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 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.     

Proper Actin Ring Formation and Septum Constriction Requires Coordinated Regulation of SIN and MOR Pathways through the Germinal Centre Kinase MST-1

Nuclear DBF2p-related (NDR) kinases constitute a functionally conserved protein family of eukaryotic regulators that control cell division and polarity. In fungi, they function as effector kinases of the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by pathway-specific germinal centre (GC) kinases. We characterized a third GC kinase, MST-1, that connects both kinase cascades. Genetic and biochemical interactions with SIN components and life cell imaging identify MST-1 as SIN-associated kinase that functions in parallel with the GC kinase SID-1 to activate the SIN-effector kinase DBF-2. SID-1 and MST-1 are both regulated by the upstream SIN kinase CDC-7, yet in an opposite manner. Aberrant cortical actomyosin rings are formed in Δmst-1, which resulted in mis-positioned septa and irregular spirals, indicating that MST-1-dependent regulation of the SIN is required for proper formation and constriction of the septal actomyosin ring. However, MST-1 also interacts with several components of the MOR network and modulates MOR activity at multiple levels. MST-1 functions as promiscuous enzyme and also activates the MOR effector kinase COT-1 through hydrophobic motif phosphorylation. In addition, MST-1 physically interacts with the MOR kinase POD-6, and dimerization of both proteins inactivates the GC kinase hetero-complex. These data specify an antagonistic relationship between the SIN and MOR during septum formation in the filamentous ascomycete model Neurospora crassa that is, at least in part, coordinated through the GC kinase MST-1. The similarity of the SIN and MOR pathways to the animal Hippo and Ndr pathways, respectively, suggests that intensive cross-communication between distinct NDR kinase modules may also be relevant for the homologous NDR kinases of higher eukaryotes.     

The meiosis-specific Sid2p-related protein Slk1p regulates forespore membrane assembly in fission yeast

Cytokinesis in all organisms involves the creation of membranous barriers that demarcate individual daughter cells. In fission yeast, a signaling module termed the septation initiation network (SIN) plays an essential role in the assembly of new membranes and cell wall during cytokinesis. In this study, we have characterized Slk1p, a protein-kinase related to the SIN component Sid2p. Slk1p is expressed specifically during meiosis and localizes to the spindle pole bodies (SPBs) during meiosis I and II in a SIN-dependent manner. Slk1p also localizes to the forespore membrane during sporulation. Cells lacking Slk1p display defects associated with sporulation, leading frequently to the formation of asci with smaller and/or fewer spores. The ability of slk1Δ cells to sporulate, albeit inefficiently, is fully abolished upon compromise of function of Sid2p, suggesting that Slk1p and Sid2p play overlapping roles in sporulation. Interestingly, increased expression of the syntaxin Psy1p rescues the sporulation defect of sid2-250 slk1Δ. Thus, it is likely that Slk1p and Sid2p play a role in forespore membrane assembly by facilitating recruitment of components of the secretory apparatus, such as Psy1p, to allow membrane expansion. These studies thereby provide a novel link between the SIN and vesicle trafficking during 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.     

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 an Actomyosin Contractile Ring in Saccharomyces cerevisiae Cytokinesis

In Saccharomyces cerevisiae, the mother cell and bud are connected by a narrow neck. The mechanism by which this neck is closed during cytokinesis has been unclear. Here we report on the role of a contractile actomyosin ring in this process. Myo1p (the only type II myosin in S. cerevisiae) forms a ring at the presumptive bud site shortly before bud emergence. Myo1p ring formation depends on the septins but not on F-actin, and preexisting Myo1p rings are stable when F-actin is depolymerized. The Myo1p ring remains in the mother–bud neck until the end of anaphase, when a ring of F-actin forms in association with it. The actomyosin ring then contracts to a point and disappears. In the absence of F-actin, the Myo1p ring does not contract. After ring contraction, cortical actin patches congregate at the mother–bud neck, and septum formation and cell separation rapidly ensue. Strains deleted for MYO1 are viable; they fail to form the actin ring but show apparently normal congregation of actin patches at the neck. Some myo1Δ strains divide nearly as efficiently as wild type; other myo1Δ strains divide less efficiently, but it is unclear whether the primary defect is in cytokinesis, septum formation, or cell separation. Even cells lacking F-actin can divide, although in this case division is considerably delayed. Thus, the contractile actomyosin ring is not essential for cytokinesis in S. cerevisiae. In its absence, cytokinesis can still be completed by a process (possibly localized cell–wall synthesis leading to septum formation) that appears to require septin function and to be facilitated by F-actin.     

The Ras/cAMP Pathway and the CDK-Like Kinase Ime2 Regulate the MAPK Smk1 and Spore Morphogenesis in Saccharomyces cerevisiae

Meiotic development (sporulation) in the yeast Saccharomyces cerevisiae is induced by nutritional deprivation. Smk1 is a meiosis-specific MAP kinase homolog that controls spore morphogenesis after the meiotic divisions have taken place. In this study, recessive mutants that suppress the sporulation defect of a smk1-2 temperature-sensitive hypomorph were isolated. The suppressors are partial function alleles of CDC25 and CYR1, which encode the Ras GDP/GTP exchange factor and adenyl cyclase, respectively, and MDS3, which encodes a kelch-domain protein previously implicated in Ras/cAMP signaling. Deletion of PMD1, which encodes a Mds3 paralog, also suppressed the smk1-2 phenotype, and a mds3-Δ pmd1-Δ double mutant was a more potent suppressor than either single mutant. The mds3-Δ, pmd1-Δ, and mds3-Δ pmd1-Δ mutants also exhibited mitotic Ras/cAMP phenotypes in the same rank order. The effect of Ras/cAMP pathway mutations on the smk1-2 phenotype required the presence of low levels of glucose. Ime2 is a meiosis-specific CDK-like kinase that is inhibited by low levels of glucose via its carboxy-terminal regulatory domain. IME2-ΔC241, which removes the carboxy-terminal domain of Ime2, exacerbated the smk1-2 spore formation phenotype and prevented cyr1 mutations from suppressing smk1-2. Inhibition of Ime2 in meiotic cells shortly after Smk1 is expressed revealed that Ime2 promotes phosphorylation of Smk1's activation loop. These findings demonstrate that nutrients can negatively regulate Smk1 through the Ras/cAMP pathway and that Ime2 is a key activator of Smk1 signaling.     

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.     

Phosphatidylcholine Supply to Peroxisomes of the Yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, phosphatidylcholine (PC), the major phospholipid (PL) of all organelle membranes, is synthesized via two different pathways. Methylation of phosphatidylethanolamine (PE) catalyzed by the methyl transferases Cho2p/Pem1p and Opi3p/Pem2p as well as incorporation of choline through the CDP (cytidine diphosphate)-choline branch of the Kennedy pathway lead to PC formation. To determine the contribution of these two pathways to the supply of PC to peroxisomes (PX), yeast mutants bearing defects in the two pathways were cultivated under peroxisome inducing conditions, i.e. in the presence of oleic acid, and subjected to biochemical and cell biological analyses. Phenotype studies revealed compromised growth of both the cho20Δopi3Δ (mutations in the methylation pathway) and the cki1Δdpl1Δeki1Δ (mutations in the CDP-choline pathway) mutant when grown on oleic acid. Analysis of peroxisomes from the two mutant strains showed that both pathways produce PC for the supply to peroxisomes, although the CDP-choline pathway seemed to contribute with higher efficiency than the methylation pathway. Changes in the peroxisomal lipid pattern of mutants caused by defects in the PC biosynthetic pathways resulted in changes of membrane properties as shown by anisotropy measurements with fluorescent probes. In summary, our data define the origin of peroxisomal PC and demonstrate the importance of PC for peroxisome membrane formation and integrity.     

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.     

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.     

Missense Mutations Allow a Sequence-Blind Mutant of SpoIIIE to Successfully Translocate Chromosomes during Sporulation

SpoIIIE directionally pumps DNA across membranes during Bacillus subtilis sporulation and vegetative growth. The sequence-reading domain (γ domain) is required for directional DNA transport, and its deletion severely impairs sporulation. We selected suppressors of the spoIIIEΔγ sporulation defect. Unexpectedly, many suppressors were intragenic missense mutants, and some restore sporulation to near-wild-type levels. The mutant proteins are likely not more abundant, faster at translocating DNA, or sequence-sensitive, and rescue does not involve the SpoIIIE homolog SftA. Some mutants behave differently when co-expressed with spoIIIEΔγ, consistent with the idea that some, but not all, variants may form mixed oligomers. In full-length spoIIIE, these mutations do not affect sporulation, and yet the corresponding residues are rarely found in other SpoIIIE/FtsK family members. The suppressors do not rescue chromosome translocation defects during vegetative growth, indicating that the role of the γ domain cannot be fully replaced by these mutations. We present two models consistent with our findings: that the suppressors commit to transport in one arbitrarily-determined direction or delay spore development. It is surprising that missense mutations somehow rescue loss of an entire domain with a complex function, and this raises new questions about the mechanism by which SpoIIIE pumps DNA and the roles SpoIIIE plays in vivo.     

Loss of the Thioredoxin Reductase Trr1 Suppresses the Genomic Instability of Peroxiredoxin tsa1 Mutants

The absence of Tsa1, a key peroxiredoxin that scavenges H₂O₂ in Saccharomyces cerevisiae, causes the accumulation of a broad spectrum of mutations. Deletion of TSA1 also causes synthetic lethality in combination with mutations in RAD51 or several key genes involved in DNA double-strand break repair. In the present study, we propose that the accumulation of reactive oxygen species (ROS) is the primary cause of genome instability of tsa1Δ cells. In searching for spontaneous suppressors of synthetic lethality of tsa1Δ rad51Δ double mutants, we identified that the loss of thioredoxin reductase Trr1 rescues their viability. The trr1Δ mutant displayed a Can^R mutation rate 5-fold lower than wild-type cells. Additional deletion of TRR1 in tsa1Δ mutant reduced substantially the Can^R mutation rate of tsa1Δ strain (33-fold), and to a lesser extent, of rad51Δ strain (4-fold). Loss of Trr1 induced Yap1 nuclear accumulation and over-expression of a set of Yap1-regulated oxido-reductases with antioxidant properties that ultimately re-equilibrate intracellular redox environment, reducing substantially ROS-associated DNA damages. This trr1Δ -induced effect was largely thioredoxin-dependent, probably mediated by oxidized forms of thioredoxins, the primary substrates of Trr1. Thioredoxin Trx1 and Trx2 were constitutively and strongly oxidized in the absence of Trr1. In trx1Δ trx2Δ cells, Yap1 was only moderately activated; consistently, the trx1Δ trx2Δ double deletion failed to efficiently rescue the viability of tsa1Δ rad51Δ. Finally, we showed that modulation of the dNTP pool size also influences the formation of spontaneous mutation in trr1Δ and trx1Δ trx2Δ strains. We present a tentative model that helps to estimate the respective impact of ROS level and dNTP concentration in the generation of spontaneous mutations.     

DNA Repair Pathway Selection Caused by Defects in TEL1, SAE2, and De Novo Telomere Addition Generates Specific Chromosomal Rearrangement Signatures

Whole genome sequencing of cancer genomes has revealed a diversity of recurrent gross chromosomal rearrangements (GCRs) that are likely signatures of specific defects in DNA damage response pathways. However, inferring the underlying defects has been difficult due to insufficient information relating defects in DNA metabolism to GCR signatures. By analyzing over 95 mutant strains of Saccharomyces cerevisiae, we found that the frequency of GCRs that deleted an internal CAN1/URA3 cassette on chrV L while retaining a chrV L telomeric hph marker was significantly higher in tel1Δ, sae2Δ, rad53Δ sml1Δ, and mrc1Δ tof1Δ mutants. The hph-retaining GCRs isolated from tel1Δ mutants contained either an interstitial deletion dependent on non-homologous end-joining or an inverted duplication that appeared to be initiated from a double strand break (DSB) on chrV L followed by hairpin formation, copying of chrV L from the DSB toward the centromere, and homologous recombination to capture the hph-containing end of chrV L. In contrast, hph-containing GCRs from other mutants were primarily interstitial deletions (mrc1Δ tof1Δ) or inverted duplications (sae2Δ and rad53Δ sml1Δ). Mutants with impaired de novo telomere addition had increased frequencies of hph-containing GCRs, whereas mutants with increased de novo telomere addition had decreased frequencies of hph-containing GCRs. Both types of hph-retaining GCRs occurred in wild-type strains, suggesting that the increased frequencies of hph retention were due to the relative efficiencies of competing DNA repair pathways. Interestingly, the inverted duplications observed here resemble common GCRs in metastatic pancreatic cancer.     

Shared and independent roles for a Galpha(i) protein and adenylyl cyclase in regulating development and stress responses in Neurospora crassa

Growth and development are regulated using cyclic AMP (cAMP)-dependent and -independent pathways in Neurospora crassa. The cr-1 adenylyl cyclase mutant lacks detectable cAMP and exhibits numerous defects, including colonial growth habit, short aerial hyphae, premature conidiation on plates, inappropriate conidiation in submerged culture, and increased thermotolerance. Evidence suggests that the heterotrimeric Gα protein GNA-1 is a direct positive regulator of adenylyl cyclase. Δgna-1 strains are female-sterile, and Δgna-1 strains have reduced apical extension rates on normal and hyperosmotic medium, greater resistance to oxidative and heat stress, and stunted aerial hyphae compared to the wild-type strain. In this study, a Δgna-1 cr-1 double mutant was analyzed to differentiate cAMP-dependent and -independent signaling pathways regulated by GNA-1. Δgna-1 cr-1 mutants have severely restricted colonial growth and do not produce aerial hyphae on plates or in standing liquid cultures. Addition of cAMP to plates or standing liquid cultures rescues cr-1, but not Δgna-1 cr-1, defects, which is consistent with previous results demonstrating that Δgna-1 mutants do not respond to exogenous cAMP. The females of all strains carrying the Δgna-1 mutation are sterile; however, unlike cr-1 and Δgna-1 strains, the Δgna-1 cr-1 mutant does not produce protoperithecia. The Δgna-1 and cr-1 mutations were synergistic with respect to inappropriate conidiation during growth in submerged culture. Thermotolerance followed the order wild type < Δgna-1 < cr-1 = Δgna-1 cr-1, consistent with a cAMP-dependent process. Taken together, the results suggest that in general, GNA-1 and CR-1 regulate N. crassa growth and development using parallel pathways, while thermotolerance is largely dependent on cAMP.