindle) is puzzling. The clue came to light from the experiment in which spindles were dramatically extended in cdc34-1 cdc6Δ cdh1Δ cells when Cdc34 function was restored by a return to the permissive temperature (Figure 27 and 28). This clearly suggests that Cdc34 function is necessary to convert a short spindle to a long spindle and argues that cdc6 mutant cells require Cdc34 function to extend their spindles. These results can be interpreted simply as the epistatic relationship between cdc6 and cdc34 mutants. However, ‘epistasis’ is an operational term that falls short of making a statement about any direct functional connections between genes. Given the narrow cellular context in which cdc6 and cdc34 mutants arrest, we prefer the suggestion that Cdc34 function is required in cdc6 cells to transform a short to a long spindle. It is noteworthy that Cdc34-mediated spindle elongation is dependent on proteasome function since spindle elongation can be abrogated by the addition of proteasome inhibitor MG132 (Figure 32). Since the MAPs are stabilized in cdc34-1 cdc6Δ cdh1Δ cells when they are returned to permissive temperature to restore Cdc34 function, it implies that while sufficient cellular levels of MAPs (due to CDH1 deletion) are essential for the assembly of a short spindle, a Cdc34-mediated enhanced stabilization of these proteins is required for spindle elongation. However, neither ectopic expression of Ase1, Cin8 or Kip1 nor expression of non-degradable version of these proteins can induce spindle elongation fully (Figure 22, 23, 24 and 25). It is possible that these unstable proteins are also targeted for proteolysis via ubiquitinindependent manner because their degradation appears not to be entirely dependent on APC. It has been reported that AURKAIP1 targets Aurora-A for degradation in a proteasome-dependent but Ub (ubiquitin)-independent manner (Lim and Gopalan 2007). It is possible that a novel proteolytic pathway contributes to Ase1, Cin8 and Kip1 degradation in addition to APCCdh1-mediated destabilization. Alternatively, Cdc34 may mediate (via its role in SCF complex) destruction of a yet-to-be identified protein that inhibit spindle elongation despite sufficient accumulation of MAPs. In conclusion, our findings implicate Cdc34 (SCF) as a new regulator of spindle dynamics. The dramatic deregulation of spindle dynamics experienced by cells that are committed to the cell cycle but fail to undergo DNA replication is a result of the interplay of four sequential cellular events: activation of the E3 ubiquitin ligase SCF, destruction of Cdk inhibitor Sic1, inactivation of another ubiquitin ligase APCCdh1and stabilization of microtubule associated proteins. The role of Cdc34 in spindle dynamics is particularly critical during the period between START and S phase in that Cdc34-mediated stabilization of Ase1 and Cin8 (or destabilization of a novel spindleelongation inhibitor) would cause premature spindle elongation in any cell that traverses START but are unable to initiate S phase. In mammalian cells, Cdc34 has been reported to associate with β-tubulin on mitotic spindle caps at anaphase (Reymond et al. 2000). This implies a direct role for Cdc34 in the ubiquitination and degradation of the unknown protein responsible for proteolysis of microtubule associated proteins required for chromosome segregation. In this context, a core component of SCF E3 ubiquitin ligase, Skp1, localizes to mitotic spindle poles during mitosis (Freed et al., 1999; Gstaiger et al., 1999). These observations strongly support a role for Cdc34 in the regulation of spindle dynamics. [...]... for immunofluorescence and Western Blot analysis B strain was arrested in nocodazole containing medium supplemented with methionine to repress transcription Cells were then released into medium containing raffinose and galactose to facilitate over-expression of non-degradable Sic1 Samples were taken at 160 minutes for immunofluorescence analysis 4. 2 .4 cdc 34 and cdc 34 cdc6 mutant cells can assemble... cdc 34- 1 CDH1-HA 3 CIP - + + Cdh1-HA3 Figure 21 Sic1 degradation promotes Cdh1 inactivation and short spindle assembly To confirm the notion that Sic1 degradation promotes Cdk1-mediated inactivation of Cdh1, we monitored the phosphorylation status of Cdh1 in cdc 34- 1 and cells expressing CDH1-HA3 from collected for immunoprecipitation and Western Blot analysis 4. 2.6 Ectopic expression of microtubule associated... contrast, cells lacking Cdc6 are also unable to undergo S phase and arrest in G1 phase (post START) but with a prematurely extended spindle This raises the possibility of Cdc 34 s involvement in regulating spindle elongation since Cdc 34 is functional in cells lacking Cdc6 Previous experimental data had shown that ectopic expression of Sic1 and Cdh1 are sufficient to prevent premature spindle elongation... bipolar spindles in the absence of Cdh1 or Sic1 but fail to elongate them Once cells have traversed START, Cdc 34 function becomes critical for G1/S transition due to its involvement in Sic1 degradation Cells deficient in Cdc 34 function are unable to initiate S phase and arrest in G1 phase (post START) with a bud and duplicated SPBs but are unable to assemble a spindle in accordance with the regulatory... cells that remained at 36˚C (Cdc 34 inactivated) This coincided with assembly of short spindles However, Ase1 and Cin8 were stabilized upon restoration of Cdc 34 function by a shift to 24 C These cells exhibited long spindles Interestingly, Kip1 was not stabilized upon restoration of Cdc 34 function at 24 C It is surprising that Cin8, Kip1 and Ase1 are unstable despite the fact that Cdh1 is abrogated It is... possible that an alternative pathway (not involving Cdh1) is responsible for the degradation of these microtubule associated proteins Nevertheless, these observations suggest that Cdc 34 can promote premature spindle elongation in Cdc6 deficient cells by protecting Ase1 and Cin8 against ubiquitin-independent degradation To confirm the observation that microtubule associated proteins can be stabilized... elongation in cdc 34- 1 and cells We treated cdc 34- 1 and cells carrying non-degradable Nomarski DAPI Anti-tubulin 36˚C 240 min 85% no spindle 15% short spindles Nomarski DAPI Anti-tubulin 36˚C 240 min 80% short spindles 20% long spindles Figure 25 Cdh1 resistant microtubule associated proteins cannot induce complete spindle elongation in cdc 34- 1 and cells We treated cdc 34- 1 and cells carrying non-degradable... spindles (data not shown) These observations support the notion that Cdh1 inactivation and short spindle assembly is tightly connected with Sic1 degradation mediated by Cdc 34 In the following section we make use of these observations and use SIC1 or CDH1 deletion to allow cdc 34- 1 mutant cells to assemble a short spindle and explore the cellular requirements for spindle elongation - cdc 34- 1 CDH1-HA 3 3... notion, we also treated cdc 34- 1 cells carrying GAL- ASE1 (US7027), GAL-CIN8 (US7023) and GAL-KIP1 (US7025) as described above We found that overexpression of microtubule associated proteins can only induce approximately 30% of the cells to assemble short spindles but cannot elicit spindle elongation (Figure 22 and 23) These observations are consistent with our hypothesis that microtubule associated... cells is not due to the lack of Cdc6 function per se The results also imply that Cdc 34 (E2 enzyme), acting in synergy with SCF and Cdc4 (F-box protein) to degrade Sic1, not only regulates G1-S transition but also facilitates Cdh1 inactivation which leads to the stabilization of microtubule associated proteins and sets up the context for the assembly of a short spindle Nomarski DAPI Anti-tubulin 36˚C 180min . premature chromosome segregation (Zhang et al. 2009). It was shown that activation of the DNA damage checkpoint leads to inactivation of Cdc5 polo kinase via Rad53-mediated phosphorylation (Zhang. cells fail to initiate DNA replication and proceed to undergo what-appears-to-be premature mitosis, causing a reductional anaphase. We also noted that the premature nuclear division is accompanied. Cdk1-mediated inactivation of Cdh1, accumulation of Cin8 and Ase1 and hence premature spindle elongation. If SCF-mediated degradation of Sic1 is an essential step in determining the fate of spindle