The nuclear kinase Lsk1p positively regulates the septation initiation network and promotes the successful completion of cytokinesis in response to perturbation of the actomyosin ring in Schizosaccharomyces pombe

Cytokinesis in fission yeast requires the function of an actomyosin-based contractile ring whose constriction is dependent on a signaling module termed the septation initiation network (SIN). In response to minor perturbation of the ring, the duration of SIN signaling is extended concurrently with a delay in nuclear cycle progression. These mechanisms require the conserved phosphatase Clp1p/Flp1p and facilitate the successful completion of cytokinesis, thereby increasing cellular viability. To isolate novel components of this cytokinesis monitoring system, we screened a genome-wide bank of protein kinase deletion mutants and identified Lsk1p, a nuclear-localized protein kinase. Similar to clp1Δ mutants, and in contrast to wild type, lsk1Δ cells are unable to maintain the integrity of the actomyosin ring upon treatment with low doses (0.3 μM) of latrunculin A. However, unlike clp1Δ mutants, lsk1Δ cells are competent to delay nuclear cycle progression after cytokinetic failure. In addition, lsk1Δ mutants suppress the lethal, multiseptate phenotype conferred by hyperactivation of the SIN, demonstrating that Lsk1p is a positive regulator of this module. In this report, we demonstrate that Lsk1p acts in parallel to Clp1p to promote actomyosin ring stability upon checkpoint activation. Our studies also establish that actomyosin ring maintenance and nuclear cycle delay in response to cytokinetic perturbation can be genetically resolved into independent pathways.     

Distinct nuclear and cytoplasmic functions of the S. pombe Cdc14-like phosphatase Clp1p/Flp1p and a role for nuclear shuttling in its regulation

Cdc14-like phosphatases regulate a variety of cell cycle events by dephosphorylating CDK sites. Their cell cycle-dependent changes in localization may be important to carry out distinct functions. Work in budding and fission yeast suggested that Cdc14-like phosphatases are inhibited by nucleolar sequestration. In S. cerevisiae, Cdc14p is released from the nucleolus by the FEAR network and Cdk1, whereas the S. pombe CDC14-like phosphatase Clp1p (also known as Flp1p) is released at mitotic entry by an unknown mechanism. The mitotic exit network (MEN) in S. cerevisiae and its homologous network, the septation initiation network (SIN), in S. pombe act through an unknown mechanism to keep the phosphatase out of the nucleolus in late mitosis. SIN-dependent cytoplasmic maintenance of Clp1p is thought to be essential for the cytokinesis checkpoint, which blocks further rounds of nuclear division until cytokinesis is completed. By targeting Clp1p to the nucleus or the cytoplasm, we demonstrate distinct functions for these pools of Clp1p in chromosome segregation and cytokinesis, respectively. Our results further suggest that the SIN does not keep Clp1p out of the nucleolus by regulating nucleolar affinity, as proposed for S. cerevisiae Cdc14p, but instead, Clp1p may be regulated by nuclear import/export.     

Cell cycle-dependent roles for the FCH-domain protein Cdc15p in formation of the actomyosin ring in Schizosaccharomyces pombe

Cell division in the fission yeast Schizosaccharomyces pombe requires the formation and constriction of an actomyosin ring at the division site. The actomyosin ring is assembled in metaphase and anaphase A, is maintained throughout mitosis, and constricts after completion of anaphase. Maintenance of the actomyosin ring during late stages of mitosis depends on the septation initiation network (SIN), a signaling cascade that also regulates the deposition of the division septum. However, SIN is not active in metaphase and is not required for the initial assembly of the actomyosin ring early in mitosis. The FER/CIP4-homology (FCH) domain protein Cdc15p is a component of the actomyosin ring. Mutations in cdc15 lead to failure in cytokinesis and result in the formation of elongated, multinucleate cells without a division septum. Here we present evidence that the requirement of Cdc15p for actomyosin ring formation is dependent on the stage of mitosis. Although cdc15 mutants are competent to assemble actomyosin rings in metaphase, they are unable to maintain actomyosin rings late in mitosis when SIN is active. In the absence of functional Cdc15p, ring formation upon metaphase arrest depends on the anillin-like Mid1p. Interestingly, when cytokinesis is delayed due to perturbations to the division machinery, Cdc15p is maintained in a hypophosphorylated form. The dephosphorylation of Cdc15p, which occurs transiently in unperturbed cytokinesis, is partially dependent on the phosphatase Clp1p/Flp1p. This suggests a mechanism where both SIN and Clp1p/Flp1p contribute to maintenance of the actomyosin ring in late mitosis through Cdc15p, possibly by regulating its phosphorylation status.     

The S. pombe Cdc14-like phosphatase Clp1p regulates chromosome biorientation and interacts with Aurora kinase

The S. pombe Cdc14-related phosphatase Clp1p/Flp1p regulates G2/M transition by antagonizing CDK activity and is essential for coordinating the nuclear division cycle with cytokinesis through the cytokinesis checkpoint. At the G2/M transition, Clp1p/Flp1p is released from the nucleolus and SPB and distributes throughout the nucleus to the spindle and the contractile ring. This early relocalization is analogous to vertebrate Cdc14 homologs and stands in contrast to S. cerevisiae Cdc14p, which is not released from the nucleolus until metaphase/anaphase transition. Here, we report that Clp1p/Flp1p localizes to kinetochores in prometaphase and functions in chromosome segregation, since deletion of clp1/flp1 causes cosegregation of sister chromatids, when sister kinetochores are prone to mono-orientation. Genetic, cytological, and biochemical experiments suggest that Clp1p/Flp1p functions together with Aurora kinase at kinetochores. Together, these results suggest that Clp1p/Flp1p has a role in repairing mono-orientation of sister kinetochores.     

Cds1 controls the release of Cdc14-like phosphatase Flp1 from the nucleolus to drive full activation of the checkpoint response to replication stress in fission yeast

The Cdc14p-like phosphatase Flp1p (also known as Clp1p) is regulated by cell cycle-dependent changes in its subcellular localization. Flp1p is restricted to the nucleolus and spindle pole body until prophase, when it is dispersed throughout the nucleus, mitotic spindle, and medial ring. Once released, Flp1p antagonizes Cdc2p/cyclin activity by reverting Cdc2p-phosphorylation sites on Cdc25p. On replication stress, ataxia-telangiectasia mutated/ATM/Rad3-related kinase Rad3p activates Cds1p, which phosphorylates key proteins ensuring the stability of stalled DNA replication forks. Here, we show that replication stress induces changes in the subcellular localization of Flp1p in a checkpoint-dependent manner. Active Cds1p checkpoint kinase is required to release Flp1p into the nucleus. Consistently, a Flp1p mutant (flp1-9A) lacking all potential Cds1p phosphorylation sites fails to relocate in response to replication blocks and, similarly to cells lacking flp1 (Deltaflp1), presents defects in checkpoint response to replication stress. Deltaflp1 cells accumulate reduced levels of a less active Cds1p kinase in hydroxyurea (HU), indicating that nuclear Flp1p regulates Cds1p full activation. Consistently, Deltaflp1 and flp1-9A have an increased percentage of Rad22p-recombination foci during HU treatment. Together, our data show that by releasing Flp1p into the nucleus Cds1p checkpoint kinase modulates its own full activation during replication stress.     

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.     

The SIN kinase Sid2 regulates cytoplasmic retention of the S. pombe Cdc14-like phosphatase Clp1

Cdc14-family phosphatases play a conserved role in promoting mitotic exit and cytokinesis by dephosphorylating substrates of cyclin-dependent kinase (Cdk). Cdc14-family phosphatases have been best studied in yeast (for review, see [1, 2]), where budding yeast Cdc14 and its fission yeast homolog Clp1 are regulated partly by their localization; both proteins are thought to be sequestered in the nucleolus in interphase. Cdc14 and Clp1 are released from the nucleolus in mitosis, and in late mitosis conserved signaling pathways termed the mitotic exit network (MEN) and the septation initiation network (SIN) keeps Cdc14 and Clp1, respectively, out of the nucleolus through an unknown mechanism [3-6]. Here we show that the most downstream SIN component, the Ndr-family kinase Sid2, maintains Clp1 in the cytoplasm in late mitosis by phosphorylating Clp1 directly and thereby creating binding sites for the 14-3-3 protein Rad24. Mutation of the Sid2 phosphorylation sites on Clp1 disrupts the Clp1-Rad24 interaction and causes Clp1 to return prematurely to the nucleolus during cytokinesis. Loss of Clp1 from the cytoplasm in telophase renders cells sensitive to perturbation of the actomyosin ring but does not affect other Clp1 functions. Because all components of this pathway are conserved, this might be a broadly conserved mechanism for regulation of Cdc14-family phosphatases.     

Nuc2p, a subunit of the anaphase-promoting complex, inhibits septation initiation network following cytokinesis in fission yeast

In most cell types, mitosis and cytokinesis are tightly coupled such that cytokinesis occurs only once per cell cycle. The fission yeast Schizosaccharomyces pombe divides using an actomyosin-based contractile ring and is an attractive model for the study of the links between mitosis and cytokinesis. In fission yeast, the anaphase-promoting complex/cyclosome (APC/C) and the septation initiation network (SIN), a spindle pole body (SPB)–associated GTPase-driven signaling cascade, function sequentially to ensure proper coordination of mitosis and cytokinesis. Here, we find a novel interplay between the tetratricopeptide repeat (TPR) domain–containing subunit of the APC/C, Nuc2p, and the SIN, that appears to not involve other subunits of the APC/C. Overproduction of Nuc2p led to an increase in the presence of multinucleated cells, which correlated with a defect in actomyosin ring maintenance and localization of the SIN component protein kinases Cdc7p and Sid1p to the SPBs, indicative of defective SIN signaling. Conversely, loss of Nuc2p function led to increased SIN signaling, characterized by the persistent localization of Cdc7p and Sid1p on SPBs and assembly of multiple actomyosin rings and division septa. Nuc2p appears to function independently of the checkpoint with FHA and ring finger (CHFR)–related protein Dma1p, a known inhibitor of the SIN in fission yeast. Genetic and biochemical analyses established that Nuc2p might influence the nucleotide state of Spg1p GTPase, a key regulator of the SIN. We propose that Nuc2p, by inhibiting the SIN after cell division, prevents further deleterious cytokinetic events, thereby contributing to genome stability.     

The localization of the integral membrane protein Cps1p to the cell division site is dependent on the actomyosin ring and the septation-inducing network in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells divide by medial fission through the use of an actomyosin-based contractile ring. Constriction of the actomyosin ring is accompanied by the centripetal addition of new membranes and cell wall material. In this article, we characterize the mechanism responsible for the localization of Cps1p, a septum-synthesizing 1,3-beta-glucan synthase, to the division site during cytokinesis. We show that Cps1p is an integral membrane protein that localizes to the cell division site late in anaphase. Neither F-actin nor microtubules are essential for the initial assembly of Cps1p to the medial division site. F-actin, but not microtubules, is however important for the eventual incorporation of Cps1p into the actomyosin ring. Assembly of Cps1p into the cell division ring is also dependent on the septation-inducing network (SIN) proteins that regulate division septum formation after assembly of the actomyosin ring. Fluorescence-recovery after-photobleaching experiments reveal that Cps1p does not diffuse appreciably within the plasma membrane and is retained at the division site by a mechanism that does not depend on an intact F-actin cytoskeleton. We conclude that the actomyosin ring serves as a spatial cue for Cps1p localization, whereas the maintenance of Cps1p at the division site occurs by a novel F-actin- and microtubule-independent mechanism. Furthermore, we propose that the SIN proteins ensure localization of Cps1p at the appropriate point in the cell cycle.     

The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast

The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3⁺ and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation.     

Vrp1p functions in both actomyosin ring-dependent and Hof1p-dependent pathways of cytokinesis

Vrp1p/verprolin/End5p is a Saccharomyces cerevisiae proline-rich protein, structurally and functionally related to human Wiskott–Aldrich syndrome protein-interacting protein. Vrp1p is required for viability at 37°C, but not 24°C. Here, we show that loss of Vrp1p ( vrp1Δ ) leads to a 3–4-fold delay in cytokinesis, wide bud necks, abnormal actomyosin rings, and aberrant septa even at 24°C. Like other mutations affecting the actomyosin ring, vrp1Δ is synthetic lethal with deletion of HOF1 (or CYK2 ), which encodes a protein related to mammalian proline serine threonine phosphatase-interacting protein and Schizosaccharomyces pombe Cdc15p required for an actomyosin ring-independent pathway of cytokinesis in S. cerevisiae . At 37°C, vrp1Δ cells rapidly cease dividing and exhibit a novel terminal phenotype: a single large bud, two well-separated nuclei, and an interphase microtubule array. The arrested cells have a persistent ring containing both actin and myosin at the bud neck. Many also exhibit some polarisation of cortical actin patches to the bud neck. Vrp1p binds an SH3-domain-containing fragment of Hof1p in vitro . Vrp1p is required in vivo for Hof1p relocalisation to a single ring at the bud neck prior to cytokinesis at 37°C, but not at 24°C. Vrp1p thus acts in both actomyosin ring formation and function, as well as in Hof1p localisation during cytokinesis.     

Mec1p is essential for phosphorylation of the yeast DNA damage checkpoint protein Ddc1p, which physically interacts with Mec3p.

Checkpoints prevent DNA replication or nuclear division when chromosomes are damaged. The Saccharomyces cerevisiae DDC1 gene belongs to the RAD17, MEC3 and RAD24 epistasis group which, together with RAD9, is proposed to act at the beginning of the DNA damage checkpoint pathway. Ddc1p is periodically phosphorylated during unperturbed cell cycle and hyperphosphorylated in response to DNA damage. We demonstrate that Ddc1p interacts physically in vivo with Mec3p, and this interaction requires Rad17p. We also show that phosphorylation of Ddc1p depends on the key checkpoint protein Mec1p and also on Rad24p, Rad17p and Mec3p. This suggests that Mec1p might act together with the Rad24 group of proteins at an early step of the DNA damage checkpoint response. On the other hand, Ddc1p phosphorylation is independent of Rad53p and Rad9p. Moreover, while Ddc1p is required for Rad53p phosphorylation, it does not play any major role in the phosphorylation of the anaphase inhibitor Pds1p, which requires RAD9 and MEC1. We suggest that Rad9p and Ddc1p might function in separated branches of the DNA damage checkpoint pathway, playing different roles in determining Mec1p activity and/or substrate specificity.     

Etd1p is a novel protein that links the SIN cascade with cytokinesis

In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast cells, ring constriction is triggered by the septum initiation network (SIN), an SPB-associated GTPase-regulated kinase cascade that coordinates exit from mitosis with cytokinesis. We have identified a novel protein, Etd1p, required to trigger actomyosin ring constriction in fission yeasts. This protein is localised at the cell tips during interphase. In mitosis, it relocates to the medial cortex region and, coincident with cytokinesis, it assembles into the actomyosin ring by association to Cdc15p. Relocation of Etd1p from the plasma membrane to the medial ring is triggered by SIN signalling and, reciprocally, relocation of the Sid2p-Mob1p kinase complex from the SPB to the division site, a late step in the execution of the SIN, requires Etd1p. These results suggest that Etd1p coordinates the mitotic activation of SIN with the initiation of actomyosin ring constriction. Etd1p peaks during cytokinesis and is degraded by the ubiquitin-dependent 26S-proteasome pathway at the end of septation, providing a mechanism to couple inactivation of SIN to completion of cytokinesis.     

The Clp1/Cdc14 phosphatase contributes to the robustness of cytokinesis by association with anillin-related Mid1

Cdc14 phosphatases antagonize cyclin-dependent kinase-directed phosphorylation events and are involved in several facets of cell cycle control. We investigate the role of the fission yeast Cdc14 homologue Clp1/Flp1 in cytokinesis. We find that Clp1/Flp1 is tethered at the contractile ring (CR) through its association with anillin-related Mid1. Fluorescent recovery after photobleaching analyses indicate that Mid1, unlike other tested CR components, is anchored at the cell midzone, and this physical property is likely to account for its scaffolding role. By generating a mutation in mid1 that selectively disrupts Clp1/Flp1 tethering, we reveal the specific functional consequences of Clp1/Flp1 activity at the CR, including dephosphorylation of the essential CR component Cdc15, reductions in CR protein mobility, and CR resistance to mild perturbation. Our evidence indicates that Clp1/Flp1 must interact with the Mid1 scaffold to ensure the fidelity of Schizosaccharomyces pombe cytokinesis.     

Schizosaccharomyces pombe Pxl1 is a paxillin homologue that modulates Rho1 activity and participates in cytokinesis

Schizosaccharomyces pombe Rho GTPases regulate actin cytoskeleton organization and cell integrity. We studied the fission yeast gene SPBC4F6.12 based on its ability to suppress the thermosensitivity of cdc42-1625 mutant strain. This gene, named pxl1(+), encodes a protein with three LIM domains that is similar to paxillin. Pxl1 does not interact with Cdc42 but it interacts with Rho1, and it negatively regulates this GTPase. Fission yeast Pxl1 forms a contractile ring in the cell division region and deletion of pxl1(+) causes a delay in cell-cell separation, suggesting that it has a function in cytokinesis. Pxl1 N-terminal region is required and sufficient for its localization to the medial ring, whereas the LIM domains are necessary for its function. Pxl1 localization requires actin polymerization and the actomyosin ring, but it is independent of the septation initiation network (SIN) function. Moreover, Pxl1 colocalizes and interacts with Myo2, and Cdc15, suggesting that it is part of the actomyosin ring. Here, we show that in cells lacking Pxl1, the myosin ring is not correctly assembled and that actomyosin ring contraction is delayed. Together, these data suggest that Pxl1 modulates Rho1 GTPase signaling and plays a role in the formation and contraction of the actomyosin ring during cytokinesis.     

Characterization of the roles of Blt1p in fission yeast cytokinesis

Spatial and temporal regulation of cytokinesis is essential for cell division, yet the mechanisms that control the formation and constriction of the contractile ring are incompletely understood. In the fission yeast Schizosaccharomyces pombe proteins that contribute to the cytokinetic contractile ring accumulate during interphase in nodes—precursor structures around the equatorial cortex. During mitosis, additional proteins join these nodes, which condense to form the contractile ring. The cytokinesis protein Blt1p is unique in being present continuously in nodes from early interphase through to the contractile ring until cell separation. Blt1p was shown to stabilize interphase nodes, but its functions later in mitosis were unclear. We use analytical ultracentrifugation to show that purified Blt1p is a tetramer. We find that Blt1p interacts physically with Sid2p and Mob1p, a protein kinase complex of the septation initiation network, and confirm known interactions with F-BAR protein Cdc15p. Contractile rings assemble normally in blt1∆ cells, but the initiation of ring constriction and completion of cell division are delayed. We find three defects that likely contribute to this delay. Without Blt1p, contractile rings recruited and retained less Sid2p/Mob1p and Clp1p phosphatase, and β-glucan synthase Bgs1p accumulated slowly at the cleavage site.     

Aim44p regulates phosphorylation of Hof1p to promote contractile ring closure during cytokinesis in budding yeast

Whereas actomyosin and septin ring organization and function in cytokinesis are thoroughly described, little is known regarding the mechanisms by which the actomyosin ring interacts with septins and associated proteins to coordinate cell division. Here we show that the protein product of YPL158C, Aim44p, undergoes septin-dependent recruitment to the site of cell division. Aim44p colocalizes with Myo1p, the type II myosin of the contractile ring, throughout most of the cell cycle. The Aim44p ring does not contract when the actomyosin ring closes. Instead, it forms a double ring that associates with septin rings on mother and daughter cells after cell separation. Deletion of AIM44 results in defects in contractile ring closure. Aim44p coimmunoprecipitates with Hof1p, a conserved F-BAR protein that binds both septins and type II myosins and promotes contractile ring closure. Deletion of AIM44 results in a delay in Hof1p phosphorylation and altered Hof1p localization. Finally, overexpression of Dbf2p, a kinase that phosphorylates Hof1p and is required for relocalization of Hof1p from septin rings to the contractile ring and for Hof1p-triggered contractile ring closure, rescues the cytokinesis defect observed in aim44∆ cells. Our studies reveal a novel role for Aim44p in regulating contractile ring closure through effects on Hof1p.