Separase, polo kinase, the kinetochore protein Slk19, and Spo12 function in a network that controls Cdc14 localization during early anaphase

In budding yeast, the phosphatase Cdc14, a key regulator of exit from mitosis, is released from its inhibitor Cfi1/Net1 in the nucleolus during anaphase. A signaling cascade, known as the mitotic exit network (MEN), controls this release. We have identified a regulatory network, the FEAR (Cdc fourteen early anaphase release) network that promotes Cdc14 release from the nucleolus during early anaphase. The FEAR network is comprised of the polo kinase Cdc5, the separase Esp1, the kinetochore-associated protein Slk19, and Spo12. We also show that the FEAR network initiates Cdc14 release from Cfi1/Net1 during early anaphase, and MEN maintains Cdc14 in the released state during late anaphase. We propose that one function of Cdc14 released by the FEAR network is to stimulate MEN activity.     

A role for the FEAR pathway in nuclear positioning during anaphase

In budding yeast, cells lacking separase function exit mitosis with an undivided nucleus localized to the daughter cell. Here we show that the inability to separate sister chromatids per se is not sufficient to cause the daughter preference. Rather, separase affects nuclear positioning as part of the Cdc14 early anaphase release (FEAR) pathway. The role of the FEAR pathway in nuclear positioning is exerted during anaphase and is not shared by the mitotic exit network. We find that the nuclear segregation defect in FEAR mutants does not stem from nonfunctional spindle poles or the absence of cytoplasmic microtubules. Instead, the concomitant inactivation of sister chromatid separation and the FEAR pathway uncovered a mother-directed force in anaphase that was previously masked by the elongating spindle. We propose that at anaphase onset, the FEAR pathway activates cytoplasmic microtubule-associated forces that facilitate chromosome segregation to the mother cell.     

Nucleolar segregation lags behind the rest of the genome and requires Cdc14p activation by the FEAR network

In order to transmit a full genetic complement cells must ensure that all chromosomes are accurately split and distributed during anaphase. Chromosome XII in S. cerevisiae contains the site of nucleolar assembly, a 1-2Mb array of rDNA genes named RDN1. Cdc14p is a conserved phosphatase, essential for anaphase progression and mitotic exit, which is kept inactive at the nucleolus until mitosis. In early anaphase, the FEAR network (Cdc Fourteen Early Anaphase Release) promotes the transient and partial release of Cdc14p from the nucleolus. The putative role of Cdc14p released by the FEAR network is thought to be the stimulation of full Cdc14p release by activation of the GTPase-driven signaling cascade (the Mitotic Exit Network or MEN) that ensures mitotic exit. Here, we show that nucleolar segregation is spatially separated and temporally delayed from the rest of the genome. Nucleolar segregation occurs during mid-anaphase and coincides with the FEAR release of Cdc14p. Inactivation of FEAR delays nucleolar segregation until late anaphase, demonstrating that one function of the FEAR network is to promote segregation of repetitive nucleolar chromatin during mid-anaphase.     

Cdc14 Early Anaphase Release,FEAR, Is Limited to the Nucleus and Dispensable for Efficient Mitotic Exit

Cdc14 phosphatase is a key regulator of exit from mitosis, acting primarily through antagonism of cyclin-dependent kinase, and is also thought to be important for meiosis. Cdc14 is released from its sequestration site in the nucleolus in two stages, first by the non-essential Cdc Fourteen Early Anaphase Release (FEAR) pathway and later by the essential Mitotic Exit Network (MEN), which drives efficient export of Cdc14 to the cytoplasm. We find that Cdc14 is confined to the nucleus during early mitotic anaphase release, and during its meiosis I release. Proteins whose degradation is directed by Cdc14 as a requirement for mitotic exit (e.g. the B-type cyclin, Clb2), remain stable during mitotic FEAR, a result consistent with Cdc14 being restricted to the nucleus and not participating directly in mitotic exit. Cdc14 released by the FEAR pathway has been proposed to have a wide variety of activities, all of which are thought to promote passage through anaphase. Proposed functions of FEAR include stabilization of anaphase spindles, resolution of the rDNA to allow its segregation, and priming of the MEN so that mitotic exit can occur promptly and efficiently. We tested the model for FEAR functions using the FEAR-deficient mutation net1-6cdk. Our cytological observations indicate that, contrary to the current model, FEAR is fully dispensable for timely progression through a series of anaphase landmarks and mitotic exit, although it is required for timely rDNA segregation. The net1-6cdk mutation suppresses temperature-sensitive mutations in MEN genes, suggesting that rather than activating mitotic exit, FEAR either inhibits the MEN or has no direct effect upon it. One interpretation of this result is that FEAR delays MEN activation to ensure that rDNA segregation occurs before mitotic exit. Our findings clarify the distinction between FEAR and MEN-dependent Cdc14 activities and will help guide emerging quantitative models of this cell cycle transition.     

DNA damage checkpoints inhibit mitotic exit by two different mechanisms

Cyclin-dependent kinase (CDK) governs cell cycle progression, and its kinase activity fluctuates during the cell cycle. Mitotic exit pathways are responsible for the inactivation of CDK after chromosome segregation by promoting the release of a nucleolus-sequestered phosphatase, Cdc14, which antagonizes CDK. In the budding yeast Saccharomyces cerevisiae, mitotic exit is controlled by the FEAR (for "Cdc-fourteen early anaphase release") and mitotic exit network (MEN) pathways. In response to DNA damage, two branches of the DNA damage checkpoint, Chk1 and Rad53, are activated in budding yeast to prevent anaphase entry and mitotic exit, allowing cells more time to repair damaged DNA. Here we present evidence indicating that yeast cells negatively regulate mitotic exit through two distinct pathways in response to DNA damage. Rad53 prevents mitotic exit by inhibiting the MEN pathway, whereas the Chk1 pathway prevents FEAR pathway-dependent Cdc14 release in the presence of DNA damage. In contrast to previous data, the Rad53 pathway negatively regulates MEN independently of Cdc5, a Polo-like kinase essential for mitotic exit. Instead, a defective Rad53 pathway alleviates the inhibition of MEN by Bfa1.     

Cdc14 and condensin control the dissolution of cohesin-independent chromosome linkages at repeated DNA

Chromosome segregation is triggered by the cleavage of cohesins by separase. Here we show that in budding yeast separation of the ribosomal DNA (rDNA) and telomeres also requires Cdc14, a protein phosphatase known for its role in mitotic exit. Cdc14 shares this role with the FEAR network, which activates Cdc14 during early anaphase, but not the mitotic exit network, which promotes Cdc14 activity during late anaphase. We further show that CDC14 is necessary and sufficient to promote condensin enrichment at the rDNA locus and to trigger rDNA segregation in a condensin-dependent manner. We propose that Cdc14 released by the FEAR network mediates the partitioning of rDNA by facilitating the localization of condensin thereto. This dual role of the FEAR network in initiating mitotic exit and promoting chromosome segregation ensures that exit from mitosis is coupled to the completion of chromosome segregation.     

FEAR but not MEN Genes are Required for Exit from Meiosis I

Exit from mitosis is regulated by Cdc14, which plays an essential role intriggering cyclin-dependent kinase inactivation. Throughout most of the cell cycle,Cdc14 is sequestered in the nucleolus where it remains inactive. After thecompletion of anaphase, an essential signaling cascade, named the Mitotic ExitNetwork, or MEN, promotes Cdc14 release. Cdc14 is also released from thenucleolus in early anaphase by another, nonessential, pathway called FEAR(CdcFourteen Early Anaphase Release). Separase (Esp1), polo kinase (Cdc5), thekinetochore protein Slk19, and Spo12, whose molecular function remains unknown,have been identified as members of the FEAR pathway. In meiosis, mutations inCDC14 and its FEAR pathway regulators, CDC5, SLK19, and SPO12, all form ascithat contain only two diploid spores because of a defect in the ability to exit meiosisI. Thus although the FEAR pathway is dispensible for mitotic exit it, is essential formeiosis I exit. The way that the genes of the Mitotic Exit Network contribute tocoordinating meiotic progression is less clear. Here, we explore this issue. Ourresults demonstrate that the orderly transition from meiosis I to meiosis II isaccomplished by eliminating MEN function and using the FEAR pathway tomodulate cyclin dependent kinase activity, in part through the actions of SIC1.     

The replication fork block protein Fob1 functions as a negative regulator of the FEAR network

BACKGROUND: The protein phosphatase Cdc14 is a key regulator of exit from mitosis in budding yeast. Its activation during anaphase is characterized by dissociation from its inhibitor Cfi1/Net1 in the nucleolus and is controlled by two regulatory networks. The Cdc14 early anaphase release (FEAR) network promotes activation of the phosphatase during early anaphase, whereas the mitotic exit network (MEN) activates Cdc14 during late stages of anaphase. RESULTS: Here we investigate how the FEAR network component Spo12 regulates Cdc14 activation. We identify the replication fork block protein Fob1 as a Spo12-interacting factor. Inactivation of FOB1 leads to premature release of Cdc14 from the nucleolus in metaphase-arrested cells. Conversely, high levels of FOB1 delay the release of Cdc14 from the nucleolus. Fob1 associates with Cfi1/Net1, and consistent with this observation, we find that the bulk of Cdc14 localizes to the Fob1 binding region within the rDNA repeats. Finally, we show that Spo12 phosphorylation is cell cycle regulated and affects its binding to Fob1. CONCLUSIONS: Fob1 functions as a negative regulator of the FEAR network. We propose that Fob1 helps to prevent the dissociation of Cdc14 from Cfi1/Net1 prior to anaphase and that Spo12 activation during early anaphase promotes the release of Cdc14 from its inhibitor by antagonizing Fob1 function.     

Nucleolar Segregation Lags Behind the Rest of the Genome and Requires Cdc14p Activation by the FEAR Network

In order to transmit a full genetic complement cells must ensure that all chromosomes are accurately split and distributed during anaphase. Chromosome XII in S. cerevisiae contains the site of nucleolar assembly, a 1-2Mb array of rDNA genes named RDN1. Cdc14p is a conserved phosphatase, essential for anaphase progression and mitotic exit, which is kept inactive at the nucleolus until mitosis. In early anaphase, the FEAR network (Cdc Fourteen Early Anaphase Release) promotes the transient and partial release of Cdc14p from the nucleolus. The putative role of Cdc14p released by the FEAR network is thought to be the stimulation of full Cdc14p release by activation of the GTPase-driven signalling cascade (the Mitotic Exit Network or MEN) that ensures mitotic exit. Here, we show that nucleolar segregation is spatially separated and temporally delayed from the rest of the genome. Nucleolar segregation occurs during mid-anaphase and coincides with the FEAR release of Cdc14p. Inactivation of FEAR delays nucleolar segregation until late anaphase, demonstrating that one function of the FEAR network is to promote segregation of repetitive nucleolar chromatin during mid-anaphase.

Links to supplemental material:

http://www.landesbioscience.com/supplement/aragonCC3-4-sup.pdf

http://www.landesbioscience.com/supplement/aragonCC3-4-supmov.mov     

Cdc15 is required for spore morphogenesis independently of Cdc14 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae exit from mitosis requires the Cdc14 phosphatase to reverse CDK-mediated phosphorylation. Cdc14 is released from the nucleolus by the Cdc14 early anaphase release (FEAR) and mitotic exit network (MEN) pathways. In meiosis, the FEAR pathway is essential for exit from anaphase I. The MEN component Cdc15 is required for the formation of mature spores. To analyze the role of Cdc15 during sporulation, a conditional mutant in which CDC15 expression was controlled by the CLB2 promoter was used. Cdc15-depleted cells proceeded normally through the meiotic divisions but were unable to properly disassemble meiosis II spindles. The morphology of the prospore membrane was aberrant and failed to capture the nuclear lobes. Cdc15 was not required for Cdc14 release from the nucleoli, but it was essential to maintain Cdc14 released and for its nucleo-cytoplasmic transport. However, cells carrying a CDC14 allele with defects in nuclear export (Cdc14-DeltaNES) were able to disassemble the spindle and to complete spore formation, suggesting that the Cdc14 nuclear export defect was not the cause of the phenotypes observed in cdc15 mutants.     

The Molecular Function of the Yeast Polo-like Kinase Cdc5 in Cdc14 Release during Early Anaphase

In the budding yeast Saccharomyces cerevisiae, Cdc14 is sequestered within the nucleolus before anaphase entry through its association with Net1/Cfi1, a nucleolar protein. Protein phosphatase PP2A^Cdc55 dephosphorylates Net1 and keeps it as a hypophosphorylated form before anaphase. Activation of the Cdc fourteen early anaphase release (FEAR) pathway after anaphase entry induces a brief Cdc14 release from the nucleolus. Some of the components in the FEAR pathway, including Esp1, Slk19, and Spo12, inactivate PP2A^Cdc55, allowing the phosphorylation of Net1 by mitotic cyclin-dependent kinase (Cdk) (Clb2-Cdk1). However, the function of another FEAR component, the Polo-like kinase Cdc5, remains elusive. Here, we show evidence indicating that Cdc5 promotes Cdc14 release primarily by stimulating the degradation of Swe1, the inhibitory kinase for mitotic Cdk. First, we found that deletion of SWE1 partially suppresses the FEAR defects in cdc5 mutants. In contrast, high levels of Swe1 impair FEAR activation. We also demonstrated that the accumulation of Swe1 in cdc5 mutants is responsible for the decreased Net1 phosphorylation. Therefore, we conclude that the down-regulation of Swe1 protein levels by Cdc5 promotes FEAR activation by relieving the inhibition on Clb2-Cdk1, the kinase for Net1 protein.     

Putting the brake on FEAR: Tof2 promotes the biphasic release of Cdc14 phosphatase during mitotic exit

The completion of chromosome segregation during anaphase requires the hypercondensation of the ~1-Mb rDNA array, a reaction dependent on condensin and Cdc14 phosphatase. Using systematic genetic screens, we identified 29 novel genetic interactions with budding yeast condensin. Of these, FOB1, CSM1, LRS4, and TOF2 were required for the mitotic condensation of the tandem rDNA array localized on chromosome XII. Interestingly, whereas Fob1 and the monopolin subunits Csm1 and Lrs4 function in rDNA condensation throughout M phase, Tof2 was only required during anaphase. We show that Tof2, which shares homology with the Cdc14 inhibitor Net1/Cfi1, interacts with Cdc14 phosphatase and its deletion suppresses defects in mitotic exit network (MEN) components. Consistent with these genetic data, the onset of Cdc14 release from the nucleolus was similar in TOF2 and tof2Δ cells; however, the magnitude of the release was dramatically increased in the absence of Tof2, even when the MEN pathway was compromised. These data support a model whereby Tof2 coordinates the biphasic release of Cdc14 during anaphase by restraining a population of Cdc14 in the nucleolus after activation of the Cdc14 early anaphase release (FEAR) network, for subsequent release by the MEN.     

Mitotic Exit in the Absence of Separase Activity

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 nonproteolytic function of separase (Esp1); and the mitotic exit network (MEN) driven by interaction between the spindle pole body and the bud cortex. Here, we evaluate the contributions of these pathways to ME kinetics. Reducing Cdk activity is critical for ME, and the MEN contributes strongly to ME efficiency. Esp1 contributes to ME kinetics mainly through cohesin cleavage: the Esp1 requirement can be largely bypassed if cells are provided Esp1-independent means of separating sister chromatids. In the absence of Esp1 activity, we observed only a minor ME delay consistent with a FEAR defect. Esp1 overexpression drives ME in Cdc20-depleted cells arrested in metaphase. We have found that this activity of overexpressed Esp1 depended on spindle integrity and the MEN. We defined the first quantitative measure for Cdc14 release based on colocalization with the Net1 nucleolar anchor. This measure indicates efficient Cdc14 release upon MEN activation; release driven by Esp1 in the absence of microtubules was inefficient and incapable of driving ME. We also found a novel role for the MEN: activating Cdc14 nuclear export, even in the absence of Net1.     

Cdc14 Inhibition by the Spindle Assembly Checkpoint Prevents Unscheduled Centrosome Separation in Budding Yeast

The spindle assembly checkpoint (SAC) is an evolutionarily conserved surveillance mechanism that delays anaphase onset and mitotic exit in response to the lack of kinetochore attachment. The target of the SAC is the E3 ubiquitin ligase anaphase-promoting complex (APC) bound to its Cdc20 activator. The Cdc20/APC complex is in turn required for sister chromatid separation and mitotic exit through ubiquitin-mediated proteolysis of securin, thus relieving inhibition of separase that unties sister chromatids. Separase is also involved in the Cdc-fourteen early anaphase release (FEAR) pathway of nucleolar release and activation of the Cdc14 phosphatase, which regulates several microtubule-linked processes at the metaphase/anaphase transition and also drives mitotic exit. Here, we report that the SAC prevents separation of microtubule-organizing centers (spindle pole bodies [SPBs]) when spindle assembly is defective. Under these circumstances, failure of SAC activation causes unscheduled SPB separation, which requires Cdc20/APC, the FEAR pathway, cytoplasmic dynein, and the actin cytoskeleton. We propose that, besides inhibiting sister chromatid separation, the SAC preserves the accurate transmission of chromosomes also by preventing SPBs to migrate far apart until the conditions to assemble a bipolar spindle are satisfied.     

The role of the polo kinase Cdc5 in controlling Cdc14 localization

In budding yeast, the protein phosphatase Cdc14 controls exit from mitosis. Its activity is regulated by a competitive inhibitor Cfi1/Net1, which binds to and sequesters Cdc14 in the nucleolus. During anaphase, Cdc14 is released from its inhibitor by the action of two regulatory networks. The Cdc Fourteen Early Anaphase Release (FEAR) network initiates Cdc14 release from Cfi1/Net1 during early anaphase, and the Mitotic Exit Network (MEN) promotes Cdc14 release during late anaphase. Here, we investigate the relationship among FEAR network components and propose an order in which they function to promote Cdc14 release from the nucleolus. Furthermore, we examine the role of the protein kinase Cdc5, which is a component of both the FEAR network and the MEN, in Cdc14 release from the nucleolus. We find that overexpression of CDC5 led to Cdc14 release from the nucleolus in S phase-arrested cells, which correlated with the appearance of phosphorylated forms of Cdc14 and Cfi1/Net1. Cdc5 promotes Cdc14 phosphorylation and, by stimulating the MEN, Cfi1/Net1 phosphorylation. Furthermore, we suggest that Cdc14 release from the nucleolus only occurs when Cdc14 and Cfi1/Net1 are both phosphorylated.     

The budding yeast GSK-3 homologue Mck1 is an essential component of the spindle position checkpoint

The spindle position checkpoint (SPOC) is a mitotic surveillance mechanism in Saccharomyces cerevisiae that prevents cells from completing mitosis in response to spindle misalignment, thereby contributing to genomic integrity. The kinase Kin4, one of the most downstream SPOC components, is essential to stop the mitotic exit network (MEN), a signalling pathway that promotes the exit from mitosis and cell division. Previous work, however, suggested that a Kin4-independent pathway contributes to SPOC, yet the underlying mechanisms remain elusive. Here, we established the glycogen-synthase-kinase-3 (GSK-3) homologue Mck1, as a novel component that works independently of Kin4 to engage SPOC. Our data indicate that both Kin4 and Mck1 work in parallel to counteract MEN activation by the Cdc14 early anaphase release (FEAR) network. We show that Mck1''s function in SPOC is mediated by the pre-replication complex protein and mitotic cyclin-dependent kinase (M-Cdk) inhibitor, Cdc6, which is degraded in a Mck1-dependent manner prior to mitosis. Moderate overproduction of Cdc6 phenocopies MCK1 deletion and causes SPOC deficiency via its N-terminal, M-Cdk inhibitory domain. Our data uncover an unprecedented role of GSK-3 kinases in coordinating spindle orientation with cell cycle progression.     

Complexity of Mitotic Exit

Completion of mitosis in budding yeast is triggered by activation of the protein phosphatase Cdc14, which is the ultimate effector of a signalling cascade, known as the mitotic exit network. Cdc14 activation leads to eradication of mitotic kinase activity, which is pivotal for mitotic exit and cytokinesis in all eukaryotes. The complexity in mitotic exit regulation is underscored by the recent discovery of a novel network, the so-called FEAR pathway that regulates early Cdc14 activation. Surprisingly, this has revealed an unexpected role for Spo12, a protein involved in meiosis, in Cdc14 activation. In this review, we will discuss these findings together with recent advances in deciphering the function of the FEAR circuit, which has unravelled an exciting new side of Cdc14.

Key Words:

Mitotic exit, Cdc14 activation, FEAR pathway, Spo12, Budding yeast     

A mathematical model of mitotic exit in budding yeast: the role of Polo kinase

Cell cycle progression in eukaryotes is regulated by periodic activation and inactivation of a family of cyclin-dependent kinases (Cdk's). Entry into mitosis requires phosphorylation of many proteins targeted by mitotic Cdk, and exit from mitosis requires proteolysis of mitotic cyclins and dephosphorylation of their targeted proteins. Mitotic exit in budding yeast is known to involve the interplay of mitotic kinases (Cdk and Polo kinases) and phosphatases (Cdc55/PP2A and Cdc14), as well as the action of the anaphase promoting complex (APC) in degrading specific proteins in anaphase and telophase. To understand the intricacies of this mechanism, we propose a mathematical model for the molecular events during mitotic exit in budding yeast. The model captures the dynamics of this network in wild-type yeast cells and 110 mutant strains. The model clarifies the roles of Polo-like kinase (Cdc5) in the Cdc14 early anaphase release pathway and in the G-protein regulated mitotic exit network.     

Dependence of Chs2 ER export on dephosphorylation by cytoplasmic Cdc14 ensures that septum formation follows mitosis

Cytokinesis, which leads to the physical separation of two dividing cells, is normally restrained until after nuclear division. In Saccharomyces cerevisiae, chitin synthase 2 (Chs2), which lays down the primary septum at the mother-daughter neck, also ensures proper actomyosin ring constriction during cytokinesis. During the metaphase-to-anaphase transition, phosphorylation of Chs2 by the mitotic cyclin-dependent kinase (Cdk1) retains Chs2 at the endoplasmic reticulum (ER), thereby preventing its translocation to the neck. Upon Cdk1 inactivation at the end of mitosis, Chs2 is exported from the ER and targeted to the neck. The mechanism for triggering Chs2 ER export thus far is unknown. We show here that Chs2 ER export requires the direct reversal of the inhibitory Cdk1 phosphorylation sites by Cdc14 phosphatase, the ultimate effector of the mitotic exit network (MEN). We further show that only Cdc14 liberated by the MEN after completion of chromosome segregation, and not Cdc14 released in early anaphase by the Cdc fourteen early anaphase release pathway, triggers Chs2 ER exit. Presumably, the reduced Cdk1 activity in late mitosis further favors dephosphorylation of Chs2 by Cdc14. Thus, by requiring declining Cdk1 activity and Cdc14 nuclear release for Chs2 ER export, cells ensure that septum formation is contingent upon chromosome separation and exit from mitosis.     

Nonredundant Requirement for Multiple Histone Modifications for the Early Anaphase Release of the Mitotic Exit Regulator Cdc14 from Nucleolar Chromatin

In Saccharomyces cerevisiae, the conserved phosphatase Cdc14 is required for the exit from mitosis. It is anchored on nucleolar chromatin by the Cfi1/Net1 protein until early anaphase, at which time it is released into the nucleoplasm. Two poorly understood, redundant pathways promote Cdc14 release, the FEAR (Cdc fourteen early release) network and the MEN (mitotic exit network). Through the analysis of genetic interactions, we report here a novel requirement for the ubiquitination of histone H2B by the Bre1 ubiquitin ligase in the cell cycle–dependent release of Cdc14 from nucleolar chromatin when the MEN is inactivated. This function for H2B ubiquitination is mediated by its activation of histone H3 methylation on lysines 4 and 79 (meH3K4 and meH3K79) but, surprisingly, is not dependent on the histone deacetylase (HDAC) Sir2, which associates with Cdc14 on nucleolar chromatin as part of the RENT complex. We also observed a defect in Cdc14 release in cells lacking H3 lysine 36 methylation (meH3K36) and in cells lacking an HDAC recruited by this modification. These histone modifications represent previously unappreciated factors required for the accessibility to and/or action on nucleolar chromatin of FEAR network components. The nonredundant role for these modifications in this context contrasts with the notion of a highly combinatorial code by which histone marks act to control biological processes.