The Fission Yeast SEL1 Domain Protein Cfh3p: A NOVEL REGULATOR OF THE GLUCAN SYNTHASE Bgs1p WHOSE FUNCTION IS MORE RELEVANT UNDER STRESS CONDITIONS*S?

In Schizosaccharomyces pombe, Bgs1/Cps1p is a β(1,3)-glucan synthase required for linear β(1,3)-glucan synthesis and primary septum formation. Here, we have studied the regulation of Bgs1p by Cfh3/Chr4p, a member of a family of conserved adaptor proteins, which resembles the chitin synthase regulator Chs4p from Saccharomyces cerevisiae and Candida albicans. cfh3Δ cells showed a genetic interaction with cps1-191, and Cfh3p co-immunoprecipitated with Bgs1/Cps1p. In the absence of cfh3⁺, cells were more sensitive to digestion by glucanases, and both Calcofluor staining and glucan synthesis were reduced. We found that in a wild-type strain, β(1,3)-glucan synthesis was reduced under stress conditions. In the cfh3Δ, cps1-191, and cfh3Δ cps1-191 strains, β(1,3)-glucan synthesis was further reduced, and growth was impaired under stress conditions, suggesting that Cfh3p and Bgs1p might play a role in ensuring growth in unfavorable environments. In a cfh3Δ mutant, Bgs1p was delocalized when the cells were distressed, but a blockade in endocytosis prevented this delocalization. Finally, we found that the SEL1 repeats are required for Cfh3p function. These results show that Cfh3p is a regulatory protein for Bgs1p and that its function is particularly necessary when the cells are undergoing stress.In Schizosaccharomyces pombe, the primary septum, composed of linear and branchedβ(1,3)-glucan, is surrounded by a secondary septum with a composition similar to that of the lateral cell wall (branched β(1,3)-glucan, β(1,6)-glucan, α(1,3)-glucan, and mannoproteins (1, 2)). Bgs1/Cps1p is the β(1,3)-glucan synthase responsible for the synthesis of linear β(1,3)-glucan and the primary septum structure (3). In the absence of this activity, the cells are able to form remedial septa that do not contain linear β(1,3)-glucan. These septa do not stain with low concentrations of Calcofluor and cannot be degraded by glucanases, so the cells remain chained, forming hyphal structures (3). In S. pombe there are four glucan synthase homologues, bgs1⁺/cps1⁺, bgs2⁺, bgs3⁺, and bgs4⁺ (4–9). In the case of the bgs1Δ cells, apical growth takes place in two opposite directions, producing branched cells with a dichotomic growth. This phenotype is not observed in bgs2Δ, bgs3Δ,or bgs4Δ mutants, thus suggesting that Bgs1p might play a specific role in the control of cell growth (3). Finally, Bgs1p is a component of the cytokinesis checkpoint, which coordinates mitosis with actomyosin ring contraction and septum synthesis (10, 11).The study of Bgs1p regulation should help us to understand the control of cell wall synthesis and cytokinesis in S. pombe. Bgs1p requires an active Septation Initiation Network (SIN), an assembled contractile actomyosin ring, and the type-V myosin Myo52p to localize properly at the division site (9, 12, 13). Regarding the regulation of biochemical activity, the PKC homologues Pck1p and Pck2p activate β(1,3)-glucan synthesis in an unknown way (14) while the Rho1p GTPase is a direct activator of the β(1,3)-glucan synthase catalytic subunit (15).The cell wall is a morphogenetic determinant, but also an essential cellular structure that protects the organism against cell lysis in hypoosmotic environments. In S. pombe, the mitogen-activated protein (MAP)³ kinase Spm1/Pmk1p pathway (also known as the cell integrity pathway) regulates growth and morphogenesis in response to multiple stresses, including hyper- or hypoosmotic conditions, nutrient limitation, and cell wall-damaging compounds (16, 17). Proper growth and morphology under hyperosmotic conditions also requires Skb1p and Skb5p, which are regulators of the Shk1/Orb2/Pak1p kinase (18, 19). However, little is known about the role of the cell wall as a protective element against hyperosmotic conditions, the presence of high concentrations of chloride ions, or nutrient limitation.Here we examine the role of the Cfh3/Chr4 protein in cell wall synthesis and response to stress. Cfh3p shares significant similarity with the Chs4 proteins from Saccharomyces cerevisiae and from Candida albicans, which are chitin synthase regulators (20, 21). In S. cerevisiae, regulation of the chitin synthase Chs3p by Chs4p is complex and still not well understood. Chs4p is required for the correct localization of Cfh3p at the bud neck by mediating its anchorage to septins through the adaptor protein Bni4p, but it also acts as a biochemical activator and is required for the stability of Chs3p at the plasma membrane (20, 22–25). In S. pombe, no chitin synthesis occurs during vegetative growth (26–29), glucan being the main cell wall component, there is no Bni4p homologue, and septins are involved in cell separation but not in septum synthesis (30, 31). We wanted to investigate whether Cfh3p played any role in cell wall synthesis and/or morphogenesis in the fission yeast. We found that in the absence of cfh3⁺, the cells showed reduced β-glucan synthesis and impaired growth under stress conditions. These phenotypes were aggravated in a double cfh3Δ cps1-191 mutant. Our results suggest that Cfh3p is a regulator of the β-glucan synthase Bgs1p whose presence is more critical when the cells are undergoing environmental stress. Cfh3p belongs to a conserved family of scaffold proteins characterized by the presence of tandem repeats of SEL1 domains, which are involved in signal transduction during different cellular processes (32). Here we found that the SEL1 domains in Cfh3p were required for its function.     

The (1,3)beta-D-glucan synthase subunit Bgs1p is responsible for the fission yeast primary septum formation

Cytokinesis is a crucial event in the cell cycle of all living cells. In fungal cells, it requires co-ordinated contraction of an actomyosin ring and synthesis of both plasmatic membrane and a septum structure that will constitute the new cell wall end. Schizosaccharomyces pombe contains four essential putative (1,3)beta-d-glucan synthase catalytic subunits, Bgs1p to Bgs4p. Here we examined the function of Bgs1p in septation by studying the lethal phenotypes of bgs1(+) shut-off and bgs1Delta cells and demonstrated that Bgs1p is responsible and essential for linear (1,3)beta-d-glucan and primary septum formation. bgs1(+) shut-off generates a more than 300-fold Bgs1p reduction, but the septa still present large amounts of disorganized linear (1,3)beta-d-glucan and partial primary septa. Conversely, both structures are absent in bgs1Delta cells, where there is no Bgs1p. The septum analysis of bgs1(+)-repressed cells indicates that linear (1,3)beta-d-glucan is necessary but not sufficient for primary septum formation. Linear (1,3)beta-d-glucan is the polysaccharide that specifically interacts with the fluorochrome Calcofluor white in fission yeast. We also show that in the absence of Bgs1p abnormal septa are formed, but the cells cannot separate and eventually die.     

Extracellular cell wall β(1,3)glucan is required to couple septation to actomyosin ring contraction

Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, β(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(1,3)glucan is required for cytokinesis to connect the cell wall with the plasma membrane and for contractile ring function, as proposed for the equivalent extracellular matrix in animal cells.     

Drc1p/Cps1p, a 1,3-beta-glucan synthase subunit, is essential for division septum assembly in Schizosaccharomyces pombe.

Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin-based contractile ring. A division septum is formed centripetally, concomitant with ring constriction. Although several genes essential for cytokinesis have been described previously, enzymes that participate in the assembly of the division septum have not been identified. Here we describe a temperature-sensitive mutation, drc1-191, that prevents division septum assembly and causes mutant cells to arrest with a stable actomyosin ring. Unlike the previously characterized cytokinesis mutants, which undergo multiple mitotic cycles, drc1-191 is the first cytokinesis mutant that arrests with two interphase nuclei. Interestingly, unlike drc1-191, drc1-null mutants proceed through multiple mitotic cycles, leading to the formation of large cells with many nuclei. drc1 is allelic to cps1, which encodes a 1,3-beta-glucan synthase subunit. We conclude that Drc1p/Cps1p is not required for cell elongation and cell growth, but plays an essential role in assembly of the division septum. Furthermore, it appears that constriction of the actomyosin ring might depend on assembly of the division septum. We discuss possible mechanisms that account for the differences in the phenotypes of the drc1-191 and the drc1-null mutants and also reflect the potential links between Drc1p and other cytokinesis regulators.     

Bgs3p,a putative 1,3-beta-glucan synthase subunit,is required for cell wall assembly in Schizosaccharomyces pombe

β-Glucans are the main components of the fungal cell wall. Fission yeast possesses a family of β-glucan synthase-related genes. We describe here the cloning and characterization of bgs3⁺, a new member of this family. bgs3⁺ was cloned as a suppressor of a mutant hypersensitive to Echinocandin and Calcofluor White, drugs that interfere with cell wall biosynthesis. Disruption of the gene is lethal, and a decrease in Bgs3p levels leads to rounded cells with thicker walls, slightly reduces the amount of the β-glucan, and raises the amount of α-glucan polymer. These cells finally died. bgs3⁺ is expressed in vegetative cells grown in different conditions and during mating and germination and is not enhanced by stress situations. Consistent with the observed expression pattern, Bgs3-green fluorescence protein (GFP-Bgs3p) was found at the growing tips during interphase and at the septum prior to cytokinesis, always localized to growth areas. We also found GFP-Bgs3p in mating projections, during the early stages of zygote formation, and at the growing pole during ascospore germination. We conclude that Bgs3p localization is restricted to growth areas and that Bgs3p is a glucan synthase homologue required for cell wall biosynthesis and cell elongation in the fission yeast life cycle.     

A Highlights from MBoC Selection: Inhibition of cell membrane ingression at the division site by cell walls in fission yeast

Eukaryotic cells assemble actomyosin rings during cytokinesis to function as force-generating machines to drive membrane invagination and to counteract the intracellular pressure and the cell surface tension. How the extracellular matrix affects actomyosin ring contraction has not been fully explored. While studying the Schizosaccharomyces pombe 1,3-β-glucan-synthase mutant cps1-191, which is defective in division septum synthesis and arrests with a stable actomyosin ring, we found that weakening of the extracellular glycan matrix caused the generated spheroplasts to divide under the nonpermissive condition. This nonmedial slow division was dependent on a functional actomyosin ring and vesicular trafficking, but independent of normal septum synthesis. Interestingly, the high intracellular turgor pressure appears to play a minimal role in inhibiting ring contraction in the absence of cell wall remodeling in cps1-191 mutants, as decreasing the turgor pressure alone did not enable spheroplast division. We propose that during cytokinesis, the extracellular glycan matrix restricts actomyosin ring contraction and membrane ingression, and remodeling of the extracellular components through division septum synthesis relieves the inhibition and facilitates actomyosin ring contraction.     

Involvement of Smi1 in cell wall integrity and glucan synthase Bgs4 localization during fission yeast cytokinesis

Cytokinesis is the final step of the cell-division cycle. In fungi, it relies on the coordination of constriction of an actomyosin contractile ring and construction of the septum at the division site. Glucan synthases synthesize glucans, which are the major components in fungal cell walls and division septa. It is known that Rho1 and Rho2 GTPases regulate glucan synthases Bgs1, Bgs4, and Ags1, and that Sbg1 and the F-BAR protein Cdc15 play roles in Bgs1 stability and delivery to the plasma membrane. Here we characterize Smi1, an intrinsically disordered protein that interacts with Bgs4 and regulates its trafficking and localization in fission yeast. Smi1 is important for septum integrity, and its absence causes severe lysis during cytokinesis. Smi1 localizes to secretory vesicles and moves together with Bgs4 toward the division site. The concentrations of the glucan synthases Bgs1 and Bgs4 and the glucanases Agn1 and Bgl2 decrease at the division site in the smi1 mutant, but Smi1 seems to be more specific to Bgs4. Mistargeting of Smi1 to mitochondria mislocalizes Bgs4 but not Bgs1. Together, our data reveal a novel regulator of glucan synthases and glucanases, Smi1, which is more important for Bgs4 trafficking, stability, and localization during cytokinesis.     

Bgs2p, a 1,3-beta-glucan synthase subunit, is essential for maturation of ascospore wall in Schizosaccharomyces pombe

Previously we have reported that Drc1p/Cps1p, a 1,3-beta-glucan synthase subunit, is essential for division septum assembly in Schizosaccharomyces pombe. In this report, we present evidence that S. pombe Bgs2p, a 1,3-beta-glucan synthase that shows 56% identity to Drc1p/Cps1p, is essential for maturation of ascospore wall in S. pombe, but is not required for vegetative growth. Diploid cells homozygous for the bgs2-null mutation, as well as homothallic bgs2-null mutant haploids undergo meiosis normally. However, a 1, 3-beta-glucan containing spore wall is not assembled in these cells. The spores resulting from meiosis of a bgs2-null mutant lyse upon release from the ascus and are therefore inviable. Using a green fluorescent protein-tagged Bgs2p, we demonstrate that Bgs2p is localized at the periphery of the ascospores during meiosis and sporulation. However, Bgs2p is not detected in vegetative cells. We conclude that Bgs2p is required for 1,3-beta-glucan synthesis during ascospore wall maturation.     

Cooperation between Paxillin-like Protein Pxl1 and Glucan Synthase Bgs1 Is Essential for Actomyosin Ring Stability and Septum Formation in Fission Yeast

In fungal cells cytokinesis requires coordinated closure of a contractile actomyosin ring (CAR) and synthesis of a special cell wall structure known as the division septum. Many CAR proteins have been identified and characterized, but how these molecules interact with the septum synthesis enzymes to form the septum remains unclear. Our genetic study using fission yeast shows that cooperation between the paxillin homolog Pxl1, required for ring integrity, and Bgs1, the enzyme responsible for linear β(1,3)glucan synthesis and primary septum formation, is required for stable anchorage of the CAR to the plasma membrane before septation onset, and for cleavage furrow formation. Thus, lack of Pxl1 in combination with Bgs1 depletion, causes failure of ring contraction and lateral cell wall overgrowth towards the cell lumen without septum formation. We also describe here that Pxl1 concentration at the CAR increases during cytokinesis and that this increase depends on the SH3 domain of the F-BAR protein Cdc15. In consequence, Bgs1 depletion in cells carrying a cdc15_ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1. On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins. In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast.     

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.     

Fission yeast Ags1 confers the essential septum strength needed for safe gradual cell abscission

Fungal cytokinesis requires the assembly of a dividing septum wall. In yeast, the septum has to be selectively digested during the critical cell separation process. Fission yeast cell wall α(1-3)glucan is essential, but nothing is known about its localization and function in the cell wall or about cooperation between the α- and β(1-3)glucan synthases Ags1 and Bgs for cell wall and septum assembly. Here, we generate a physiological Ags1-GFP variant and demonstrate a tight colocalization with Bgs1, suggesting a cooperation in the important early steps of septum construction. Moreover, we define the essential functions of α(1-3)glucan in septation and cell separation. We show that α(1-3)glucan is essential for both secondary septum formation and the primary septum structural strength needed to support the physical forces of the cell turgor pressure during cell separation. Consequently, the absence of Ags1 and therefore α(1-3)glucan generates a special and unique side-explosive cell separation due to an instantaneous primary septum tearing caused by the turgor pressure.     

Polarity determinants Tea1p, Tea4p, and Pom1p inhibit division-septum assembly at cell ends in fission yeast

Correct positioning of the cell-division plane is crucial for cell function in all organisms. The fission yeast Schizosaccharomyces pombe divides by utilizing an actomyosin-based contractile ring and is an attractive model for the study of cytokinesis. The metazoan anillin-related protein Mid1p stimulates medial assembly of the division septum by recruiting actomyosin-ring components to the medial cortex. Here, we describe an inhibitory mechanism, involving the cell-end-localized polarity determinants Tea1p, Tea4p/Wsh3p, and Pom1p (tip complex), which prevents division-septum assembly at the cell ends. While Mid1p and the tip complex are dispensable for cell viability, their simultaneous loss leads to lethality. The FER/CIP homology protein Cdc15p, which organizes the actomyosin ring and cell membranes during cytokinesis, is a candidate for regulation by the tip complex. Since dual regulation of division-site placement is also seen in nematodes, such regulation might be a general feature of eukaryotic cytokinesis.     

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.     

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.     

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.     

Verprolin cytokinesis function mediated by the Hof one trap domain

In budding yeast, partitioning of the cytoplasm during cytokinesis can proceed via a pathway dependent on the contractile actomyosin ring, as in other eukaryotes, or alternatively via a septum deposition pathway dependent on an SH3 domain protein, Hof1/Cyk2 (the yeast PSTPIP1 ortholog). In dividing yeast cells, Hof1 forms a ring at the bud neck distinct from the actomyosin ring, and this zone is active in septum deposition. We previously showed the yeast Wiskott-Aldrich syndrome protein (WASP)-interacting protein (WIP) ortholog, verprolin/Vrp1/End5, interacts with Hof1 and facilitates Hof1 recruitment to the bud neck. A Vrp1 fragment unable to interact with yeast WASP (Las17/Bee1), localize to the actin cytoskeleton or function in polarization of the cortical actin cytoskeleton nevertheless retains function in Hof1 recruitment and cytokinesis. Here, we show the ability of this Vrp1 fragment to bind the Hof1 SH3 domain via its Hof one trap (HOT) domain is critical for cytokinesis. The Vrp1 HOT domain consists of three tandem proline-rich motifs flanked by serines. Unexpectedly, the Hof1 SH3 domain itself is not required for cytokinesis and indeed appears to negatively regulate cytokinesis. The Vrp1 HOT domain promotes cytokinesis by binding to the Hof1 SH3 domain and counteracting its inhibitory effect.     

Contributions of turgor pressure, the contractile ring, and septum assembly to forces in cytokinesis in fission yeast

A paradigm of cytokinesis in animal cells is that the actomyosin contractile ring provides the primary force to divide the cell [1]. In the fission yeast Schizosaccharomyces pombe, cytokinesis also involves a conserved cytokinetic ring, which has been generally assumed to provide the force for cleavage [2-4] (see also [5]). However, in contrast to animal cells, cytokinesis in yeast cells also requires the assembly of a cell wall septum [6], which grows centripetally inward as the ring closes. Fission yeast, like other walled cells, also possess high (MPa) turgor pressure [7-9]. Here, we show that turgor pressure is an important factor in the mechanics of cytokinesis. Decreasing effective turgor pressure leads to an increase in cleavage rate, suggesting that the inward force generated by the division apparatus opposes turgor pressure. The contractile ring, which is predicted to provide only a tiny fraction of the mechanical stress required to overcome turgor, is largely dispensable for ingression; once septation has started, cleavage can continue in the absence of the contractile ring. Scaling arguments and modeling suggest that the large forces for cytokinesis are not produced by the contractile ring but are driven by the assembly of cell wall polymers in the growing septum.     

The 14-3-3 protein rad24p modulates function of the cdc14p family phosphatase clp1p/flp1p in fission yeast

Schizosaccharomyces pombe cells divide through the use of an actomyosin-based contractile ring. In response to perturbation of the actomyosin ring, S. pombe cells delay in a "cytokinesis-competent" state characterized by continuous repair and maintenance of the actomyosin ring and a G2 delay. This checkpoint mechanism requires the function of the Cdc14p-family phosphatase Clp1p/Flp1p and the septation initiation network (SIN). In response to cytokinetic defects, Clp1p, normally nucleolar in interphase, is retained in the cytoplasm until completion of cell division in a SIN-dependent manner. Here, we show that a phosphorylated form of Clp1p binds the 14-3-3 protein Rad24p and is retained in the cytoplasm in a Rad24p-dependent manner in response to cytokinesis defects. This physical interaction depends on the function of the SIN component, Sid2p. In the absence of Rad24p, cells are unable to maintain SIN signaling and lose viability upon mild cytokinetic stress. The requirement of Rad24p in this checkpoint is bypassed by ectopic activation of the SIN. Furthermore, SIN-dependent nuclear exclusion of Clp1p is dependent on Rad24p function. We conclude that Rad24p-mediated cytoplasmic retention of Clp1p/Flp1p is important for cell viability upon stress to the division apparatus.     

The Saccharomyces cerevisiae actin cytoskeletal component Bsp1p has an auxiliary role in actomyosin ring function and in the maintenance of bud-neck structure

Iqg1p is a component of the actomyosin contractile ring that is required for actin recruitment and septum deposition. Cells lacking Iqg1p function have an altered bud-neck structure and fail to form a functional actomyosin contractile ring resulting in a block to cytokinesis and septation. Here it is demonstrated that increased expression of the actin cytoskeleton associated protein Bsp1p bypasses the requirement for contractile ring function. This also correlates with reduced bud-neck width and remedial septum formation. Increased expression of this protein in a temperature-sensitive iqg1-1 background causes remedial septum formation at the bud neck that is reliant upon chitin synthase III activity and restores cell separation. The observed suppression correlates with a restoration of normal bud-neck structure. While Bsp1p is a component of the contractile ring, its recruitment to the bud neck is not required for the observed suppression. Loss of Bsp1p causes a brief delay in the redistribution of the actin cytoskeleton normally observed at the end of actin ring contraction. Compromise of Iqg1p function, in the absence of Bsp1p function, leads to a profound change in the distribution of actin and the pattern of cell growth accompanied by a failure to complete cytokinesis and cell separation.