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.     

Fission yeast Clp1p phosphatase regulates G2/M transition and coordination of cytokinesis with cell cycle progression.

Background: In Saccharomyces cerevisiae the mitotic-exit network (MEN) functions in anaphase to promote the release of the Cdc14p phosphatase from the nucleolus. This release causes mitotic exit via inactivation of the cyclin-dependent kinase (Cdk). Cdc14p-like proteins are highly conserved; however, it is unclear if these proteins regulate mitotic exit as in S. cerevisiae. In Schizosaccharomyces pombe a signaling pathway homologous to the MEN and termed the septation initiation network (SIN) is required not for mitotic exit, but for initiation of cytokinesis and for a cytokinesis checkpoint that inhibits further cell cycle progression until cytokinesis is complete.Results: We have identified the S. pombe Cdc14p homolog, Clp1p, and show that it is not required for mitotic exit but rather functions together with the SIN in coordinating cytokinesis with the nuclear-division cycle. As cells enter mitosis, Clp1p relocalizes from the nucleolus to the spindle and site of cell division. Clp1p exit from the nucleolus does not depend on the SIN, but the SIN is required for keeping Clp1p out of the nucleolus until completion of cytokinesis. Clp1p, in turn, may promote the activation of the SIN by antagonizing Cdk activity until cytokinesis is complete and thus ensuring that cytokinesis is completed prior to the initiation of the next cell cycle. In addition to its roles in anaphase, Clp1p regulates the G2/M transition since cells deleted for clp1 enter mitosis precociously and cells overexpressing Clp1p delay mitotic entry. Unlike Cdc14p, Clp1p appears to antagonize Cdk activity by preventing dephosphorylation of Cdc2p on tyrosine.Conclusions: S. pombe Clp1p affects cell cycle progression in a markedly different manner than its S. cerevisiae homolog, Cdc14p. This finding raises the possibility that related phosphatases in animal cells will prove to have important roles in coordinating the onset of cytokinesis with the events of mitosis.     

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.     

Crm1-Mediated Nuclear Export of Cdc14 is Required for the Completion of Cytokinesis in Budding Yeast

The mitotic exit network (MEN) controls the exit from mitosis in budding yeast. The proline-directed phosphatase, Cdc14p, is a key component of MEN and promotes mitotic exit by activating the degradation of Clb2p and by reversing Cdk-mediated mitotic phosphorylation. Cdc14p is sequestered in the nucleolus during much of the cell cycle and is released in anaphase from the nucleolus to the nucleoplasm and cytoplasm to perform its functions. Release of Cdc14p from the nucleolus during anaphase is well understood. In contrast, less is known about the mechanism by which Cdc14p is released from the nucleus to the cytoplasm. Here we show that Cdc14p contains a leucine-rich nuclear export signal (NES) that interacts with Crm1p physically. Mutations in the NES of Cdc14p allow Clb2p degradation and mitotic exit, but cause abnormal morphology and cytokinesis defects at non-permissive temperatures. Cdc14p localizes to the bud neck, among other cytoplasmic structures, following its release from the nucleolus in late anaphase. This bud neck localization of Cdc14p is disrupted by mutations in its NES and by the leptomycin B-mediated inhibition of Crm1p. Our results suggest a requirement for Crm1p-dependent nuclear export of Cdc14p in coordinating mitotic exit and cytokinesis in budding yeast.     

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.     

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.     

Disruption of centrosome structure,chromosome segregation,and cytokinesis by misexpression of human Cdc14A phosphatase

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and gamma-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.     

Cytokinesis in the C. elegans embryo: regulating contractile forces and a late role for the central spindle

Genetic and molecular studies in the nematode Caenorhabditis elegans have identified multiple essential pathways that regulate and execute cytokinesis in early embryonic cells. These pathways influence both the microfilament cytoskeleton and the microtubule cytoskeleton. Microfilaments are enriched throughout the cell cortex at all times during the cell cycle in embryonic cells. Cortical microfilaments are required for multiple processes in embryonic cells, including polar body extrusion during meiosis, anterior-posterior axis specification by the sperm-donated microtubule-organizing center, and cytokinesis during mitosis. In addition to contractile apparatus proteins that are required positively for cleavage furrow ingression, the Nedd8 ubiquitin-like protein modification pathway negatively regulates contractile forces outside the cleavage furrow during cytokinesis. Another pathway that acts positively during cytokinesis involves the mitotic spindle. The central spindle, where anti-parallel non-kinetochore microtubules overlap and are cross-linked, is required for a late step in cytokinesis, and other pathway(s) involved in membrane addition during cytokinesis may also require the central spindle. The amenability of C. elegans to classical genetics, the ease of reducing gene function with RNA interference, the completion of the genome sequence, and the availability of transgenic GFP fusion proteins that render the cytoskeleton fluorescent, all serve to make the early worm embryo an especially promising system for further advances in the identification of cytokinesis pathways, and in defining their interactions.     

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     

Analysis of the mitotic exit control system using locked levels of stable mitotic cyclin

Cyclin‐dependent kinase (Cdk) both promotes mitotic entry (spindle assembly and anaphase) and inhibits mitotic exit (spindle disassembly and cytokinesis), leading to an elegant quantitative hypothesis that a single cyclin oscillation can function as a ratchet to order these events. This ratchet is at the core of a published ODE model for the yeast cell cycle. However, the ratchet model requires appropriate cyclin dose–response thresholds. Here, we test the inhibition of mitotic exit in budding yeast using graded levels of stable mitotic cyclin (Clb2). In opposition to the ratchet model, stable levels of Clb2 introduced dose‐dependent delays, rather than hard thresholds, that varied by mitotic exit event. The ensuing cell cycle was highly abnormal, suggesting a novel reason for cyclin degradation. Cdc14 phosphatase antagonizes Clb2–Cdk, and Cdc14 is released from inhibitory nucleolar sequestration independently of stable Clb2. Thus, Cdc14/Clb2 balance may be the appropriate variable for mitotic regulation. Although our results are inconsistent with the aforementioned ODE model, revision of the model to allow Cdc14/Clb2 balance to control mitotic exit corrects these discrepancies, providing theoretical support for our conclusions.     

Mutual Dependence of Mob1 and the Chromosomal Passenger Complex for Localization during Mitosis

The spatial and temporal coordination of chromosome segregation with cytokinesis is essential to ensure that each daughter cell receives the correct complement of chromosomal and cytoplasmic material. In yeast, mitotic exit and cytokinesis are coordinated by signaling cascades whose terminal components include a nuclear Dbf2-related family kinase and a noncatalytic subunit, Mps one binding (Mob) 1. There are five human Mob1 isoforms, all of which display redundant localization patterns at the spindle poles and kinetochores in early mitosis, and the spindle midzone during cytokinesis. Mob1 shares similar localization patterns to Polo-like kinase (Plk1) and the chromosomal passenger complex (CPC), and although depletion of Plk1 resulted in a loss of Mob1 from the spindle poles, Mob1 recruitment to kinetochores was unaffected. Conversely, disruption of CPC signaling resulted in a loss of Mob1 from kinetochores without disrupting recruitment to the spindle poles. In Mob1-depleted cells, the relocalization of the CPC and mitotic kinesin-like protein (MKLP) 2 to the spindle midzone was delayed during early anaphase, and as a consequence, the midzone recruitment of MKLP1 also was affected. Together, these results suggest that Mob1 and the other mammalian orthologues of the mitotic exit network regulate mitotic progression by facilitating the timely mobilization of the CPC to the spindle midzone.     

The nucleolar phosphatase Cdc14B is dispensable for chromosome segregation and mitotic exit in human cells

In yeast, the protein phosphatase Cdc14 promotes chromosome segregation, mitotic exit, and cytokinesis by reversing M-phase phosphorylations catalyzed by Cdk1. A key feature of Cdc14 regulation is its sequestration within the nucleolus, which restricts its access to potential substrates for much of the cell cycle. Mammals also possess a nucleolar Cdc14 homolog, termed Cdc14B, but its roles during mitosis and cell division remain speculative. Here we analyze Cdc14B’s subcellular dynamics during mitosis and rigorously test its functional contributions to cell division through homozygous disruption of the Cdc14B locus in human somatic cells. While Cdc14B is initially released from nucleoli at the start of mitosis, the phosphatase quickly redistributes onto segregating sister chromatids during anaphase. This relocalization is mainly driven by Cdk1 inactivation, as pharmacologic inhibition of Cdk1 in prometaphase cells redirects Cdc14B onto chromosomes. However, in sharp contrast to yeast cdc14 mutants, human Cdc14BΔ/Δ cells were viable and lacked defects in spindle assembly, anaphase progression, mitotic exit, and cytokinesis, and continued to segregate ribosomal DNA repeats with near-normal proficiency. Our findings reveal substantial divergence in mitotic regulation between yeast and mammalian cells, as the latter possess efficient mechanisms for completing late M-phase events in the absence of a nucleolar Cdc14-related phosphatase.     

Functions of the mitotic B-type cyclins CLB1, CLB2, and CLB3 at mitotic exit antagonized by the CDC14 phosphatase

In the budding yeast Saccharomyces cerevisiae, cell cycle progression and cytokinesis at mitotic exit are proposed to be linked by CDC14 phosphatase antagonizing the function of mitotic B-type cyclin (CLBs). We have isolated a temperature-sensitive mutant, cdc14(A280V), with a mutation in the conserved phosphatase domain. Prolonged arrest in the cdc14(A280V) mutant partially uncoupled cell cycle progression from the completion of cytokinesis as measured by bud re-emergence, in the form of elongated apical projections, and DNA re-replication. In contrast to previous mitotic exit mutants, cdc14(A280V) mutants displayed a strong bias for the first apical projection to form in the mother cell body. Using cdc14(A280V) mutant phenotypes, the functions of the B-type cyclins at mitotic exit were investigated. The preference in mother-daughter apical projection formation was observed to be independent of any individual CLB function. However, cdc14(A280V)clb1Δ cells displayed a pronounced increase in apical projections, while cdc14(A280V)clb3Δ cells were observed to form round cellular chains. While cdc14(A280V) cells arrested at mitotic exit, both cdc14(A280V)clb1Δ and cdc14(A280V)clb3Δ cells completed cytokinesis, but failed cell separation. cdc14(A280V)clb2Δ cells displayed a defect in actin ring assembly. These observations differentiate the functions of CLB1, CLB2, and CLB3 at mitotic exit, and are consistent with the hypothesis that CLB activities are antagonized by the CDC14 phosphatase in order to couple cell cycle progression with cytokinesis at mitotic exit.     

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.     

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.     

Dma1 prevents mitotic exit and cytokinesis by inhibiting the septation initiation network (SIN)

In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) triggers cytokinesis after mitosis. We investigated the relationship between Dma1p, a spindle checkpoint protein and cytokinesis inhibitor, and the SIN. Deletion of dma1 inactivates the spindle checkpoint and allows precocious SIN activation, while overexpressing Dma1p reduces SIN signaling. Dma1p seems to function by inhibiting the SIN activator, Plo1p kinase, since dma1 overexpression and deletion phenotypes suggest that Dma1p antagonizes Plo1p localization. Furthermore, failure to maintain high cyclin-dependent kinase (CDK) activity during spindle checkpoint activation in dma1 deletion cells requires Plo1p. Dma1p itself localizes to spindle pole bodies through interaction with Sid4p. Our observations suggest that Dma1p functions to prevent mitotic exit and cytokinesis during spindle checkpoint arrest by inhibiting SIN signaling.     

SPOC alert--when chromosomes get the wrong direction

The asymmetrically dividing budding yeast relies upon the alignment of the mitotic spindle along the mother to daughter cell polarity axis for the fidelity of chromosome segregation during mitosis. In the case of spindle misalignment, a surveillance mechanism named the spindle position checkpoint (SPOC) prevents cells from exiting mitosis through the inhibition of the mitotic exit network (MEN). MEN is a signal transduction pathway that mediates mitotic exit through fully activation of the Cdk-counteracting phosphatase Cdc14. In this mini-review, we briefly describe the mechanisms leading to mitotic exit in budding yeast cells focusing on the control of MEN by the SPOC. In addition, we summarize the recent advances in the molecular understanding of SPOC regulation and discuss whether similar checkpoints may exist in higher eukaryotic cells that undergo asymmetric divisions.     

PRC1 controls spindle polarization and recruitment of cytokinetic factors during monopolar cytokinesis

The central spindle is a postanaphase array of microtubules that plays an essential role in organizing the signaling machinery for cytokinesis. The model by which the central spindle organizes the cytokinetic apparatus is premised on an antiparallel arrangement of microtubules, yet cells lacking spindle bipolarity are capable of generating a distal domain of ectopic furrowing when forced into mitotic exit. Because protein regulator of cytokinesis (PRC1) and kinesin family member 4A (KIF4A) are believed to play a principal role in organizing the antiparallel midzone array, we sought to clarify their roles in monopolar cytokinesis. Although both factors localized to the distal ends of microtubules during monopolar cytokinesis, depletion of PRC1 and KIF4A displayed different phenotypes. Cells depleted of PRC1 failed to form a polarized microtubule array or ectopic furrows following mitotic exit, and recruitment of Aurora B kinase, male germ cell Rac GTPase-activating protein, and RhoA to the cortex was impaired. In contrast, KIF4A depletion impaired neither polarization nor ectopic furrowing, but it did result in elongated spindles with a diffuse distribution of cytokinetic factors. Thus, even in the absence of spindle bipolarity, PRC1 appears to be essential for polarizing parallel microtubules and concentrating the factors responsible for contractile ring assembly, whereas KIF4A is required for limiting the length of anaphase microtubules.