S. pombe cdc11p, together with sid4p, provides an anchor for septation initiation network proteins on the spindle pole body.

BACKGROUND: The signal for the onset of septum formation in the fission yeast Schizosaccharomyces pombe is transduced by the septation initiation network (SIN). Many of the components of the SIN are located on the spindle pole body during mitosis, from where it is presumed that the signal for septum formation is delivered. Cdc11 mutants are defective in SIN signaling, but the role of cdc11 in the pathway has remained enigmatic. RESULTS: We have cloned the cdc11 gene by a combination of chromosome walking and transfection of cosmids into a cdc11 mutant. Cdc11p most closely resembles Saccharomyces cerevisiae Nud1p and is essential for septum formation. Cdc11p is a phosphoprotein, which becomes hyperphosphorylated during anaphase. It localizes to the spindle pole body at all stages of the cell cycle, in a sid4p-dependent manner, and cdc11p is required for the localization of all the known SIN components, except sid4p, to the SPB. Cdc11p and sid4p can be coimmunoprecipitated from cell extracts. Finally, like its S. cerevisiae ortholog Nud1p, cdc11p is involved in the proper organization of astral microtubules during mitosis. CONCLUSIONS: We propose that cdc11p acts as a bridge between sid4p and the other SIN proteins, mediating their association with the spindle pole body.     

Mitotic hyperphosphorylation of the fission yeast SIN scaffold protein cdc11p is regulated by the protein kinase cdc7p

The fission yeast septation initiation network (SIN) triggers the onset of septum formation and cytokinesis. SIN proteins signal from the spindle pole body (SPB), to which they bind in a cell cycle-dependent manner, via the scaffold proteins sid4p and cdc11p. cdc11p becomes hyperphosphorylated during anaphase, when the SIN is active. We have investigated the phosphorylation state of cdc11p during mitosis in various mutant backgrounds. We show that association of cdc11p with the spindle pole body is required for its phosphorylation and that ectopic activation of the SIN results in hyperphosphorylation of cdc11p. We demonstrate that mitotic hyperphosphorylation of cdc11p requires the activity of cdc7p and that its dephosphorylation at the end of mitosis requires PP2A-par1p. Furthermore, spindle checkpoint arrest prevents cdc11p hyperphosphorylation. Finally, we show that the septation inhibitor byr4p interacts preferentially with hypophosphorylated cdc11p. We conclude that cdc11p hyperphosphorylation correlates with activation of the SIN and that this may be mediated primarily by cdc7p in vivo.     

Sid4p-Cdc11p assembles the septation initiation network and its regulators at the S. pombe SPB

The Schizosaccharomyces pombe septation initiation network (SIN) triggers actomyosin ring constriction, septation, and cell division. It is organized at the spindle pole body (SPB) by the scaffold proteins Sid4p and Cdc11p. Here, we dissect the contributions of Sid4p and Cdc11p in anchoring SIN components and SIN regulators to the SPB. We find that Sid4p interacts with the SIN activator, Plo1p, in addition to Cdc11p and Dma1p. While the C terminus of Cdc11p is involved in binding Sid4p, its N-terminal half is involved in a wide variety of direct protein-protein interactions, including those with Spg1p, Sid2p, Cdc16p, and Cdk1p-Cdc13p. Given that the localizations of the remaining SIN components depend on Spg1p or Cdc16p, these data allow us to build a comprehensive model of SIN component organization at the SPB. FRAP experiments indicate that Sid4p and Cdc11p are stable SPB components, whereas signaling components of the SIN are dynamically associated with these structures. Our results suggest that the Sid4p-Cdc11p complex organizes a signaling hub on the SPB and that this hub coordinates cell and nuclear division.     

The Scw1 RNA-binding domain protein regulates septation and cell-wall structure in fission yeast

Loss of the nonessential RNA-binding domain protein, Scw1, increases resistance to cell-wall-degrading enzymes in fission yeast. Surprisingly, scw1 null mutations also suppress the lethality of mutations (cdc11-136, cdc7-24, cdc14-118, sid1-239, sid2-250, sid3-106, sid4-A1, and mob1-1) at all levels of the sid pathway. This pathway forms part of the septation initiation network (SIN), which regulates the onset of septum formation and ensures the proper coupling of mitosis to cytokinesis. In contrast, scw1(-) mutations do not suppress ts alleles of the rng genes, cdc12 or cdc15. These mutations also prevent the formation of a septum and in addition block assembly and/or function of the contractile acto-myosin ring. sid mutants exhibit a hyper-sensitivity to cell-wall-degrading enzymes that is suppressed by loss of Scw1. Furthermore, scw1(-)-mediated rescue of sid mutants is abolished in the presence of calcofluor white, a compound that interferes with cell-wall synthesis. These data suggest that Scw1 acts in opposition to the SIN as a negative regulator of cell-wall/septum deposition. Unlike components of the SIN, Scw1 is predominantly a cytoplasmic protein and is not localized to the spindle pole body.     

The spindle pole body protein Cdc11p links Sid4p to the fission yeast septation initiation network

The Schizosaccharomyces pombe septation initiation network (SIN) signals the onset of cell division from the spindle pole body (SPB) and is regulated by the small GTPase Spg1p. The localization of SIN components including Spg1p to the SPB is required for cytokinesis and is dependent on Sid4p, a constitutive resident of SPBs. However, a direct interaction between Sid4p and other members of the SIN has not been detected. To understand how Sid4p is linked to other SIN components, we have begun to characterize an S. pombe homolog of the Saccharomyces cerevisiae SPB protein Nud1p. We have determined that this S. pombe Nud1p homolog corresponds to Cdc11p, a previously uncharacterized SIN element. We report that Cdc11p is present constitutively at SPBs and that its function appears to be required for the localization of all other SIN components to SPBs with the exception of Sid4p. The Cdc11p C terminus localizes the protein to SPBs in a Sid4p-dependent manner, and we demonstrate a direct Cdc11p-Sid4p interaction. The N-terminus of Cdc11p is required for Spg1p binding to SPBs. Our studies indicate that Cdc11p provides a physical link between Sid4p and the Spg1p signaling pathway.     

The role of the sid1p kinase and cdc14p in regulating the onset of cytokinesis in fission yeast

Coordination of mitosis and cytokinesis is crucial for ensuring proper chromosome segregation and genomic stability. In Schizosaccharomyces pombe, the sid genes (cdc7, cdc11, cdc14, spg1, sid1, sid2 and sid4) define a signaling pathway that regulates septation and cytokinesis. Here we describe the characterization of a novel protein kinase, Sid1p. Sid1p localizes asymmetrically to one spindle pole body (SPB) in anaphase. Sid1p localization is maintained during medial ring constriction and septum synthesis and disappears prior to cell separation. Additionally, we found that Cdc14p is in a complex with Sid1p. Epistasis analysis places Sid1p-Cdc14p downstream of Spg1p-Cdc7p but upstream of Sid2p. Finally, we show that cyclin proteolysis during mitosis is unaffected by inactivating the sid pathway; in fact, loss of Cdc2-cyclin activity promotes Sid1p-Cdc14p association with the SPB, possibly providing a mechanism that couples cytokinesis with mitotic exit.     

Mob1p interacts with the Sid2p kinase and is required for cytokinesis in fission yeast

A great deal is now known about how cells regulate entry into mitosis, but only recently have the mechanisms controlling exit from mitosis and cytokinesis begun to be revealed. In the budding yeast Saccharomyces cerevisiae, Mob1p interacts with the Dbf2p kinase and cells containing mutations in these genes arrest in late anaphase [1] [2]. Proteins related to Mob1p are present in both plants and animals, but information about Mob1p function has been obtained only from budding yeast. Here, we describe the identification and characterization of Mob1p from Schizosaccharomyces pombe. Mob1p associates with the Sid2p kinase and like Sid2p, Mob1p is required for the initiation of cytokinesis, but not for mitotic exit. Mob1p localizes to the spindle pole body (SPB) and to the cell-division site during cell division, suggesting that it might be involved in transducing the signal to initiate cell division from the SPB to the division site. Mob1p is required for Sid2p localization, and Mob1p localization requires the function of the cdc7, cdc11, cdc14, spg1, sid1, sid2, and sid4 genes, suggesting that together with Sid2p, Mob1p functions at the end of the signaling cascade required to regulate the onset of cytokinesis at the end of mitosis.     

Localization of Fission Yeast Type II Myosin, Myo2, to the Cytokinetic Actin Ring Is Regulated by Phosphorylation of a C-Terminal Coiled-Coil Domain and Requires a Functional Septation Initiation Network

Myo2 truncations fused to green fluorescent protein (GFP) defined a C-terminal domain essential for the localization of Myo2 to the cytokinetic actin ring (CAR). The localization domain contained two predicted phosphorylation sites. Mutation of serine 1518 to alanine (S(1518)A) abolished Myo2 localization, whereas Myo2 with a glutamic acid at this position (S(1518)E) localized to the CAR. GFP-Myo2 formed rings in the septation initiation kinase (SIN) mutant cdc7-24 at 25 degrees C but not at 36 degrees C. GFP-Myo2S(1518)E rings persisted at 36 degrees C in cdc7-24 but not in another SIN kinase mutant, sid2-250. To further examine the relationship between Myo2 and the SIN pathway, the chromosomal copy of myo2(+) was fused to GFP (strain myo2-gc). Myo2 ring formation was abolished in the double mutants myo2-gc cdc7.24 and myo2-gc sid2-250 at the restrictive temperature. In contrast, activation of the SIN pathway in the double mutant myo2-gc cdc16-116 resulted in the formation of Myo2 rings which subsequently collapsed at 36 degrees C. We conclude that the SIN pathway that controls septation in fission yeast also regulates Myo2 ring formation and contraction. Cdc7 and Sid2 are involved in ring formation, in the case of Cdc7 by phosphorylation of a single serine residue in the Myo2 tail. Other kinases and/or phosphatases may control ring contraction.     

The fission yeast septation initiation network (SIN) kinase, Sid2, is required for SIN asymmetry and regulates the SIN scaffold, Cdc11

The Schizosaccharomyces pombe septation initiation network (SIN) is an Spg1-GTPase-mediated protein kinase cascade that triggers actomyosin ring constriction, septation, and cell division. The SIN is assembled at the spindle pole body (SPB) on the scaffold proteins Cdc11 and Sid4, with Cdc11 binding directly to SIN signaling components. Proficient SIN activity requires the asymmetric distribution of its signaling components to one of the two SPBs during anaphase, and Cdc11 hyperphosphorylation correlates with proficient SIN activity. In this paper, we show that the last protein kinase in the signaling cascade, Sid2, feeds back to phosphorylate Cdc11 during mitosis. The characterization of Cdc11 phosphomutants provides evidence that Sid2-mediated Cdc11 phosphorylation promotes the association of the SIN kinase, Cdc7, with the SPB and maximum SIN signaling during anaphase. We also show that Sid2 is crucial for the establishment of SIN asymmetry, indicating a positive-feedback loop is an important element of the SIN.     

Correct regulation of the septation initiation network in Schizosaccharomyces pombe requires the activities of par1 and par2

In Schizosaccharomyces pombe, the initiation of cytokinesis is regulated by a septation initiation network (SIN). We previously reported that deletion of par1 and par2, two S. pombe genes encoding B' regulatory subunits of protein phosphatase 2A, causes a multiseptation phenotype, very similar to that seen in hyperactive SIN mutants. In this study, we examined the genetic interactions between par deletions and mutations in the genes encoding components of SIN and found that deletion of par1 and par2 suppressed the morphological and viability defects caused by overproduction of Byr4p and rescued a loss-of-function allele of spg1. However, par deletions could not suppress any mutations in genes downstream of spg1 in the SIN pathway. We showed further that, in suppressing the lethality of a spg1 loss-of-function allele, the correct localization of Cdc7p to the spindle pole body (SPB), which is normally lost in spg1 mutant cells, was restored. The fact that par mutant cells themselves exhibited a symmetric localization of Cdc7p to SPBs indicated a hyperactivity of SIN in such cells. On the basis of our epistasis analyses and cytological studies, we concluded that par genes normally negatively regulate SIN at or upstream of cdc7, ensuring that multiple rounds of septation do not occur.     

Ppc89 links multiple proteins, including the septation initiation network, to the core of the fission yeast spindle-pole body

The spindle-pole body (SPB), the yeast analog of the centrosome, serves as the major microtubule (MT) organizing center in the yeast cell. In addition to this central function, the SPB organizes and concentrates proteins required for proper coordination between the nuclear-division cycle and cytokinesis. For example, the Schizosaccharomyces pombe septation-initiation network (SIN), which is responsible for initiating actomyosin ring constriction and septation, is assembled at the SPB through its two scaffolding components, Sid4 and Cdc11. In an effort to identify novel SIN interactors, we purified Cdc11 and identified by mass spectrometry a previously uncharacterized protein associated with it, Ppc89. Ppc89 localizes constitutively to the SPB and interacts directly with Sid4. A fusion between the N-terminal 300 amino acids of Sid4 and a SPB targeting domain of Ppc89 supplies the essential function of Sid4 in anchoring the SIN. ppc89Delta cells are inviable and exhibit defects in SPB integrity, and hence in spindle formation, chromosome segregation, and SIN localization. Ppc89 overproduction is lethal, resulting primarily in a G2 arrest accompanied by massive enlargement of the SPB and increased SPB MT nucleation. These results suggest a fundamental role for Ppc89 in organization of the S. pombe SPB.     

The protein phosphatase 2A B'-regulatory subunit par1p is implicated in regulation of the S. pombe septation initiation network.

In order to identify regulators of the Schizosaccharomyces pombe septation initiation network (SIN), which signals the onset of cell division, we have isolated extragenic suppressors of mutations in the GTPase spg1p, which is a central element in this pathway. One of these encodes the protein phosphatase 2A (PP2A) B'-regulatory subunit par1p. Loss of par1p function rescues mutants in cdc11, cdc7, and spg1, but no other SIN mutants. Our data suggest that PP2A-par1p acts as a negative regulator of SIN signalling.     

Sid2p, a spindle pole body kinase that regulates the onset of cytokinesis.

The fission yeast Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin contractile ring. Precisely at the end of anaphase, the ring begins to constrict and the septum forms. Proper coordination of cell division with mitosis is crucial to ensure proper segregation of chromosomes to daughter cells. The Sid2p kinase is one of several proteins that function as part of a novel signaling pathway required for initiation of medial ring constriction and septation. Here, we show that Sid2p is a component of the spindle pole body at all stages of the cell cycle and localizes transiently to the cell division site during medial ring constriction and septation. A medial ring and an intact microtubule cytoskeleton are required for the localization of Sid2p to the division site. We have established an in vitro assay for measuring Sid2p kinase activity, and found that Sid2p kinase activity peaks during medial ring constriction and septation. Both Sid2p localization to the division site and activity depend on the function of all of the other septation initiation genes: cdc7, cdc11, cdc14, sid1, spg1, and sid4. Thus, Sid2p, a component of the spindle pole body, by virtue of its transient localization to the division site, appears to determine the timing of ring constriction and septum delivery in response to activating signals from other Sid gene products.     

Timely Septation Requires SNAD-dependent Spindle Pole Body Localization of the Septation Initiation Network Components in the Filamentous Fungus Aspergillus nidulans

In the filamentous fungus Aspergillus nidulans, cytokinesis/septation is triggered by the septation initiation network (SIN), which first appears at the spindle pole body (SPB) during mitosis. The coiled-coil protein SNAD is associated with the SPB and is required for timely septation and conidiation. We have determined that SNAD acted as a scaffold protein that is required for the localization of the SIN proteins of SIDB and MOBA to the SPB. Another scaffold protein SEPK, whose localization at the SPB was dependent on SNAD, was also required for SIDB and MOBA localization to the SPB. In the absence of either SEPK or SNAD, SIDB/MOBA successfully localized to the septation site, indicating that their earlier localization at SPB was not essential for their later appearance at the division site. Unlike their functional counterparts in fission yeast, SEPK and SNAD were not required for vegetative growth but only for timely septation. Furthermore, down-regulation of negative regulators of the SIN suppressed the septation and conidiation phenotypes due to the loss of SNAD. Therefore, we conclude that SPB localization of SIN components is not essential for septation per se, but critical for septation to take place in a timely manner in A. nidulans.     

Fission yeast cdc31p is a component of the half-bridge and controls SPB duplication

The fission yeast spindle pole body (SPB) is a nucleus-associated organelle that duplicates once each cell cycle during interphase. Duplicated SPBs serve as the poles of an intranuclear mitotic spindle after their insertion into the nuclear envelope in mitosis (Ding et al., Mol. Biol. Cell 8, 1461-1479). Here, we report the identification and characterization of Schizosaccharomyces pombe cdc31p, a member of the conserved calcium-binding centrin/CDC31 family. Immunofluorescence and immunoelectron microscopy show that cdc31p is a SPB component localized at the half-bridge structure of the SPB. cdc31 is an essential gene and Deltacdc31 cells and cdc31 conditional mutant cells arrest in mitosis with a monopolar mitotic spindle organized from a single SPB. EM analysis demonstrates that mutant cdc31 cells fail to duplicate the SPB. In addition, cdc31p exhibits genetic interactions with the SPB component sad1p and is required for sad1p localization. Finally, cdc31 mutant can undergo single or multiple rounds of septation before the exit from mitosis, suggesting that cdc31p activity or SPB duplication may be required for the proper coordination between the exit from mitosis and the initiation of septation.     

Initiation of cytokinesis is controlled through multiple modes of regulation of the Sid2p-Mob1p kinase complex

The Sid2p-Mob1p kinase complex is an important component of the septation initiation network (SIN) in the fission yeast Schizosaccharomyces pombe. However, regulation of this complex is still elusive. Here we show that Mob1p is required not only for the subcellular localization of Sid2p but also for its kinase activity. We identified a region at the amino terminus of Sid2p that is required for Mob1p binding and spindle pole body (SPB) localization. Deletion of this region abolishes Mob1p binding and diminishes SPB localization, whereas this region alone is sufficient to associate with Mob1p and SPBs. We further show that a similar region of the N terminus of the Sid2p-related protein kinase Orb6p binds to the Mob1p-related protein Mob2p, suggesting that this may be a conserved mode of interaction for this family of kinases. Phosphorylation of Ser402 and especially Thr578 is important for Sid2p function. Sid2p with a mutation of Thr578 to Ala (T578A) can no longer rescue sid2-250 mutant cells, and this results in reduction of Mob1p binding. Sid2p mutants mimicking phosphorylation at this site (T578D and T578E) can rescue sid2-250 cells, enhance Sid2p kinase activity, and partially rescue growth defects of upstream sin mutants. Interestingly, Sid2p, but not Mob1p, is self-associated. Our experiments suggest that self-associated Sid2p is inactive. This self-association is mediated by a region that overlaps with Mob1p and SPB binding sites. Overexpression of Mob1p is able to disrupt the self-association of Sid2p. Taken together, our results suggest that Sid2p kinase may utilize multiple modes of regulation including self-association, Mob1p binding, and phosphorylation to achieve its full activity at an appropriate time and place in the cell.     

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.     

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.     

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.