Regulation of the Bfa1p-Bub2p complex at spindle pole bodies by the cell cycle phosphatase Cdc14p

The budding yeast mitotic exit network (MEN) is a GTPase-driven signal transduction cascade that controls the release of the phosphatase Cdc14p from the nucleolus in anaphase and thereby drives mitotic exit. We show that Cdc14p is partially released from the nucleolus in early anaphase independent of the action of the MEN components Cdc15p, Dbf2p, and Tem1p. Upon release, Cdc14p binds to the spindle pole body (SPB) via association with the Bfa1p-Bub2p GTPase activating protein complex, which is known to regulate the activity of the G protein Tem1p. Cdc14p also interacts with this GTPase. The association of the MEN component Mob1p with the SPB acts as a marker of MEN activation. The simultaneous binding of Cdc14p and Mob1p to the SPB in early anaphase suggests that Cdc14p initially activates the MEN. In a second, later step, which coincides with mitotic exit, Cdc14p reactivates the Bfa1p-Bub2p complex by dephosphorylating Bfa1p. This inactivates the MEN and displaces Mob1p from SPBs. These data indicate that Cdc14p activates the MEN in early anaphase but later inactivates it through Bfa1p dephosphorylation and so restricts MEN activity to a short period in anaphase.     

The differential roles of budding yeast Tem1p, Cdc15p, and Bub2p protein dynamics in mitotic exit

In the budding yeast Saccharomyces cerevisiae the mitotic spindle must be positioned along the mother-bud axis to activate the mitotic exit network (MEN) in anaphase. To examine MEN proteins during mitotic exit, we imaged the MEN activators Tem1p and Cdc15p and the MEN regulator Bub2p in vivo. Quantitative live cell fluorescence microscopy demonstrated the spindle pole body that segregated into the daughter cell (dSPB) signaled mitotic exit upon penetration into the bud. Activation of mitotic exit was associated with an increased abundance of Tem1p-GFP and the localization of Cdc15p-GFP on the dSPB. In contrast, Bub2p-GFP fluorescence intensity decreased in mid-to-late anaphase on the dSPB. Therefore, MEN protein localization fluctuates to switch from Bub2p inhibition of mitotic exit to Cdc15p activation of mitotic exit. The mechanism that elevates Tem1p-GFP abundance in anaphase is specific to dSPB penetration into the bud and Dhc1p and Lte1p promote Tem1p-GFP localization. Finally, fluorescence recovery after photobleaching (FRAP) measurements revealed Tem1p-GFP is dynamic at the dSPB in late anaphase. These data suggest spindle pole penetration into the bud activates mitotic exit, resulting in Tem1p and Cdc15p persistence at the dSPB to initiate the MEN signal cascade.     

Spindle pole bodies exploit the mitotic exit network in metaphase to drive their age-dependent segregation

Like many asymmetrically dividing cells, budding yeast segregates mitotic spindle poles nonrandomly between mother and daughter cells. During metaphase, the spindle positioning protein Kar9 accumulates asymmetrically, localizing specifically to astral microtubules emanating from the old spindle pole body (SPB) and driving its segregation to the bud. Here, we show that the SPB component Nud1/centriolin acts through the mitotic exit network (MEN) to specify asymmetric SPB inheritance. In the absence of MEN signaling, Kar9 asymmetry is unstable and its preference for the old SPB is disrupted. Consistent with this, phosphorylation of Kar9 by the MEN kinases Dbf2 and Dbf20 is not required to break Kar9 symmetry but is instead required to maintain stable association of Kar9 with the old SPB throughout metaphase. We propose that MEN signaling links Kar9 regulation to SPB identity through biasing and stabilizing the age-insensitive, cyclin-B-dependent mechanism of symmetry breaking.     

Regulation of the localization of Dbf2 and mob1 during cell division of saccharomyces cerevisiae.

The mitotic exit network (MEN) governs Cdk inactivation. In budding yeast, MEN consists of the protein phosphatase Cdc14, the ras-like GTPase Tem1, protein kinases Cdc15, Cdc5, Dbf2 and Dbf2-binding protein Mob1. Tem1, Dbf2, Cdc5 and Cdc15 have been reported to be localized at the spindle pole body (SPB). Here we report changes of the localization of Dbf2 and Mob1 during cell division. Dbf2 and Mob1 localize to the SPBs in anaphase and then moves to the bud neck, just prior to actin ring assembly, consistent with their role in cytokinesis. The neck localization, but not SPB localization, of Dbf2 was inhibited by the Bub2 spindle checkpoint. Cdc14 is the downstream target of Dbf2 in Cdk inactivation, but we found that the neck localization of DbP2 and Mob1 was dependent on the Cdc14 activity, suggesting that Dbf2 and Mob1 function in cytokinesis at the end of the mitotic signaling cascade.     

Nud1p, the yeast homolog of Centriolin, regulates spindle pole body inheritance in meiosis

Nud1p, a protein homologous to the mammalian centrosome and midbody component Centriolin, is a component of the budding yeast spindle pole body (SPB), with roles in anchorage of microtubules and regulation of the mitotic exit network during vegetative growth. Here we analyze the function of Nud1p during yeast meiosis. We find that a nud1-2 temperature-sensitive mutant has two meiosis-related defects that reflect genetically distinct functions of Nud1p. First, the mutation affects spore formation due to its late function during spore maturation. Second, and most important, the mutant loses its ability to distinguish between the ages of the four spindle pole bodies, which normally determine which SPB would be preferentially included in the mature spores. This affects the regulation of genome inheritance in starved meiotic cells and leads to the formation of random dyads instead of non-sister dyads under these conditions. Both functions of Nud1p are connected to the ability of Spc72p to bind to the outer plaque and half-bridge (via Kar1p) of the SPB.     

Mitotic exit network controls the localization of Cdc14 to the spindle pole body in Saccharomyces cerevisiae

Budding yeast Cdc14 phosphatase plays essential roles in mitotic exit. Cdc14 is sequestered in the nucleolus by its inhibitor Net1/Cfi1 and is only released from the nucleolus during anaphase to inactivate mitotic CDK. It is believed that the mitotic exit network (MEN) is required for the release of Cdc14 from the nucleolus because liberation of Cdc14 by net1/cfi1 mutations bypasses the essential role of the MEN. But how the MEN residing at the spindle pole body (SPB) controls the association of Cdc14 with Net1/Cfi1 in the nucleolus is not yet understood. We found that Cdc14-5GFP was released from the nucleolus in the MEN mutants (tem1, cdc15, dbf2, and nud1), but not in the cdc5 cells during early anaphase. The Cdc14 liberation from the nucleolus was inhibited by the Mad2 checkpoint and by the Bub2 checkpoint in a different manner when microtubule organization was disrupted. We observed Cdc14-5GFP at the SPB in addition to the nucleolus. The SPB localization of Cdc14 was significantly affected by the MEN mutations and the bub2 mutation. We conclude that Cdc14 is released from the nucleolus at the onset of anaphase in a CDC5-dependent manner and that MEN factors possibly regulate Cdc14 release from the SPB.     

A role for cell polarity proteins in mitotic exit

The budding yeast mitotic exit network (MEN) is a signal transduction cascade that controls exit from mitosis by facilitating the release of the cell cycle phosphatase Cdc14 from the nucleolus. The G protein Tem1 regulates MEN activity. The Tem1 guanine nucleotide exchange factor (GEF) Lte1 associates with the cortex of the bud and activates the MEN upon the formation of an anaphase spindle. Thus, the cell cortex has an important but ill-defined role in MEN regulation. Here, we describe a network of conserved cortical cell polarity proteins that have key roles in mitotic exit. The Rho-like GTPase Cdc42, its GEF Cdc24 and its effector Cla4 [a member of the p21-activated kinases (PAKs)] control the initial binding and activation of Lte1 to the bud cortex. Moreover, Cdc24, Cdc42 and Ste20, another PAK, probably function parallel to Lte1 in facilitating mitotic exit. Finally, the cell polarity proteins Kel1 and Kel2 are present in complexes with both Lte1 and Tem1, and negatively regulate mitotic exit.     

Temporal Coupling of Spindle Disassembly and Cytokinesis is Disrupted by Deletion of LTE1 in Budding Yeast

The mitotic exit network (MEN) is a signal transduction cascade that controls exit from mitosis in budding yeast by triggering the nucleolar release and hence activation of the Cdc14 phosphatase. Activation of the MEN is tightly coordinated with spindle position in such a way that Cdc14 is only fully released upon spindle pole body (SPB) migration into the daughter cell. This temporal regulation of the MEN has been proposed to rely in part on the spatial separation of the G-protein Tem1 at the SPB and its nucleotide exchange factor Lte1 confined to the daughter cell cortex. However, the dispensability of LTE1 for survival has raised questions regarding this model. Here using real-time microscopy we show that lte1? mutants not only delay exit from mitosis but also uncouple the normal coordination between spindle disassembly and contraction of the actomyosin ring at cell division. These mitotic defects can be suppressed by a bub2? mutation or by Cdc14 over-expression suggesting that they are caused by compromised MEN activity. Thus Lte1 function is important to fine-tune the timing of mitotic exit and to couple this event with cytokinesis in budding yeast.     

A novel functional domain of Cdc15 kinase is required for its interaction with Tem1 GTPase in Saccharomyces cerevisiae

Exit from mitosis requires the inactivation of cyclin-dependent kinase (CDK) activity. In the budding yeast Saccharomyces cerevisiae, a number of gene products have been identified as components of the signal transduction network regulating inactivation of CDK (called the MEN, for the mitotic exit network). Cdc15, one of such components of the MEN, is an essential protein kinase. By the two-hybrid screening, we identified Cdc15 as a binding protein of Tem1 GTPase, another essential regulator of the MEN. Coprecipitation experiments revealed that Tem1 binds to Cdc15 in vivo. By deletion analysis, we found that the Tem1-binding domain resides near the conserved kinase domain of Cdc15. The cdc15-LF mutation, which was introduced into the Tem1-binding domain, reduced the interaction with Cdc15 and Tem1 and caused temperature-sensitive growth.The kinase activity of Cdc15 was not so much affected by the cdc15-LF mutation. However, Cdc15-LF failed to localize to the SPB at the restrictive temperature. Our data show that the interaction with Tem1 is important for the function of Cdc15 and that Cdc15 and Tem1 function in a complex to direct the exit from mitosis.     

Exit from exit: resetting the cell cycle through Amn1 inhibition of G protein signaling

In S. cerevisiae cells undergoing anaphase, a ras-related GTPase, Tem1, is located on the spindle pole body that enters the daughter cell and activates a signal transduction pathway, MEN, to allow mitotic exit. MEN activation must be reversed after mitotic exit to reset the cell cycle in G1. We find that daughter cells activate an Antagonist of MEN pathway (AMEN) in part through induction of the Amn1 protein that binds directly to Tem1 and prevents its association with its target kinase Cdc15. Failure of Amn1 function results in defects of both the spindle assembly and nuclear orientation checkpoints and delays turning off Cdc14 in G1. Thus, Amn1 is part of a daughter-specific switch that helps cells exit from mitotic exit and reset the cell cycle.     

Mutual regulation of cyclin-dependent kinase and the mitotic exit network

The mitotic exit network (MEN) is a spindle pole body (SPB)–associated, GTPase-driven signaling cascade that controls mitotic exit. The inhibitory Bfa1–Bub2 GTPase-activating protein (GAP) only associates with the daughter SPB (dSPB), raising the question as to how the MEN is regulated on the mother SPB (mSPB). Here, we show mutual regulation of cyclin-dependent kinase 1 (Cdk1) and the MEN. In early anaphase Cdk1 becomes recruited to the mSPB depending on the activity of the MEN kinase Cdc15. Conversely, Cdk1 negatively regulates binding of Cdc15 to the mSPB. In addition, Cdk1 phosphorylates the Mob1 protein to inhibit the activity of Dbf2–Mob1 kinase that regulates Cdc14 phosphatase. Our data revise the understanding of the spatial regulation of the MEN. Although MEN activity in the daughter cells is controlled by Bfa1–Bub2, Cdk1 inhibits MEN activity at the mSPB. Consistent with this model, only triple mutants that lack BUB2 and the Cdk1 phosphorylation sites in Mob1 and Cdc15 show mitotic exit defects.     

Asymmetric spindle pole localization of yeast Cdc15 kinase links mitotic exit and cytokinesis

The inactivation of mitotic cyclin-dependent kinases (CDKs) during anaphase is a prerequisite for the completion of nuclear division and the onset of cytokinesis [1, 2]. In the budding yeast Saccharomyces cerevisiae, the essential protein kinase Cdc15 [3] together with other proteins of the mitotic exit network (Tem1, Lte1, Cdc5, and Dbf2/Dbf20 [4-7]) activates Cdc14 phosphatase, which triggers cyclin degradation and the accumulation of the CDK inhibitor Sic1 [8]. However, it is still unclear how CDK inactivation promotes cytokinesis. Here, we analyze the properties of Cdc15 kinase during mitotic exit. We found that Cdc15 localized to the spindle pole body (SPB) in a unique pattern. Cdc15 was present at the SPB of the mother cell until late mitosis, when it also associated with the daughter pole. High CDK activity inhibited this association, while dephosphorylation of Cdc15 by Cdc14 phosphatase enabled it. The analysis of Cdc15 derivatives indicated that SPB localization was specifically required for cytokinesis but not for mitotic exit. These results show that Cdc15 has two separate functions during the cell cycle. First, it is required for the activation of Cdc14. CD14, in turn, promotes CDK inactivation and also dephosphorylates of Cdc15. As a consequence, Cdc15 binds to the daughter pole and triggers cytokinesis. Thus, Cdc15 helps to coordinate mitotic exit and cytokinesis.     

In vitro regulation of budding yeast Bfa1/Bub2 GAP activity by Cdc5

The Cdc5 protein of budding yeast is a polo-like kinase that has multiple roles in mitosis including control of the mitotic exit network (MEN). MEN activity brings about loss of mitotic kinase activity so that the mitotic spindle is disassembled and cytokinesis can proceed. Activity of the MEN is regulated by a small GTPase, Tem1, which in turn is controlled by a two-component GTPase-activating protein (GAP) formed by Bfa1 and Bub2. Bfa1 has been identified as a regulatory target of Cdc5 but there are conflicting deductions from indirect in vivo assays as to whether phosphorylation inhibits or stimulates Bfa1 activity. To resolve this question, we have used direct in vitro assays to observe the effects of phosphorylation on Bfa1 activity. We show that when Bfa1 is phosphorylated by Cdc5, its GAP activity with Bub2 is inhibited although its ability to interact with Tem1 is unaffected. Thus, in vivo inactivation of Bfa1-Bub2 by Cdc5 would have a positive regulatory effect by increasing levels of Tem1-GTP so stimulating exit from mitosis.     

The Bub2p spindle checkpoint links nuclear migration with mitotic exit

Bfa1p and Bub2p are spindle checkpoint proteins that likely have GTPase activation activity and are associated with the budding yeast spindle pole body (SPB). Here, we show that Bfa1p and Bub2p bind the Ras-like GTPase Tem1p, a component of the mitotic exit network, to the cytoplasmic face of the SPB that enters the bud, whereas the GDP/GTP exchange factor Lte1p is associated with the cortex of the bud. Migration of the SPB into the bud probably allows activation of Tem1p through Lte1p, thereby linking nuclear migration with mitotic exit. Since components of the Bub2p checkpoint are conserved in other organisms, we propose that the position of the SPB or mammalian centrosome controls the timing of mitotic exit.     

The study of Bfa1p(E438K) suggests that Bfa1 control the mitotic exit network in different mechanisms depending on different checkpoint-activating signals

During mitosis, genomic integrity is maintained by the proper coordination of anaphase entry and mitotic exit via mitotic checkpoints. In budding yeast, mitotic exit is controlled by a regulatory cascade called the mitotic exit network (MEN). The MEN is regulated by a small GTPase, Tem1p, which in turn is controlled by a two-component GAP, Bfa1p-Bub2p. Recent results suggested that phosphorylation of Bfa1p by the polo-related kinase Cdc5p is also required for triggering mitotic exit, since it decreases the GAP activity of Bfa1p-Bub2p. However, the dispensability of GEF Lte1p for mitotic exit has raised questions about regulation of the MEN by the GTPase activity of Tem1p. We isolated a Bfa1p mutant, Bfa1p(E438K), whose overexpression only partially induced anaphase arrest. The molecular and biochemical functions of Bfa1p(E438K) are similar to those of wild type Bfa1p, except for decreased GAP activity. Interestingly, in BFA1(E438K) cells, the MEN could be regulated with nearly wild type kinetics at physiological temperature, as well as in response to various checkpoint-activating signals, but the cells were more sensitive to spindle damage than wild type. These results suggest that the GAP activity of Bfa1p-Bub2p is responsible for the mitotic arrest caused by spindle damage and Bfa1p overproduction. In addition, the viability of cdc5-2 delta bfa1 cells was not reduced by BFA1(E438K), suggesting that Cdc5p also regulates Bfa1p to activate mitotic exit by other mechanism(s), besides phosphorylation.     

Mitotic exit control: a space and time odyssey

The mitotic exit network (MEN), a protein kinase cascade under the switch-like control of the small GTPase Tem1, triggers exit from mitosis in budding yeast. Now it emerges that signals from both Tem1 and the yeast Polo kinase Cdc5 converge onto the MEN kinase Cdc15 to accurately restrict MEN activation to late mitosis.     

Cdc5-dependent asymmetric localization of bfa1 fine-tunes timely mitotic exit

In budding yeast, the major regulator of the mitotic exit network (MEN) is Tem1, a GTPase, which is inhibited by the GTPase-activating protein (GAP), Bfa1/Bub2. Asymmetric Bfa1 localization to the bud-directed spindle pole body (SPB) during metaphase also controls mitotic exit, but the molecular mechanism and function of this localization are not well understood, particularly in unperturbed cells. We identified four novel Cdc5 target residues within the Bfa1 C-terminus: (452)S, (453)S, (454)S, and (559)S. A Bfa1 mutant in which all of these residues had been changed to alanine (Bfa1(4A)) persisted on both SPBs at anaphase and was hypo-phosphorylated, despite retaining its GAP activity for Tem1. A Bfa1 phospho-mimetic mutant in which all of these residues were switched to aspartate (Bfa1(4D)) always localized asymmetrically to the SPB. These observations demonstrate that asymmetric localization of Bfa1 is tightly linked to its Cdc5-dependent phosphorylation, but not to its GAP activity. Consistent with this, in kinase-defective cdc5-2 cells Bfa1 was not phosphorylated and localized to both SPBs, whereas Bfa1(4D) was asymmetrically localized. BFA1(4A) cells progressed through anaphase normally but displayed delayed mitotic exit in unperturbed cell cycles, while BFA1(4D) cells underwent mitotic exit with the same kinetics as wild-type cells. We suggest that Cdc5 induces the asymmetric distribution of Bfa1 to the bud-directed SPB independently of Bfa1 GAP activity at anaphase and that Bfa1 asymmetry fine-tunes the timing of MEN activation in unperturbed cell cycles.     

A dynamical model of the spindle position checkpoint

The orientation of the mitotic spindle with respect to the polarity axis is crucial for the accuracy of asymmetric cell division. In budding yeast, a surveillance mechanism called the spindle position checkpoint (SPOC) prevents exit from mitosis when the mitotic spindle fails to align along the mother‐to‐daughter polarity axis. SPOC arrest relies upon inhibition of the GTPase Tem1 by the GTPase‐activating protein (GAP) complex Bfa1–Bub2. Importantly, reactions signaling mitotic exit take place at yeast centrosomes (named spindle pole bodies, SPBs) and the GAP complex also promotes SPB localization of Tem1. Yet, whether the regulation of Tem1 by Bfa1–Bub2 takes place only at the SPBs remains elusive. Here, we present a quantitative analysis of Bfa1–Bub2 and Tem1 localization at the SPBs. Based on the measured SPB‐bound protein levels, we introduce a dynamical model of the SPOC that describes the regulation of Bfa1 and Tem1. Our model suggests that Bfa1 interacts with Tem1 in the cytoplasm as well as at the SPBs to provide efficient Tem1 inhibition.     

Saccharomyces cerevisiae Mob1p is required for cytokinesis and mitotic exit

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G(1) by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G(1) transition to control cyclin-CDK inactivation and cytokinesis.