The protein kinase Kin4 inhibits exit from mitosis in response to spindle position defects

Accurate nuclear position is essential for each daughter cell to receive one DNA complement. In budding yeast, a surveillance mechanism known as the spindle position checkpoint ensures that exit from mitosis only occurs when the anaphase nucleus is positioned along the mother-bud axis. We identified the protein kinase Kin4 as a component of the spindle position checkpoint. KIN4 prevents exit from mitosis in cells with mispositioned nuclei by inhibiting the mitotic exit network (MEN), a GTPase signaling cascade that promotes exit from mitosis. Kin4 is active in cells with mispositioned nuclei and predominantly localizes to mother cells, where it is ideally situated to inhibit MEN signaling at spindle pole bodies (SPBs) when anaphase spindle elongation occurs within the mother cell.     

Tem1 localization to the spindle pole bodies is essential for mitotic exit and impairs spindle checkpoint function

The mitotic exit network (MEN) is a signaling cascade that triggers inactivation of the mitotic cyclin-dependent kinases and exit from mitosis. The GTPase Tem1 localizes on the spindle pole bodies (SPBs) and initiates MEN signaling. Tem1 activity is inhibited until anaphase by Bfa1-Bub2. These proteins are also part of the spindle position checkpoint (SPOC), a surveillance mechanism that restrains mitotic exit until the spindle is correctly positioned. Here, we show that regulation of Tem1 localization is essential for the proper function of the MEN and the SPOC. We demonstrate that the dynamics of Tem1 loading onto SPBs determine the recruitment of other MEN components to this structure, and reevaluate the interdependence in the localization of Tem1, Bfa1, and Bub2. We also find that removal of Tem1 from the SPBs is critical for the SPOC to impede cell cycle progression. Finally, we demonstrate for the first time that localization of Tem1 to the SPBs is a requirement for 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.     

Kin4 kinase delays mitotic exit in response to spindle alignment defects

For many polarized cells, it is critical that the mitotic spindle becomes positioned relative to the polarity axis. This is especially important in yeast, where the site of cytokinesis is predetermined. The spindle position checkpoint (SPOC) therefore delays mitotic exit of cells with a mispositioned spindle. One component of the SPOC is the Bub2-Bfa1 complex, an inhibitor of the mitotic exit network (MEN). Here, we show that the Kin4 kinase is a component of the SPOC and as such is essential to delay cell cycle progression of cells with a misaligned spindle. When spindles are correctly oriented, Kin4 and Bub2-Bfa1 are asymmetrically localized to opposite spindle pole bodies (SPBs). Bub2-Bfa1 then becomes inhibited by Cdc5 polo kinase with anaphase onset, a prerequisite for mitotic exit. In response to spindle misalignment, Kin4 and Bub2-Bfa1 are brought together at both SPBs. Kin4 now maintains Bub2-Bfa1 activity by counteracting Cdc5, thereby inhibiting mitotic exit.     

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.     

Timing is everything: regulation of mitotic exit and cytokinesis by the MEN and SIN

Proper completion of mitosis requires careful coordination of numerous cellular events. It is crucial, for example, that cells do not initiate spindle disassembly and cytokinesis until chromosomes have been properly segregated. Cells have developed numerous safeguards or checkpoints to delay exit from mitosis and initiation of the next cell cycle in response to defects in late mitosis. In this review, we discuss recent work on two homologous signaling pathways in budding and fission yeast, termed the mitotic exit network (MEN) and septation initiation network (SIN), respectively, that are essential for coordinating completion of mitosis and cytokinesis with other mitotic events.     

Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

The asymmetrically dividing yeast S. cerevisiae assembles a bipolar spindle well after establishing the future site of cell division (i.e., the bud neck) and the division axis (i.e., the mother-bud axis). A surveillance mechanism called spindle position checkpoint (SPOC) delays mitotic exit and cytokinesis until the spindle is properly positioned relative to the mother-bud axis, thereby ensuring the correct ploidy of the progeny. SPOC relies on the heterodimeric GTPase-activating protein Bub2/Bfa1 that inhibits the small GTPase Tem1, in turn essential for activating the mitotic exit network (MEN) kinase cascade and cytokinesis. The Bub2/Bfa1 GAP and the Tem1 GTPase form a complex at spindle poles that undergoes a remarkable asymmetry during mitosis when the spindle is properly positioned, with the complex accumulating on the bud-directed old spindle pole. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. The mechanism driving asymmetry of Bub2/Bfa1/Tem1 in mitosis is unclear. Furthermore, whether asymmetry is involved in timely mitotic exit is controversial. We investigated the mechanism by which the GAP Bub2/Bfa1 controls GTP hydrolysis on Tem1 and generated a series of mutants leading to constitutive Tem1 activation. These mutants are SPOC-defective and invariably lead to symmetrical localization of Bub2/Bfa1/Tem1 at spindle poles, indicating that GTP hydrolysis is essential for asymmetry. Constitutive tethering of Bub2 or Bfa1 to both spindle poles impairs SPOC response but does not impair mitotic exit. Rather, it facilitates mitotic exit of MEN mutants, likely by increasing the residence time of Tem1 at spindle poles where it gets active. Surprisingly, all mutant or chimeric proteins leading to symmetrical localization of Bub2/Bfa1/Tem1 lead to increased symmetry at spindle poles of the Kar9 protein that mediates spindle positioning and cause spindle misalignment. Thus, asymmetry of the Bub2/Bfa1/Tem1 complex is crucial to control Kar9 distribution and spindle positioning during mitosis.     

Functional characterization of Dma1 and Dma2, the budding yeast homologues of Schizosaccharomyces pombe Dma1 and human Chfr

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, dma1Delta dma2Delta 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.     

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.     

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.     

The yeast centrosome translates the positional information of the anaphase spindle into a cell cycle signal

The spindle orientation checkpoint (SPOC) of budding yeast delays mitotic exit when cytoplasmic microtubules (MTs) are defective, causing the spindle to become misaligned. Delay is achieved by maintaining the activity of the Bfa1-Bub2 guanosine triphosphatase-activating protein complex, an inhibitor of mitotic exit. In this study, we show that the spindle pole body (SPB) component Spc72, a transforming acidic coiled coil-like molecule that interacts with the gamma-tubulin complex, recruits Kin4 kinase to both SPBs when cytoplasmic MTs are defective. This allows Kin4 to phosphorylate the SPB-associated Bfa1, rendering it resistant to inactivation by Cdc5 polo kinase. Consistently, forced targeting of Kin4 to both SPBs delays mitotic exit even when the anaphase spindle is correctly aligned. Moreover, we present evidence that Spc72 has an additional function in SPOC regulation that is independent of the recruitment of Kin4. Thus, Spc72 provides a missing link between cytoplasmic MT function and components of the SPOC.     

Chromosome passenger complexes control anaphase duration and spindle elongation via a kinesin-5 brake

During mitosis, chromosome passenger complexes (CPCs) exhibit a well-conserved association with the anaphase spindle and have been implicated in spindle stability. However, their precise effect on the spindle is not clear. In this paper, we show, in budding yeast, that a CPC consisting of CBF3, Bir1, and Sli15, but not Ipl1, is required for normal spindle elongation. CPC mutants slow spindle elongation through the action of the bipolar kinesins Cin8 and Kip1. The same CPC mutants that slow spindle elongation also result in the enrichment of Cin8 and Kip1 at the spindle midzone. Together, these findings argue that CPCs function to organize the spindle midzone and potentially switch motors between force generators and molecular brakes. We also find that slowing spindle elongation delays the mitotic exit network (MEN)-dependent release of Cdc14, thus delaying spindle breakdown until a minimal spindle size is reached. We propose that these CPC- and MEN-dependent mechanisms are important for coordinating chromosome segregation with spindle breakdown and mitotic exit.     

SIN and the art of splitting the fission yeast cell

The septation initiation network (SIN) triggers the onset of cytokinesis in the fission yeast Schizosaccharomyces pombe by promoting contraction of the medially placed F-actin ring. SIN signaling is regulated by the polo-like kinase plo1p and by cdc2p, the initiator of mitosis, and its activation is co-ordinated with other events in mitosis to ensure that cytokinesis does not begin until chromosomes have been separated. Though the SIN controls the contractile ring, the signal originates from the poles of the mitotic spindle. Recent studies suggest that the spindle pole body may act as a dynamic assembly site for active SIN signaling complexes. In the budding yeast Saccharomyces cerevisiae the counterpart of the SIN, called the MEN, mediates both mitotic exit and cytokinesis, in part through regulating activation of the phosphoprotein phosphatase Cdc14p. Flp1p, the S. pombe ortholog of Cdc14p, is not essential for mitotic exit, but may contribute to an orderly mitosis-G1 transition by regulating the destruction of the mitotic inducer cdc25p.     

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.     

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.     

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.     

The MEN mediates the effects of the spindle assembly checkpoint on Kar9-dependent spindle pole body inheritance in budding yeast

Many asymmetrically dividing cells segregate the poles of the mitotic spindle non-randomly between their two daughters. In budding yeast, the protein Kar9 localizes almost exclusively to the astral microtubules emanating from the old spindle pole body (SPB) and promotes its movement toward the bud. Thereby, Kar9 orients the spindle relative to the division axis. Here, we show that beyond perturbing Kar9 distribution, activation of the spindle assembly checkpoint (SAC) randomizes SPB inheritance. Inactivation of the B-type cyclin Clb5 led to a SAC-dependent defect in Kar9 orientation and SPB segregation. Furthermore, unlike the Clb4-dependent pathway, the Clb5- and SAC-dependent pathways functioned genetically upstream of the mitotic exit network (MEN) in SPB specification and Kar9-dependent SPB inheritance. Together, our study indicates that Clb5 functions in spindle assembly and that the SAC controls the specification and inheritance of yeast SPBs through inhibition of the MEN.     

The MEN mediates the effects of the spindle assembly checkpoint on Kar9-dependent spindle pole body inheritance in budding yeast

Many asymmetrically dividing cells segregate the poles of the mitotic spindle non-randomly between their two daughters. In budding yeast, the protein Kar9 localizes almost exclusively to the astral microtubules emanating from the old spindle pole body (SPB) and promotes its movement toward the bud. Thereby, Kar9 orients the spindle relative to the division axis. Here, we show that beyond perturbing Kar9 distribution, activation of the spindle assembly checkpoint (SAC) randomizes SPB inheritance. Inactivation of the B-type cyclin Clb5 led to a SAC-dependent defect in Kar9 orientation and SPB segregation. Furthermore, unlike the Clb4-dependent pathway, the Clb5- and SAC-dependent pathways functioned genetically upstream of the mitotic exit network (MEN) in SPB specification and Kar9-dependent SPB inheritance. Together, our study indicates that Clb5 functions in spindle assembly and that the SAC controls the specification and inheritance of yeast SPBs through inhibition of the MEN.     

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.     

Lte1, Cdc14 and MEN‐controlled Cdk inactivation in yeast coordinate rDNA decompaction with late telophase progression

The mechanism of chromatin compaction in mitosis has been well studied, while little is known about what controls chromatin decompaction in early G1 phase. We have localized the Condensin subunit Brn1 to a compact spiral of rDNA in mitotic budding yeast cells. Brn1 release and the resulting rDNA decompaction in late telophase coincided with mitotic spindle dissociation, and occurred asymmetrically (daughter cells first). We immunoprecipitated the GTP‐exchange factor Lte1, which helps activate the mitotic exit network (MEN) in anaphase, with mitotic Brn1. In lteΔ cells Brn1 release was delayed, even at temperatures that do not impair mitotic exit. Mutations in MEN pathway components that act downstream of Lte1 similarly delayed rDNA decompaction. We found that Brn1 release in wild‐type cells coincided with the release of Cdc14 phosphatase from the nucleolus and with mitotic CDK inactivation, yet it could be selectively delayed by perturbation of the MEN pathway. This may argue that different levels of Cdk inactivation control spindle disassembly and chromatin decompaction. Mutation of lte1 also impaired rotation of the nucleus in early G1.