Mitotic Exit in the Absence of Separase Activity

doi:10.1091/MBC.E08-10-1042

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Mitotic Exit in the Absence of Separase Activity

Journal Article•DOI•

Mitotic Exit in the Absence of Separase Activity

Ying Lu¹, Frederick R. Cross¹•Institutions (1)

Rockefeller University¹

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.

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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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Journal Article•DOI•

Phosphatases: providing safe passage through mitotic exit

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Claudia Wurzenberger¹, Daniel W. Gerlich¹•Institutions (1)

ETH Zurich¹

01 Aug 2011-Nature Reviews Molecular Cell Biology

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.

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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.

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294 citations

Journal Article•DOI•

Periodic cyclin-Cdk activity entrains an autonomous Cdc14 release oscillator.

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Ying Lu¹, Frederick R. Cross¹•Institutions (1)

Rockefeller University¹

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.

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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.

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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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Journal Article•DOI•

Inhibition of Cdc42 during mitotic exit is required for cytokinesis

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Benjamin D. Atkins¹, Satoshi Yoshida², Satoshi Yoshida¹, Koji Saito³, Chi-Fang Wu³, Daniel J. Lew³, David Pellman¹, David Pellman⁴ - Show less +4 more•Institutions (4)

Harvard University¹, Brandeis University², Duke University³, Howard Hughes Medical Institute⁴

22 Jul 2013-Journal of Cell Biology

TL;DR: A decrease in Cdc42 activation during mitotic exit is necessary to allow localization of key cytokinesis regulators and proper septum formation.

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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.

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79 citations

Journal Article•DOI•

Global analysis of Cdc14 phosphatase reveals diverse roles in mitotic processes.

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Joanna Bloom¹, Joanna Bloom², Ileana M. Cristea², Andrea L. Procko², Veronica Lubkov², Brian T. Chait², Michael Snyder¹, Frederick R. Cross² - Show less +4 more•Institutions (2)

Yale University¹, Rockefeller University²

18 Feb 2011-Journal of Biological Chemistry

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.

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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.

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79 citations

Journal Article•DOI•

From START to FINISH: computational analysis of cell cycle control in budding yeast.

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Pavel Kraikivski¹, Katherine C. Chen¹, Teeraphan Laomettachit², T. M. Murali¹, John J. Tyson¹ - Show less +1 more•Institutions (2)

Virginia Tech¹, King Mongkut's University of Technology Thonburi²

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.

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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.

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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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Journal Article•DOI•

Comprehensive Identification of Cell Cycle–regulated Genes of the Yeast Saccharomyces cerevisiae by Microarray Hybridization

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Paul T. Spellman¹, Gavin Sherlock², Gavin Sherlock¹, Michael Q. Zhang², Vishwanath R. Iyer¹, Kirk R. Anders¹, Michael B. Eisen¹, Patrick O. Brown³, Patrick O. Brown¹, David Botstein¹, Bruce Futcher² - Show less +7 more•Institutions (3)

Stanford University¹, Cold Spring Harbor Laboratory², Howard Hughes Medical Institute³

01 Dec 1998-Molecular Biology of the Cell

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.

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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...

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5,176 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 Article•DOI•

Cohesins: chromosomal proteins that prevent premature separation of sister chromatids

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Christine Michaelis¹, Rafal Ciosk¹, Kim Nasmyth¹•Institutions (1)

Research Institute of Molecular Pathology¹

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.

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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.

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1,545 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 Article•DOI•

Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1

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Frank Uhlmann¹, Friedrich Lottspeich², Kim Nasmyth¹•Institutions (2)

Research Institute of Molecular Pathology¹, Max Planck Society²

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.

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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

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1,021 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)....
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...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 Article•DOI•

Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression

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Dominik Mumberg¹, Rolf Müller, Martin Funk•Institutions (1)

University of Marburg¹

01 Jan 1994-Nucleic Acids Research

1,011 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 Article•DOI•

The B-type cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevisiae

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Etienne Schwob¹, Thomas Böhm¹, Michael D. Mendenhall², Kim Nasmyth¹•Institutions (2)

Research Institute of Molecular Pathology¹, University of Kentucky²

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.

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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.

...read moreread less

951 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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