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Journal ArticleDOI

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.

AbstractIn 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...

Topics: Mitotic exit (67%), Separase (61%), Polo-like kinase (58%), Anaphase (57%), Cdc14 (57%)

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Citations
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Journal ArticleDOI
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.

275 citations


Journal ArticleDOI
16 Apr 2010-Cell
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.
Abstract: One oscillation of Cyclin-dependent kinase (Cdk) activity, largely driven by periodic synthesis and destruction of cyclins, is tightly coupled to a single complete eukaryotic cell division cycle. Tight linkage of different steps in diverse cell-cycle processes to Cdk activity has been proposed to explain this coupling. Here, we demonstrate an intrinsically oscillatory module controlling nucleolar release and resequestration of the Cdc14 phosphatase, which is essential for mitotic exit in budding yeast. We find that this Cdc14 release oscillator functions at constant and physiological cyclin-Cdk levels, and is therefore independent of the Cdk oscillator. However, the frequency of the release oscillator is regulated by cyclin-Cdk activity. This observation together with its mechanism 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. This concept may have broad implications for the structure and evolution of eukaryotic cell-cycle control.

91 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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Journal ArticleDOI
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.
Abstract: Cdc14 phosphatase regulates multiple events during anaphase and is essential for mitotic exit in budding yeast. Cdc14 is regulated in both a spatial and temporal manner. It is sequestered in the nucleolus for most of the cell cycle by the nucleolar protein Net1 and is released into the nucleus and cytoplasm during anaphase. To identify novel binding partners of Cdc14, we used affinity purification of Cdc14 and mass spectrometric analysis of interacting proteins from strains in which Cdc14 localization or catalytic activity was altered. To alter Cdc14 localization, we used a strain deleted for NET1, which causes full release of Cdc14 from the nucleolus. To alter Cdc14 activity, we generated mutations in the active site of Cdc14 (C283S or D253A), which allow binding of substrates, but not dephosphorylation, by Cdc14. Using this strategy, we identified new interactors of Cdc14, including multiple proteins involved in mitotic events. A subset of these proteins displayed increased affinity for catalytically inactive mutants of Cdc14 compared with the wild-type version, suggesting they are likely substrates of Cdc14. We have also shown that several of the novel Cdc14-interacting proteins, including Kar9 (a protein that orients the mitotic spindle) and Bni1 and Bnr1 (formins that nucleate actin cables and may be important for actomyosin ring contraction) are specifically dephosphorylated by Cdc14 in vitro and in vivo. Our 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.

75 citations


Journal ArticleDOI
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.

68 citations



References
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Journal ArticleDOI
TL;DR: A comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle is created, and it is found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins.
Abstract: We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures sync...

5,072 citations


"Mitotic Exit in the Absence of Sepa..." refers background in this paper

  • ...We confirmed that Pds1 was not degraded despite expression of GAL-SIC1-4A (in fact, its level increased [Supplemental Figure 1C], perhaps due to increased transcription after mitosis [Spellman et al., 1998])....

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  • ...developed by Sullivan and Uhlmann (2003). Cells were depleted of Cdc20 and arrested in metaphase, by using a methionine-suppressible MET3-CDC20 construct (Sullivan and Uhlmann, 2003)....

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Journal ArticleDOI
03 Oct 1997-Cell
TL;DR: Three chromosmal proteins that prevent premature separation of sister chromatids in yeast are described, two of which are members of the SMC family, which are putative ATPases with coiled-coil domains.
Abstract: Cohesion between sister chromatids opposes the splitting force exerted by microtubules, and loss of this cohesion is responsible for the subsequent separation of sister chromatids during anaphase. We describe three chromosmal proteins that prevent premature separation of sister chromatids in yeast. Two, Smc1p and Smc3p, are members of the SMC family, which are putative ATPases with coiled-coil domains. A third protein, which we call Scc1p, binds to chromosomes during S phase, dissociates from them at the metaphase-to-anaphase transition, and is degraded by the anaphase promoting complex. Association of Scc1p with chromatin depends on Smc1p. Proteins homologous to Scc1p exist in a variety of eukaryotic organisms including humans. A common cohesion apparatus might be used by all eukaryotic cells during both mitosis and meiosis.

1,474 citations


"Mitotic Exit in the Absence of Sepa..." refers background in this paper

  • ...The temperature-sensitive scc1-73 cohesin allele (Michaelis et al., 1997) allows sister chromatid separation without Esp1 activity at restrictive temperature (Uhlmann et al....

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  • ...The temperature-sensitive scc1-73 cohesin allele (Michaelis et al., 1997) allows sister chromatid separation without Esp1 activity at restrictive temperature (Uhlmann et al., 1999)....

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Journal ArticleDOI

977 citations


"Mitotic Exit in the Absence of Sepa..." refers background in this paper

  • ...The potential lethality of this allele was overcome by mildly overexpressing Esp1 under a truncated GAL1 promoter (Mumberg et al., 1994), GALS-ESP1....

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Journal ArticleDOI
01 Jul 1999-Nature
TL;DR: It is shown that Esp1 causes the dissociation of Scc1 from chromosomes by stimulating its cleavage by proteolysis, and a mutant SCC1 is described that is resistant to Esp1-dependent cleavage and which blocks both sister-chromatid separation and the dissociations from chromosomes.
Abstract: Cohesion between sister chromatids is established during DNA replication and depends on a multiprotein complex called cohesin Attachment of sister kinetochores to the mitotic spindle during mitosis generates forces that would immediately split sister chromatids were it not opposed by cohesion Cohesion is essential for the alignment of chromosomes in metaphase but must be abolished for sister separation to start during anaphase In the budding yeast Saccharomyces cerevisiae, loss of sister-chromatid cohesion depends on a separating protein (separin) called Esp1 and is accompanied by dissociation from the chromosomes of the cohesion subunit Scc1 Here we show that Esp1 causes the dissociation of Scc1 from chromosomes by stimulating its cleavage by proteolysis A mutant Scc1 is described that is resistant to Esp1-dependent cleavage and which blocks both sister-chromatid separation and the dissociation of Scc1 from chromosomes The evolutionary conservation of separins indicates that the proteolytic cleavage of cohesion proteins might be a general mechanism for triggering anaphase

971 citations


"Mitotic Exit in the Absence of Sepa..." refers background in this paper

  • ...The temperature-sensitive scc1-73 cohesin allele (Michaelis et al., 1997) allows sister chromatid separation without Esp1 activity at restrictive temperature (Uhlmann et al., 1999)....

    [...]

  • ...At 37°C, 35% initially unbudded cells elongated the spindle in spite of galactose addition, suggesting inefficient expression of GAL1-SCC1-RRDD at 37°C....

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  • ...A MYO1-GFP TUB1-GFP strain lacking GAL1-SCC1-RRDD (BD78-2C) was treated in parallel as a control, pooling bud-to-bud data for mothers and daughters....

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  • ...In these experiments, blocking sister chromatid separation does not block Esp1 activation, because endogenous Scc1 is cleaved on schedule even in the presence of ectopic Scc1RRDD (Uhlmann et al., 1999)....

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  • ...This question has been examined previously with the use of the noncleavable version of Scc1 expressed from the GAL1 promoter (GAL1-SCC1-RRDD) (Uhlmann et al., 1999)....

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Journal ArticleDOI
21 Oct 1994-Cell
TL;DR: It is shown that DNA replication also requires activation of Cdc28 by B-type (Clb) cyclins, and proteolysis of a cyclin-specific inhibitor of CDC28 is an essential aspect of the G1 to S phase transition.
Abstract: When yeast cells reach a critical size, they initiate bud formation, spindle pole body duplication, and DNA replication almost simultaneously. All three events depend on activation of Cdc28 protein kinase by the G1 cyclins Cln1, -2, and -3. We show that DNA replication also requires activation of Cdc28 by B-type (Clb) cyclins. A sextuple clb1-6 mutant arrests as multibudded G1 cells that resemble cells lacking the Cdc34 ubiquitin-conjugating enzyme. cdc34 mutants cannot enter S phase because they fail to destroy p40SIC1, which is a potent inhibitor of Clb but not Cln forms of the Cdc28 kinase. In wild-type cells, p40SIC1 protein appears at the end of mitosis and disappears shortly before S phase. Proteolysis of a cyclin-specific inhibitor of Cdc28 is therefore an essential aspect of the G1 to S phase transition.

942 citations


"Mitotic Exit in the Absence of Sepa..." refers background in this paper

  • ...DNA replication in the next cell cycle did not occur, presumably because of stable Sic1 accumulation ( Schwob et al., 1994; Verma et al., 1997)....

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  • ...DNA replication in the next cell cycle did not occur, presumably because of stable Sic1 accumulation (Schwob et al., 1994; Verma et al., 1997)....

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