A study on premature segregation of unreplicated chromosomes 5

Chapter A search for Cdc34-mediated up- or downregulated proteins that mediate spindle elongation 5.1 Background As described above, we have observed that Cdc34 can induce premature spindle elongation by regulating spindle dynamics. Although both cdc34-1 and cdc6Δ MET-CDC6 cells arrest in G1 phase and cannot undergo S phase, they both demonstrate very different spindle phenotypes. cdc34-1 mutant (with functional Cdc6) cannot assemble a spindle and duplicated spindle poles remain connected by a bridge; however cdc6Δ MET-CDC6 cells (with functional Cdc34) not only assemble a spindle but also untimely extend it to cause segregation of unreplicated chromosomes. This raised the possibility that Cdc34 plays a central role in inducing spindle elongation. In Chapter 3, we have presented evidence suggesting that Cdc34 stimulates spindle elongation by stabilizing microtubule associated proteins. To elucidate the nature of Cdc34’s role in spindle extension, we employed a strategy which involved labeling of cells with stable amino acid isotopes (SILAC) (Ong et al. 2002) which would help to identify proteins that are up- or down-regulated through quantitative mass spectrometry (de Godoy et al. 2008). SILAC is a metabolic labeling method that utilizes a cell’s machinery to incorporate exogenous heavy isotopes of amino acid residues into expressed proteins. This labeling strategy facilitates subsequent mass spectrometric analysis to differentiate between peptide signals from labeled or unlabeled source. 5.2 Results 5.2.1 Cdc34 promotes up-regulation of the polo-like kinase Cdc5 during premature spindle elongation For SILAC mass spectrometry, cdc34-1 cdc6Δ MET-CDC6 lys1Δ strain (US7048) was used. Two overnight cultures were grown; one in medium supplemented with normal unlabelled lysine (refer to Section 2.2.15 for more details) and the other in medium supplemented with labeled H-lysine. The next day, both cultures were diluted and synchronized in mitosis using nocodazole and methionine was added to repress and deplete CDC6. Subsequently, the unlabelled lysine cells were released into medium supplemented with methionine at 36ºC. Three hours later, these cells were collected for protein extraction. In contrast, the labeled Hlysine cells were released into medium containing methionine at 24˚C for three hours. Cells were again collected for the preparation of total protein extracts. Subsequently, both ‘total protein extracts’ from 36˚C and 24˚C samples were subjected to mass spectrometric analysis as described in Section Error! Reference source not found As shown in Figure 33A, the spectra produced by mass spectrometry analysis identified 2781 proteins. Of these, 187 were up-regulated, 2390 were unchanged and 204 were down-regulated. Cdc5, which was up-regulated two fold upon restoration of Cdc34 function, specifically drew our attention because when overexpressed, Cdc5 has been shown to promote, spindle formation in spindle assembly-defective cdc28Y19E cells. Additionally, Cdc5 is also reported to play a synergistic role with Cdk1 to phosphorylate Cdh1 at multiple sites leading to its inactivation (Crasta et al. 2008). This allows accumulation of microtubule associated proteins and, consequently spindle formation. Up-regulated Proteins: Protein IDs YJR090C   YMR001C   Gene Names Molecular Weight (kDa) Ratio H/L Normalized Ratio H/L Significance SCF  E3  ubiquitin   ligase  complex  F-­‐ box  protein  Grr1   GRR1 132.73   18.714   1.04E-­‐19   Cell  cycle   serine/threonine-­‐ protein  kinase   Cdc5   CDC5   81.03   2.6913   0.0020991   Gene Names Molecular Weight (kDa) Ratio H/L Normalized Ratio H/L Significance KIP3   91.089   0.51741   0.044283   Protein Names Down-regulated Proteins: Protein IDs YGL216W   Protein Names Kinesin-­‐like   protein  Kip3   Table 6: Cdc34 mediated Up- or Down-regulated proteins with functions relevant to SCF and spindle dynamics. To confirm the above observations, we attempted to monitor the stability of Cdc5 in the absence and presence of Cdc34 function. We treated two separate cultures of cdc34-1 cdc6Δ MET-CDC6 cdh1Δ cells (US6938) with nocodazole to arrest them in mitosis in medium supplemented with methionine at 24˚C to repress CDC6 transcription and to ensure complete Cdc6 depletion. Subsequently, both cultures were released into medium containing methionine at 36˚C to inactivate Cdc34 and arrest at Cdc34-execution point until 210 minutes. At this juncture, one culture was kept in 34˚C while the other was shifted to 24˚C to allow restoration of Cdc34 function. Samples were taken every 30 minutes for Western Blot analysis. Similar to microtubule associated proteins such as Ase1 and Cin8, Cdc5 is unstable in the absence of Cdc34, even when CDH1 is deleted. This suggests that, Cdc5 may be targeted for proteolysis via the ubiquitin-independent pathway. Upon restoration of Cdc34, Cdc5 is stabilized (Figure 33B). A Peptide sequence: FFTTQICGAIK Two-fold increase in Cdc5 ∆m/z=4.01 150 120 90 60 30 210 150 +met 36˚C 180 90 120 30 +met 36˚C 60 +met NOC -met cyc B Cdc5 150 120 90 60 30 210 180 +met 24˚C 150 90 120 60 +met 36˚C 30 -met cyc +met NOC G6PD Cdc5 G6PD Figure 33. Cdc34 promotes up-regulation of the polo-like kinase Cdc5 during premature spindle elongation A. For SILAC mass spectrometry, strain was used. Two overnight cultures were grown; one in medium supplemented with normal unlabelled lysine (refer to Section 2.2.15 for more details) and the other in medium supplemented with labeled H-lysine. The next day, both cultures were diluted and synchronized in mitosis using nocodazole and methionine was added to repress and deplete . Subsequently, the unlabelled lysine cells were released into medium supplemented with methionine at 36ºC. Three hours later, these cells were collected for protein extraction. In contrast, the labelled H-lysine cells collected for the preparation of total protein extracts. Subsequently, both ‘total protein extracts’ Section 2.2.15. As shown is the spectrum of Cdc5 produced by mass spectrometry analysis. B. To confirm the above observations, we attempted to monitor the stability of Cdc5 in the absence and presence of Cdc34 function. We treated two separate cultures of cells with nocodazole to arrest them in mitosis in medium supplemented transcription and to ensure complete Cdc6 depletion. Samples were taken every 30 minutes for Western Blot analysis. 5.2.2 Ectopic expression of Cdc5 can induce spindle formation and elongation Our initial investigations in cdc34-1 and cdc34-1 cdc6Δ MET-CDC6 cdh1Δ cells have provided evidence that ectopic expression of Ase1, Cin8 and Kip1 can induce spindle formation and spindle elongation in a large majority of cells, although not in all cells. Since Cdc5 stabilization is mediated by Cdc34, similar to that observed for microtubule associated proteins, and its stabilization coincides with spindle elongation, we tested if Cdc5 overexpression can affect the fate of spindles. To test this possibility, cdc34-1 and cdc34-1cdh1Δ cells each carrying GAL-CDC5 and GAL-CDC5 (N209A-kinase dead) (US7044, US7046, US7045 and US7047) were first synchronized in G2-M by nocodazole treatment in raffinose medium supplemented with methionine. These cells were then subjected to second synchronization in the subsequent G1 phase by α-factor treatment in medium containing raffinose and methionine. The cells were pre-induced with galactose for hour to express Cdc5 or kinase-dead Cdc5 and then released into medium containing raffinose and galactose at 34˚C. As shown in Figure 34, over-expression of Cdc5 allowed 30% of cdc34-1 cells to form short spindles at non-permissive temperature. Moreover, over-expression of Cdc5 can induce spindle elongation in 60% of cdc34-1 cdh1Δ cells. It should be noted that these cells can assemble short spindles without Cdc5 over-expression due to lack of Cdh1 function. As a control, we have overexpressed kinase-dead Cdc5 and have observed that kinase-dead Cdc5 fails to promote any spindle formation or spindle elongation. The data suggests that Cdc5 can contribute to the regulation of spindle dynamics and can promote spindle elongation. cdc34-1 GALCDC5 DAPI Nomarski Anti-tubulin DAPI Nomarski Anti-tubulin 70% no spindles 40% short spindles 30% short spindles 60% long spindles cdc34-1 GALCDC5(N209A) Nomarski DAPI 100% no spindles Anti-tubulin Nomarski DAPI Anti-tubulin 100% short spindles Figure 34. Ectopic expression of Cdc5 can induce spindle formation and elongation. cdc34-1 GAL-CDC5 GAL-CDC5 - - 5.2.3 Cdc5 is unstable in the absence of Cdc34 function Our previous investigations showed that Ase1 and Cin8 are highly unstable in cdc34-1 cells compared to that in cdc6Δ cells. This explains the inability of cdc34-1 cells to assemble a spindle. Both Ase1 and Cin8 become more stable when CDH1 is deleted in cdc34-1 cells and they contribute to short spindle assembly. Since Cdc5 overexpression cannot induce spindle formation and spindle elongation in all the cdc34-1 and cdc34-1 cdh1Δ cells, it is possible that Cdc5 is in low abundance in Cdc34 deficient cells due to enhanced proteolysis. To test this, cdc34-1, cdc6Δ MET-CDC6, cdc34-1 cdh1Δ and cdc6Δ MET-CDC6 cdh1Δ cells carrying myc6-tagged CDC5 under the control of the GAL1 promoter (US7044, US7046, US7049 and US7050) were grown overnight in medium lacking methionine. The overnight cultures were washed and inoculated into raffinose and galactose medium supplemented with methionine at 36˚C. Cells were kept in this medium for hours to pre-induce Cdc5 and then released into their respective non-permissive conditions in glucose medium with 0.1mg/ml cycloheximide (to inhibit de novo synthesis of Cdc5) and the fate of the protein pulse was monitored. As shown in Figure 35, protein pulse was not detected in cdc34-1 cells indicating that Cdc5 is extremely unstable. However, Cdc5 was remarkably stable in cdc6Δ cells, correlating with premature spindle elongation. Moreover, Cdc5 was more stable in cdc34-1 cdh1Δ cells as compared to cdc34-1 cells (Figure 35, lower panels). This suggests that Cdc5 may be degraded via an ubiquitin-independent pathway in the absence of Cdc34. Overall, Cdc5 protein pulse appeared most stable in cdc6Δ cells due to the presence of Cdc34. This strongly supports the notion that Cdc34 is involved in regulating the stability of spindle elongation effectors such as Ase1, Cin8 and Cdc5. Cdc34 + Cdc34 - 80 100 60 40 +MET Glu cycloheximide 36ºC 20 100 -MET cyc 80 60 40 +MET Glu cycloheximide 36ºC 20 -MET cyc cdc34-1 GALCDC5-myc6 (Cdc34 -) 120 100 80 60 40 +MET Glu cycloheximide 36ºC 20 -MET cyc (Cdc34 +) 120 100 80 60 40 +MET Glu cycloheximide 36ºC 20 -MET cyc Figure 35. Cdc5 is unstable in the absence of Cdc34 function. cdc34-1 myc6 CDC5 cdc34-1 GAL1 - 5.3 Discussion Using SILAC mass spectrometry, we have identified a few up- or down-regulated proteins with functions closely related to spindle elongation upon restoration of Cdc34 function. Amongst those proteins, Cdc5 seems to be a plausible candidate as it has been reported to play a synergistic role with Cdk1 to inactivate Cdh1, thus allowing the accumulation and upregulation of microtubule associated proteins such as Cin8 and Kip1. These proteins presumably generate a ‘sliding force’ to induce spindle elongation regardless of which cell cycle stage the cells are in. Cdc34 appears to be also responsible for stabilization and upregulation of Cdc5. Once stabilized in G1 phase, Cdc5 can contribute to spindle elongation in cells that fail to undergo S phase. This is because prolonged delay in G1 phase will allow Cdc5 to accumulate; consequently Cdh1 is inactivated, thus resulting in the accumulation of microtubule associated proteins and triggering premature spindle elongation. Our mass spectrometric analysis has also identified Kip3 as one the proteins that is, unlike Cdc5, downregulated upon the restoration of Cdc34 function. Kip3, a kinesin-8 family member, undergoes plus-end directed motility and can perform plus-end microtubule depolymerization (Du et al.), (Walczak 2006), (Gupta et al. 2006). It is possible that Kip3 is subjected to negative regulation by Cdc5. 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[...]...cleavage of cohesins by separase (Alexandru et al 2001) Cdc5 has also been implicated in regulation of APC/C and in the degradation of cyclin B during anaphase (Charles et al 1998; Shirayama et al 1998) As a component of the FEAR pathway, Cdc5 mediates the release of Cdc14 phosphatase in early anaphase through phosphorylation (Visintin et al 2003; Rahal et al 2008) Furthermore, Cdc5 phosphorylates Bfa1,... Ase1) and eventually premature extension of the spindle Since at this stage, chromosomes are unreplicated, kinetochore duplication, bi-orientation and cohesins are unable to resist the dramatic spindle extension, as they collectively do during normal S phase, G2 and metaphase, hence spindles form and prematurely elongate (i.e premature chromosome segregation) Thus initiation of DNA replication that allows... point’ also set in motion the spindle assembly and elongation machinery It is through the initiation of DNA replication (via kinetochore duplication, cohesin-loading and biorientation) that cells restrain spindle elongation and avoid premature chromosome segregation In other words, cells that have traversed START and completed Cdc34 execution step must initiate DNA replication or face extreme chromosomal... that untimely segregation of unreplicated chromosomes is perhaps a common property of G1 cells that traverse START but are unable to initiate DNA replication However, cdc34 mutant appears to be an exception to this tentative rule Cells deficient in Cdc34 function traverse START and are unable to initiate DNA replication but yet do not undergo precocious chromosome segregation It can be argued that... Yeast Saccharomyces: Life Cycle and Inheritance Strathern JN, Jones EW and Broach JR Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 59 -96 Calzada A, Sacristan M, Sanchez E and Bueno A (2001) Cdc6 cooperates with Sic1 and Hct1 to inactivate mitotic cyclin-dependent kinases Nature 412(6844): 355 - 358 Calzada A, Sanchez M, Sanchez E and Bueno A (2000) The stability of the Cdc6 protein is regulated... Regulation of the transcription factor Gcn4 by Pho 85 cyclin PCL5 Mol Cell Biol 22( 15) : 53 955 404 Shirayama M, Toth A, Galova M and Nasmyth K (1999) APC(Cdc20) promotes exit from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5 Nature 402(6 758 ): 203-207 Shirayama M, Zachariae W, Ciosk R and Nasmyth K (1998) The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and... Surana U (2006) Cdk1 regulates centrosome separation by restraining proteolysis of microtubule-associated proteins EMBO J 25( 11): 255 1- 256 3 Crasta K, Lim HH, Giddings TH, Jr., Winey M and Surana U (2008) Inactivation of Cdh1 by synergistic action of Cdk1 and polo kinase is necessary for proper assembly of the mitotic spindle Nat Cell Biol 10(6): 6 65- 6 75 Crasta K, Lim HH, Zhang T, Nirantar S and Surana... Podtelejnikov AV, Mann M and Peters JM (2000) Mitotic regulation of the APC activator proteins CDC20 and CDH1 Mol Biol Cell 11 (5) : 155 5- 156 9 Krishnan V, Nirantar S, Crasta K, Cheng AY and Surana U (2004) DNA replication checkpoint prevents precocious chromosome segregation by regulating spindle behavior Mol Cell 16 (5) : 687-700 Krishnan V and Surana U (20 05) Taming the spindle for containing the chromosomes. .. century, abnormal chromosome number – also known as aneuploidy – has been recognized as a near ubiquitous feature of human cancers Study of colorectal cancers has shown that approximately 85% display aneuploidy errors, and contain cells with an average of 60 to 90 chromosomes (Pellman 2001) Moreover, high clinical grades tumours are closely associated with greater degrees of aneuploidy In normal cells,... (cdh1-m1 1A) causes cells to arrest in G2 with unseparated SPBs (Crasta et al 2006) Failure to break the intra-SPB bridge can be attributed to very low levels of Cin8, Kip1and Ase1 Further analysis revealed that Cdc28/Cdk1-Clb kinase and Cdc5 acted in synergy to fully inactivate Cdh1 via phosphorylation at multiple sites during normal cell cycle to allow accumulation of MAPs and the assembly of a short . separase (Alexandru et al. 2001). Cdc5 has also been implicated in regulation of APC/C and in the degradation of cyclin B during anaphase (Charles et al. 1998; Shirayama et al. 1998). As a component. overexpressed kinase-dead Cdc5 and have observed that kinase-dead Cdc5 fails to promote any spindle formation or spindle elongation. The data suggests that Cdc5 can contribute to the regulation of spindle. cells are in. Cdc34 appears to be also responsible for stabilization and up- regulation of Cdc5. Once stabilized in G1 phase, Cdc5 can contribute to spindle elongation in cells that fail to