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1.  Budding Yeast Greatwall and Endosulfines Control Activity and Spatial Regulation of PP2ACdc55 for Timely Mitotic Progression 
PLoS Genetics  2013;9(7):e1003575.
Entry into mitosis is triggered by cyclinB/Cdk1, whose activity is abruptly raised by a positive feedback loop. The Greatwall kinase phosphorylates proteins of the endosulfine family and allows them to bind and inhibit the main Cdk1-counteracting PP2A-B55 phosphatase, thereby promoting mitotic entry. In contrast to most eukaryotic systems, Cdc14 is the main Cdk1-antagonizing phosphatase in budding yeast, while the PP2ACdc55 phosphatase promotes, instead of preventing, mitotic entry by participating to the positive feedback loop of Cdk1 activation. Here we show that budding yeast endosulfines (Igo1 and Igo2) bind to PP2ACdc55 in a cell cycle-regulated manner upon Greatwall (Rim15)-dependent phosphorylation. Phosphorylated Igo1 inhibits PP2ACdc55 activity in vitro and induces mitotic entry in Xenopus egg extracts, indicating that it bears a conserved PP2A-binding and -inhibitory activity. Surprisingly, deletion of IGO1 and IGO2 in yeast cells leads to a decrease in PP2A phosphatase activity, suggesting that endosulfines act also as positive regulators of PP2A in yeast. Consistently, RIM15 and IGO1/2 promote, like PP2ACdc55, timely entry into mitosis under temperature-stress, owing to the accumulation of Tyr-phosphorylated Cdk1. In addition, they contribute to the nuclear export of PP2ACdc55, which has recently been proposed to promote mitotic entry. Altogether, our data indicate that Igo proteins participate in the positive feedback loop for Cdk1 activation. We conclude that Greatwall, endosulfines, and PP2A are part of a regulatory module that has been conserved during evolution irrespective of PP2A function in the control of mitosis. However, this conserved module is adapted to account for differences in the regulation of mitotic entry in different organisms.
Author Summary
In all eukaryotic cells chromosome partition during mitosis requires a number of processes, including the formation of the mitotic spindle, i.e. the machinery that drives chromosome segregation to the daughter cells. Mitotic entry requires a delicate balance between protein phosphorylation, driven by cyclin-dependent kinases (CDKs), and protein dephosphorylation, carried out by specific phosphatases that counteract CDK activity. A critical threshold in CDK activity is indeed required for mitotic entry. In the past few years the Greatwall kinase has also been implicated in mitotic entry through phosphorylation of proteins of the endosulfine family, which in turn inhibit the activity of the PP2A phosphatase that would otherwise dephosphorylate CDK targets. Whether Greatwall and endosulfines have a mitotic function in budding yeast, where PP2A promotes, rather than inhibits, mitotic entry has not been established. Here we show that the Greatwall-endosulfine-PP2A regulatory module is conserved also in budding yeast and that endosulfines from different species are interchangeable for their mitotic function. However, in budding yeast cells endosulfines contribute to full activation and proper localization of PP2A, suggesting that they act as both inhibitors and activators of PP2A. Our data emphasize how the same regulatory module is adapted to meet specific mitotic features in different organisms.
PMCID: PMC3701715  PMID: 23861665
2.  Budding Yeast Dma Proteins Control Septin Dynamics and the Spindle Position Checkpoint by Promoting the Recruitment of the Elm1 Kinase to the Bud Neck 
PLoS Genetics  2012;8(4):e1002670.
The first step towards cytokinesis in budding yeast is the assembly of a septin ring at the future site of bud emergence. Integrity of this ring is crucial for cytokinesis, proper spindle positioning, and the spindle position checkpoint (SPOC). This checkpoint delays mitotic exit and cytokinesis as long as the anaphase spindle does not properly align with the division axis. SPOC signalling requires the Kin4 protein kinase and the Kin4-regulating Elm1 kinase, which also controls septin dynamics. Here, we show that the two redundant ubiquitin-ligases Dma1 and Dma2 control septin dynamics and the SPOC by promoting the efficient recruitment of Elm1 to the bud neck. Indeed, dma1 dma2 mutant cells show reduced levels of Elm1 at the bud neck and Elm1-dependent activation of Kin4. Artificial recruitment of Elm1 to the bud neck of the same cells is sufficient to re-establish a normal septin ring, proper spindle positioning, and a proficient SPOC response in dma1 dma2 cells. Altogether, our data indicate that septin dynamics and SPOC function are intimately linked and support the idea that integrity of the bud neck is crucial for SPOC signalling.
Author Summary
During asymmetric cell division, spindle positioning is critical for ensuring the unequal inheritance of polarity factors. In budding yeast, the constriction between mother cell and bud, the bud neck, determines the cleavage plane. Cytoskeletal proteins called septins form a ring at the bud neck that serves as scaffold for the recruitment of proteins required for cytokinesis. The septin ring also interacts with cytoplasmic microtubules to orient the spindle along the mother–bud axis and to ensure that each daughter cell inherits one full set of chromosomes. A surveillance device called Spindle Position Checkpoint (SPOC) oversees this process and delays mitotic exit and cytokinesis until the spindle is properly oriented along the division axis, thus ensuring genome stability. Feedback mechanisms responding to spindle mispositioning likely exist also in higher eukaryotes. In this manuscript, we show that two budding yeast ubiquitin ligases are involved in the regulation of septin dynamics and the SPOC by promoting the bud neck localization of the Elm1 protein kinase, which in turn participates in both processes. Altogether our data indicate that bud neck integrity and septins are important for SPOC signalling and that the two processes are controlled by a common regulatory module.
PMCID: PMC3343086  PMID: 22570619
3.  The RSC chromatin-remodeling complex influences mitotic exit and adaptation to the spindle assembly checkpoint by controlling the Cdc14 phosphatase 
The Journal of Cell Biology  2010;191(5):981-997.
Rsc2 promotes Cdc14 release from the nucleolus to free cells from mitotic arrest.
Upon prolonged activation of the spindle assembly checkpoint, cells escape from mitosis through a mechanism called adaptation or mitotic slippage, which is thought to underlie the resistance of cancer cells to antimitotic drugs. We show that, in budding yeast, this mechanism depends on known essential and nonessential regulators of mitotic exit, such as the Cdc14 early anaphase release (FEAR) pathway for the release of the Cdc14 phosphatase from the nucleolus in early anaphase. Moreover, the RSC (remodel the structure of chromatin) chromatin-remodeling complex bound to its accessory subunit Rsc2 is involved in this process as a novel component of the FEAR pathway. We show that Rsc2 interacts physically with the polo kinase Cdc5 and is required for timely phosphorylation of the Cdc14 inhibitor Net1, which is important to free Cdc14 in the active form. Our data suggest that fine-tuning regulators of mitotic exit have important functions during mitotic progression in cells treated with microtubule poisons and might be promising targets for cancer treatment.
PMCID: PMC2995168  PMID: 21098112
4.  Cdc14 Inhibition by the Spindle Assembly Checkpoint Prevents Unscheduled Centrosome Separation in Budding Yeast 
Molecular Biology of the Cell  2009;20(10):2626-2637.
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.
PMCID: PMC2682603  PMID: 19339280
5.  Comment on “A Centrosome-Independent Role for γ-TuRC Proteins in the Spindle Assembly Checkpoint” 
Science (New York, N.Y.)  2007;316(5827):982.
Müller et al. (Reports, 27 October 2006, p. 654) showed that inhibition of the γ-tubulin ring complex (γ-TuRC) activates the spindle assembly checkpoint (SAC), which led them to suggest that γ-TuRC proteins play molecular roles in SAC activation. Because γ-TuRC inhibition leads to pleiotropic spindle defects, which are well known to activate kinetochore-derived checkpoint signaling, we believe that this conclusion is premature.
PMCID: PMC2590763  PMID: 17510347
6.  The budding yeast PP2ACdc55 protein phosphatase prevents the onset of anaphase in response to morphogenetic defects 
The Journal of Cell Biology  2007;177(4):599-611.
Faithful chromosome transmission requires establishment of sister chromatid cohesion during S phase, followed by its removal at anaphase onset. Sister chromatids are tethered together by cohesin, which is displaced from chromosomes through cleavage of its Mcd1 subunit by the separase protease. Separase is in turn inhibited, up to this moment, by securin. Budding yeast cells respond to morphogenetic defects by a transient arrest in G2 with high securin levels and unseparated chromatids. We show that neither securin elimination nor forced cohesin cleavage is sufficient for anaphase in these conditions, suggesting that other factors contribute to cohesion maintainance in G2. We find that the protein phosphatase PP2A bound to its regulatory subunit Cdc55 plays a key role in this process, uncovering a new function for PP2ACdc55 in controlling a noncanonical pathway of chromatid cohesion removal.
PMCID: PMC2064206  PMID: 17502422
7.  Accumulation of Mad2–Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae 
The Journal of Cell Biology  2006;174(1):39-51.
The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. In this study, we show that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a mad2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. Our biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution.
PMCID: PMC2064158  PMID: 16818718
8.  Disappearance of the budding yeast Bub2–Bfa1 complex from the mother-bound spindle pole contributes to mitotic exit 
The Journal of Cell Biology  2006;172(3):335-346.
Budding yeast spindle position checkpoint is engaged by misoriented spindles and prevents mitotic exit by inhibiting the G protein Tem1 through the GTPase-activating protein (GAP) Bub2/Bfa1. Bub2 and Bfa1 are found on both duplicated spindle pole bodies until anaphase onset, when they disappear from the mother-bound spindle pole under unperturbed conditions. In contrast, when spindles are misoriented they remain symmetrically localized at both SPBs. Thus, symmetric localization of Bub2/Bfa1 might lead to inhibition of Tem1, which is also present at SPBs. Consistent with this hypothesis, we show that a Bub2 version symmetrically localized on both SPBs throughout the cell cycle prevents mitotic exit in mutant backgrounds that partially impair it. This effect is Bfa1 dependent and can be suppressed by high Tem1 levels. Bub2 removal from the mother-bound SPB requires its GAP activity, which in contrast appears to be dispensable for Tem1 inhibition. Moreover, it correlates with the passage of one spindle pole through the bud neck because it needs septin ring formation and bud neck kinases.
PMCID: PMC2063644  PMID: 16449187
9.  The spindle position checkpoint in budding yeast: the motherly care of MEN 
Cell Division  2006;1:2.
Mitotic exit and cytokinesis must be tightly coupled to nuclear division both in time and space in order to preserve genome stability and to ensure that daughter cells inherit the right set of chromosomes after cell division. This is achieved in budding yeast through control over a signal transduction cascade, the mitotic exit network (MEN), which is required for mitotic CDK inactivation in telophase and for cytokinesis. Current models of MEN activation emphasize on the bud as the place where most control is exerted. This review focuses on recent data that instead point to the mother cell as being the residence of key regulators of late mitotic events.
PMCID: PMC1459270  PMID: 16759408
10.  Functional Characterization of Dma1 and Dma2, the Budding Yeast Homologues of Schizosaccharomyces pombe Dma1 and Human ChfrD⃞ 
Molecular Biology of the Cell  2004;15(8):3796-3810.
Proper transmission of genetic information requires correct assembly and positioning of the mitotic spindle, responsible for driving each set of sister chromatids to the two daughter cells, followed by cytokinesis. In case of altered spindle orientation, the spindle position checkpoint inhibits Tem1-dependent activation of the mitotic exit network (MEN), thus delaying mitotic exit and cytokinesis until errors are corrected. We report a functional analysis of two previously uncharacterized budding yeast proteins, Dma1 and Dma2, 58% identical to each other and homologous to human Chfr and Schizosaccharomyces pombe Dma1, both of which have been previously implicated in mitotic checkpoints. We show that Dma1 and Dma2 are involved in proper spindle positioning, likely regulating septin ring deposition at the bud neck. DMA2 overexpression causes defects in septin ring disassembly at the end of mitosis and in cytokinesis. The latter defects can be rescued by either eliminating the spindle position checkpoint protein Bub2 or overproducing its target, Tem1, both leading to MEN hyperactivation. In addition, dma1Δ dma2Δ cells fail to activate the spindle position checkpoint in response to the lack of dynein, whereas ectopic expression of DMA2 prevents unscheduled mitotic exit of spindle checkpoint mutants treated with microtubule-depolymerizing drugs. Although their primary functions remain to be defined, our data suggest that Dma1 and Dma2 might be required to ensure timely MEN activation in telophase.
PMCID: PMC491838  PMID: 15146058
11.  Budding yeast PAK kinases regulate mitotic exit by two different mechanisms 
The Journal of Cell Biology  2003;160(6):857-874.
We report the characterization of the dominant-negative CLA4t allele of the budding yeast CLA4 gene, encoding a member of the p21-activated kinase (PAK) family of protein kinases, which, together with its homologue STE20, plays an essential role in promoting budding and cytokinesis. Overproduction of the Cla4t protein likely inhibits both endogenous Cla4 and Ste20 and causes a delay in the onset of anaphase that correlates with inactivation of Cdc20/anaphase-promoting complex (APC)–dependent proteolysis of both the cyclinB Clb2 and securin. Although the precise mechanism of APC inhibition by Cla4t remains to be elucidated, our results suggest that Cla4 and Ste20 may regulate the first wave of cyclinB proteolysis mediated by Cdc20/APC, which has been shown to be crucial for activation of the mitotic exit network (MEN). We show that the Cdk1-inhibitory kinase Swe1 is required for the Cla4t-dependent delay in cell cycle progression, suggesting that it might be required to prevent full Cdc20/APC and MEN activation. In addition, inhibition of PAK kinases by Cla4t prevents mitotic exit also by a Swe1-independent mechanism impinging directly on the MEN activator Tem1.
PMCID: PMC2173773  PMID: 12642613
cytokinesis; mitotic exit network; Cla4; spindle checkpoint; Swe1
12.  Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast 
The Journal of Cell Biology  2001;155(5):711-718.
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.
PMCID: PMC2150857  PMID: 11724813
anaphase; anaphase-promoting complex; microtubules; securin; spindle
13.  Budding Yeast Bub2 Is Localized at Spindle Pole Bodies and Activates the Mitotic Checkpoint via a Different Pathway from Mad2  
The Journal of Cell Biology  1999;145(5):979-991.
The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle. In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest. Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins. We investigated the role of Bub2 in budding yeast mitotic checkpoint. The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions. Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.
PMCID: PMC2133126  PMID: 10352016
budding yeast; Bub2; mitotic checkpoint; anaphase; anaphase-promoting complex
14.  The β4 Integrin Interactor p27BBP/eIF6 Is an Essential Nuclear Matrix Protein Involved in 60S Ribosomal Subunit Assembly  
The Journal of Cell Biology  1999;144(5):823-838.
p27BBP/eIF6 is an evolutionarily conserved protein that was originally identified as p27BBP, an interactor of the cytoplasmic domain of integrin β4 and, independently, as the putative translation initiation factor eIF6. To establish the in vivo function of p27BBP/eIF6, its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae. In epithelial cells containing β4 integrin, p27BBP/eIF6 is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of β4 integrin. Surprisingly, in the absence and in the presence of the β4 integrin subunit, p27BBP/eIF6 is in the nucleolus and associated with the nuclear matrix. Deletion of the IIH S. cerevisiae gene, encoding the yeast p27BBP/eIF6 homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit. Furthermore, human p27BBP/eIF6 can rescue the lethal effect of the iihΔ yeast mutation. The data obtained in vivo suggest an evolutionarily conserved function of p27BBP/eIF6 in ribosome biogenesis or assembly rather than in translation. A further function related to the β4 integrin subunit may have evolved specifically in higher eukaryotic cells.
PMCID: PMC2148184  PMID: 10085284
epithelial cells; yeast; nucleolus; hemidesmosomes; intermediate filaments

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