Mitotic Exit in the Absence of Separase Activity
01 Mar 2009-Molecular Biology of the Cell (American Society for Cell Biology)-Vol. 20, Iss: 5, pp 1576-1591
TL;DR: The first quantitative measure for Cdc14 release based on colocalization with the Net1 nucleolar anchor is defined, indicating efficient CDC14 release upon MEN activation; release driven by Esp1 in the absence of microtubules was inefficient and incapable of driving ME.
Abstract: In budding yeast, three interdigitated pathways regulate mitotic exit (ME): mitotic cyclin–cyclin-dependent kinase (Cdk) inactivation; the Cdc14 early anaphase release (FEAR) network, including a n...
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TL;DR: This work has shown that the key mitotic exit phosphatase in budding yeast, Cdc14, is now well understood, and in animal cells, it is now emerging that mitoticexit relies on distinct regulatory networks, including the protein phosphatases PP1 and PP2A.
Abstract: The mitosis-to-interphase transition involves dramatic cellular reorganization from a state that supports chromosome segregation to a state that complies with all functions of an interphase cell. This process, termed mitotic exit, depends on the removal of mitotic phosphorylations from a broad range of substrates. Mitotic exit regulation involves inactivation of mitotic kinases and activation of counteracting protein phosphatases. The key mitotic exit phosphatase in budding yeast, Cdc14, is now well understood. By contrast, in animal cells, it is now emerging that mitotic exit relies on distinct regulatory networks, including the protein phosphatases PP1 and PP2A.
294 citations
TL;DR: An intrinsically oscillatory module controlling nucleolar release and resequestration of the Cdc14 phosphatase is demonstrated, which is essential for mitotic exit in budding yeast and suggests that the intrinsically autonomous CDC14 release cycles are locked at once-per-cell-cycle through entrainment by the Cdk oscillator in wild-type cells.
97 citations
Cites methods from "Mitotic Exit in the Absence of Sepa..."
...Cdc14 release was quantified as the coefficient of variation (CV, standard deviation divided by mean) of Cdc14-YFP pixel intensities (mother and bud treated separately), divided by the Net1-mCherry CV....
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...We determined the response of the Cdc14 release cycle to fixed cyclin-Cdk levels (Drapkin et al., 2009), using a quantitative, single cell measurement for Cdc14 localization based on variation of cellular Cdc14-YFP pixel intensities, standardized to variation of nucleolar Net1-mCherry (Lu and Cross, 2009) (Experimental Procedures; Figure 1A)....
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...…cycle to fixed cyclin-Cdk levels (Drapkin et al., 2009), using a quantitative, single cell measurement for Cdc14 localization based on variation of cellular Cdc14-YFP pixel intensities, standardized to variation of nucleolar Net1-mCherry (Lu and Cross, 2009) (Experimental Procedures; Figure 1A)....
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..., 2009), using a quantitative, single cell measurement for Cdc14 localization based on variation of cellular Cdc14-YFP pixel intensities, standardized to variation of nucleolar Net1-mCherry (Lu and Cross, 2009) (Experimental Procedures; Figure 1A)....
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TL;DR: A decrease in Cdc42 activation during mitotic exit is necessary to allow localization of key cytokinesis regulators and proper septum formation.
Abstract: The role of Cdc42 and its regulation during cytokinesis is not well understood. Using biochemical and imaging approaches in budding yeast, we demonstrate that Cdc42 activation peaks during the G1/S transition and during anaphase but drops during mitotic exit and cytokinesis. Cdc5/Polo kinase is an important upstream cell cycle regulator that suppresses Cdc42 activity. Failure to down-regulate Cdc42 during mitotic exit impairs the normal localization of key cytokinesis regulators-Iqg1 and Inn1-at the division site, and results in an abnormal septum. The effects of Cdc42 hyperactivation are largely mediated by the Cdc42 effector p21-activated kinase Ste20. Inhibition of Cdc42 and related Rho guanosine triphosphatases may be a general feature of cytokinesis in eukaryotes.
79 citations
TL;DR: The findings suggest the dephosphorylation of the formins may be important for their observed localization change during exit from mitosis and indicate that Cdc14 targets proteins involved in wide-ranging mitotic events.
79 citations
10 Dec 2015
TL;DR: This work crafted a new mathematical model of cell cycle progression in yeast that exploits a natural separation of time scales in the cell cycle control network to construct a system of differential-algebraic equations for protein synthesis and degradation, post-translational modifications, and rapid formation and dissociation of multimeric complexes.
Abstract: In the cell division cycle of budding yeast, START refers to a set of tightly linked events that prepare a cell for budding and DNA replication, and FINISH denotes the interrelated events by which the cell exits from mitosis and divides into mother and daughter cells. On the basis of recent progress made by molecular biologists in characterizing the genes and proteins that control START and FINISH, we crafted a new mathematical model of cell cycle progression in yeast. Our model exploits a natural separation of time scales in the cell cycle control network to construct a system of differential-algebraic equations for protein synthesis and degradation, post-translational modifications, and rapid formation and dissociation of multimeric complexes. The model provides a unified account of the observed phenotypes of 257 mutant yeast strains (98% of the 263 strains in the data set used to constrain the model). We then use the model to predict the phenotypes of 30 novel combinations of mutant alleles. Our comprehensive model of the molecular events controlling cell cycle progression in budding yeast has both explanatory and predictive power. Future experimental tests of the model's predictions will be useful to refine the underlying molecular mechanism, to constrain the adjustable parameters of the model, and to provide new insights into how the cell division cycle is regulated in budding yeast.
69 citations
Additional excerpts
...In this case, anaphase proceeds normally, activating the MEN while the FEAR pathway remains blocked.(42) In agreement with Lu and Cross,(42) our simulations (Figure 3) show that MEN activity is absolutely needed for mitotic exit, and FEAR is dispensable....
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References
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TL;DR: It is shown that Cdc14p contains a leucine-rich nuclear export signal (NES) that interacts with Crm1p physically and is involved in coordinating mitotic exit and cytokinesis in budding yeast.
Abstract: The mitotic exit network (MEN) controls the exit from mitosis in budding yeast. The proline-directed phosphatase, Cdc14p, is a key component of MEN and promotes mitotic exit by activating the degradation of Clb2p and by reversing Cdk-mediated mitotic phosphorylation. Cdc14p is sequestered in the nucleolus during much of the cell cycle and is released in anaphase from the nucleolus to the nucleoplasm and cytoplasm to perform its functions. Release of Cdc14p from the nucleolus during anaphase is well understood. In contrast, less is known about the mechanism by which Cdc14p is released from the nucleus to the cytoplasm. Here we show that Cdc14p contains a leucine-rich nuclear export signal (NES) that interacts with Crm1p physically. Mutations in the NES of Cdc14p allow Clb2p degradation and mitotic exit, but cause abnormal morphology and cytokinesis defects at non-permissive temperatures. Cdc14p localizes to the bud neck, among other cytoplasmic structures, following its release from the nucleolus in late anaphase. This bud neck localization of Cdc14p is disrupted by mutations in its NES and by the leptomycin B-mediated inhibition of Crm1p. Our results suggest a requirement for Crm1p-dependent nuclear export of Cdc14p in coordinating mitotic exit and cytokinesis in budding yeast.
55 citations
"Mitotic Exit in the Absence of Sepa..." refers background in this paper
...Mutations of that sequence cause Cdc14 to fail to localize to the bud neck during mitotic exit and also cause defects in cytokinesis (Bembenek et al., 2005)....
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...Because Cdc14-YFP also localizes to the SPB and bud-neck (Yoshida et al., 2002; Bembenek et al., 2005), which could be misinterpreted as a nucleolar signal in r value calculation, we exclude the bud-neck region in segmentation, and exclude Cdc14-YFP signal at SPB by removing the YFP pixels whose…...
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TL;DR: Transport-defective mutations in two karyopherins can bypass cdc15Δ, suggesting that the function of the MEN is to promote mitotic exit by regulating nuclear transport.
Abstract: The Mitotic Exit Network (MEN) proteins – including the protein kinase Cdc15 and the protein phosphatase Cdc14 – are essential for exit from mitosis in Saccharomyces cerevisiae. To identify downstream targets of the MEN, we sought telophase arrest bypassed (tab) mutations that bypassed the essential requirement for CDC15. Previous studies identified net1
tab2-1 and CDC14
TAB6-1 as mutations in the RENT complex subunits Net1 and Cdc14, respectively, and revealed that the MEN acts by promoting release of Cdc14 from its nucleolar Net1 anchor during anaphase. However, the remaining tab mutants were not characterized. Fourteen out of fifteen tab mutants were mapped to three recessive (tab1-tab3) and three dominant (TAB5-TAB7) linkage groups. We show that net1
tab2-1 enables growth of tem1Δ, cdc15Δ, dbf2Δ dbf20Δ, and mob1Δ, but not cdc5Δ or cdc14Δ, arguing that whereas the essential task of the first four genes is to promote exit from mitosis, CDC5 possesses additional essential function(s). net1
tab2-1 but not CDC14
TAB6-1 resulted in a high rate of chromosome loss, indicating that Net1 promotes accurate chromosome segregation in addition to its multiple known roles. Finally, TAB1 was shown to be MTR10, a gene encoding nuclear transport receptor/adaptor. In some of the tab mutants including mtr10
tab1-1, defective nuclear export of the ribosomal protein Rpl11b was observed. Furthermore, the transport-defective -31 allele of the karyopherin SRP1, but not the transport competent -49 allele, exhibited a tab phenotype. Transport-defective mutations in two karyopherins can bypass cdc15Δ, suggesting that the function of the MEN is to promote mitotic exit by regulating nuclear transport.
45 citations
"Mitotic Exit in the Absence of Sepa..." refers background in this paper
...Mutations in the nuclear transport factors SRP1 and KAP104 alleviate the telophase arrest of MEN mutants (Shou and Deshaies, 2002); it is possible that our finding of MENdependent Cdc14 nuclear export is related to these observations, although the critical cargo responsible for this genetic effect…...
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TL;DR: It is shown that separation of sister chromatids is not sufficient for proper spindle integrity during elongation, and successful spindle elongation and stability require both sister Chromatid separation and anaphase-promoting complex activation.
Abstract: At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.
42 citations
"Mitotic Exit in the Absence of Sepa..." refers background in this paper
...Because premature cohesin cleavage can activate the Mad2dependent spindle checkpoint (Severin et al., 2001), we carried out these experiments in a mad2 background....
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TL;DR: Observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress, and it is proposed that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication.
Abstract: The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide depletion by preventing replication fork collapse and the segregation of unreplicated chromosomes Although the block to chromosome segregation has been thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 mutants undergo cycles of spindle extension and collapse after hydroxyurea treatment that are distinct from anaphase cells Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest Kinetochore–spindle attachments are required to prevent spindle extension during replication blocks, and chromosomes with two centromeres or an origin of replication juxtaposed to a centromere rescue the rad53 checkpoint defect These observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension
40 citations
"Mitotic Exit in the Absence of Sepa..." refers background in this paper
...Subsequently, we observe a gradual increase of large budded cells with 2C DNA content (Supplemental Figure 2) and unseparated chromosome dots (Bachant et al., 2005) (Figure 2C)....
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TL;DR: The results demonstrate that the subcellular localization of the mitotic cyclin Clb2 is one of the key determinants of its biological function, and a direct role of the cytoplasmic pool of Clb1,2-Cdk1 in terminating cytoskeleton and growth polarization, independently of G1 cyclin transcriptional regulation.
Abstract: Orderly progression through the eukaryotic cell cycle is a complex process involving both regulation of cyclin dependent kinase activity and control of specific substrate-Cdk interactions. In Saccharomyces cerevisiae, the mitotic cyclin Clb2 has a central role in regulating the onset of anaphase and in maintaining the cellular shape of the bud by inhibiting growth polarization induced in G1. However, how Clb2 and the partially redundant cyclin Clb1 confer specificity to Cdk1 in these processes still remains unclear. Here, we show that Clb2 mutants impaired in nuclear import or export are differentially affected for subsets of Clb2 functions while remaining fully functional for others. Our data support a direct role of the cytoplasmic pool of Clb1,2-Cdk1 in terminating cytoskeleton and growth polarization, independently of G1 cyclin transcriptional regulation. By contrast, the nuclear form of the cyclin is required for timely initiation of anaphase. Clb2 localization influences its stage-specific degradation as well. We report that Clb2 trapped in the cytoplasm is stabilized during anaphase but not at the time of mitotic exit. Altogether, our results demonstrate that the subcellular localization of the mitotic cyclin Clb2 is one of the key determinants of its biological function.
31 citations
"Mitotic Exit in the Absence of Sepa..." refers background in this paper
...High Clb-Cdk may block cytokinesis and rebudding directly (Padmashree and Surana, 2001; Eluere et al., 2007)....
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