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•

APC/C-Cdh1-mediated degradation of the Polo kinase Cdc5 promotes the return of Cdc14 into the nucleolus (Genes and Development (2008) 22 (79-90))

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Clara Visintin, Brett N. Tomson, Rami Rahal, Jennifer Paulson, Michael B. Cohen, Jack Taunton, Angelika Amon, Rosella Visintin - Show less +4 more

01 Jun 2008-Genes & Development

TL;DR: In this paper, the authors show that activation of a ubiquitin ligase known as the Anaphase-Promoting Complex or Cyclosome (APC/C) bound to the specificity factor Cdh1 triggers the degradation of the Polo kinase Cdc5, a key factor in releasing Cdc14 from its inhibitor in the nucleolus.

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Abstract: In the budding yeast Saccharomyces cerevisiae, the protein phosphatase Cdc14 triggers exit from mitosis by promoting the inactivation of cyclin-dependent kinases (CDKs). Cdc14's activity is controlled by Cfi1/Net1, which holds and inhibits the phosphatase in the nucleolus from G1 until metaphase. During anaphase, two regulatory networks, the Cdc14 Early Anaphase Release (FEAR) network and the Mitotic Exit Network (MEN), promote the dissociation of Cdc14 from its inhibitor, allowing the phosphatase to reach its targets throughout the cell. The molecular circuits that trigger the return of Cdc14 into the nucleolus after the completion of exit from mitosis are not known. Here we show that activation of a ubiquitin ligase known as the Anaphase-Promoting Complex or Cyclosome (APC/C) bound to the specificity factor Cdh1 triggers the degradation of the Polo kinase Cdc5, a key factor in releasing Cdc14 from its inhibitor in the nucleolus.

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6 citations

Journal Article•DOI•

Nucleolar asymmetry and the importance of septin integrity upon cell cycle arrest.

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Urvashi Rai¹, Fadi J. Najm², Alan M. Tartakoff¹•Institutions (2)

Case Western Reserve University¹, Harvard University²

24 Mar 2017-PLOS ONE

TL;DR: The present investigation was undertaken to learn how other features of nuclear organization are affected upon depletion of the APC activator, Cdc20, and found that the nucleolus remains in the mother domain, marking the polarity of the nucleus.

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Abstract: Cell cycle arrest can be imposed by inactivating the anaphase promoting complex (APC). In S. cerevisiae this arrest has been reported to stabilize a metaphase-like intermediate in which the nuclear envelope spans the bud neck, while chromatin repeatedly translocates between the mother and bud domains. The present investigation was undertaken to learn how other features of nuclear organization are affected upon depletion of the APC activator, Cdc20. We observe that the spindle pole bodies and the spindle repeatedly translocate across the narrow orifice at the level of the neck. Nevertheless, we find that the nucleolus (organized around rDNA repeats on the long right arm of chromosome XII) remains in the mother domain, marking the polarity of the nucleus. Accordingly, chromosome XII is polarized: TelXIIR remains in the mother domain and its centromere is predominantly located in the bud domain. In order to learn why the nucleolus remains in the mother domain, we studied the impact of inhibiting rRNA synthesis in arrested cells. We observed that this fragments the nucleolus and that these fragments entered the bud domain. Taken together with earlier observations, the restriction of the nucleolus to the mother domain therefore can be attributed to its massive structure. We also observed that inactivation of septins allowed arrested cells to complete the cell cycle, that the alternative APC activator, Cdh1, was required for completion of the cell cycle and that induction of Cdh1 itself caused arrested cells to progress to the end of the cell cycle.

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6 citations

Journal Article•DOI•

Protease-Dead Separase Is Dominant Negative in the C. elegans Embryo

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Diana M. Mitchell¹, Lindsey R. Uehlein-Klebanow¹, Joshua N. Bembenek¹•Institutions (1)

University of Tennessee¹

22 Sep 2014-PLOS ONE

TL;DR: It is shown that protease-dead separase causes embryo lethality, and that proteases- dead separase cannot rescue separase mutants, and suggested that prote enzyme-deadseparase interferes with endogenous separase function.

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Abstract: Separase is a protease that promotes chromosome segregation at anaphase by cleaving cohesin. Several non-proteolytic functions of separase have been identified in other organisms. We created a transgenic C. elegans line that expresses protease-dead separase in embryos to further characterize separase function. We find that expression of protease-dead separase is dominant-negative in C. elegans embryos, not previously reported in other systems. The C. elegans embryo is an ideal system to study developmental processes in a genetically tractable system. However, a major limitation is the lack of an inducible gene expression system for the embryo. We have developed two methods that allow for the propagation of lines carrying dominant-negative transgenes and have applied them to characterize expression of protease-dead separase in embryos. Using these methods, we show that protease-dead separase causes embryo lethality, and that protease-dead separase cannot rescue separase mutants. These data suggest that protease-dead separase interferes with endogenous separase function, possibly by binding substrates and protecting them from cleavage.

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5 citations

Cites background from "Mitotic Exit in the Absence of Sepa..."

...Additional non-proteolytic functions of separase have been identified, including anaphase exit [11] and Cdc14 early anaphase release (FEAR) pathway activation [12,13], and polar body extrusion in mouse oocytes [6]....
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Journal Article•DOI•

Efficient and rapid exact gene replacement without selection.

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Frederick R. Cross¹, Kresti Pecani¹•Institutions (1)

Rockefeller University¹

01 Feb 2011-Yeast

TL;DR: Eliminating the need for selection (over days) of rare recombinants removes concerns about second‐site suppressor mutations and also allows direct phenotypic analysis, even of lethal gene replacements, without the need of a method to make the lethality conditional or to employ regulated promoters of unknown strength compared to the endogenous promoter.

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Abstract: We describe a highly efficient method for exact gene replacement in budding yeast. Induction of rapid and efficient recombination in an entire cell population results in at least 50% of the recombinants undergoing a switch of the endogenous copy to a specific mutated allele, with no remaining markers or remnant of foreign DNA, without selection. To accomplish this, a partial copy of the replacement allele, followed by an HO cut site, is installed adjacent to the wild-type locus, in a GAL-HO MATa-inc background. HO induction results in near-quantitative site cleavage and recombination/gene conversion, resulting in either regeneration of wild-type or switch of the endogenous allele to the mutant, with accompanying deletion of intervening marker sequences, yielding an exact replacement. Eliminating the need for selection (over days) of rare recombinants removes concerns about second-site suppressor mutations and also allows direct phenotypic analysis, even of lethal gene replacements, without the need of a method to make the lethality conditional or to employ regulated promoters of unknown strength compared to the endogenous promoter. To test this method, we tried two known lethal gene replacements, substituting the non-essential CDH1 gene with a dominantly lethal version mutated for its Cdk phosphorylation sites and substituting the essential CDC28 gene with two recessively lethal versions, one containing an early stop codon and another inactivating Cdc28 kinase activity. We also tested a gene replacement of unknown phenotypic consequences: replacing the non-essential CLB3 B-type cyclin with a version lacking its destruction box. Copyright © 2010 John Wiley & Sons, Ltd.

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5 citations

Cites background from "Mitotic Exit in the Absence of Sepa..."

...However, finding such conditional constructs required considerable prior knowledge of the function of the stabilized lethal protein and, further, the conditional strains were not wild-type under permissive conditions (e.g. elongated G1 for GAL-SIC1, and poor viability for GAL-ESP1 cells unless a very attenuated GAL1 promoter was used)....
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...In another example, lethal removal of the destruction box of the PDS1 securin could be accomplished by modest conditional overexpression of the target of securin inhibition, the separase ESP1 (Lu and Cross, 2009)....
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...In our previous work, we found that GAL-SIC1 expression could make CLB2-db replacements viable, and similarly for GAL-ESP1 and PDS1-db, and GAL-ACM1 for CDH1–11m (Lu and Cross, 2009; Robbins and Cross, 2010; Wäsch and Cross, Copyright 2010 John Wiley & Sons, Ltd. Yeast 2011; 28: 167–179....
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Mitotic Exit: Thresholds and Targets

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Andrea Geoghegan Procko¹•Institutions (1)

Rockefeller University¹

01 Jan 2011

4 citations

Cites methods from "Mitotic Exit in the Absence of Sepa..."

...(A) Previous experiments (Lu and Cross, 2009) show that when cells lacking CDH1 are arrested in metaphase (by depletion of Cdc20) and released by the expression of separase (ESP1) to anaphase, Cdc14 is released for a sustained period before cell cycle progression....
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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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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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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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